FORESTRY SCIENCES
In preparation:
edited by
1.M. BONGA
Maritime Forest Research Centre, Fredericton, Canada
and
D.l. DURZAN
University of California, Davis, U.S.A.
1982
SPRINGER-SCIENCE+BUSINESS MEDIA, B.V.
Library of Congress Cataloging_.in Publication Data
Main entry under title:
Tissue culture in forestry.
(Forestry sciences)
Includes indexes.
1. Plant tissue culture. 2. Forests and forestry.
I. Bonga, J. M. II. Durzan, D. J. III. Series.
SD403.5.T57 634.9'56 82-6292
ISBN 978-90-481-8272-5 ISBN 978-94-017-3538-4 (eBook) AACR2
DOI 10.1007/978-94-017-3538-4
All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or
transmitted in any form or by any means, mechanical, photocopying, recording, or otherwise,
without the prior written permission of the publishers, SpIinger-Science+Business Media, B. V.
v
TABLE OF CONTENTS
1. INTRODUCTION 1
1. INTRODUCTION
2. LABORATORY ORGANIZATION
2.1. General layout
2.2. Facilities for tissue excision and transfer
2.3. Dish washing
2.4. Glassware and chemical storage facilities
2.5. Water purification
2.5.1. Distillation
2.5.2. Deionization
2.5.3. Reverse osmosis
2.5.4. Storage
2.6. Glassware and media sterilization
2.7. Shakers and fermentors
2.8. In~ubation facilities
3. MEDIA PREPARATION
3.1. Functions of some media components
3.1.1. Agar and its substitutes
3.1.2. Minerals, ratios, and concentrations
3.1.3. Osmoticums
3.1.4. Charcoal
3.1.5. EDTA
3.1.6. Buffers
3.2. Culture vessels and closures
3.3. Storage of nutrient media
4. PREPARATION OF CULTURES
4.1. Condition of plant material
4.2. Collection and storage
4.3. Surface sterilization
4.4. Excision and transfer of tissues
4.5. Pre-culture treatments
4.6. Incubation environment
4.7. Transfer to soil
5. CONCLUSION
1. INTRODUCTION
2. PRODUCTION CYCLE
3. GENETIC RESOURCES
3.1. Energy and fuel-wood species
3.2. Multiple-use species
3.3. Tropical legumes
3.4. Fiber and pulpwood species
4. PROPAGATION SYSTEMS
4.1. Seed orchards
4.2. ~ vitro vegetative propagation
VI
1. INTRODUCTION
2. ORGANOGENESIS IN CALLUS AND SUSPENSION CULTURES
OF GYMNOSPERMS
3. MORPHOGENESIS IN CULTURES OF ORGANS AND ORGAN
SECTIONS
3.1. Axillary bud formation
3.2. Adventitious bud formation
3.2.1. Shoot formation on embryos and cotyledons
3.2.2. Shoot formation along the hypocotyl
3.2.3. Shoot formation on needles
3.3. Embryogenesis
3.4. Formation of shoots
3.4.1. Elongation of shoots from dormant buds
3.4.2. Elongation of shoots from adventitious
and axillary buds
3.5. Root formation
4. REGENERATION FROM EXPLANTS FROM MATURE PLANTSi
REJUVENATION
5. ESTABLISHMENT OF PROPAGULES IN SOIL
6. CONCLUSIONS
1. INTRODUCTION
2. USE OF CONVENTIONAL METHODS OF VEGETATIVE
PROPAGATION IN PRODUCTION FORESTRY
2.1. Past practices and utility
2.2. Modified approaches and applications
2.3. Economic considerations using conventional
or modified propagation techniques
3. VEGETATIVE PROPAGATION VIA TISSUE AND ORGAN CULTURES
3.1. Brief historical account of organogenesis in
woody dicots
3.2. Types of cultures and their application to large
scale commercial propagation
3.2.1. Callus cultures
3.2.2. Organ cultures
3.2.3. Plantlet formation via embryogenesis in
cell suspensions
4. ECONOMIC CONSIDERATIONS
4.1. Cost comparisons of seedlings produced by tissue
culture techniques versus seedlings produced
from seed.
5. PROBLEMS ENCOUNTERED IN PROPAGATION OF TREES
USING TISSUE CULTURE TECHNIQUES
VII
1. INTRODUCTION
2. VALUE OF PALMS AND PROBLEMS ASSOCIATED WITH
THEIR DEVELOPMENT
2.1. Sources of nutrition
2.1.1. Source of edible oils
2.1.2. Source of carbohydrate
2.2. Ornamental use
2.3. Present methods of cultivation and propagation
2.3.1. Coconut palm (Cocos nucifera L.)
2.3.2. Date palm (Phoenrx-dactylifera L.)
2.3.3. Oil palm (Elaeis guineensis Jacq.)
2.3.4. Ornamental palms
3. SOLVING PROBLEMS WITH TISSUE CULTURE - CURRENT
STATUS OF RESEARCH
VIII
1 . INTRODUCTION
2. PATHOGEN CLASSIFICATIONS
2.1. Viruses
2.2. Bacteria
2.3. Nematodes
2.4. Insects
2.5. Fung i
2.5.1. Dual and axenic culture studies
2.5.2. Disease resistance studies with fungi
3. CONCLUSION
1. INTRODUCTION
2. AUXINS
2 . 1. Background
2.2. Indole-3-acetic acid (IAA)
2.3. Indole-3-butyric acid (IBA)
2.4. Naphthaleneacetic acid (NAA)
2.5. 2,4-dichlorophenoxyacetic acid (2,4-D)
2.6. Other auxins
3. CYTOKININS
3.1. Background
3.2. Kinetin
3 . 3. 6-benzylaminopurine (BAP)
3.4. Other cytokinins
4. GIBBERELLINS
4.1. Background
4.2. Effects of gibberellins
5. OTHER GROWTH - REGULATING SUBSTANCES
6. CONCLUSIONS
1. INTRODUCTION
2. IMPORTANCE OF NITROGEN METABOLISM
2.1. Range of naturally occurring nitrogenous
components in forest trees
2.2. Gene expression and mapping
2.3. Metabolic changes in organized and unorganized
systems
2.4. Nitrogen and nutrition
2.5. Aspects of intermediary nitrogen metabolism
3. NITROGEN METABOLISM IN GROWTH AND DEVELOPMENT
3.1. Precultural factors
3.2. Callus formation
3.3. Cell suspensions
3.3.1. Conifers
3.3.2. Acer
3.4. Morphogenesis
3.4.1. Nitrogen metabolism of natural embryos
3.4.2. Somatic embryogenesis
3.4.2.1. Sweetgum (Liquidambar styraciflua)
3.4.2.2. Douglar-fir and loblolly pine
3.4.3. Organogenesis
4. OUTLOOK
1. INTRODUCTION
2. NUTRITIONAL ASPECTS
3. CARBOHYDRATE UPTAKE
4. CARBOHYDRATE METABOLISM
4.1. Sucrose degradation
4.2. Metabolism of other carbon sources
4.3. Hexose mobilization and metabolism
4.3.1. Cell cycle studies
4.3.2. Growth studies
4.3.3. Organized development
4.4. Cell wall biogenesis
4.4.1. Primary cell walls
4.4.2. Cell wall turnover
4.4.3. Secondary cell walls
4.5. Carbon skeleton utilization
5. OSMOTIC ROLE
6. CONCLUDING THOUGHTS
1. INTRODUCTION
2. IN VITRO SELECTION
2.1. Natural variation
2.2. Induction of variation
2.3. Selection techniques
2.4. Plant regeneration
2 . 5. Applications
x
3. SOMATIC HYBRIDIZATION
3.1. Protoplast techniques
3.2. Graft hybridization
4. GENETIC TRANSFORMATION
4.1. Principles
4.2. Procedures
4.2.1. DNA uptake
4.2.2. Transformation using biological vectors
4.2.3. Pollen as a vector in genetic transformation
5. CONCLUSIONS
1. INTRODUCTION
2. JUVENILITY-MATURITY
2.1. Definitions
2.2. Determination in meristems
2.3. Juvenile zones
2.4. Clonal aging
2.5. Genetic stability
2.6. Mechanisms of maturation
2.7. Mechanisms of juvenility retention
2.8. Mechanisms of genetic stability
2.9. Mechanisms of rejuvenation
2.10. Sexual rejuvenation
3. SIGNIFICANCE FOR PROPAGATION BY TISSUE CULTURE
3.1. Choice of explants
3.1.1. Flower parts
3.1.2. Vegetative buds
3.1.3. Roots
3.1.4. Root-shoot junction
3.2. Chemical and physical methods of reducing
organelles
4. SUMMARY AND CONCLUSION
LIST OF CONTRIBUTORS
Over the past few decades tissue culture has rapidly evolved
into one of the major research tools in biology and medicine. It
has presently reached a level of sophistication where its adapta-
tion to large-scale industrial use has become possible in some
areas of agriculture, horticulture, and drug manufacturing. In
forestry, the commercial application of tissue culture is still
in its infancy, but the first inroads have been made, and further
developments can be expected.
The term "Tissue Culture" was coined in the days when the
technique was mainly restricted to the culture of pieces of tis-
sue. However, over the years the term has become somewhat of a
misnomer, because presently not only tissue pieces, but also free
cells, protoplasts, organs, and embryos are cultured.
From an experimental point of view, in vitro systems (tissues
excised from the organism and cultured in isolation) have many
advantages over in vivo ones (tissues left within the organism),
for example: 1) In the living plant the behavior of each part of
tissue is strongly influenced by correlative controls imposed by
the rest of the plant. By isolating a plant part, and culturing
it in vitro, the nature of some of these correlative controls can
be determined. 2) The isolated plant part may be free to
express potentialities that are normally suppressed in vivo, the
most obvious examples being organogenesis and embryogenesis. 3)
All in vitro experimentation is carried out under aseptic condi-
tions and therefore, the tissues and cells are not destroyed by
microorganisms. Furthermore, many chemicals can be applied over
long periods of time without these chemicals being metabolized or
degraded by microorganisms. 4) The physical environment of the
cultures is generally easy to manipulate. Most cultures are
2
trees, because the forest tree literature simply does not provide
the required information. However, it was attempted to keep ref-
erences dealing with organisms other than trees to a minimum, and
to use literature dealing with tree species preferentially.
Tissue culture of forest trees has lagged behind that of many
agricultural crops. The main reasons for that are: 1) 'I'he long
life cycle of trees. 2) If one wishes to use mature trees, rather
than embryos or small seedlings, greenhouse material is hardly
ever available, and explants have to be taken from field grown
trees. Consequently, considerable physiological variation in
explants can be expected because of site differences and annual
fluctuations in climate. 3) Because of breeding problems, gene-
tic variation in trees is generally greater than in agricultural
crops, again resulting in variability and unpredictability in the
experiments. 4) Tissues from mature trees are often morphogenet-
ically unresponsive to the currently used experimental treat-
ments. As a consequence, obtaining in vitro veget~tive propaga-
tion is, for many tree species, still impossible or difficult. 5)
Endogenous microbial contaminants are often present, especially
in tissues of field grown material. Removal of these contaminants
is often difficult or impossible and high contamination rates are
common.
Obviously, problems still abound, and routine application of
tissue culture in forest research and industry has been lagging
as a result of it. However, as the various chapters in this book
demonstrate, the area of tree tissue culture is rapidly advanc-
ing, and new solutions for some of these problems can be expected
in the next decade or so.
4
J.M. BONGA
1. INTRODUCTION
Tissue culture is a technique in which small tissue pieces or organs are
removed from a donor plant and cultured aseptically on a nutrient medium. By
manipulating the chemical composition of the nutrient medium and other environ-
mental parameters, the growth and development of the tissues in culture can be
directed into different channels.
Tissue culture techniques are often plagued by unknown variables. Con-
sequently results obtained in one experiment are not always reproducible in
subsequent ones, or results which can easily be duplicated in one laboratory,
sometimes are not reproducible in another. Problems may also arise when
successful routines established in small-scale initial experiments are modified
to produce the same results on a larger scale, more efficiently, and at lower
cost. Such new routines may mean slightly modified methods of media prepara-
tion, different types of culture vessels, larger growth cabinets, etc., with
each of these steps possibly introducing unsuspected unknown changes, signifi-
cantly affecting the results.
Most tissue culture techniques described in the literature are applicable
universally, although minor modifications may have to be worked out to adapt thl
technjques to local conditions. For example, in laboratories located in areas
with a warm humid climate, or in buildings with high dust levels or air drafts,
precautions to maintain asepsis may have to be much more stringent than in othel
laboratories.
Over the last few decades, there has been a steady trend towards more sophis-
ticated equipment. In a few instances this has led to easier and faster rou-
tines. For example, the weighing of chemicals, which a few decades ago was a
difficult and time consuming process is simple and fast with modern balances.
However, sophistication and automation of equipment is not always a substitute
for experience or dexterity (11, 46, 109), i.e., good results are often obtaine(
with simple, cheap equipment.
5
2. LABORATORY ORGANIZATION
2.1. General layout
Even though equipment has been modernized and some of the techniques have
changed, a modern tissue culture laboratory is still largely organized as de-
scribed by White in his classical work "The Cultivation of Animal and Plant
Cells" (198). However, if one wishes to consult more recent sources, layouts for
a modern tree propagation laboratory and greenhouse have been published (179).
Ideally, a tissue culture laboratory should have one or more sterile rooms
for tissue excision and transfer, a culture medium preparation room, separate
areas for dishwashing and chemical and glassware storage, a cold room for bulk
storage of plant material and prepared culture media, and a temperature control-
led culture room with illuminated shelves or small growth cabinets. However,
space and finances often do not allow this type of laboratory layout. In fact,
it is not uncommon to find several technicians and graduate students working in
one laboratory room without the benefit of several of the amenities mentioned
above, turning out large numbers of "clean" cultures free of contamination.
6
Therefore, simple working conditions are not always an impediment to good work.
Of course this only applies to laboratories involved in the traditional, and
simpler kinds of tree tissue culture. Those where more specialized research i~
carried out, especially in areas such as recombinant DNA and hazardous product~
will require more complex, expensive equipment and strict guidelines for opera-
tion.
2.2. Facilities for tissue excision and transfer
If the tissue culture unit is located in a building with relatively high
levels of airborne microbial spores, or if it is part of a large commercial
enterprise, proper sterile rooms may be a necessity. These are small rooms int
which air is injected through a filtering system designed to remove airborne
dust and spores. They generally have no windows (36, 168), have smooth easily
washed walls and other surfaces, and often are provided with bactericidal
ultraviolet lights to sterilize the room when not in use (36, 99, 198). SteriJ
rooms have several drawbacks; they occupy space, are expensive to build, and
most important, many staff members have misgivings about working regularly in
such a confined, featureless, and windowless environment. In many laboratorieE
therefore, sterile rooms are being replaced by laminar-flow-hoods, which are
suitable for most operations (46, 109, 168). For manipulations requiring only
small work area, a simple box without laminar-air-flow, is often sufficient
(198) (Fig. 1).
To keep sources of microbial spores and dust in the laboratory to a minimum,
petri plates, flasks, and test tubes with contaminated nutrient medium or cul-
tures should be autoclaved unopened, and cleaned as soon as possible. Sources
of microbes and small insects, such as potted plants or other plant material,
may have to be removed from the laboratory if the contamination rate of the
cultures is persistently high.
2.3. Dishwashing
Most laboratories presently have automatic dishwashers in which glassware iE
cleaned by powerful hot-water-detergent jets (46). Most of these machines rinE
in tapwater and in distilled or demineralized water to remove the detergent.
For difficult-to-clean glassware, electric ashing as a means of cleaning has
been suggested (104). For sensitive cell suspension cultures, glassware may
have to be cleaned in a chromic acid - sulfuric acid mixture. This procedure
requires strict safety precautions (168).
7
Fig. 1. A hood for tissue excision and transfer. The hood (90 x 60 x 45 cm) is
made of wood with one slanted glass panel. In this glass panel, near the open
front of the box, are two eyepieces of a dissecting microscope (m) protruding
through a square (15 x 15 cm) hole. Around the eye pieces the hole is sealed
with a small piece of plastic film, taped to the sides of the hole. The plastic
film is flexible enough to allow up and down movement of the optics for focuss-
ing. A small alcohol flame or electric incinerator to sterilize instruments and
the mouths of glassware is placed in one of the back corners under an aluminum
heat shield (h) and ventilation hole (v). The heat shield is required to pre-
vent heat-cracks in the glass. The hood is easily sterilized by occasionally
washing its interior surfaces with 70% alcohol. Preferably, the hood is placed
on a laboratory bench in a draft-free area. To avoid excessive convection
currents inside the hood, it should not be much larger than the one shown here.
This means that the hood is suited only for routines requiring limited space.
ped in paper before autoclaving, the volatiles being generated from the paper b)
the hi gh temperature steam (9, 198). Another problem can be the formation of
volatile inhibitors from rubber stoppers and tubin g during autoclaving (22).
The media should not be autoclaved in large volume in one vessel, but in
small volumes in several vessels. The larger the volume of the medium, the
lower the surface to volume ratio, and the poorer the heat exchange. For exam-
ple, several litres of medium in one flask will not reach the maximum tempera-
ture of the autoclave if autoclaved for the usual 15 min. If autoclaved longer ,
to reach hi g her temperatures, there is the danger of violent boiling during
11
Fig. 2. A bench top sterilizer with a propeller (p) to keep chemicals in solu-
tion during autoclaving, and with an entry port (e) to add filter-sterilized
chemicals. A dispensing pump (d) is used to transfer the nutrient to the cul-
ture vessels.
Many chemicals will partially decompose when autoclaved. For example, carbo-
hydrates, particularly at a slightly acid pH, will undergo some degree of hy-
drolysis and further breakdown when autoclaved (13, 17, 122, 130, 141, 142,
168). Fructose will produce small amounts of toxic furfurals in normal auto-
claving (122, 130, 141). Sugar decomposition is stimulated if the sucrose is
autoclaved together with iron and phosphate ions (170), and sugars interact with
12
amino acids when heated together (122, 130). Most vitamins (82) and gibberellic
acid (28, 139) are heat- labile, but the commonly used auxins (except indoleace-
tic acid) and cytokinins are relatively stable (53, 136).
However, even though autoclaving induces chemical changes in the nutrient
medium, it is still the preferred method of sterilization, except for a few very
heat sensitive chemicals. The main reasons for this preference are: 1) The
operation is simple and effective. 2) As long as the duration of autoc1aving is
not extended past the usual 15 or 20 minutes at about 120C, the chemical chang-
es are small and generally have little or no noticeable effect on growth of the
cultures (202). 3) The autoclaving effect is not always a neutral or negative
one;-for example, Ball (13) found better growth of Sequoia sempervirens callus
on a medium with autoclaved sucrose than with filter sterilized sucrose. In
some media, inhibitors are inactivated by autoclaving (122).
Heat-labile chemicals, such as glutamine and some of the vitamins, are
cold-sterilized and added to the autoclaved portion of the medium (70, 194).
Cold-sterilization is sometimes carried out by dissolving the chemical in a
small amount of solvent, generally dimethylsulfoxide (91) or ethanol (53, 125,
135). However, ethanol may not be a good choice for this purpose. Concentra-
tions of 1% (135) and lower (53, 125) in the medium will inhibit callus growth
and have been found to inhibit embryogenesis (181). The more common method of
cold-sterilization is by filtration through membrane filters. Filtration tech-
niques have evolved rapidly over the last few decades and have found many indus-
trial and laboratory applications. As a consequence, a large variety of fil-
ters, primarily membrane filters, are now commercially available to remove
microorganisms from solutions. Most filters are made of cellulose acetate and
have 0.20 ~m pores. The most popular method of filtration is vacuum filtra-
tion in which the solution is placed in a filter funnel and sucked through the
membrane into a vacuum flask. Before use, the filter, its funnel, and the vacu-
um flask are sterilized by autoclaving or with alcohol (27, 106), the latter
method being quicker and more convenient. Vacuum filtration has a few disadvan-
tages. If a water run vacuum aspirator is used, irregularities in the water
flow may cause a backflow of air or water into the vacuum flask, introducing
contaminants to the filtrate. Furthermore, if the filtrate contains organics it
may foam in vacuum and some of the more volatile organics may partly be removed
by evaporation. To avoid these complications, it is probably better to use a
pressure rather than a vacuum filtration system (Fig. 3).
There are some problems associated with membrane filters. Often these fil-
13
ters contain a small amount of detergent, some of which is released into the
filtrate. Most cell cultures will not be affected by trace amounts of detergent
in the nutrient, but the possibility that the growth of some sensitive cell
populations could be influenced cannot be ruled out. Rinsing of the membrane
filter in water will remove the detergent, but reduces filtration speed (34,
38). Adsorption of proteins and some other media components to the filter and
oxidation of sensitive chemicals may occur (84, 122). Furthermore, 0.20 ~m
polycarbonate filters may pass some bacterial species; cellulose ester filters
of the same pore size will remove these bacteria (131).
should be protected from air currents. Slow growing cultures can be placed in-
side clear polyethylene bags; for faster growing cultures the bags may have to
be punctured to allow a more rapid air exchange. Polyethylene is an excellent
barrier to water vapor, but allows exchange of carbon dioxide and a less rapid
exchange of other atmospheric gasses (151).
Volatiles in the culture room atmosphere may create some problems for extra
sensitive cultures, especially if ventilation of the rooms is restricted. There
are various sources of volatiles; air conditioning units may leak refrigerant
(freon or ammonia), fluorescent lights produce ethylene (200), and gasses
emanate from electric motors and insulation material (33). However, normally
these volatiles will not reach levels high enough to have a significant effect
on the cultures.
One problem often encountered in culture rooms is contamination of cultures
by fungi carried by mites (Ill). Placing the cultures in tightly closed poly-
ethylene bags does not exclude the mites. Presumably they are attracted by
volatiles, produced by the cultures, that pass through the plastic of the bag.
The mites appear to be capable of drilling through the plastic and crawling into
the culture tubes carrying fungi with them. To rid the culture room of mites,
bench tops and shelves should be washed with 70% alcohol and floors and walls
wi th a sodium hypochlori te cleaner. Hanging a few "Vapona" (d ichlorvos, Shell)
insecticide strips in the culture room for a few weeks will further eliminate
the insects without noticeably affecting the cultures. Several other miticides
of low phytotoxicity are available (Ill, 143, 162).
3. MEDIA PREPARATION
Tree tissue cultures have been maintained on a large variety of different
nutrient media. The chemical comp0sition of these is not discussed in detail
here, because several of the later chapters in this volume will deal at length
with the currently used nutrient formulas, the functions of nitrogen, carbohy-
drates, and hormones in the media, and other aspects of nutrition. Complete
nutrient formulas can also be found in textbooks on plant tissue culture (71,
166, 170) and reviews of nutrient media (70, 86, 145). Therefore, this discus-
sion will focus mainly on a few principles and problems not always emphasized in
the general literature.
3.1. Functions of some media components
The primary function of most components of the medium is nutritional, i.e.,
they provide energy or serve as building blocks for other essential molecules in
16
the plant cells. However, some components have functions that are mostly non-
nutritional, and sometimes more physical than chemical in nature. These non-
nutritional functions have received less attention in the literature than the
nutritional ones, and therefore will be emphasized in this section.
3.1.1. Agar and its substitutes. As was pointed out earlier, liquid suspen-
sion cultures are often preferred over semi-solid agar cultures. However, for
many tissues, techniques for cell suspension cultures have not yet been perfec-
ted and the tissues still survive or grow better (50, 117, 174), or, as is the
case in cultures of Eucalyptus (52), are more morphogenetically active, if cul-
tured on an agar medium. The reasons are not clear, but the following may be
suspected: 1) Loss of vital chemicals from the cells by leaching may be more
severe in liquid medium. 2) Agar, besides providing a solid support for the
tissues, could be beneficial because it has an adsorptive capacity (199), and
like charcoal, may remove some cellular waste products. 3) Cells in agitated
liquid medium are prone to mechanical damage.
One advantage of cultures on semi-solid agar media over liquid suspension
cultures is that they do not require expensive and bulky shakers. On the other
hand, because agar is a natural product, one may expect differences in growth
response of the cultures, depending on the degree of purification of the agar
(146), and with different batches. Furthermore, because of dwindling supplies,
good quality agar is sometimes difficult to obtain and is becoming expensive,
although the cost can be reduced by recycling agar from old cultures (10).
Another problem with agar is that it is a source of many minerals, in particular
sodium (89, 132) and possibly some vitamins (132, 199, 202) and toxins (102,
132), which may complicate metabolic and nutritional studies.
Therefore, several agar substitutes have been investigated, but so far, none
have been widely accepted. The most interesting of recently developed substi-
tutes are; positively charged dextran microspheres (107), "Plantgar", a starch
co-polymer (44), polyac rylamide (86, 119), silica (144) and "Ficoll", a sucrose
polymer (184). Instead of gelling agents, glass beads (47), filterpaper, glass
fiber or polyester (40, 86) are sometimes used to support the tissues. Glass
fiber supports should be washed in acid to remove chemical contaminants before
they are used (176). When using agar, its concentration is important; morpho-
genesis as well as callus growth rates are influenced by its concentration (102,
146,184,199).
3.1.2. Minerals, ratios, and concentations. If one looks at the macro-ele-
ment composition of different nutrient media one will notice large differences
17
nutrient media enclosed in plastic bags will reduce desiccation and keep out
dust and contaminants. Slow chemical changes will occur in refrigerated cultur,
media (122). L-glutamine in liquid medium is largely destroyed in about four
weeks (7). Some stock solutions for plant tissue culture media can be stored
froze~ (70). A few chemicals are highly unstable in liquid solutions. For
example, ascorbic acid was lost at a rate of 4% per day from nutrient media
stored at DoC, while at room temperature its half life was only 15.5 h (63).
Indole acetic acid decomposes in solution, especially if exposed to light (18,
23).
4. PREPARATION OF CULTURES
4.1. Condition of Plant Material
In tree tissue cultures results often vary greatly from year to year, even
when the ex plants are obtained from material collected from the same tree at th,
same time each year. This probably is partly caused by climatic cycles and
fluctuations in other environmental factors. For example, annual growth in
conifers is affected by spring rainfall, and annual nitrogen uptake by rainfall
in June; both these processes are correlated with climatic cycles lasting from
4.4 to 42 years (116). Such cycles in the physiological conditions of field
grown trees could account for some of the year-to-year variation in culture
response. A very short cycle has been observed in rooting response of genet-
ically uniform Pinus radiata cuttings; however, which environmental component
controls this cycle is not known (161). In conifer cultures the rate of adven-
titious bud induction on cotyledons is correlated with the growth rate of the
parent tree (123) and with seed size (3), both of which are largely determined
by environmental factors.
In seedlings or small sized plants, variation in the physiological condition
of the plants can be reduced by raising them in controlled environments, i.e.,
in greenhouses or growth chambers, instead of in the field. The morphogenetic
capability of greenhouse raised plants is improved by adherence to optimal
light, temperature, and fertilization schedules (64). However, if the objectiv(
is to culture tissues from mature trees, greenhouse material is only rarely
available, and field grown material will have to be used. In such material,
environmentally induced variations in the physiological condition are unavoid-
able.
4.2. Collection and storage
In the early days of tree tissue culture, most experiments were carried out
21
with callus cultures from cambial zone explants. White's (198) and Gautheret's
(71) textbooks and some other early references (76, 199) give extensive descrip-
tions of the collection and subsequent handling of material for the preparation
of cambial zone cultures. However, over the years the interest in cambial zone
cultures has waned, because these, though providing good callus growth, often
have a low morphogenetic capability. Therefore, interest has shifted to cul-
tures of embryo or young seedling sections, shoot tips, buds, microsporophylls,
anthers, pollen, and female gametophytes.
Good quality seeds to provide embryos, female gametophytes, or small seed-
lings can be obtained commercially or are often available from government for-
estry institutions. Dry seeds of most trees can be stored in sealed containers
at 3 to -18C for several years without loss of viability (190) .
Branches with vegetative buds or generative structures, to provide shoot
tips, young foliage, microsporophylls, anthers, or pollen, are collected in the
field and transported to the laboratory in plastic bags. One problem is that
microsporophylls, anthers, and, in some cases, vegetative shoots are suitable
for culture only if excised from material collected durin g specific short
periods each year. For example, balsam fir shoots are morphogenetic only if
excised from buds collected shortly before the spring bud break, i.e., during
the short period when the lateral meristems are formed inside the bud (26), or
if excised from late summer or early fall buds, i.e., during the peri od when the
needle primordia are formed (Bonga, unpublished) .
Therefore, if one intends to use such material on a year-round basis, proper
storage methods are imperative. Conifer branches will remain viable for several
months if stored at 4C in paper bags placed inside plastic bags (Bonga, unpub-
lished). The function of the paper bags is to absorb excess surface moisture
from the branches, thus reducing fungal contamination. Extensive studies have
been carried out with regard to fruit and vegetable storage, involving gaseous
mixtures (35), reduced air pressure (108), fungicides (83), specific relative
humidities (19), and growth regulator treatment (79). Some of these methods
have been used for storing tree seedlings (189). For cold storage of tree seed-
lings it has been recommended to use Kraft paper bags with a semipermeable
polyethylene lining, which restricts moisture loss but allows sufficient gas
exchange to maintain the seedlings in a healthy state (113, 189). To reduce
moulding, the seedlings are sometimes wrapped in damp sphagnum before being
placed inside the storage bags (189). Apple shoots have been stored in vitro at
low temperature for 12 months (110). Storage of normally short-lived citrus
22
pollen was improved by using a nitrogen atmosphere and low temperatures (152).
When material is stored at low temperature under conditions of restricted gas
exchange or altered atmospheric conditions, slow, degenerative physiological
changes occur. Pine seedlings lost carbohydrate in cold storage (112, 189),
while in other plants, alcohol, acetaldehyde, and abscisic acid levels increas-
ed, and chloroplasts and mitochondria were inactivated (41). Therefore, one can
expect explants from stored material to behave differently in culture than
explants from fresh material.
Cold storage is not always detrimental. In some cases it is required to
break dormancy, while in others, it has a beneficial effect in non-dormant
material. For example, 6 to 8 weeks of cold storage of non-dormant branches of
balsam fir before excision and culture of young shoots stimulated the formation
of adventitious structures on the expanding needles (26).
A technique that is presently being developed, mainly for germplasm storage,
is cryopreservation, i.e., storage of tissues in liquid air or liquid nitrogen.
With tree tissues, such storage has been achieved for poplar callus (154) and
suspension cultured sycamore cells (172).
4.3. Surface Sterilization
It is often difficult to surface sterilize material without simultaneously
damaging or killing the tissues. In fact, when extra sensitive tissues, such as
shoot apical meristems, are to be excised it is sometimes better not to use any
sterilant (146). Sometimes microorganisms are endogenous, e.g., in seeds (173),
in dormant winter buds but not in the spring flush of pecan trees (98), and on
the bud scales but not the leaf primordia of apple trees (4). Tree tissue cul-
tures free of such microbes can often only be obtained if the explant is limited
to the shoot apical meristem (24).
The most common surface sterilants are calcium and sodium hypochlorite, with
the calcium salt being less toxic to tissues than the sodium salt (173). How-
ever, calcium hypochlorite reacts with carbon dioxide in the atmosphere, and,
therefore, is chemically unstable (156). Hypochlorite has a negligible activity
at pH over 8 (17); buffered at about pH 6 it is much more effective (173).
Generally a drop of detergent is added. However, in a surface sterilization
experiment with white spruce twigs it was found that adding detergent, though
lowering the contamination rate, increased the toxicity of hypochlorite to the
explants (25). A short exposure to hypochlorite followed by a 2-min wash in 70%
ethanol has been effective for conifer buds and shoots (12, 26). All hypochlor-
ite should be removed from the tissues, because trace amounts left behind will
23
interfere with amino acid uptake and metabolism. Washing several times in water
does not always remove it sufficiently; washing in dilute hydrochloric acid may
be required (1, 2).
Mercuric chloride in 50% alcohol has been used to surface sterilize pine
brachyblasts (51), and in water, has been used to treat shoots of sandalwood
(160). If used to sterilize conifer seeds, mercuric chloride stimulates imbibi-
tion and consequently germination. However, mercury ions are adsorbed to the
seedcoat and have to be removed by washing in potassium chloride (177).
Alcohol without other sterilants has been used to surface sterilize pine
seedlings (188) and poplar shoots (37), but generally alcohol is used in combi-
nation with other chemicals. The effectiveness of alcohol depends on chain
length of the molecule and the presence or absence of water (137).
Hydrogen peroxide has been used to surface sterilize shoots of saltbush (201)
and poplar (37). For stem sections of some conifer species, it was more satis-
factory than sodium hypochlorite (76). Various simple acids and bases were
effective in surface sterilizing carrot roots (157); merthiolate was more effec-
tive than sodium hypochlorite and hydrogen peroxide for green alder seeds (88).
4.4. Excision and transfer of tissues
When tissues are cut, the cut surfaces often turn brown within a few minutes
because of oxidation of phenols to toxic quinones (118) in the damaged cells.
The smaller the explant, the higher the cut surface to volume ratio, and thus
the higher the degree of damage. This probably is one reason why it is often
difficult to achieve survival of small explants. To keep cell damage (crushing)
to a minimum the scalpels used for excision should be as sharp as possible.
Therefore, to sterilize them, they should not be heated unnecessarily, because
overheating dulls the edge. The common procedure is to dip the scalpel in
alcohol, which is then removed by igniting it. The combination of the steriliz-
ing effect of the alcohol and the mild heat created when it is burned off is
enough to sterilize the scalpel without damaging its sharp edge (175).
Additional treatments are often required to prevent browning. Excised tis-
sues of coffee (118), palm (180), and balsam fir (26) were treated with various
antioxidants, tissues of peach with the herbicide "Dieca" (121), and those of
teak and jojoba with polyvinyl-pyrrolidone (78, 150). Keeping tissues submerged
in water during excision of the explants results in less browning and improved
survival rates of the cultures, presumably by excluding air from the cut sur-
faces (26).
Browning is not the only problem associated with excision. There is a short
24
period of rapid loss of adenine nucleotides and potassium from freshly excised
tissues, which can be counteracted by washing in calcium chloride (77), and of
intercellular carbon dioxide (103). Various types of stress caused by injury,
and recovery from these stresses have been described (69, 103). Whenever excis-
ing tissues, these stresses should, if possible, be identified and counteracted
as much as possible. In addition, for very delicate and small explants, special
tools and excision methods may be required to keep cell damage to a minimum (48,
175). However not all wounding effects are negative, e.g., in cultures of
jojoba (150) and apple (165), rooting was induced by wounding.
After excision and transfer of the explants to the culture vessel it is
customary to flame the mouth of the vessel in a small alcohol or gas flame
before recapping the vessel. However, it has been found that this procedure
introduces a significant amount of ethylene into the culture vessels (15).
Therefore, if tissues that are extra sensitive to ethylene are to be excised and
transferred, it may be advisable to sterilize the mouth of the vessel with an
electric incinerator (Fig. 2) instead of a flame.
4.5. Pre-culture treatments
Sometimes additional pre- or post-excision treatments are carried out before
transfer of the explant to the nutrient medium. In cultures of shoots excised
from balsam fir buds, higher survival rates and better morphogenesis were
obtained if the shoots were forced to bud break in an EDTA solution. Morphogene-
sis was also stimulated by a short soak of the explant in malonic acid, a res-
piratory inhibitor, before transfer to the nutrient (26). Surface sterilized
eucalyptus branch tips were treated for two hours in an ascorbic acid solution
and then stored in water before excision of the nodes (52). Embryogenesis in
coffee leaf explants was stimulated by keeping the explants in the dark on a
saline-sugar agar for 36 h before transfer to the nutrient (163).
4.6. Incubation environment
Many cultures grow well within a wide range of photoperiods and light inten-
sities. Illumination by fluorescent lights (Gro-Lux, or similar type) at about
1000 lux for 16 h per day appears to be satisfactory for most cultures (124).
Light quality is often important. In embryo derived callus cultures of Douglas
fir, red light stimulated adventitious bud formation (90). However, red light
stimulation of shoot induction is not universal; for example, in tobacco callus
cultures, blue is more effective than red in stimulating shoot production (158).
Green-yellow radiation repressed the growth of ginkgo pollen suspension cultures
(96), and spruce cultures grew better under Gro-Lux than under Warm-White fluo-
25
for transfer, they may have become dormant and require a low temperature rest
period before transfer. For a more detailed discussion of these problems and
other technical aspects of transfer to soil the reader is referred to other pub
lications (114, 179).
5. CONCLUSION
Tissue culture of forest trees is rapidly entering a new era. From being
purely a research tool it is increasingly becoming an integral part of various
forest operations. Already it is applied in tree breeding, selection and propa'
gation programs, and the prospects are bright for its future use in tree diseas
control and the production of metabolites, drugs, and other valuable compounds.
For forestry application, it is essential to standardize and optimize the
tissue culture procedures as much as possible. Reliable methods that consis-
tently produce uniform results are needed because in large-scale commercial
applications even a temporary failure of the techniques is costly and therefore
not acceptable. As was outlined throughout this chapter, there are countless,
often unknown, factors that interfere with even the most standardized tissue
culture procedures. In the past, the main emphasis was on the establishment of
the major factors controlling growth in tissue culture. However, the point has
now been reached where the more minor controlling factors should also be deter-
mined and standardized to make large-scale research and commercial application
possible on a reliable basis.
6. ACKNOWLEDGEMENTS
I wish to thank Dr. W. K. Coleman, Canada Department of Agriculture,
Fredericton, N.B., Canada, for reviewing the manuscript.
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161. SMITH DR, TA THORPE 1976 Rooting in cuttings of Pinus radiata seedlings:
fluctuations in relation to selection day. Bot Gaz 137: 128-132
162. SMITH RS 1967 Control of tarsonemid mites in fungal cultures. Mycologia
59: 600-609
163. SONDAHL MR, WR SHARP 1977 High frequency induction of somatic embryos in
cultured leaf explants of Coffea arabica L. Z Pflanz.enphysiol 81: 395-408
164. SOPORY SK, SC MAHESHWARI ~Similar effects of iron-chelating agents
and cytokinins on the production of haploid embryos from the pollen grains
of Datura innoxia. Z Pflanzenphysiol 69: 97-99
165. SRISKANDARAJAH S, MG MULLINS 1981 Micropropagation of Granny Smith apple:
factors affecting root formation in vitro. J. Hort Sci 56: 71-76
166. STREET HE 1966 The nutrition and metabolism of plant tissue and organ cul-
tures. In EN Willmer, ed, Cells and Tissues in Culture, Methods, Biology
and Physiology, Vol 3, Academic Press, London, New York, pp 533-629
167. STREET HE 1966 Growth, differentiation and organogenesis in plant tissue
and organ cultures. In EN Willmer, ed, Cells and Tissues in Culture,
Methods, Biology and Physiology, Vol 3, Academic Press, London, New York,
pp 631-689
34
D.J. DURZAN
1. INTRODUCTION
The currently ava ilable body of knowledge of cell and
tissue culture of forest trees is mainly a product of
scientific curiosity rather than nec essi ty. Once the basic
techniques were established in the 1950's and applied to the
propagation of horti c ultural a nd agricultural plants,
managers enga g ed in tree improvement programs realized that
similar methods could even tuall y be u sed commercially in the
forest industry. However, although cell and tissue culture
was the strategic new technique needed to bring the study of
trees into the laboratory (10), tissues of many mature trees
r e main somewhat recalcitrant in response to most standard
practices . Furthermore, the technique coul d not progress
until:
A. Methods were availab le for the genetic selection
of trees, cells and tissues.
B. Synthetic plant growth regulators (e .g.,
a-naphthalene acetic acid, N6 -benzylaminopurine,
were ava il ab l e and proven effective in controlling
t he growth and development of a wide range of
spec ie s (Fig. 1).
C. Ce llular nutrition could be impr oved and control-
led through al l aspects of the propagation pro-
cess .
Shortages of food , energy, materials, shelter and
transportation have forced governments, private individuals
and commercial enterprises to intens ify the ir search for new
ways to sustain and improve the prof it ab ility of l oca l
37
III
~
a:
w
D..
III
o
Z
BENZYLAMINOPURINE
~20
C)
~
III
iii
w
zw
C)
o
it 10
a:
o NAPHTHALENE ACETIC ACID
~
u..
o
~
a:
oD..
w
a: 10- 4 10-5 10-6 10-7 10-8 10-9
MOLES
FIGURE 1. The relationship between reports of successful
morphogenesis 6 reported in gymnosperms and the molar concen-
trations of N -benzylaminopurine, c(-naphthalene acid,
(3-indolebutyric acid (IBA) and f3-indole acetic acid (IAA)
added to nutrient media. Data are collected from the
literature to 1980 .
If
Character- genetic
istics gain Then the effect
Tree under is at may be an increase
species improvement least: in measure of:
" 1-"
~e~
-"II
SEEDS Ij
~H
COMMERCIAL" } ~
PRIVATE \ BULK HEAT END
fEDERAL LAND , MATERIALS WASTES USE
l. ". ~
L---r-~ DOMESTICATION ,/' \ '
\ I ",' \
, /' DEFORMATION MANUFACTURING
PRODUCTION UTILIZATION
1. Plant breeding and genet- Strengthen tools of genetic worldwide potential is immense.
ic manipulation limited manipulation: plant breed- Production increases from most
by life cycle and complex ing and "classical" genetics; lines of research expected in
nature of trees. cell biology; genetic stocks; 10 to 20 years.
vegetative propagation via
cell and tissue culture
methods; population genetics.
3. Tree growth rate and form. Improve photosynthetic effici- Higher yields of trees, partic-
ency to depos i t more wood ularly in the marginal areas;
relative to the available N. substantial increases in poten-
Increase shade tolerance and tial yields after 15 years or
photosynthesis in major species. more.
Consider reducing photorespir-
ation. Select and develop
trees with greater assimilative
surfaces (roots, shoots) with
distribution of leaves to optim-
ize use of incident light, and
with superior cambial and wood
development.
4. Resistance to environment- Improve resistance of tree crops Larger and more stable yields
tal stresses so that trees to drought, temperature extremes, in 10 to 15 years. Trees can
could survive and grow on deficiencies of acid soils, salt be grown in new locations.
poorer soi Is and in severe tolerance. Develop rapid scre-
climates. ening techniques, shorter rota-
tion, larger root systems, better
use of soil fungi. Introduce
tree-farming systems.
5. Pest management to over- Reduce preharvest losses due Large and pervasive losses due
come losses due to in- to pests; integrate pest to pests can be minimized in
sects, diseases, and management; utilize specific short run by adapting known
fires. contro l mechanisms, e.g., techno logy and in long run by
mixed species plantations. biological control techniques.
6. Weather and climate (es- Improve techniques for predic- Substantial risk reduction in
pecially where multiple ing weather and climate and use short run, especially for
constraints on growth are information to reduce weather nursery stock; site selection
imposed by light, temper- damage; develop baseline infor- can increase payoffs substan-
ature. and moisture). mation about inventories cor- tially.
related with site and climate
through remote sensing.
7. Management of forest soils Improve management of soils to Annual production on some lands
to maintain nutrient bal- increase productivity; soil can be increased by 150 to 200
ance and minimize under- classification; land clearing percent.
story competition. methods; correction of soil
deficiencies; maintenance of
desired soil characteristics;
sui table cropping systems and
technologies .
8. Irrigation and water man- Improve management of water May markedly improve yields in
agement to minimize pro- supplies; adjust tree-farming some areas and make capi tal in-
ductivity losses. systems and irrigation for vestments more effective.
optimal supply to trees; adapt
operations to water availabil-
i ty; study transpiration re-
tardants and acid rain.
9. Fertilizer supplements and Improve cost/return ratios of New and more efficient fertil-
mycorrhizal associations chemical ferti lizers: develop izers may have great effect on
to overcome nutrient im- new methods of producing slow- production on poor sites.
balances. release nitrogen and phosphorus
fertilizer; develop new fertili-
zers tailored to forest condi-
tions; select species that util-
ize nutrients efficiently; study
nutrient cycling.
43
3. GENETIC RESOURCES
According to Zobel (98), genetic improvement of forest
resources consists of:
A. Locating and using the correct species.
45
Table 4. Comparison of properties of two breeding systems aimed at propagating trees. The
first system involves the need for a seed orchard and the other a viable cellu"'I"ar
cloning cycle. The latter assumes that one day trees may be regenerated from cells
Q3l.
Numbers to
1) det e ct mutation 10 ~ trees 10 7 cells
2) detect linke d traits 10 trees 10 3 cells
Time to produce seed or 8-13 years, minimum of 2-3 1-2 years estimated (10 6 /
soma tic embryo month) (somatic embryos)
~;~~~lO~r t~~~:S ?rnom se~~li~;:
chards over 16 years)
Seed pro duction predictabi Ii ty Var iable s e ed years Controlled production in vitro
on demand - --
Types of breeding system Selection most applicable All breeding systems can be
mutation Hybrids possible for certain exploited with cells and their
inbreeding species: other methods slow protoplasts; field testing
hybridization and require 8-10 year genera- r e quired for certification of
backcross tion times quality and "true to type"
selection
*cf. Proc. 17th Canadian Tree Improvement Association, Gander, Newfoundland, August 27-31, 1979.
49
1{ }
STIIUCTURAl
MICROOAGANI" INTEGRATION IFEEDeACK AND OU ... LlTY
..... IC ... l DOMIN"'NCE , OISE .... SCREENING,
GIRDLING , WOUNOING , NUTRITIOfIIAl ,
""',"0::"0 ",.",...,.,
--"'--". =i:: ...
CLIENT
NEEOS
~ ~~~.::~: --~
HI(OETUIMINATlOfli
(MERISTU'MQOlfIEOI
GENETICINl'VT
'"OVEN , M... TUIIE SPECIMENS
P'ROTEIN5YNTHEII5,
'ACKAGING AND QUALITY
ICREENING OF
~Y
CALLUS~
INOUCTION
INI'UT (AUXINfCYTOKININI
CAUTIONS
_ JUVENILITY INTER'RETATION
,..
CHARCO ... L
....-"5 ACHIEVEO'
4. PROPAGATION SYSTEMS
In forestry, three levels of tree selection are in use
and controlled: the origin of wild seeds, seed production
areas, and seed orchards (58, 95) In seed orchards,
aggressive forest-tree breeding should be established to
produce new genotypes.
4.1. Seed orchards
Seed orchards will be a source of genetically improved
seed for the forest industry for years to come (41, 58, 88,
95, 98). Where trees of high value are scarce, vegetative
propagation can be used but the returns would have to
50
6. CONSTRAINTS
There are still many yet unproven culture systems and
recalci trant species, especially for more valuable forest
trees (50). We must ask why the current technology does not
apply and how to foster greater success. This concern is
important because plant cells are far more complex than
microbial cells. We still have a long way to go in
controlling the reactions in our culture systems (42).
We cannot yet prescribe the rules that limit the
capacity of plants to fabricate biomass. Even when there is
no organ ized shoot tip or meristem or no newly generated
engineered genetic system, the tissue culturist knows that
cells may still respond metabolically to the external
variables of light, temperature and nutrition. Better
control over organized growth and morphogenesis is of high
priority and a prerequisite for the application of genetic
engineering technologies can be applied to cells of woody
species.
Difficult problems ln cell and tissue culture and their
associated technologies still limit the application of these
methods on a large scale. For example, in the genetic
e ngine er ing of plant cells, the efficiency of gene transfer
and express ion is not yet very high and is restricted to a
few host systems (42) . For production purposes the genetic
stability of the cloning vehicles themselves is unknown.
Whil e landmarks in engineering have been achieved with
bacterial systems, the transfer of a set of 17 nitro-
gen-fixing genes to a more complex cell such as yeast did
not lead to nitrogen fixation (23). Gene transfer is
clearly not enough. Furthermore, plant storage protein~ are
usually a collection of subunits, each coded by genes that
63
8. ACKNm.oJLEDGMENTS
The author thanks the Institute of Paper Chemistry for
the use of Figures 2, 3, 5, 6, 7 and 8.
REFERENCES
1. AD HOC PANEL. 1980 . Firewood Crops . Shrub and tree
species for energy production. Report of an Advisory
Commi ttee on Technology Innovation. Board on Science
and Technology for International Development. National
Academy of Sciences. Washington D.C.
2. 1979a. Microbial processes : Promising
technologies for developing countries. Ibid .
3. 1979b. Tropical legumes: Resources for
the future. Ibid.
4. --r977a. Methane generation from human,
animal and agricultural wastes. Ibid.
5. 1977b. Expansion deS ressources en eau
dans les zones arides. Ibid.
6. 1976. Energy for rural development.
Renewable resources and alternative technologies for
developing countries. Ibid .
7. . 1975. Underexploited tropical plants with
-p-r~o~m~i~s~i~n~g---e~conomic value. Ibid.
8 . ALEXANDER M. 1980. Biodegradation of chemicals of
environmental concern. Science 211:132-138.
9. ASAOKA H. 1981. Research and development in Japan .
Present and future. In: The role of fundamental
research in papermaking. 7th Fundamental Research
Symposium, Cambridge, UK, Session 8, 25 pp.
10. BAILEY IW, Spoehr HA. 1929. The role of research in the
development of forestry in North America. New York,
MacMillan .
11. . 1980. Communications--modes, media, meanings.
-::;:I'-n-:---::;:P:-a-p-er science and technology, the cutting edge.
Proc. Conf. 50th Anniversary, The Institute of Paper
Chemistry, Appleton, WI, pp. 209-222.
12. BAKER WOo 1981. Using materials sci e nce. Science 211:
359-363.
13. BAUER WD. 1981. Infection of legumes by Rhizobia. Ann.
Rev. Plant Physiol . 32:407-449.
14. BAULE H, FRICKER C. 1970. The fertilizer treatment of
forest trees. Germany, BLV Verlagsgesellschaft mbH
Munchen.
67
A. DAVID
1. INTRODUCTION
Most gymnosperms are propagated sexually through seeds. With regard to
genetic improvement of a species, sexual reproduction will preserve some of the
mean traits of selected families. Vegetative propagation, on the other hand,
is a procedure with the potential to maintain the best characteristics of se-
lected individuals, and therefore often is the preferred method of propaga-
tion.
Vegetative propagation generally involves the formation of new meristems
from differentiated tissues. If cuttings are kept under favorable conditions,
they sometimes can be transformed into a new organism, possessing the charac-
teristics of the parent individual.
In spite of extensive experimentation during the last several years, many
aspects of vegetative propagation remain poorly understood. The age of the
tree from which the cuttings are taken, their position on the tree, the time of
their removal, and the seasonal variations in rooting capacity are all factors
in vegetative propagation that require further study. Vegetative propagation
of many species, especially those of the gymnosperms, is still a major problem
to be solved.
The establishment of continuous cambial cultures as reviewed by Gautheret
(38) gave a new stimulus to research.
Research with several herbacious plants has shown that whenever it is pos-
sible to maintain cell multiplication of a callus, one can, under certain nu-
tritional conditions, initiate the formation of organs. For some species,
plants can be regenerated from single, isolated cells in culture (51), or hap-
loid plants can be obtained from anthers in culture (55). Sometimes "vegeta-
tive embryos" are produced with various types of cultivars (56).
Although the first research in tree tissue culture was carried out a long
time ago, it did not incite much enthusiasm initially . It was not till 1950,
that work with gymnosperms started seriously, probably because of encouraging
results obtained by Ball with Sequoia sempervirens (4). Since then, tissues of
73
many gymnosperms have been cultured, resulting, in some cases, in the isolation
of continuous cultures, and occasionally in organogenesis. In 1975, Brown and
Sommer abstracted all the work carried out with in vitro cultures of gymno-
sperms (14).
This chapter is based largely on a review published by David and Thomas
(33), updated with more recent information. The first part will deal primarily
with organogenesis in callus cultures. Direct morphogenesis in organs or organ
sections will be detailed in the second part. The establishment of plants from
vegetative material obtained from mature specimens will be discussed. The
various publications dealing with each of these will be discussed in chrono-
logical order (Bibliography ending March 1981) and summarized. Finally, a few
new working methods, arising from this survey, will be presented.
Webb and Street (76) found that cultures of embryos removed from unripe
seeds of Pinus contorta and Picea sitchensis' developed callus with a shoot
forming capacity on a medium with a mixture of auxins and cytokinins (IAA, IBA,
2-iP, BAP). This capacity for shoot formation was maintained after transfer to
fresh medium.
75
Reilly and Washer (63) found that cotyledons of Pinus radiata, as long as
they were in contact with a shoot inducing medium, produced a callus with
meristematic cells that formed buds. The calli remained mitotic for several
months while retaining their shoot forming potential.
Groups of cells resembling the first stages of embryos were observed in cell
suspension cultures of calli developing from needles of young seedlings of
Pseudotsuga menziesii and Pinus taeda (78). Precursors of indoleacetic acid
oxidase favored embryoid formation (45).
Kadkade and Jopson (46) investigated the effect of illumination on shoot
formation in callus from embryos of Pseudotsuga menziesii. Callus formation
was stimulated by wavelengths of 550 and 660 nm, and five times more buds were
formed in cultures exposed to 660 nm light (0.42 m W/cm2) than in those kept
in the dark. The authors are of the opinion that light stimulates shoot initi-
ation, but not elongation.
Durzan (34) reported globular embryos in cell suspension cultures of
Pseudotsuga menziesii shoot tips from 3-4 year old saplings from the green-
house. These structures became polarized along a root-shoot axis, and eventu-
ally developed cotyledons (Fig. 2).
Embryos of Pinus wallichiana cultured on a modified Murashige Skoog medium
containing NAA (0.1 ppm) gave rise to calli that were regularly subcultured.
Transfer of these calli to a medium containing BAP (1 ppm) resulted in bud
initiation. After replacing the cytokinin by coconut milk these buds elongated
into shoots (49).
Following the establishment of gymnosperm tissue cultures, the first mani-
festations of organogenesis, as described by Ball and later by Norstog and
Rhamstine, did not arouse the interest that these observations deserved. Many
years passed before further studies in this area were initiated and encouraging
results were obtained.
A survey of the literature indicates two categories of development in callus
and suspension cultures. In the case of callus, the exact origin of the new
shoots is not precisely known especially because histological studies were
generally not carried out. The original explant may have had an effect on the
formation of buds in the unorganized, newly formed callus.
Several authors have noted the retention of organogenetic potential after
subculture. Such investigations should form the basis for further studies to
determine if this morphogenetic potential can maintain itself indefinitely, or
whether it diminishes progressively in successive subcultures (most experiments
76
have not been carried out beyond a few subcultures). Whatever the outcome, the
induction of organogenesis in unorganized callus still appears to be only par-
tially achieved. Minocha (54) has attempted to solve this problem by exposing
callus, obtained from embryos of Pinus strobus, to more than 60 combinations of
auxin and cytokinin. However, this did not result in the formation of buds or
roots.
Therefore, unlike many herbacious angiosperm callus cultures, those of
gymnosperms have so far generally not expressed the organogenetic potential of
the cells. However, the fact remains that if we could obtain organogenesis,
either in unorganized callus associated with the original explant, or in cell
colonies that have gone through several subcultures, the door would be opened
to a new way of producing unlimited numbers of propagules (Figs. 1 and 2).
c
Figure 2. Formation of structures that possibly could develop into embryos in
suspension cultures of Pseudotsuga menziesii (Durzan).
A. Globular stage; B. Polarized development; C. Development of cotyle
dons (v = vascular region; c = cotyledons; e = epidermal layer).
However, genetic stability will have to be assured during the various phases of
regeneration of plants from unorganized cell masses. Meanwhile, one should not
reject multiplication directly from the original explant, because at present it
is still the most effective method of propagation of existing genetic traits.
77
mEq 1-1 of potassium in the medium. The initiation of the buds by exogenous
cytokinin was stimulated by the presence of cotyledons.
Rancillac (60, 61) using similar explants of Pinus pinaster, showed that
addition of lAA, IBA, or NAA did not stimulate the growth of cytokinin induced
axillary buds. On the contrary, the auxins stimulated callus formation. Gib-
berellic acid also had a negative effect. On the other hand, a medium rich in
minerals (like in a Murashige Skoog medium) stimulated organogenesis in the
explants.
Figure 3. Bud development (~) in the axil of cotyledons and juvenile leaves
of Pinus pinaster (David).
Figure 4. The same explant (Fig. 3) after a few weeks in culture ; note the
development of several shoots from the axillary buds .
Figure 5. A brachyblast of Pinus pinaster with one pair of needles shortly
after transfer to the nutrient and before transformation of the vegetative apex
to a bud (David) .
Figure 6 . A bud (~) formed at the apex of a brachyblast under the influence of
a cytokinin-auxin combination .
79
Thus, several authors working with various species have induced the
formation of axillary buds in vitro. The reacting cells are resting, primary
meristematic cells located at the axils of the cotyledons and juvenile leaves,
or at the apex of brachyblasts (Figs. 3, 4, 5, and 6).
Because systematic studies of mineral nutritional requirements are lacking,
it is impossible to recommend specific nutrient formulas. However, it appears
that high potassium promotes morphogenesis. Amongst the cytokinins that are
generally used (kinetin, zeatin, and BAP), BAP is the most active one if used
at concentrations of about 10-5 or 5 x 10-5 mol. An auxin (NAA) is often
used at the same time at about 10-8 mol. The hormonal combination is applied
over a period, which, depending on the concentrations used, varies from a few
days to a few weeks.
The intensity of the response depends on the physiological state and the age
of the parent at the time of explant excision. Presumbly all explants have the
same morphogenetic potential, regardless of the age of the material, but in
explants from mature trees this potential remains suppressed, as will be
discussed later.
80
Two methods for the multiplication of shoots of Pinus pinaster have been
developed by David (30). In the first of these, the axillary bud development
that was described earlier is being made use of. After each new bud has
elongated into a short stem with needles, they are exposed to cytokinin to
induce additional axillary buds. One can thus obtain successive generations of
shoots. In the second method, new apical buds are induced on brachyblasts
(short shoots) by BAP. These elongate and can then be stimulated to form
additional shoots by cytokinin treatment. Furthermore, new brachyblasts are
often formed, each of which may form new apical buds.
Boulay has described a process for the multiplication of shoots of Sequoia
sempervirens (10), and Pseudotsuga menziesii (11) similarly based on the
principle of a succession of axillary buds.
On the other hand, Reilly and Brown (62) found that the composition of the
mineral solution did not play a fundamental role in shoot formation on embryos
of Pinus radiata.
Cotyledons of Tsuga heterophylla, cultured by Cheng (24), produced buds if
treated with lAA, IBA and BAP (2.5 to 5 ~mol).
5 II IDol BAP and NAA, or only auxin, many cells divided, a callus was formed,
and the original structure of the explant dissappeared completely.
The site of shoot formation depended on the positioning of the explant on
the nutrient. For example, David (30) and Isemukali (41) observed that if
cotyledons of Pinus pinaster were placed vertical, cell division occurred in a
subterminal position. If the explants were placed horizontal (Fig. 7), all of
the surface became covered with buds. If a nutrient rich in ammonium (10.3
mEq 1- 1 instead of 2.6 mEq 1- 1 ) is used, the ammonium acted as a deter-
minant in shoot formation. It appears that this level of ammonium favors cell
division and creates the conditions required before cytokinin can induce
shoots. The mitotic effect of ammonium was already shown earlier by David
(29). Rancillac (60), culturing embryos of Pinus pinaster, confirmed the
superiority of BAP over kinetin in shoot formation (Fig. 8).
Shoot formation in very young plants (a few days old) and on cotyledons
(Fig. 9) of Pinus sylvestris have been studied by Tranvan and Thomas (69, and
in press). They observed that morphogentic activity does not occur till after
the metabolism of the embryo is activated (after imbibition). The optimal BAP
concentration for shoot formation on the cotyledons ranged from 5 x 10- 6
mol to 10- 4 mol, depending on the age of the cotyledons (excised from
plants 2-10 days old). For the development stage for which 5 x 10- 5 mol is
optimal, a minimum of 3 days is required for shoot induction, the optimum being
6 days. If the cytokinin is applied longer, abnormal structures will develop.
The buds are generally formed in the subterminal part of the cotyledons from
subepidermal cells close to procambial cells that will become involved in
linking the buds to the vascular system of the cotyledons. The authors conclu-
ded that shoot formation did not depend on small, specialized cell groups.
The first indications of the biochemical mechanisms involved in shoot
formation were presented by Hasegawa et al. (40), who found that cytokinin
stimulated the synthesis of low molecular weight (16,000 to 20,000 daltons)
proteins during the first days of application.
Minocha (54) obtained more than 12 buds per Pinus strobus embryo on a medium
with 1 to 2 mg 1- 1 BAP and 1 mg 1- 1 triiodobenzoic acid. These buds were
formed at the apex of the cotyledons.
Finally, buds were induced by Konar and Singh (49) in embryos of Pinus
wallichiana cultured on a Murashige and Skoog medium with BAP and a greatly
reduced amount of ammonium.
83
3.2.2. Shoot formation along the hypocotyl. Isikawa (42) cultured hypocotyl
sections of Cryptomeria japonica on a nutrient low in organics (meso-inositol
10 mg 1-1, pyridoxine 0.1 mg 1-1, sucrose 20 g 1-1) and obtained buds in
12% of the cultures. Addition of NAA (0.02 mg 1-1) did not stimulate bud
formation, but increased their elongation rate. The addition of vitamins, malt
extract (500 mg 1-1), and abscisic acid (1 mg 1-1), or BAP (10 mg 1-1),
or NAA (0.1 mg 1-1) with BAP (10 mg 1-1) increased bud formation to 25% of
the cultures.
84
Figure 11. A shoot developing near the needle base of Picea abies. The
divisions which give rise to the meristemoids from which the shoot buds are
produced, have their origin in the epidermis (Jansson and Bornman).
hormone, before excision of the explants. It has also been observed, that the
morphogenetic capacity of cotyledons rapidly diminishes during the development
of the parent. Therefore, with increasing age of the material, increasingly
stronger hormonal stimuli are required to initiate morphogenesis. These
observations show, that, whatever the nature of the explant or age of the
parent plant, it is the degree of differentiation of certain cells in the
cultured organ that determines its shoot forming potential.
Within each species, the ability to form buds varies between clones. It
would be of interest to determine if this ability could be related to specific
characteristics of vigor, thus allowing proper selection at an early stage,
e.g., in controlled pollination experiments.
Because of lack of systematic studies, there are only a few indications of
what minerals are required to stimulate adventitious bud formation in the ex-
plants. However, it appears that the mineral requirements may not be very
specific. On the contrary, the hormonal requirements and mode of application
for shoot formation have been studied in detail. Amongst the various cyto-
kinins, BAP at about 5 x 10- 5 mol is the most effective. An auxin, gener-
ally NAA at a low concentration, is often used together with the cytokinin.
However, since the hormonal dose required to induce morphogenesis varies be-
tween species, type of explant, and stage of development of the explant, opti-
mal concentrations for bud formation have to be established in each case.
Several studies have shown that an excess of cytokinin does not disturb cellu-
lar dedifferentiation, but interferes with the normal later development of the
buds. Hormones generally have to be applied for a few days to initiate bud
formation; subsequent organization occurs on a medium free of hormones. The
cytokinin concentration appears to determine the number of buds per explant,
the auxin concentration the number of explants that form buds.
The buds originate from cell layers that are exterior to the mesophy11 (in
cotyledons and needles) and the cortex (in hypocotyls). Under the influence of
the hormones, cells that are relatively little differentiated return to a
primary meristematic condition, and thus regain the capacity of division and
bud initiation.
The formation of buds from more or less differentiated cells suggests an
important modification of the behavior of the cell. Because the cells pass
through a stage of unorganized division, one should question the stability of
the genome. Only one study (79) has been carried out on that topic and it
indicated that the chromosome numbers were stable. More work in that direction
88
should be done.
3.3. Embryogenesis
In 1965, Konar and Oberoi (48) cultured embryos of Biota orientalis on
various nutrient media and found what they termed embryos, developing on the
cotyledons. However, later studies by Thomas et al. (68) showed, that actually
these structures were adventitious buds.
Norstog (57) produced embryoids on embryos and female gametophytes of Zamia
integrifolia on a Whi tes' mineral medium wi th extra phosphorus and with
glutamine and alanine. Banerjee and Radforth (5) cultured embryos of Pinus
resinosa at various stages of development. A nutrient medium was used, that
was low in minerals, but supplemented with extracts of the female gametophyte
of Ginkgo. In a few cases, embryos developed from the suspensor.
In 1977, Bonga (6) obtained embryo-like structures on needles of buds of
adult Abies balsamea. Before transfer to the culture medium, the explants were
soaked for 15 minutes in a solution containing 1 g 1-1 IBA, N-dimethylamino-
succanimic acid, or 1-phenyl-3-methyl-5-pyrazolone. Embryoids were also formed
on needles of adult Picea glauca (7), but these structures did not develop past
the cotyledonary stage without becoming disorganized.
In conclusion, only a few publications indicate embryo formation, and some of
these claims may have been based on inadequate identification. Only detailed
histological studies or normal subsequent development of the observed structures
will certify that they are embryos.
stimulates elongation.
Several authors have indicated that shoot elongation does not occur till
after root formation. This demonstrates the influence of the root system on
the function of the shoot apex, and suggests that early root induction could be
a means to obtain shoot elongation. Furthermore, rooted buds would benefit
from the juvenile influence exerted by the roots. This effect is of consider-
able importance in propagation experiments with material from mature trees, as
will be discussed later.
Finally, it should be pointed out that the elongation rates of shoots in
vitro is always much lower than those in situ. This shows that the culture
medium does not compensate for the absence of the root system, and that culture
conditions are not optimal yet.
It serves no useful purpose to perpetually produce new generations of buds
if these cannot be stimulated to grow into normal shoots, and more research to
solve this problem is necessary. The technique of axillary bud formation per-
mits accumulation of genetically homogenous bud populations, and thus provides
suitable material for future studies.
Boulay and Franclet (12) obtained roots in shoots (Fig. 14) that developed
from dormant buds excised from Pseudotsuga menziesii plants 6 months, 2, and 4
years old, and from epicotyls from plants 4-6 weeks old. The media used con-
tained one-half or one-third of the mineral concentrations used for shoot
growth. The most effective auxin for rooting was lAA at high concentrations
(10-20 mg 1-1). Rooting is influenced by the age of the parent plant at
the time the buds were excised. The best results were obtained with parent
plants less than 2 years old. When dealing with epicotyl sections, auxins were
not required. The authors also emphasized the importance of the substrate in
root formation and advised that substrates should allow good aeration.
Shoots of Pinus pinaster that had developed from axillary buds were cul-
tured by David and David (31) in a medium with IBA (10- 7 mol), vitamins,
and amino acids. A few of the shoots formed roots.
Root formation was influenced by the origin of the explants that formed
shoots. Coleman and Thorpe (28) noted that in the presence of IBA (5.10- 5
mol) 50% of the shoots that formed on cotyledon explants formed roots,
while in shoots induced on explants of 4- to 10-year-old trees, only 11% did
so. They also showed that the physiological state of the tissues not only in-
fluences bud induction, but also the subsequent behavior of the newly formed
buds.
Cheng and Voqui (26) obtained rooting in 80% of the adventitious shoots that
developed from cotyledon sections of Pseudotsuga menziesii. The young shoots
were cultured on an agar medium containing minerals, a low concentration of
sucrose (0.5%), and NAA (0.25 ~mol). They noted that at a temperature of
19C satisfactory rooting occurred and that the plant lets developed normally.
If the temperature was raised to 24C, callus was formed and the plantlets
showed abnormal growth. They concluded, that depending on the temperature, the
auxin affected different types of cells.
Webb and Street (76) demonstrated that rooting of adventitious shoots of
Pinus contorta and Picea sitchensis was influenced by the type of cytokinin
that had been used for induction of the shoots. If 2-iP or kinetin had been
used for shoot induction, more roots were formed than if BAP had been used.
They also noted that cytokinins with a strong shoot inducing capacity inhibited
both rooting and stem elongation of the shoots.
Rooting of adventitious shoots of Pinus radiata was obtained by Reilly and
Washer (63) on a medium low in minerals, supplemented with NAA (0.5 ppm) and
IBA (2 ppm). Root elongation was subsequently stimulated by a 2-3 week soak in
distilled water.
Chalupa (18) found that shoots that had developed from dormant buds of Picea
abies rooted easier if the buds had been excised from trees less than 2 years
old, than if they had been excised from older trees.
Root formation (Fig. 16) was induced in shoots of Pinus pinaster after
soaking for 24 h in an IBA solution followed by transfer to a well aerated,
sterile mixture of perlite and peatmoss (32). Rooting occurred in 80% of the
shoots from axillary buds of 20-day-old Pinus pinaster plants. Only 50% of the
shoots from the apex of brachyblasts of 2- to 3-year-old trees rooted.
Using the same species, Rancillac (60) found that the auxin concentration
affected the quality of the roots. NAA, at 10- 6 mol, induced many roots at
the base of the shoots, but also callus, which often prevented the establish-
ment of vascular connections between roots and shoots. On the other hand, a
concentration of 10- 7 mol resulted in thin, normal roots, and no callus at
the base of the shoot. A temperature of 20C resulted in better root growth
than 25C. A nutrient, low in minerals, was best for root growth. Transfer to
94
a greenhouse was possible after the roots had grown for 2-3 months on the
sterile medium.
Shoots from dormant buds of Pseudotsuga menziesii were rooted by Boulay
(11), but rooting occurred only if the buds were taken from trees less than
4-years old.
Minocha (54) found that some adventitious buds of Pinus strobus, formed 1
or 2 roots if treated with 0.01-2 mg 1-1 IBA and subsequently cultured on
agar, or on a solidified mineral solution. The author concluded that the low
rooting percentages were the result of an excess of endogenous hormones. High
concentrations of sucrose (3-6%) favored rooting.
Bornman and Jansson (8) obtained rooting in 35% of short shoots of Pinus
sylvestris treated with coumarin (10 ~mol).
-fungicide solution. The shoots were then planted on a mixture of perlite and
vermiculite (4:1) and maintained in a damp atmosphere. About one month later,
up to 80% of the shoots had rooted. The results varied, depending on the vigor
of the shoots at the time of root induction.
Adventitious root formation was also studied by Faye et al. (in press).
They noted formation of two types of roots. In one case, roots elongated from
pointed meristems, in the other, short dichotomously branching roots developed
from rounded meristems. The short roots were similar to roots with mycorrhiza
found on field grown trees. Therefore, the formation of roots resembling those
with mycorrhiza does not depend on the symbiotic fungus.
Root formation in gymnosperms (Figs. 14, 15, and 16) has been studied by
only a few investigators. Root formation has generally only been possible if
the shoots originated from embryos or plants from a few days to a few years
old. Many investigators have observed spontaneous rooting without the aid of
exogenous growth hormones, but the rooting percentages were generally low
(about 1%).
Generally, NAA and/or IBA are used to induce root meristems. It appears
that the duration of application (from 24 h to continuous) required is inver-
sely proportional to the concentration. As in shoot formation, the quality of
the adventitious root system depends on the intensity of the induction stimu-
lus. A strong activation results in many primordia, but in some species the
roots formed from these may not be functional.
Generally, a lowering of the mineral concentration to one-half or one-third
of the concentration used for shoot formation, favored root development. Some
investigators found that cytokinin combinations that favored shoot induction,
subsequently inhibited the formation of new roots. The cytokinin effect was
residual, i.e., it was still noticeable several weeks after removal of the
cytokinin from the medium. Furthermore, the rooting ability like the shoot
forming and stem elongating ability was reduced with increasing age of the
parent plant from which the explants were taken.
Although auxins are generally used for root induction, Smith and Thorpe (64)
have shown that in addition some amino acids and phenylpropanoids were involved
in the formation of root primordia in hypocotyls of Pinus radiata. Haissig
(39) postulated that root formation occurs only if auxins, certain phenolics,
specific oxidases, and activators of these enzymes are present together at a
non-specific site. These substances act synergistically in the induction of
root primordia.
96
6. CONCLUSIONS
In summary, regeneration of plants has been obtained with several species.
In most cases the explants were embryos, and organs, or organ sections from
specimens up to a few years old. The applications of such techniques are
numerous. Of particular interest are studies of genotype-environment inter-
actions, testing of clones for cold and pest resistance, and the rapid multi-
plication of controlled crosses.
99
Figure 19. A Pinus pinaster plant, that originated from an axillary bud of a
very young plant, growing in a nursery bed (David).
Figure 20. A Pinus pinaster plant, that originated from an adventitious bud on
a cotyledon, growing in a nursery bed (David).
However, a tree does not express its full genetic potential till it reaches
the mature stage. During this maturation process several physiological changes
take place in the tree that affect the in vitro behavior of the explants taken
from the tree at various times. This effect has been demonstrated in the
ability to form adventitious and axillary buds, in the rate of shoot elonga-
tion, and rooting. Because of the decreased morphogenetic ability of mature
material it is generally not possible to apply the techniques developed for
juvenile material to mature plants.
Present research is showing that treatment of the plants before excision of
the explants and in vitro culture, as well as manipulation of the environment
after regeneration, will occasionally result in the reappearance of juvenile
behavior. These results raise the expectation, that soon the systematic multi-
plication of selected mature trees will become possible.
Vegetative propagation produces uniform copies, and thus provides a tool for
the propagation of genetically improved specimens. However, the classical
100
procedures have two major problems; low efficiency under nursery conditions,
and difficulties in propagating mature trees. Because tissue culture research
has lead to new, efficient methods of vegetative propagation of several angio-
sperms, a similar approach has been attempted with gymnosperms, but so far with
only limited success.
In comparison with the classical propagation techniques, the experimenter
has greater manipulative control over cells and tissues with in vitro tech-
niques. Furthermore, the classical methods have always suffered from seasonal
constraints. In vitro techniques may remove some of these, and thus improve
the annual propagation rates.
With many gymnosperm species, in vitro propagation presently is possible
only if juvenile material is used. This often provides axillary or adventi-
tious buds, which, if they properly elongate and form roots, will grow into
viable new plants. The ability to form buds, elongated shoots, and roots de-
pends on the species; for example, in some cases shoot elongation is exces-
sively slow, in others rooting is difficult. These problems indicate, that the
optimal conditions for development during each of these phases have not yet
been defined, and that further research is required. Following the in vitro
culture phase it is essential to transfer the new plants to a proper substrate
and to maintain temporarily a high atmospheric humidity.
Methods similar to those used for the propagation of juvenile material could
eventually lead to in vitro vegetative reproduction of mature trees, selected
for their superior phenotype. However, to date, the results with mature trees
have generally been only partially successful. The difficulties encountered
are largely caused by physiological changes that occur during maturation of the
trees. Presumably in vitro culture removes most internal correlations, but
this is not sufficient to rejuvenate the cells in explants from mature trees.
Although some treatments applied during the in vitro phase may initiate par-
tial rejuvenation, the condition of the donor plant at the time of explant
excision is also important. Therefore, research regarding the proper condi-
tioning of the donor plant should be intensified.
Foreseeing the possibility of somatic hybridization, and genetic transforma-
tion of cells by incorporation of organelles, or molecules (see Chapter 12),it
becomes even more essential to master the morphogenetic control mechanisms of
the cells. When techniques are developed to raise new plants from such genet-
ically modified cells, it should be established whether or not the character-
istics acquired through genetic manipulations, are stable.
101
7. REFERENCES
1. ABO EL-NIL M, ZS WOCROK 1978 Morphogenetic heterogeneity of cultured
Douglas fir cotyledons and its correlation with seed weight. Proceedings
of the 4th international congress of plant tissue and cell culture, Univer-
sity Calgary, Alberta, Canada, 515
2. AITKEN J, KJ REILLY 1978 Tissue culture of Radiata pine - Streamlining
techniques for plantlet production. Proceedings of the 4th congress of
plant tissue and cell culture, University of Calgary, Alberta, Canada, 515
3. AL-TALIB K, JG TORREY 1959 The aseptic culture of isolated buds of
Pseudotsuga taxifolia. Plant Physiol 34 : 630-637
4. BALL E 1950 Differentiation in a callus culture of Sequoia sempervirens.
Growth 14 : 295-325
5. BANERJEE SN, NW RADFORTR 1969 In vitro studies on the developing embryos of
Pinus resinosa. Bot Mag 82 : 329-~
6. BONGA JM 1977 Organogenesis in in vitro cultures of embryonic shoots of
Abies balsamea (Balsam fir). In-Vitro 13 : 41-48
7. BONGA JM 1978 Morphogenetic effects of C02 in bud culture of conifers.
Proceedings of the 4th international congress of plant tissue and cell cul-
ture, University of Calgary, Alberta, Canada, 515
8. BORNMAN CR, E JANSSON 1980 Organogenesis in cultured Pinus sylvestris tis-
sue. Z Pflanzenphysiol 96 : 1-6 - - -
9. BOULAY M 1976 Recherches sur la proliferation du Douglas par culture in
vitro. Ann Rech Sylvicoles. AFOCEL, Domaine de l'Etan~on, 77370 Nangi~
France, 84-145
10. BOULAY M 1979 Multiplication et clonage rapide du Sequoia sempervirens par
la culture in vitro. Etudes et Recherches, AFOCEL, Domaine de l'Etan~on,
77370 Nangi~ France 12 : 49-55
11. BOULAY M 1979 Propagation in vitro du Douglas par micropropagation de ger-
mination aseptique et culture~ourgeons dormants. Etudes et Recherches,
AFOCEL, Domaine de l'Etan~on 77370 Nangis, France, 12 : 67-75
12. BOULAY M, A FRANCLET 1977 Recherches sur la propagation vegetative du
Douglas: Pseudotsuga menziesii (Mirb.) Franco. Possibilites d'obtention de
plants viables a partir de la culture in vitro de bourgeons de pieds-meres
juveniles. CR Acad Sci 284 : 1405-1407---
13. BOULAY M, C MELIN, D LAFFRAY 1974 Recherches preliminaires sur la culture
in vitro de bourgeons de Douglas. Ann Rech Sylvicoles. AFOCEL, Domaine de
I'Etan~on, 7730 Nangis, France, 47-145
14. BROWN CL, HE SOMMER 1975 An atlas of Gymnosperms cultured in vitro:
1924-1974. Georgia Forest Research Council, Macon, Georgia - - - -
15. CAMPBELL RA, DJ DURZAN 1975 Induction of multiple buds and needles in tis-
sue cultures of Picea glauca. Can J Bot 53 : 1652-1657
16. CAMPBELL RA, DJ DURZAN 1976 Vegetative propagation of Picea glauca by
tissue culture. Can J For Res 6 : 240-243
17. CHALUPA V 1975 Induction of organogenesis in forest tree tissue cultures.
Commun Inst For 9 : 39-50
18. CHALUPA V 1977 Development of isolated Norway spruce and Douglas fir buds
in vitro. Commun Inst For Cech 10 : 71-78
102
19. CHALUPA V 1977 Organogenesis in Norway spruce and Douglas fir tissue cul-
tures. Commun Inst For Cech 10 : 79-87
20. CHALUPA V, DJ DURZAN 1973 Growth of Norway spruce (Picea abies) tissue and
cell cultures. Commun Inst For Cech 8 : 111-125
21. CHALUPA V, DJ DURZAN 1973 Growth and development of resting buds of coni-
fers in vitro. Can J For Res 3 : 196-208
22. CHEAH~~CHENG 1978 Histological analysis of adventitious bud forma-
tion in cultured Douglas fir cotyledon. Am J Bot 65 : 845-849
23. CHENG TY 1975 Adventitious bud formation in culture of Douglas Fir
(Pseudotsuga menziesii Mirb.) Franco. Plant Sci Lett 5 : 97-103
24. CHENG TY 1976 Vegetative propagation of western hemlock (Tsuga hetero-
phylla) through tissue culture. Plant Cell Physiol 17 : 1347-13~
25. CHENG TY 1977 Factors affecting adventitious bud formation of cotyledon
culture of Douglas fir. Plant Sci Lett 9 : 179-187
26. CHENG TY, TH VOQUI 1977 Regeneration of Douglas fir plant1ets through tis-
sue culture. Science 198 : 306-307
27. COLEMAN W, TA THORPE 1976 Induction of buds in tissue cultures of four dif-
ferent conifers. Plant Physiol Supp 57 : 67
28. COLEMAN W, TA THORPE 1977 In vitro culture of western red cedar (Thuja
plicata Donn.). I. Plantler-formation. Bot Gaz 138 : 298-304
29. DAVID A 1972 Effets de diverses solutions minerales sur 1a proliferaton de!
tissus de Pin maritime en culture in vitro CR Acad Sc 275 : 2857-2860
30. DAVID A 1979 Manifestations de diverses potentia1ites organogenes et micro-
propagation vegetative chez Ie Pin maritime (Pinus pinaster Sol.). Ann Recl
Sylvicoles, AFOCEL, Domaine de l'Etan~on, 77370 Nangis, France, 57-75
31. DAVID A, H DAVID 1977 Manifestations de diverses potentia1ites organogenes
d' organes ou de fragments d' organes de Pin mari time (Pinus pinaster Sol.)
en culture in vitro. CR Acad Sc 284 : 627-630 -----
32. DAVID H, K ISEMUKALI, A DAVID 1978 Obtention de plants de Pin maritime
(Pinus pinaster Sol.) a partir de brachyblastes ou d'apex caulinaires de
tres jeunes sujets cultives in vitro. CR Acad Sc 287 : 245-248
33. DAVID A, MJ THOMAS 1979 Organogenese et multiplication vegetative in vitro
chez les Gymnospermes. L'annee BioI 18 : 381-416 -- -----
34. DURZAN DJ 1979 Progress and Promise in forest genetics. Proceedings of the
50th anniversary conference, "Paper Science and Technology - the Cutting
Edge", Appleton, Wisconsin, The Institute of Paper chemistry, 31-60
35. FRANC LET A 1979 Rajeunissement des arbres adultes en vue de leur propaga-
tion vegetative. Etudes et Recherches. AFOCEL, Domaine de l'Etan~on, 77370
Nangis, France, 12 : 3-18
36. FRANCLET A, A DAVID, H DAVID, M BOULAY 1980 Premiere mise en evidence mor-
phologique d'un rajeunissement de meristemes primaires caulinaires de Pin
maritime (Pinus pinaster Sol.). CR Acad Sc 290 : 927-929
37. FRIDBORG G~EDERSEN, L LANDSTROM, T ERIKSSON 1978 The effect of activa-
ted charcoal on tissue cultures : adsorption of metabolites inhibiting mor-
phogenesis. Physiol plant 43 : 104-106
38. GAUTHERET RJ 1959 La culture des tissus vegetaux. Techniques et realisa-
tions, Masson, Paris
39. HAISSIG BE 1974 Influences of auxins and auxin synergists on adventitious
root primordium initiation and development. NZ J FOR SCI 4 : 311-323
40. HASEGAWA PM, T YASUDA, TY CHENG 1979 Effect of auxin and cytokinin on new1)
synthesized proteins of cultured Douglas fir cotyledons. Physiol Plant 46
211-217
41. ISEMUKALI K 1979 Manifestation de diverses potentia1ites organogenes et
Micropropagation vegetative chez Ie Pin maritime (Pinus pinaster Sol).
These de Doctorat de 3e cycle, Laboratoire de Physiologie Vegetale,
103
64. SMITH DR, TA Thorpe 1977 Root initiation in cuttings of Pinus radiata seed-
lings : effects of aromatic amino acids and simple phenyl propanoids. Bot
Gaz 138 : 434-437
65. SOMMER HE 1975 Differentiation of adventitious buds on Douglas fir embryos
in vitro. Proc Int Plant Prop Soc 25 : 125-127
66. SOMMER HE, CL BROWN 1974 Plantlet formation in pine tissue cultures. Am J
Bot Supp 61 : 11
67. SOMMER HE, CL BROWN, PP Kormanik 1975 Differentiation of plantlets in long-
leaf pine (Pinus palustris Mill.) tissue cultured in vitro. Bot Gaz 136 :
196-200 -- - --
68. THOMAS MJ, E DUHOUX, J VAZART 1977 In vitro organ initiation in tissue cul-
tures of Biota orientalis and other species of the Cupressaceae. Plant Sci
Lett 8 : 395-400
69. TRANVAN H 1979 In vitro adventitious bud formation on isolated seedlings of
Pinus silvestriS-L. BioI Plant 21 : 230-233
70. VAZART J, M DA CONCEICAO, MJ THOMAS 1979 Structure anatomique et cytolo-
gique de l'hypocotyle du Biota orientalis L. au stade de l'etalement des
cotyledons. Rev Cytol BioI Veget 2 : 83-96
71. VON ARNOLD S, T ERIKSSON 1978 Induction of adventitious buds on embryos of
Norway spruce grown in vitro. Physiol Plant 44 : 283-287
72. VON ARNOLD S, T ERIKSSON 1979 Induction of adventitious buds on buds of
Norway spruce (Picea abies) grown in vitro. Physiol Plant 45 : 29-34
73. VON ARNOLD S, T ERIKSSON 1979 Bud induction on isolated needles of Norway
spruce (Picea abies) grown in vitro. Plant Sci Lett 15 : 363-372
74. WEATHERHEAD MA~URDON, GC-HENSHAW 1978 Some effects of activated char-
coal as an additive to plant tissue culture media. Z Pflanzenphysiol 89
141-147
75. WEATHERHEAD MA, J BURDON, GG HENSHAW 1979 Effects of activated charcoal as
an additive to plant tissue culture media : Part 2. Z Pflanzenphysiol 94
399-405
76. WEBB KJ, HE STREET 1977 Morphogenesis in vitro of Pinus and Picea. Acta
Hortic 78 : 259-269 - -- -- --
77. WINTON LL, SA VERHAGEN 1977 Shoots from Douglas fir cultures. Can J Bot 55
: 1246-1250
78. WINTON LL, S VERHAGEN 1977 Embryoids in suspension cultures of Douglas fir
and Loblolly pine. Tappi, Paper Chemistry, Appleton, Wisconsin. 21-24
79. WOCHOK ZS, M ABO EI-NIL 1977 Conifer tissue culture. Proc Int Plant Prop
Soc 27 : 131-135
!O5
APPENDIX
CYCADALES
GNETALES
CONIFERALES
Pinaceae
Continued
Continued
Taxodiaceae
Continued
Cupressaceae
1. INTRODUCTION
Vegetative propagation of woody dicots possessing one
or ~ore desirable genetic traits has been practiced by man
for centuries. Numerous species of ornamental, fruit, or
nut trees among diverse genera have been cloned successfully
by rooted cuttings, layering, or budding and grafting depend-
ing upon species response, utility and needs (48, 29).
Asexual propagation has been used infrequently in commercial
forest production because most forest trees are difficult to
root past their juvenile stage and the millions of plants
needed in reforestation programs each year have been more
easily produced from seed. with ~nprecendented progress in
tissue culture technology during the past decade, coupled
with the continued spiraling demand for cellulose throughout
the world, one can predict with certainty that asexually
produced trees will comprise a significant portion of forest
planting stock in the future.
eM
eM
4. ECONOMIC CONSIDERATIONS
4.1. Cost comparisons of seedlings produced by tissue culture
techniques versus seedlings produced from seed
In attempting to make direct economic comparisons
between different systems of producing plantable seedlings
for reforestation one is always confronted with making
certain assumptions within the limits of recognized constraints.
In order to obtain some reasonably reliable comparisons
between seedlings produced via tissue culture and those
produced by conventional nursery techniques one is confronted,
with three broad considerations, namely: (1) capital
outlay in the form of land, buildings, facilities, and
equipment; (2) total direct production costs including
supervisory personnel, labor, taxes, insurance, supplies and
expendibles, equipment operation, heating and cooling,
lighting, and etc. which continue to escalate continuously;
and (3) the potential value of the product produced within a
given time period, in our case, the yield of forest products
at the end of the rotation.
For simplification purposes only, assume that capital
outlays and depreciation rates are comparable for both
systems even though the large acreages required for commer-
cial seed production in seed orchards, plus considerable
nursery acreage required for seedling production, offices,
warehouses for seedling packaging and storage, outlays for
heavy equipment, tractors and etc., would in all probability,
far exceed a tissue culture facility consisting of one
building containing approximately 3000 square feet of usable
space with a controlled temperature and light facility of
only 400 square feet, a small greenhouse (ca. 1200 sq. ft.)
and attendant lathhouse space of ca. 20,000 square feet for
use in acclimating seedlings prior to planting. Furthermore,
to be more conservative we may also disregard the value of
the final product produced although by fixing the genotype
of the best selected genetically superior clones one could
certainly expect greater yields from cloned material then
130
6. FUTURE OUTLOOK
6.1. Use of shoot-tip cultures
Of the present strategies used for vegetative propagation
of economically important dicot trees, the use of shoot tip
136
7. REFERENCES
1. ANDERSON WC, GW MEAGHER 1977 Cost of propagating
broccoli plants through tissue culture. Hort Sci 12:
543-544
2. ANDERSON WC, GW MEAGHER 1978 Cost of producing
plants through tissue culture using Lilies as an example.
Proc Oregon State University Ornamentals Short Course
Mimeographed
3. BERBEE FM, JG BERBEE, AC HILDEBRANT 1972 Introduction
of callus and trees from stem tip cultures of a hybrid
poplar. In Vitro 7: 269
4. BERBEE JG, JO OMUEMU, RR MARTIN, JD CASTELLO 1976
Detection and elimination of virus in Populus. In
Intensive Plantation Culture. USFS-USDA Tech Report NC
21 N Cent For Exp Sta, pp 91
5. BHATNAGAR HP 1974 Vegetative propagation rooting
practices with forest trees in India. NZ J For Sci 4:
170-176
6. BILAN MV 1974 Rooting of Liquidambar styraciflua
cuttings. NZ J For Sci 4: 177-180
7. BONGA JM 1977 Applications of tissue culture in
forestry. In J Reinert, YPS Bajaj, eds, Applied and
Fundamentar-Aspects of Plant Cell, Tissue, and Organ
Culture. Springer-Verlag Berlin, pp 93-108
8. BROWN CL 1976 Forests as energy sources in the year
2000: What man can imagine, man can do. J For 74: 1-6
9. BROWN CL 1980 Application of tissue culture technology
to production of woody biomass. International Energy
Agency Proc, Brighton England, Oct 30-Nov 1 1980 (In press)
138
Tree Year
Species age a Explant Source Cultured Reference
Tree Year
Species age a Explant Source Cultured Reference
Table 3. Some Dicotyledonous Species from which Embryoids have been Regenerated from Tissue
or Organ Culture a
a This list is incomplete with respect to other cultivated woody plants, notably Citrus
Carica, Hevea, Vitis etc.
bMS - Murashige and Skoog; HE - Heller; WH - White; LS - Linsmaier and Skoog; BL - Blades
147
Approximate
System Costs at Production Site
Bare root, 1-0
Nursery Stock From
Unimproved Seed $65.00 a
Containerized Seedlings
Using Unimproved Seed $75.00 b
Containerized Seedlings
From Shoot Tip Cultures $123.00 c
~
\0
150
Grafting techniques that have been successful with Eucalyptus are: approach
grafting, side grafting, splice grafting, cleft grafting, whip grafting, bark
grafting, ring-veneer grafting, herbaceous grafting, crown grafting and budding
(71).
Species of some systematic groups graft easily onto their own rootstock
whereas other species take better when grafted onto a rootstock of a different
species (108). In addition, the establishment of a graft union is not necessar-
ily related to the future growth and compatability of the graft. Delayed incom-
patability is common in grafts of Eucalyptus and makes this means of vegetative
propagation uneconomical. For example, in grafts of E. deglupta, there were no
incompatability symptoms until shortly before the death of the tree which oc-
cured in trees several years old (38). In addition, grafts of ~ grandis scions
onto ~ deglupta rootstocks died even though an effective graft union was es-
tablished and the scion had been growing actively for several months (71). Most
154
3. TISSUE CULTURE
Callus formation in tissue culture of Eucalyptus has been reported in a
number of species (Table 1). The different plant parts that have been shown to
develop callus include seeds, hypocotyls, seedling roots, stem segments, pet-
ioles, leaf blades, apical shoots, lignotubers, anthers, bark explants and
pollen grains (67).
In early studies (75, 115), coconut milk was found to be essential in the
culture medium to sustain growth of Eucalyptus callus. However, it was shown
(39) that coconut milk was not necessary and that ~ bancroftii callus can grow
vigorously on a completely defined medium and ~ grandis, ~ laevopinea, E.
melliodora and E. nicholli on a medium with casein hydrolysate.
Callus from adult ~ grandis and E. urnigera was grown (49) on a defined
medium with neither coconut milk nor casein hydrolysate. A black exudate arose
in many callus cultures and caused problems (39, 51, 67, 75).
Sussex (115), using single cell cultures of E. camaldulensis that had passed
through 36 passages during three years, and Piton (104), using callus cultures
of E. camaldulensis initiated 10 years previously, both showed that the ploidy
level (2n=22 chromosomes) in this species did not change during prolonged
periods of culture and subculture. However, it was also found that although
mitoses were normal until metaphase, there were often anomalies during anaphase
and telophase in the young dividing cells in the external layers of the callus
(104).
156
Species Author
Eucalyptus alba Reinw. ex. B1. Kitahara and Ca1das (77); Goncalves (66).
E. bancroftii (Maid.) Maid. de Fossard (39); de Fossard et al (45); Lee
and de Fossard (81).
E. cama1dulensis Delnh. Jacquiot (75); Sussex (115); Piton (104);
Goncalves (66).
E. citriodora Hook. Aneja and Atal (2); Lakshima Sita (79);
Lakshima Sita and Vai1yanathan (80).
E. cladocalyx F. Muell. Jacquiot (75).
~ gomphocephala DC. Jacquiot (75).
E. grandis Hill ex. Maid. Cresswell and de Fossard (29); de Fossard
(39); de Fossard et al (45); Goncalves
(66); Kitahara and Caldas (77).
E. gunnii Hook f. Jacquiot (75).
~ ~inea R.T. Pak . ' de Fossard (39); de Fossard et a1 (45).
~ macu1ata Hook. Goncalves et al (67).
~ melliodora A. Cunn. ex Schau. de Fossard (39); de Fossard et a1 (45).
~ nichollii Maid. and Plake1y de Fossard et a1 (45).
~ nova-anglica Deane and Maid. Winton (117).
~ ob1iqua L'Herit. Blake (9).
~ polybractea de Fossard (39).
E:- robusta Sm. Goncalves (66).
~ saligna Sm. Goncalves (66).
E:- tereticornis Sm. Jacquiot (75); Goncalves (66).
~ x trabuti (hybrid Marcavi1aca and Monta1di (85).
-- botyroides x camaldulensis)
E. urnigera Hook. f. de Fossard et a1 (45).
~ urophylla S.T. Blake Goncalves et a1 (67).
~ viminalis Labill. Blake (9).
4. ORGAN CULTURE
Contrary to tissue culture where simple cells are cultured, in organ culture
differentiated tissues such as leaves, stems and roots are placed in a control-
led system of nutrients and environment. Roots and/or buds are induced on the
explant either directly or after the formation of a callus. The number of tree
species, including Eucalyptus, which can be propagated by organ culture tech-
niques is increasing (1, 71, 92). These organ culture techniques are now often
being used in preference to the traditional methods of vegetative propagation
of trees because of the very high multiplication rates that are possible (some-
times millions per year) (71).
Preliminary experiments on organ culture of Eucalyptus, were undertaken in
1970 (27, 28). Initially, cultures of apical tips from adult field grown ~
Nodes from adult trees are much more difficult to root and to develop into
plantlets. However, success has been obtained from E. grandis plants up to 4
years old, a S year old ~ dalrympleana tree (49, SO, S1), and ~ ficifolia (6,
40). No success with rooting was obtained from nodes of adult ~ urnigera, ~
obtained (49) presumably because the micro-organisms were present within the
tissues.
After surface sterilization by the method of Cresswell and Nitsch (31), fol-
lowed by two to four weeks of culture, bacteria or fungi developed either from
the cut ends of the explants or from the abscission layers of the petiole on
the node when using field-grown plants. To reduce this type of contamination,
surface disinfection (30, 31) was extended by soaking the branches overnight in
running tap water or in a weak solution of calcium hypochlorite prior to the
routine surface sterilization technique. There are many variations of this
technique but success depends on the state of damage of the tissues to be
cultured.
Obtaining aseptic plant tissues threatens to be a major stumbling block for
the adoption of the organ culture technique for routine vegetative propagation
of field-grown plants. Nevertheless, one advantage of the organ culture tech-
nique developed for ~ grandis (31) is that actively growing tissues are most
suitable for root initiation. Tissues which are relatively young should have
suffered less damage than older tissues and thus should be easier to disinfect.
To obtain large numbers of young shoots, glasshouse-grown 18 months old E.
grandis trees were pruned (43). The new growth was protected by covering with
glacine bags, which further reduced the subsequent contamination rate in vitro.
The branches probably should be treated with a fungicide and/or insecticide
before pruning and bagging in order to destroy insect eggs. Although this
reduces contamination of induced coppice shoots on adult trees, the subsequent
effect of the insecticide on the growth of the explant in vitro is unknown and
it still only partially resolves the problem of obtaining aseptic tissues from
adult trees.
Often all cultures or even subcultures suddenly became contaminated by
creamy-white bacteria either as a liquid on the surface of the medium or as a
cloud within the medium. This contamination has been identified at AFOCEL as
Bacillus lentus, which is transmitted by alcohol on the forceps or other
implements used in sterile culture work.
4.2.2. Brown exudate. Various methods of eliminating brown exudate or
preventing its formation by woody species in vitro have been tried but rarely
with complete success (75, 96, 109). The darkening of explants, callus and
medium used in the culture of Eucalyptus tissues has been reported (30, 31, 39,
51, 67, 75).
160
In E. grandis organ cultures, there were found to be two exudates (49). One
exudate, produced as a result of wounding, appeared within one hour of excisiol
of the explants and was aggravated by certain constituents of the culture
medium such as high sucrose, serine, chlorogenic acid, cytokinins (49), a high
boron concentration (67), and light (Table 2). This initial exudate, which
increases with age and woody character of the tissues, led to death of the
cultured explants. The second exudate appeared near the end of the incubation
period and seemed to be a product of dying cells.
Complete darkness o
Diffuse light (1,500 lux) +
Short days 8 hr light/16 hr dark ++
Long days 16 hr light/8 hr dark ++++
With callus of ~ bancroftii and ~ nichollii, it was observed (39) that the
increased amount of exudate corresponded to a decrease in the fresh weight of
the callus produced during the culture period. Similary, it was found (67) that
with ~ grandis anther callus, the callus with the least growth at the end of
the culture period, had the highest polyphenoloxidase (PPO) activity. An analy-
sis of the actual values indicated that the increase in PPO activity was of. the
same magnitude as the increase in fresh weight of the tissues. The presence of
polyvinylpyrrolidone, tyrosine and cysteine were inhibitory to growth and did
not reduce PPO activity (67).
The initial exudate in E. grandis cultures could be reduced and eliminated
(51) in most cases if:
a) healthy tissues of a stem diameter less than 5 mm were chosen and the
surface sterilization process did not damage the tissues;
b) a soaking pretreatment (dissected explants are soaked in sterile dis-
tilled water in the light for three hours before planting) was done and the
cultures were initially incubated in darkness (8 days for E. grandis organ
cultures).
The water in which the ~ grandis tissues were soaked was examined (51) and
it was found that the soak water was inhibitory in the cress seed germination
bios say and in the tomato rooting test. However, after treatment of the soak
161
Table 3. Phenolic content of extracts from leaf disks and nodes of E. grandis
(24 explants/20 ml sterile dist'illed water). Polyphenols expressed as
~g gallic acid equivalents/ml (49).
There were more phenols produced by leaf disks than by nodes possibly
because leaf disks had a greater percentage of damaged tissue from the dissec-
tion.
The types of phenols in the soak water were then analyzed following the
technique of Marigo (87). Since it had been observed (49) that dark prepared
extracts had little or no effect on the cress seed germination bioassay and the
tomato rooting test, both light (8,000 lux) and dark prepared extracts were
analyzed. As can be seen in Table 4, at the time of extraction (To) there were
more phenols and especially flavonoids present in light prepared extracts than
in dark prepared ones.
These results lead to the question: were the flavonoids the inhibitors? In
the bioassays, there was inhibition for the first 2-6 days before rooting or
germination, depending on the concentration of the leachates. In addition,
observation of the extracts (after removal of the tissues) revealed a change ir
color from yellow to brown with time. The light and dark prepared extracts
were analyzed for their phenol content at To and after storage of the extract
at 25C in the light for 13 days (Table 4). This revealed that in the dark
prepared extracts, there was an increase in flavonoids in the light over the 1~
days period whereas in the light made extracts, where initially the flavonoid
~ontent was high, the flavonoids were completely degraded and disappeared.
Although the procedure of estimating the flavonoid content is limited (only the
flavonoids with 3 hydroxyl groups on the A nucleus like phloroglucinol are
precipitated by formaldehyde (110, it is tempting to postulate an inhibitory
role of flavonoids in the exudates, especially since four rooting inhibitors,
presumed to be derived from phloroglucinol, have been isolated from adult
leaves of ~ grandis (33, 34, 48, 101). It has also been reported (94, 95) that
some inhibitors are decomposed in water.
Flavonoids had little effect on rooting in ~ grandis tissue cultures
because the long time required for root initiation (8 days for cuttings and 2
weeks for nodes in vitro) permitted the flavonoids to be degraded.
These studies (49) have revealed that phenols are present in the leachates
from newly-dissected ~ grandis tissues and that these phenols, particularly
the flavonoid category, are inhibitory to the processes of seed germination and
elongation of tomato roots. In order to obtain tissue and organ cultures of E.
grandis free from initial brown exudate, they must be cultured under conditions
which do not favour phenol synthesis. For example, in the dark or low intensit)
light on a medium devoid of phenol precursers. It was also seen (49) that the
physiological state of the parent plant is extremely important with respect to
exudate formation. Less exudate is produced in culture if the branches are
submitted to a cold pretreatment (5C for several days before dissection) or
the parent plants are grown under short days.
4.2.3. Rooting inhibitors. There are several reports of specific rooting
inhibitors in Eucalyptus. Namely, the three G inhibitors isolated and chemi-
cally identified from adult ~ grandis (32, 34, 94, 95, 99, 100, 113,) and
inhibitor from a crude extract of adult ~ deglupta (37) and more recently,
Grandinol from adult leaves of E. grandis (33).
163
~ deglupta forms roots readily on cuttings from trees up to one year old
but does not produce roots on tissues from five year old trees. The presence of
an inhibitor in a methanol extract of adult ~ deglupta leaves and stems was
established (37) using the cress seed germination bioassay.
The concentration of rooting inhibitors was found to increase (94, 95) in
successively older leaves of E. grandis. This increase in concentration of
inhibitors was correlated with a decrease in rooting ability of cuttings taken
from successively higher internodes (100). The G inhibitors, which are presumed
to be derived from phloroglucinol, have been found in high concentrations in
adult ~ grandis leaves and in low concentrations in juvenile leaves of ~
activation of growth. Similarly, the requirements for active growth of the tis-
sues is demonstrated by the fact that cuttings taken from seedlings and young
epicormic shoots are more likely to root than cuttings from adult trees. To
study seasonal effects in rooting of nodes, in vitro cuttings from hedged E.
grandis plants were grown under completely controlled temperature and photoper-
iodic conditions in the Phytotrons at Gif-sur-Yvette. Many different combina-
tions of temperatures and daylengths were tested and as expected, the growth of
the parent plant and the rooting ability of its tissues changed depending on
the conditions. Best rooting of leaf disks, nodes and apices generally occurred
in those of plants which were the most actively growing at the time of dissec-
tion.
It was found that there was a seasonal trend in the number of nodes that
rooted and that the best rooting conditions were not always the same (Table 5).
January 33 33 83 9
April o o 25 50
A high percentage of rooting was obtained initially when the plant was
placed in a new environment because growth of the whole plant was stimulated.
Several months later, there was a decrease in rooting because the plant had
become accustomed to the environmental conditions and zones of metabolic act iv-
ity had been established within the plant. Thus it appeared that the shock of
changing a plant from one set of environmental conditions to another affected
the metabolism of the plant and stimulated rooting. This was also demonstrated
by another experiment (51), in which the rooting of ~ grandis nodes was
determined before transfer of the plants and 3 and 9 weeks after transfer of
the plants to the new environment. Eight plants (regenerated from nodes from 2
plants i.e. two genotypes) were transferred from 24/17 D c L.D. into 4 different
environmental conditions (Table 6); two seedlings were left unchanged. In table
6, there was no significant difference between environmental conditions but
there was a significant increase in rooting of nodes from plants kept between 0
165
and 9 weeks in the new environment. Nodes of the young seedlings which did not
change environment maintained a steady rate of root initiation. Hence the
active growth stimulated by the change in environment for adult tissues and the
active growth of the seedlings, led to a positive rooting response.
Weeks
o 4 0 0 0 58.5
3 12.5 44 8 16.5 66.5
9 29 37.5 21.5 17 64
(effect of horizontal node level), as gauged by the growth of the ,l eaf on the
node. Best rooting occurred in nodes where a functional yellow/green to green
leaf was present and where the axillary bud had not commenced growth before
culture. When nodes with very young leaves less than two-thirds of the final
size, or nodes with senescing leaves or from which the leaves had already
abscised, were cultured roots were rarely initiated under the test conditions.
Choice of replicates should not consist of the last 3 to 4 nodes of each branch
but of higher numbered nodes (apex = 1) and should depend on the length of the
branch, presence of axillary buds and the physiological state of the leaves.
For natural cold trials, the boxes were taken to the site sufficiently early
for the plants to be hardened off before the first cold. After two winters in
the field the most resistant and most vigorous plants were cut back, cuttings
were grafted onto young seedlings and the new growth from this stock was sent
to the laboratory at Etan~on for mass propagation in vitro. Thus in July 1980,
this technique of mass production was done with the new growth of 13 individ-
uals from winter selection in 1978/79 and 1979/80.
1 "Melfert" is the registered name for special containers designed for the
planting out of in vitro produced plantlets.
170
It is important to underline that even though the clones were entering their
3rd year, they were still relatively juvenile because a continual supply of
juvenile sprouts was obtained by the cutting back operation. Trials using parts
of branches taken from the same clones which were not cut back failed but not
from lack of reactivity but because of infection. The tissues from the cut
back plants are the origin of the actual industrial cultures. Thus this justi-
fies the work being done on the stimulation of rejuvenalized growth on scions
of adult trees by spraying with cytokinins (89).
Plants sprayed with solutions of methanol/water with 50 mg/l of benzylamino-
purine produced an abundance of apparently axillary and perhaps adventive buds
whose morphology appears to be promising even for horticultural type cuttings
in situ. These results are in agreement with those recently published (89) for
E. ficifolia and for Pinus pinaster (35, 63).
5.2. Introduction of clones in vitro
The following methods are presently being used at AFOCEL. As soon as the
branches are taken from the mother plant, the cut ends are sealed with paraffin
and dipped for 8 minutes in filtered calcium hypochlorite solution. Then the
branches are rinsed two times in sterile water and cut into segments consisting
of one node and 1/3rd of each of 2 leaves. Each cutting is planted upright in
an agar medium in a 25 x 150 mm tube with a cellulose stopper. The culture
medium used (medium I) is that of Murashige and Skoog (1962) modified by a
reduction of the calcium by one third. There is a high cytokinin/auxin balance,
BAP 1 mg/l, NAA 10-2 mg/l. In order to avoid browning of the tissues and
the medium, the tissues must be excised and immediately placed in the dark in a
culture room at 25C (50). If the plants have been grown in a glasshouse and
sprayed with the fungicide benomyl 24 to 48 hours before the branches are
taken, then less than 40% of the cuttings become contaminated in vitro.
After 8 days darkness, the cultures are transferred to a culture room with
photo-thermoperiod of 16 hours light (2 x 40 W Sylvania Gro-Lux tubes 30 cm
from the cultures) at 25C and 8 hours darkness at 20C.
After 2 or 3 weeks on medium I, axillary and proventive buds multiply and
develop. Generally, the proventive buds give the best results in the multipli-
cation stages. These buds are separated and transplanted as soon as possible
before the development of suffocating callus from the abscission layers on the
petiole.
5.3. Multiplication of shoots in vitro
Cytokinins were not used in the medium for the propagation of very young
171
seedlings (50, 82). Multiplication resulted from the repeated elongation and
division of the axillary buds. The technique adopted for 1 to 2 year old clones
selected for cold hardiness resembles the one used by de Fossard et al (44).
Shoots isolated from the primary cultures are transferred either in the first,
third or fourth subculture depending on the clone, onto the multiplication
medium (medium II). This differs from medium I by the vitamins (44) and by a
reduction of BAP from 1 mg/l to 0.1 mg/l with NAA at 10- 2 mg/l in both
media.
If the multiplication is continued on medium I, the tissues very rapidly
become transparent with fragile leaves and stems. Multiplication on medium II
results in intense axillary budding. The leaves become small and thick and have
a cotyledonary morphology and a purple colored lower surface. The very short
internodes thicken and bunches of buds grow in all directions even within the
agar itself. Reduction of the BAP prescribed for medium II, leads to a reduc-
tion in the multiplication rate but allows a stability of the physiological
state of the stock in relation to the rhythm of the subcultures. With monthly
subcultures and stabilized climatic conditions, a multiplication rate of more
than ten per month can be obtained. Contrary to the method of other workers
(50, 82) which requires a careful dissection of the small shoots into nodes, in
this method the clumps of buds are simply divided into ten subcolonies. Further
dissection of these colonies easily produces a propagation rate of more than 30
per culture (Fig. 6).
5.4. Elongation of the shoots
To stimulate elongation of the shoots and to obtain a favourable leaf mor-
phology, two factors, activated charcoal and gibberellic acid were tested, at
first separately, then together. Gibberellic acid in the range 0.01 mg/l to 10
mg/l was added to the medium II before autoclaving. All the concentrations
higher than 1 mg/l lead to a considerable elongation of the internodes of the
transferred shoots. Growth of 30 to 40 mm in one week was not rare, but the
elongated shoots had a undesired morphology; the round leaves became longer and
pointed and their dimensions were reduced from the base to the apex of the
stem. At the same time, the internodes increased from 1 mm to more than 15 rom.
These phenomena occurred in less than 15 days, after this time the apex of the
shoots died and the leaves became fragile and fell at the slightest shock. If
transplanted earlier, the cultures can be rooted and placed again on medium II
but death occurs in 90% of the cases. However, media containing less than 0 .5
mg/l (with an optimum at 0.1 mg/l) gibberellin, though causing less elongation,
172
6. CONCLUSION
The described method permits a maximum multiplication rate of 30 buds per
culture if the buds are carefully dissected at the end of the multiplication
phase. In practice, this rate is reduced to 10 per month by a quicker random
dissection of the colonies of buds. This is generally sufficient to provide
enough plant material. Cold storage did not stimulate the multiplication rate;
there was no noticeable change in reactions of the buds after cold storage for
6 months.
The culture of buds on a slowly agitated liquid medium has also been tested.
Eventually such cultures could lead to multiplication of shoots of Eucalyptus
clones in chemostats on an industrial scale (15). Dissected shoots could then
be elongated on an agar medium containing gibberellin and activated charcoal in
order to eliminate the unfavourable after effects of the strong concentrations
of cytokinins used in phases 1 and 2.
For sanitary reasons, all operations were done in individual 25 mm test
tubes. However, the rooting phase which lasts only 2 weeks can be done more
economically in 500 ml bottles. Present research suggests that by using special
Table 7. Expenses of the present techniques of in vitro mass production of Eucalyptus
Type of operation Transplanted Preparation Preparation Placed in con- Culture in Yield Cost
under sterile of vessels and distribu- tainers and greenhouse per of
conditions tion of media planted out and harden- plant material
ing off
- .J
u.
-
176
7. REFERENCES
1. ABBOT AJ 1977 Propagating temperate woody species in tissue culture. Sci
Hortic 28: 155-162
2. ANEJA S, ATAL CK 1969 plantlet formation in tissue cultures from ligno-
tubers of Eucalyptus citriodora Hook. Curr Sci (Bangalore) 38: 69
3. ASAHIRA T, NITSCH JP 1968 Effect of polarity and kinetin on the browning
reaction of Dioscorea batatas and D. japonica. Planta (Berl) 84: 292-294
4. BACHELARD EP, STOWE BB 1963 Rooting of cuttings of Acer rubrum L. and
Eucalyptus camaldulensis Dehn. Aust J BioI Sci 16: 751-7-6-7----
5. BACHE LARD EP, STOWE BB 1963 Growth in vitro of roots of Acer rubrun L.
and Eucalyptus camaldulensis Dehn. Physiol Plant 16: 20-~ ------
6. BARKER PK, DE FOSSARD RA, BOURNE RA 1977 Progress towards clonal propaga-
tion of Eucalyptus species by tissue culture techniques. Internat Plant
Propagators' Soc Combined Proc 27: 546-556
7. BATCHELLER OAJ 1973 New concepts in budding and grafting Eucalyptus.
Internat Plant Propagators' Soc Combined Proc 23: 195-200
8. BHATNAGAR HP, JOSHI DN 1973 Vegetative propagation of E. tereticornis Sm.
(mysore de lignotubers) Indian For 99: 509-519
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34. CROW WD, OSAWA T, PLATZ KM, SUTHERLAND DS 1976 Root inhibitors in
Eucalyptus grandis II. Synthesis of the inhibitors and origin of the
peroxide linkage. Aust J Chern 29: 2525-2531
35. DAVID A, DAVID H, MATEILLE T, JARLET E 1981 Bourgeonnement adventif in
vitro sur des cotyledons et aiguilles de Pin maritime (Pinus pinaster-
Sol). Ann Rech Sylvicoles AFOCEL 1980, pp 45-55 --
36. DAVIDSON J 1974 Grafting Eucalyptus deglupta. NZ J For Sci 4: 204-210
37. DAVIDSON J 1974 Reproduction of Eucalyptus deglupta by cuttings. NZ J For
Sci 4: 191-203
38. DAVIDSON J 1977 Problems of vegetative propagation of Eucalyptus. FAO
Third World Consultation on Forest Tree Breeding. Canberra, Australia, pp
857-882
39. DE FOSSARD RA 1974 Tissue culture of Eucalyptus. Aust For 37: 43-54
40. DE FOSSARD RA 1978 Tissue culture propagation of Eucalyptus ficifolia F.
Muell. In Proceedings of a symposium on Plant Tissue Culture. Science
Press, Peking, pp 425-438
41. DE FOSSARD RA, BOURNE RA 1976 Vegetative propagation of Eucalyptus fici-
folia F. Muell. by nodal culture in vitro. Internat Plant Propagatorsr-Soc
Combined Proc 26: 373-378 -- -----
42. DE FOSSARD RA, BOURNE RA 1977 Clonal propagation of Eucalyptus by nodal
culture. FAO Third World Consultation on Forest Tree Breeding. Canberra,
Australia, pp 1023-1030
43. DE FOSSARD RA, BARKER PK, BOURNE RA 1977 The organ culture of nodes of
four species of Eucalyptus. Acta Hortic 78: 157-165
44. DE FOSSARD RA, BENNETT MT, GORST JR, BOURNE RA 1978 Tissue culture propa-
gation of Eucalyptus ficifolia F. Muell. Internat Plant Propagators' Soc
Combined Proc 28: 427-435
45. DE FOSSARD RA, NITSCH C, CRESSWELL RJ, LEE HCM 1974 Tissue and organ cul-
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47. DESTREMAU DX 1980 Quelques generalites sur les Eucalyptus. AFOCEL-ARMEF
Informations Foret 144: 23-30
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a plant growth regulator in Eucalyptus and other Myrtaceae. Planta
146: 419-422
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culture. PH D Thesis University of New England NSW, Australia
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propagtion d'arbres forestiers. AFOCEL - Etudes et Recherches n-r2, pp
57-66
51. DURAND-CRESSWELL R, NITSCH C 1977 Factors influencing the regeneration of
Eucalyptus grandis by organ culture. Acta Hortic 78: 149-155
52. EVANS J 1980 Prospects for Eucalyptus as forest trees in Great Britain.
Forestry 53: 129-142
53. FAZIO S 1964 Propagating Eucalyptus from cuttings. Internat Plant
Propagtors' Soc Combined Proc 14: 288-290
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55. FIELDING JM 1954 Methods of raising Monterey pine from cuttings in the
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56. FILHO WS, YONEZAWA FJ 1974 Survival of Eucalyptus saligna grafted by
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179
J.F.REYNOLDS
1. INTRODUCTION
The use of products produced from palms has dramatically
increased. The following chapter will deal with the current
world utilization of palms as a natural resource and the
problems associated with their development. Unfortunately,
traditional breeding methods have not been adequate to fully
expand the potential use of palm products. Palms, as with
most woody species, are difficult to breed largely due to
the time it takes to evaluate test crosses. Plant cell
organ and tissue culture procedures may be a key to speed
breeding programs, and introduction of new hybrids or varieties,
and may aid in the study of palm diseases. In addition,
early in vitro response of palm tissue may be correlated to
increased vigor or heterosis of hybrids, reducing the time
necessary for cross evaluation.
Tissue culture studies of palms are uncommon due to the
difficulties in obtaining and culturing palm tissues. In
vitro studies using Coconut palm tissue were first initiated
in the early 1950's (12). Since this study few new species
have been examined using in vitro systems. Significant
advances in the regeneration of palm tissue in vitro have
only been observed in the past few years (45), (57), (58),
(59)
The following will review most tissue culture studies
of palms and illustrate the difficulties associated with
their culture and progress toward a commercial use of in
vitro techniques.
183
nutrition.
2.2. Ornamental use
Palms are important in the ornamental plant ~nQu~try.
plants (11).
5.2. Inflorescence reversion
The physiology of inflorescence reversion from generative
to vegetative meristem would be particularly useful to palm
breeding programs. Reversion of generative meristems have
been noted in vivo (14) and more recently in culture (46).
This phenomenon has also been noted with species of the
Liliaceae (15). In vitro culture systems may be useful in
studying factors influencing this reversion.
5.3. Breeding programs
Tissue culture can be used in a variety of ways to
supplement current breeding programs of food palms. Propa-
gation of Fl hybrids difficult to obtain could be more
easily done. Haploid production via anther culture would
be useful due to the extreme heterozygosity associated with
coconut, date, and oil palms. Embryo culture would be im-
portant in obtaining Makapuno coconut trees, since Makapuno
embryos normally abort. In addition, hybrids with abortive
embryos. due to endosperm abnormality or incompatibility
could be rescued. Proper evaluation of hybrid coconut or oil
palm trees cannot be done until trees have developed fruit.
In these crops such evaluation may take 10 years or more.
Embryo culture may aid in early identification of crosses
with hybrid vigor, assuming that rapid vegetative growth
can be correlated with yield.
5.4. Disease investigations
In ,vitro culture techniques for disease studies of palms
have been reported by Fisher and Tsai (21). These workers
speculated on methods useful in understanding lethal yel-
lowing disease (LY) of coconuts. LY has been affecting
coconut palms in the Carribean since the early 1960's.
Florida was influenced by LY in the early 1970's. The cause
of the disease has been speculated to be a mycoplasm, which
can be suppressed by antibiotics. Transport of the anti-
biotic Oxytetracycline HCl was studied by McCoy (30) with
the aim of controlling LY. The mode of transmission of the
203
6. REFERENCES
1. ABRAHAM A, KJ THOMAS 1962 A note on the in vitro culture
of excised coconut embryos. Indian Coconut J. 15:
84-87
2. AMMAR S, A BENBADIS 1977 Multiplication v~g~tative du
palmier dattier (Phoenix dactylifera L) par la culture
de tissus de jeunes plantes de semis. CR Acad Sci 284:
1789-1792
3. BAJAJ YPS, J REINERT 1977 Cryobiology of plant cell
cultures and establishment of gene-banks. In J Reinert,
YPS Bajaj eds, Applied and Fundamental Aspects of Plant
Cell, Tissue, and Organ Culture. Springer-Verlag, Berlin,
Heidelberg, New York, pp 757-789
4. BALAGA HY 1975 Induction of branching in coconut.
Kalikasan, Philipp J. Biol 4: 135-140
5. BALAGA HY, EV DE GUZMAN 1972 The growth and development
of coconut 'Makapuno' embryos in vitro. II Increased
root incidence and growth in response to media composition
and to sequential culture from liquid to solid medium.
Philipp Agric 53: 551-565
6. BALICK MJ 1979 Amazonian oil palms of promise: a survey.
Econ Bot 33: 11-28
7. BOUVINET J, H RABECHAULT 1965 Effets de l'acide
gibberellique sur les embryons de palmier a
~uile
(Elaeis guineesis Jacq var dura) en culture in vitro.
CR Acad Sci 260: 5336-5338 --
8. BRIDGEN MP, GL STABY 1981 Low pressure and low oxygen
storage of Nicotiana tabacum and Chrysanthemum X Morifolium
tissue cultures. Plant Sci Lett 22: 177-186
9. CAPLIN SM, FC STEWARD 1952 Investigations on growth and
metabolism of plant cells. II Variables affecting the
growth of tissue explants and the development of a
quantitative method using carrot root. Ann Bot 16: 219-234
204
1. INTRODUCTION
This volume is primarily devoted to tissue culture as
related to forestry. Application of tissue culture to forest
species has only a short history, thus little information is
available on its use for in vitro forest pathology. However, the
potentials of in vitro cultures for the study of phytopathology
have been demonstrated with horticultural and agricultural plants
which have a rich tissue culture tradition. It is worth our time
to become aware of these potentials and technologies because the
in vitro approach may find its best expression in forestry where
the study of pathology is hampered by tree size, rugged terrain
and variable environment over the long forest life.
It is our intention in this chapter to highlight, without
exhaustive review, the tissue culture-plant pathology studies
involving viruses, bacteria, fungi, nematodes, and insects.
Where possible, reference will be made to research dealing with
tree species. We will not expound on the work done in
agriculture and horticulture for this is discussed in detail in
recent reviews (43, 44, 45, 47). These reviews are excellent
works and the major points and examples used in this chapter are
further elaborated in these articles. We hope that inclusion of
this topic in a book on forest tissue culture will acquaint new
readers with in vitro pathology and show what research tools are
and may become available.
209
2. PATHOGEN CLASSIFICATIONS
2.1. Viruses
Perhaps no other area of plant pathology has found tissue
culture more useful than has virology. This area has benefited
greatly from tissue culture in both applied and basic research
areas. Applied benefits have been realized through virus
elimination research, which has the production of virus free
(disease-free) plants as its goal. Basic resesarchers initially
viewed tissue culture as promising for a convenient and
environmentally regulated means of obtaining the host cells
necessary to sustain viruses. However, conventional callus
cultures proved difficult because infection establishment was
inconsistent, and even when infection was obtained, the titre
frequently decreased (43). Infection of callus generally
depended on abrading the callus surface and inoculations were
unquantified (46). The use of plant protoplasts (cells devoid of
cell walls) currently is proving workable for virus studies, and
providing the benefits of convenience and regulation originally
perceived by researchers. Due to the absence of the cell wall on
protoplasts, plant virologists are now able to get synchronous,
efficient in vitro infections which are amenable to quantitative
study (46).
Protoplast techniques are not yet available for all plant
species, and in some cases obtaining and maintaining protoplasts
is difficult. However, despite tissue culture difficulties the
use of protoplasts for viral infections has become widespread,
and several reviews (44, 45, 89, 90, 100, 102) are available on
protoplast-virus interactions.
The efficiency of protoplast infection relative to infection
of cells with intact cell walls is currently fostering study into
several areas: (a) Initial infection studies with protoplasts
which have examined viral uptake as a function of pinocytosis
(17) or plasmalemma injury (11, 12), (b) Double infection
studies,where protoplasts were simultaneously inoculated with 2
viruses (76) or where systemically infected protoplasts were
inoculated with a second virus (4, 5). Works such as these
should produce better understanding of cross protection. (c)
211
2.2. Bacteria
Studies dealing with bacteria in tissue culture systems are
not so numerous as those reported for fungal and viral
associations, but for several bacterial pathogens, studies on
disease establishment and host resistance are receiving
attention. Persistent effort has been applied since the 1950's
(13) to in vitro study of the tumorigenic bacterium Agrobacterium
tumefaciens, but recently the attention has shifted to the
molecular study of the DNA plasmid vector of the disease.
Studies on association of nitrogen fixing bacteria with plant
cells (18) and symbiotic relationships with tissues and organs
(91) have recently been reviewed. This work is also shifting
substantially toward the genes involved, but the fundamental
aspects of symbiosis remain unclear. Tumorigenesis and nitrogen
fixation are special cases for special purposes, but they helped
to form the methodology for studying bacteria in tissue culture
systems .
Infection and toxin induced disease symptoms were
demonstrated in vitro in 1953 with soft rot bacteria and callus
-- -----
213
crown gall disease can occur on some tree species (see Butcher,
14), it is mentioned in this chapter mainly due to its importance
as an example of a plasmid capable of transfer of foreign DNA to
a higher plant (19). The Ti plasmid may be viewed as a model
system to act as a guide for those who seek genetic engineering
of forest trees.
2.3. Nematodes
Dual or monoxenic culture of plant parasitic nematodes and
host tissues had its foundation in the early 1900's, but Mountain
(69) in 1955 provided impetus for further work by growing
Pratylenchus minyus in excised root cultures of tobacco and corn.
Since that work, reports of nematodes in cultures of excised
roots have been many, and the culture of certain nematodes on
callus tissues is routine (21, 59, 86). In general, migratory
nematodes can be routinely maintained in callus cultures, but
sedentary endoparasitic nematodes can not yet be fully maintained
on callus alone (53). Interest in the in vitro dual culture of
nematodes grew through the 1960's and early 1970's and
information was gained about parasitic nematodes and their
disease interaction with host cells, but attempts to refine
cultural methods to the axenic level produced little success. As
a result, few plant parasitic nematodes as yet can be maintained
in axenic culture free of host tissues (70, 92). In contrast to
the parasitic nematodes, free living nematodes are more amenable
to axenic culture and methodology for the culture of the free
living species has been reviewed (73, 80). The information
gained from studies with free living forms will provide base
information to future axenic studies with plant parasitic forms.
Currently, dual cultures of plant tissues and nematodes are
important in producing large stock quantities of nematodes for
experiments, or in maintaining "nematode banks" of defined
nematode races or populations (8). Dual cultures also have been
useful in the study of host-parasite interactions, especially
with regards to plant growth regulators which apparently affect
both the nematode and the host. Schroeder and Jenkins (86)
demonstrated better reproduction of Pratylenchus penetrans in
215
2.4. Insects
Judging from the paucity of literature in the area, it
appears that insect-plant dual cultures are little used in plant
pathology. Ingram (43) in reviewing plant parasites in tissue
culture cited only 5 papers which dealt with both insects and
plant tissues in vitro. In later reviews Ingram (44, 45) either
eliminated consideration of insect-plant tissue culture work or
maintained without expansion his previous consideration.
Recently, however, Mott et al. (68) and Nappen (72) have used
loblolly pine tissue culture to study interactions with beetles.
Mott et al. (68) demonstrated that southern pine beetles
Dendroctonus frontalis could be reared from eggs in vitro on a
216
3. CONCLUSION
In a chapter such as this where the topic is very broad and
the purpose is mostly elaboration of research approaches or
tools, it is difficult to decide when to stop adding topics for
consideration. By no means have we covered the overall area of
in vitro pathology. Topics such as parasitic higher plants are
not discussed here nor are well studied areas such as phytoalexin
or mycorrhizal research considered. However, the reader is
224
plasts in
88. SHEPARD JF, D BIDNEY, E SHAHIN 1980 Potato proto
crop improv ement . Scien ce 208: 17-24
virolo gy.
89. TAKEBE I 1975 The use of proto plasts in plant
Ann Rev Phyto patho l 13: 105-12 5
plant virus
90. TAKEBE I 1977 Proto plasts in the study of
replic ation . In HH Fraen kel-C onrat, RR Wagne r, eds, Com-
pp 237-28 3
prehe nsive Virolo gy, Vol. 11, Plenum , New York, symbi osis.
ds in the study of
91. TORREY JG 1978 In Vitro metho Cultu re 1978,
In TA Thorp e, ed,~r ontie rs of Plant Tissue
ry,
Intern ation al Assoc . for Plant Tissue Cultu re, Calga
Canad a, pp 373-38 0
iving , plant -
92. VANFLETEREN JR 1978 Axenic cultur e of free-l Rev
paras itic and insec t-para sitic nemat odes. Ann
Phyto patho l 16: 131-15 7
Destr uction of
93. VOLCANI Z, AJ RIKER, AC HILDERBRANDT 1953 ria. Phyto -
variou s tissue s in cultur e by certai n bacte
patho logy 43: 92-94
elimi nation .
94. WALKEY DGA 1978 In vitro metho ds for virus Cultu re 1978,
In TA Thorp e, ed, Front iers of Plant Tissue
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Intern ation al Assoc for Plant Tissue Cultu re, Calga
Canad a, pp 245-25 4
plants by tissue
95. WALKEY, DGA 1980 Produ ction of virus -free Cultu re
cultu re. In DS Ingram , JP Helge son, eds, Tissue
ds for-P lant Patho logist s, Black well Scien tific
Metho
Publi cation s, Oxfor d, Boston pp 109-11 7
Callus cultu re of
96. WALKINSHAW CH, FF JEWELL, NM WALTER 1965 Rep
fusifo rm rust-i nfect ed slash pine. Plant Dis
49: 616-61 8 cultur e in
97. WARREN RS, DG ROUTLEY 1970 The use of tissue to
the study of single gene resist ance of tomato
266-26 9
Phyto phtho ra infest ans. J Amer Soc Hort Sci, 95: plant
D LOWE 1966 The effec t of the synth etic
98. WEBSTER JM, on the
growth substa nce, 2,4-di chloro pheno xyace tic acid,
host- paras ite relati onshi ps of some plant -para sitic gy
nemat odes on monox enic callus cultu res. Paras itiolo
56: 313-32 2
ative growth of
99. WILLIAMS PG, KJ SCOTT, JL KUHL 1966 Veget Phyto pathol ogy
Pucci nia grami nis f. sp. tritic i in vitro .
56: 1418-1 419
cation of
100. WOOD KR, MI BOULTON, AJ MAULE 1980a Appli
proto plasts in plant virus resear ch. In F Sala, B Paris i,
ts
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and Persp ective s, Elsev ier, New York, pp 405-41
ion of
101. WOOD KR, MI BOULTON, AJ MAULE 1980b The infect (CMV)
cucum ber proto plasts with cucum ber mosaic virus
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Scien tific
Cultu re Methods~or Plant Patho logist s, Black well
Publi cation s, Oxfor d, Bosto n, pp 79-86
plasts and
102. ZAITLIN M, R BEACHY 1974 The use of proto
separt ed cells in plant virus resear ch. Adv Virus
Res 19: 1-35
103. ZUCKERMAN BM 1971 Gnoto biolog y. In BM Zucke rman, WF Mai,
Academ ic
RA Rohde , eds, Plant Paras itic Nemat odes, Vol. II,
Press , New York, pp 159-18 4
231
1. INTROoocrION
All tissue and organ culture systems used with forest species make
use of natural or artificial plant growth regulators. Without added
hormones, nost tissues do oot remain viable, nuch less grow in the
manner we wish. Unfortunately, few studies have dealt with the
mechanism of hormone action; instead, effective hormones and their
concentration have been derived empirically. we are therefore left
with a wide range of hormones that have been applied to a number of
species with sometimes conflicting results. The purpose of this
chapter is to summarize some of these results, particularly those from
recent papers, and arrive at generalizations where possible. Since
extensive reviews are available elsewhere (9, 13, 89), 00 attempt will
be made here to list every paper which has reported use of a growth
regulator in an aseptic culture system of a forest species.
one of the nost striking features of plant hormones is their
multiplicity of effects. Indole-3-acetic acid (IAA), an endogenous
hormone in higher plants, causes cell elongation, but it also affects
xylem formation (78), germination (94), and a variety of other
physiological processes. Gibberellins also cause cell elongation, but
in addition they affect certain flowering processes and seed
germination. Cytokinins are thought of primarily as expediters of cell
division, but they also exert strong control over norphogenetic
processes, as we shall see later. This nultiplicity of effects is
intriguing, but it complicates attempts at explaining how growth
regulators operate.
Tissue culturists have been concerned primarily with norphogenetic
effects of growth regulators. As we look nore closely at the various
classes of these regulators on the following pages, we should keep in
232
mind that these substances may react differently in intact plants than
in culture systems.
2. AUXINS
2.1 Background
A variety of compounds have been classed as auxins by physiol-
ogists. In this chapter we shall consider auxins to be oompounds which,
like some natural hormones, cause cell enlargement in intact tissues.
This classification is based on the morphological response of tissues
rather than on the mode of action of the chemical. '!hus, the
well-known herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) is
considered to be an auxin even though it is rot endogenous to plants.
In culture systems, though, it often produces results similar to those
produced by indole-3-acetic acid (IAA) and is used po substitute for
1M because it is more stable and sometimes more effective. Other
man-made substitutes for IAA such as naphthaleneacetic acid (NAA) or
indole-3-butyric acid (IBA) are also oommonly used for the same
reasons.
Auxins are often used in aseptic culture systems to induce callus
tissue, although the early work of Gautheret (40) with a variety of
tree species clearly indicates that auxin is rot essential in such
cultures. Of course, the large plant p3.rts used by Gautheret may have
contained sufficient native auxin so that there was ro need for an
exogenous supply. Auxins stimulate callus formation, but the optimal
concentration varies with species (43). Auxin is also used to maintain
callus cultures (13). Q'lce callus is obtained, auxins may be used in
conjunction with cytokinins to induce organogenesis (8, 29). It seems
curious that auxins are regularly called upon to promote
dedifferentiation of cells, and then to promote differentiation of the
dedifferentiated tissue. This dual vole of auxins has puzzled
physiologists for many years, and its matter-of-fact acceptance by
current researchers has rot helped to clarify our understanding of
auxin action.
or explants l: NAA {14, 17, 18, 22, 23, 30, 32, 37, 38, 74, 75, 82, 85,
100, 103, 105, 116}.
In some cases, lx::Mever, removal of NAA fran the culture !Tedium
resulted in induction of roots {29}. NAA has been found to aid in
praroting shoots in a few species such as Pinus radiata {75}, Picea
abies {52}, Thuja plicata {33}, Pseudotsuga menziesii {24, 29}, and
Santalum album {6}, rut it also inhibits shoot formation in sane cases
{IS, 17, 37, 116}. Cheng and Voqui {29} found that NAA inhibited root
formation on cotyledon explants of Pseudotsuga menziesii. Konar {58}
reported stimulation of suspension cultures of Pinus gerardiana, and
David and David {35} found an increased yield of protoplasts \'Alen
cotyledon segments of Pinus pinaster were pretreated with NAA. Similar
results were reported with protoplasts of Pseudotsuga menziesii {55}.
NAA is rot a naturally occurring plant honnone, rut it has found
extensive use in the hands of tissue culturists. Even though we do rot
understand its mode of action, without this compound many of the recent
advances in the technology of tissue culture probably would rot have
been forthcaning.
Table 1. Some forest species funning callus upon treatment with 2,4-0.
Angiosperms Gyrmosperms
Citation Citation
Huhtinen and zeki (47) found similar results with Betula pendula.
Mapes (unpublished results) found that a high concentration (2.5 mg/l)
of 2,4-0 caused root primordia to fonn in profusion on hypocotyl
segments of Tsu<;,!a heteroEhylla and that rooting of adventitious
shoots could be cbtained by using 2,4-0. On the other hand, Vieitez et
al. (101) found no effect of 2,4-0 on differentiation in castanea sativa
cultures, and WOlter (116) noted that it inhibited root formation in
Pq>ulus tremuloides callus. Konar (58) reported that 2,4-0 caused
cells in suspension cultures of Pinus <;,!erardiana to proliferate but
that it inhibited root formation on oolid nedia. Chalupa and rurzan
(20) found only inhibition of shoot growth with 2,4-0 in cultures of
Picea <;,!lauca.
'!his brief surrrnary leads to a rather cbvious conclusion: 2,4-0
does not affect plant cells like other auxins, even though it is
238
3. CY'IOKININS
3.1 Background
Even before cytokinins were known, it was recognized that a growth
factor other than auxin was required for anbryo development. Coconut
milk was often used to supply that unknown growth factor (73). After
its discovery, kinetin (67) or similar substances soon replaced coconut
milk in many culture systems. The advantages of using defined media
were soon evidenced by rapid progress in aseptic culture technology.
The "hormone balance" ooncept of Skoog and Miller (88), who showed that
the ratio between cytokinin and auxin in the medium was critical in
determining whether shoots or roots were initiated on tobacco callus,
has emerged as a guiding principle for tissue culturists. It is now
recognized that cytokinins are an essential oamponent for culture of
most species and that an appropriate ratio of cytokinins to auxins
often leads to organogenesis.
Cytokinins are closely associated with processes of cell division.
At one time it was thought that cytokinins nodulated tRNA functioning,
but that explanation does rot agree with recent evidence. A IlOre
likely explanation is that cytokinins act directly upon enzymes and
control enzyme activity rather than enzyme synthesis (102). Varnell
and Vasil (99) suggest that kinetin may act on nucleus-based events or
on rrembranes in the cytoplasm in the apical neristem. Whatever the
site of action, recognition of the close tie between cytokinins and
enzyme activity and of the powerful effects of cytokinins on cell
differentiation and IlOrphogenesis has resulted in this group of plant
hormones becoming better understood than any other. The expression of
cytokinin activity is influenced by structural variations in the cyto-
kinins. Spatial oonfiguration as well as the type of atoms on the
N6 substituent determine cytokinin activity. Recognition of this fact
does rot nean, however, that the effects of cytokinins on plant tissues
240
can be pr-edicted. '!he ooly reliable rrethod for learning row any given
tissue is likely to respond to any given cytokinin is by rreans of an
empirical experiment. Recent studies with crown gall disease 00
3.2 Kinetin
'!he first cytokinin discovered was 6-furfurylaminopurine, or
kinetin (67). It has been a a::rnponent of many rredia recipes used with
a wide range of forest tree species during the past two decades (14),
even though it is not a natural cytokinin. It stimulates growth of
callus tissue in gymnosperms (12, 43, 80) and angiosperms (51, 61).
In cell suspension cultures of PSeudotsuga menziesii, Winton (113)
reported that kinetin caused a d::>ubling of cell volume weekly. On the
other hand, Chalupa et al. (23) found that kinetin did not improve
callus formation 00 explants of Pinus banksiana, and Risser and \'bite
(77) reported that kinetin inhibited growth of tumors of Picea glauca.
Zajaczkowski (118) found kinetin to have little effect 00 growth of new
xylem in Pinus sylvestris. Numerous "negative" results with other
species have undoubtedly been left unreported.
Because of its IlOrphogenetic effects in tobacco callus (88), kine-
tin has been a logical candidate for use in shoot induction experiments
with forest trees. Either low levels of kinetin alone or kinetin plus
auxin have induced roots in cultures of Castanea sativa (101), Biota
orientalis (95), and POpulus ussuriensis (2). A IlOre OOITIllOn organoge-
netic effect of kinetin, OOwever, has been induction of shoots. Shoots
or buds have been induced by kinetin in cultures of Picea abies (4),
Picea sitchensis (103), Pinus contorta (103), Pinus taeda (65),
Pseudotsuga menziesii (27), Betula pendula (47), Eucalyptus ficifolia
(7), POpulus nigra var. italica (100), ~. nigra var. robusta (19), ~.
The reason for the greater effectiveness of BI\P may lie in the
abilities of plant tissues to metabolize the natural hormones more
rapidly than artificial growth regulators. or BI\P could induce
production of natural hormones such as zeatin within the tissues and
243
4. GIBBERELLINS
4.1 Background
Gibberellins are another class of potent growth hormones. They
exert strong control 00 cell elongation and have been associated with
the flowering response. The mechanism of action of gibberellins is
probably 00 cell membranes or at least 00 some cellular component
associated with membranes. Longer-term regulatory effects may occur
through modification of RNA and protein synthesis (102).
in the shoots did rot dlange during imposed donnancy. von Arnold and
Eriksson (3, 4) found ro effect of gibberellins en ruds or embryos of
Picea abies, and Olalupa and D..Irzan (21) found ro stimulation of ruds of
Picea glauca. Similarly, tumors of Picea glauca did rot respond to
GA (77) . Gibberellins had little or ro effect on excised embryos of
Pinus strobus (68) or en cambial segments of Pinus sylvestris (118) or
Pinus monticola (44). Apical neristems of Pinus elliottii did rot
respond to gibberellins (99), ror did ootyledon explants of Pinus
pinaster (34), excised ruds of Pseudotsuga menziesii (1), or callus of
Pinus sylvestris (79).
The failure of aseptic cultures to respond to treatment with gib-
berelin parallels the results of application to intact trees. Except
for inducing flowering in certain species under certain oonditions,
gibberellins elicit little or ro effect when applied to forest trees,
either in vivo or in vitro. FUrthermore, Murashige (70) showed that
gibberellins increased in callus cultures and that they apparently
inhibited norphogenesis (69). We can oonclude that either gibberellins
are rot important in morphogenetic processes or that the endogenous
levels of this hormone are sufficient in most tissues without an
exogenous supply. lance et al. (60) have begun to shed light en this
problem by making careful neasurements of endogenous levels of
gibberellins in culture systems.
6. CONCLUSIONS
It is clear that growth horJl'Ones playa key role in determining
the course of norphogenesis in higher plants. we have seen in the
above discussion how the various plant growth horJl'Ones produce a wide
variety of effects in aseptic cultures of forest trees. Although
auxins and cytokinins appear to be essential for norphogenesis, we have
seen examples of norphogenesis without the use of exogenous growth
regulators, tx:>ssibly because endogenous levels were sufficient.
Manmade analogs of growth regulators often are nore effective than the
endogenous compounds themselves. The auxin/cytokinin ratio usually
247
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axenic tissue cultures of 12 conifer species. can J Bot
47:547-549.
44. Harvey AE, Grasham JL, W:lldron CC (1971) 'lbe effects of grCMth
regulating oampounds on healthy and blister rust infected tissue
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45. Hecht 91 (1980) Probing the cytokinin receptor site(s). IN: Skoog,
F, ed, Plant grCMth substances. Proc. lOth Int. Conf. Plant
GrCMth Subst. New York: Springer-Verlag, p 144-158.
46. Hricova D, Strmen J, Bauer S (1974) Preparation and
characterization of the tissue culture of spruce (Picea excelsa
Link. ) BioI Plant 16: 118-122. --
47. Huhtinen 0, Yahydoglu Z (1974) Das Fruhe Bluhen von aus
Kalluskulturen herausgezogenen Pflanzen bei der Birke (Betula
pendula R:>th.). Silvae Genet 23:32-34. ---
48. Isikawa H (1974) In vitro formation of adventitious rods and roots
on the hypocotyl of"""'Cryp~ria japonica. Bot M:lg 86:73-77.
49. JaCX}Uiot C (1964) Application de la technique de culture des
tissues vegetaux a l'etude de quelques problemes de la physiologie
de l'arbre. Ann Sci For 21:309-473.
50. JaCX}Uiot C (1966) Plant tissues and excised organ cultures and
their significance in forest research. J Inst WXx:l Sci (London)
16:22-34.
51. Jacquiot C (1969) Effect of some p..!rine derivatives on the grCMth
and organogenesis in tree tissues cultivated in vitro. Ann Sci
For 26:131. ---
52. Jansson E, Bornmann CH (1980) In vitro phyllanorphic regeneration
of shoot rods and shoots in picea-abies. Physiol Plant 49:105-111.
53. Johnson MA, carlson JA (197~ndoleacetic acid oxidase and
related enzymes in cultured and seedling Douglas-fir. Biochem
Physiol Pflanzen 174:115-127.
54. Kevers C, Col.DllanS M, De Greef W, Hofinger M, Gaspar 'II. (1981)
Habituation in sugarbeet callus: Auxin content, auxin protectors,
peroxidase pattern and inhibitors. Physiol Plant 51:281-286.
55. Kirby El3, Cheng T-Y (1979) Colony formation fran protoplasts
derived fran Douglas-fir cotyledons. Plant Sci Lett 14:145-154.
56. Kitahara EH, caldas IS (1975) Shoot and root formation in
hypocotyl callus cultures of Eucalyptus alba. For Sci 21:242-243.
57. Konar RN (1974) In vitro studies on Pinus. I. Establishment
and grCMth of callus. Physiol Plant 32:193-197.
58. Konar RN (1975) In vitro studies on Pinus. II. 'lbe grCMth and
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Phytomorphology 25:55-59.
59. Konar RN, Singh r-N (1980) Irrluction of shoot rods from tissue
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60. Lance B, Rei~'lborpe T (1976) Endogenous gibberellins and
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253
61. Lee 01, De Fossard RA (1974) '!he effects of various auxins and
cytokinins on the in vitro culture of stem and ligno tuber tissues
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Pflanzenphysiol 70: 406-413. --
63. ux> Sol, Wang FlI (1943) '!he culture of young oonifer anbryos in
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64. Mathes r1: (1964) '!he in vitro formation of plantlets fran isolated
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69. Murashige T (1964) Analysis of the inhibition of organ
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70. Murashige T (1974) Plant propagation through tissue cultures.
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254
1. INTRODUCTION
In this chapter, the nitrogen metabolism of cells and
tissues of economically important forest trees is interpre-
ted in the light of our limited knowledge of the specific
genetic gains to be captured by cell and tissue culture
technology.
There are basically two levels to the vegetative
propagation of trees with desired traits. One deals with
organogenesis to regenerate missing parts on cuttings, e.g.,
rooting of needle fascicles or adventitious buds. The other
relies on controlling of the development of cells and
protoplasts for the production of the complete organism
(50). Both have advantages and disadvantages and all
involve nitrogenous compounds.
Emphasis will be placed on the commercially important
coniferous species, Douglas-fir (Pseudotsuga menziesii) and
loblolly pine (Pinus taeda), and on the existing literature
on the suspension cultures of sycamore (Acer
pseudoplatanus). This is only because most information we
have is on these species. The importance of nitrogen
metabolism and the recent advances in molecular biology as
they relate to the propagation of forest trees will be
reviewed. This will be followed by a discussion of nitrogen
metabolism in the physiological and biochemical processes
associated with the control of growth, differentiation and
morphogenesis as seen through nitrogen metabolism.
257
Hl
CHLOROPHYLL
_---_-~r~~Oin 2
thioredoXin .....
reductase _ ~
~ 10
2
enzyme control
.... " H~
.......... " ATP ADP
ferredoxin .;
_~-:-_".~I N02-----~---- -..,.-l""'--'-_ glutamine
nitrate nitrate
reductase reductase
CYTOSOL
ferredoxin
glutamate synthase
a-ketoglutarate
CHLOROPLAST transaminase
~
PROTEIN
KEY
I Primary N acceptor I
Product of reaction
TRYPTOPHAN AUXIN
TYROSINE
PHENYLALANINE
HISTIDINE
C02---91~Y1--1I.-ta--------P_h_ot_oS~y_n~--_is--------------~F-~~ ~
'glutamine
t
glyo.ylate
TTP
pentose phosphata path
6
~ erythrose-4-phosphat8
"'-A--U---N-IN--'E
> ~:~~7~E
glYCOlYSiS!
~~~~~~NE ~Pyruvata
TTP
~
<
NAD+ CO 2
CoA
CN- I NA~~+ I
Acetyl CoA IC 2 fragment)
J
ASPARAGINE
>
A~!~~TATE \ ___N_A-'--~-'--H_+H--;-+~o.alo.cetata IC4 ) IC 6)
Citrata\
KREBS CYCLE
HOMOSERINE*
I -J NAD+
t C, t '-----.
=,~~~',~i ."",0",,, -\ "_m ..~:*:
SuCCinic-~-,?
N
..mialdehYddl GLUTA~111
GLUTAMINE I
~-AMINOBUTYRATE ARGININE
CO2 PROLINE
A
II I---
-tm>KtN~"':=~N--:- -- -- -- -- -- -- - "\
i~ threshold of integrated correlations
I
I
1 '
NUCLEOTIDES
~s
~
ENZYME FORMING SYSTEM .C ~i:
I
=t I
INDUCTION--+...!,..+---- REPRESSION
E N Z Y M E " DEGRADATION
(PRIMARY AMINO
~ACT'VATOON~ t--'NH'."'ON "'0'1
I f I
MEVALONATE / AROMATIC, GLUTAMATE, ASPARTATE, ALANINE, GLYCINE ,
OA
I
A$$CISICACID
1- -d~I!li~.~~.c pt_ -1;.[llllt-- - - _./
FIGURE 3. The relationship of enzyme-forming systems
originating from information in DNA to nitrogen assimila-
tion, to the conversion of substrate to amino acid products,
and to the production of certain plant growth regulators.
Table 1. Exalrples of amino acid conjugates of natural and synthetic plant gra.>t:h regulators.
a -Naphthalene aoetic acid .6 -D-Glucose and aspartyl conjugates in 1979 J Riov et a!.,
conjugates Pinus sp. Physio!. Plant 46:
o
O)'CH2-CII-R
133.
RIBONUCLEOTIDE
REDUCTASE
(THIOREDOXINI
CHROMONEMA R"'E.'"'LlCA=T'":':ON'1
f':1 -1 % HETERO-
ii
NUCLEAR
!e
RNA
CHROMATID ORGANIZATION
(mRNA
PRECUAmRt
1i1l
!~
CENTROMERE DtVISION
CHROMOSOME I REPLICATION I
/
CYTOKINESIS
.mM
POLAR MIGRATION OF DAUGHTER CHROMOSOMES
CEllI REPLICATION I
NUCLEAR I REPLICATION I
A B
FIGURE 4. Nitrogen metabolism and the mitotic cycle in
higher plants (A) relate the behavior of DNA in chromatin to
the replication of cells. Daughter cells contribute to the
life cycle of the tree and to apomictic or sexual (meiosis)
reproduction. The stages of the cell cycle are Gl, S (DNA
synthesis), G2, and mitosis. B depicts a single strand of
chromatin and shows how and where messenger RNA is believed
to be formed. The integrity of the strand of chromatin is
shown as a function of increasing ionic strength. C empha-
sizes the flow of nitrogenous compounds via ribonucleotide
precursors into deoxyribonucleotides for the synthesis of DNA
and into ribonucleotides for the synthesis for all types of
RNA. However, in cytokinin-directed organogenesis, DNA in
cytochemical studies seems to have several fates. DNA levels
can be reduced, and sections can be eliminated, amplified,
rearranged, and modified. Changes in DNA levels during the
organogenesis of Pinus coulteri have been described by Patel
and Berlyn (112).-----
- Arginine + Arginine
cell yield/flask
(grams by wt) 0.29 9.11 0.18 7.13
272
3S
.....
CD
..
~
CD
.
U
..
"0
c
:J
,g
2
31
phenol
142
143
urea ...
...
Q)
III
~
23 112 ...<i
Q)
8 10
(J
III
3
0
c
118
4 ...::s
III
9 119 .Q
13
phenol , e.J
FIGURE 6. _Autoradiograph of a paper I~romatogram revealing
the radioactive metabolic products of C-carbamyl phosphate
applied to the top of a white spruce callus as in Fig. 5
(Durzan and Lopushanski, unpublished). The l~allus was
extracted after 0.5 h of exposure of 10 ~Ci/g C-carbamyl
phosphate. Carbamyl phosphate is the large chromatographic
spot at the extreme right and moving in the direction of
acid butanol. Other unidentified products are associated
with this spot. Key to the identity of radioactive spots is
the same as in Fig . 5.
When cytokinins or purine derivatives are added to
medium, the pattern of N metabolism is usually significantly
altered . Purines have several fates, including degradation
to urea and salvage of the products for nucleotide synthe-
sis. Purines also have physiological effects that may
influence morphogenesis (14,59).
As for auxins, Riov et al. (122) found that incubation
of tissues of Pinus ~. with NAA_1_ 14 C resulted in the
formation of l-Q-naphthylacetyl-~-D-glucose and ~-naphthyl
acetyl aspartate . Unpublished work at The Institute of
Paper Chemistry indicates that similar conjugates of N6 _
benzylaminopurine are formed in cell suspensions and in
cultured excised embryos of Douglas-fir.
275
into the cells and the nucleus (146). In older cells the
14C-label was localized in the thick cell wall suggesting
that the rate of protein synthesis varies with age of
cultured Acer cells. The hydroxyproline-rich glycoprotein
is synthesized in the cytoplasm and then transported to the
cell wall (39).
Another study involving Acer suspension cultures
indicated that "gas shock," or abrupt changes in gas concen-
tration, may affect cellular nitrogen metabolism (150).
Changes in the cellular uptake of the substrates leucine,
methionine, and adenine were noted after the culture flasks
were opened and the suspension cells were filtered. These
activi ties were presumed to change the composition of the
gases to which the cells were exposed and produce a gas
shock in the metabolism of the cells. This response to
culture vessel opening and sampling may become an important
consideration in the analysis of growth and biochemical
parameters of suspension cells.
The work with Acer and conifers (Appendix 1) contrib-
utes to our knowledge and to control of in vitro culture of
trees, and it has permitted a scale-up of cell suspension
cultures. These advances provide new opportunities for work
in applied areas of plant research that in the long run may
include vegetative propagation, secondary product formation
and even genetic engineering.
3.4. Morphogenesis. Visual or chemical markers are
needed to follow developmental patterns before we can obtain
a better understanding of the role of cellular and subcellu-
lar organization in cellular and tissue development (68, 85,
128). Subjects now being studied are the mechanisms of cell
division, the emergence of pattern and shape (11, 133), the
biophysical role of cell walls in pattern determination
(71), the evolving structure and function of subcellular
organelles, and the transformation of membrane systems, such
as rough endoplasmic reticulum (ER) to a smooth ER (93, 105,
109) . All these physiological activities in one way or
another involve growth regulators (64, 103, 134).
287
4. OUTLOOK
Since nitrogen is often a factor limiting growth in
situ ana in vitro, all aspects of vegetative propagation and
silviculture could benefit from the understanding and
application of nitrogen. Apart from the use of slow-release
fertilizers, the opportunities and technologies to do this
economically are not immediately obvious (25). However,
recent aevelopments, particularly in the provision of
controlled rates of active substances and use of monoclonal
antibodies, may help remove these shortcomings (159).
One opportunity lies with the introduction and control
of symbiotic nitrogen fixation (10, 16, 17). It may be
possibl e to genetically engineer plant cells, as has been
done with microorganisms. Although this goal may be real-
ized someday, the value of this technology to forestry
remains to be proven. Using lectins, we may be able to bind
nitrogen-fixing organisms not only to the rhizosphere but
also to the aerial parts (e.g., needles) of trees. It may
also be possible to make improvements in the cloning cycle
(50, 54, 71), such as the formation of artificial seeds where
the somatic embryo is coated to form a pellet; this would
proviae ease of handling, protection, and the slow release
of nutrients (103). Also possible may be the use of
301
REFERENCES
1. ABO EL-NIL MM. 1980. Embryogenesis of gymnosperm forest
trees. US Pat. 4,217,730, August 19, 11 claims, 6 pp.
2. ATKINSON DE. 1977. Cellular energy metabolism and its
regulation. New York, Academic Press.
3. BALL EA. 1978. Cloning in vitro of Sequoia sempervi-
rens. Abstract 1726 In:- Frontiers of Plant Tissue
Culture, TA Thorpe, ed., University of Calgary, Cal-
gary, Canada.
4. BANDURSKI RS, SCHULZE A. 1977. Concentration of indole-
3-acetic acid and its derivatives in plants. Plant
Physiol. 60:211-213.
5. BANERJEE SN, RADFORTH NW. 1969. In vitro studies on the
developing embryos of Pinus reslnosa. Bot. Mag. Tokyo
82:329-340.
6. BARONDES SH. 1981. Lectins: their multiple endogenous
cellular functions. Annu. Rev. Biochem. 50:207-231.
7. BARTELS H. 1971. Genetic control of multiple esterases
from needles and macrogametophytes of Picea abies.
Planta (Berl.) 99:283-289.
8. BAULE H, FRICKER C. 1970. The fertilizer treatment of
forest trees. Munchen, Germany, BLV Verlagsgesellschaft.
9. BENGSTON GW. 1973. Fertilizer use in forestry. Proc.
IUFRO-FAO Int. Symp. on Forest Fertilization Ministere
de l'Agriculture, Dec. 3 to 7, Paris, pp. 97-168.
10. BAUER WD. 1981. Infection of legumes by Rhizobia. Annu.
Rev. Plant Physiol. 32:4707-449.
11. BERLYN CP, BECK RC. 1980. Tissue culture as a technique
for studying meristematic activity. Proc. IUFRO Symp.
Control of shoot growth in trees. CHA Little, ed.,
Maritines Forest Research Centre, Environment, Canada,
pp. 305-324.
12. BERLYN GP, MIKSCHE JP. 1965. Growth of excised pine
embryos and the role of cotyledons during germination
in vitro. Ann. J. Bot. 52:730-736.
13. BORCHERT R. 1968. Spontane diploidisierung in Gewebkul-
turen des Megagametophyten von Pinus lambertiana. Z.
Pflanzenphysiol. 59:389-392.
14. BRAUN AC. 1980. Genetic and biochemical studies on the
suppression of and recovery from the tumorous state in
higher plants. In vitro (Rockville) 16:38-48.
15. BRIGHT SW, NORTHCOTE DH. 1974. Protoplast regeneration
from normal and bromodeoxyuridine-resistant sycamore
cells. J. Cell Sci. 16:445-463.
16. BRILL WR. 1979. Nitrogen fixation: basic to applied.
Am. Sci. 67:458-466.
17. BRILL WR. 1981. Agricultural microbiology. Am. Sci.
245:199-215.
18. BRINK RA. 1962. Phase change in higher plants and
somatic cell heredity. Q. Rev. BioI. 37:1-22.
19. BROWN CL, GIFFORD ME, Jr. 1958. The relation of coty-
ledons to root development of pine embryos grown in
vitro. Plant Physiol. 33:57-64.
303
A. Gymnosperms
B. Angiosperms
TREVOR A. THORPE
1. INTRODUCTION
Although great progress is being made in the production of
photoautotrophic tissues in vitpo (e.g., see 10, 181), cultured
plant cells, tissues and organs, even those that turn green in
light, generally require a carbon source for successful in vitpo
culture (50, 51, 145). As a matter of fact a carbon/energy
source is considered one of the five classes of essential sub-
stances needed to bring about growth and organized development
in vitpo (36, 50).
The importance of a carbon/energy source in the medium has
been recognized for a long time. The pioneering studies with
excised root cultures in the 1930's (175) led to the examination
of the types of carbohydrates suitable for root cultures, their
mode of uptake, metabolism, etc. (145). The carbohydrate re-
quirements for callus culture were first studied experimentally
in the 1940's using carrot root callus (53, 54). These early
studies led to the conclusion that sucrose was the best carbon
source for in vitpo cultures, although a variety of other cou-
pounds could be utilized by plant tissues in culture.
The capacity to utilize various carbon sources for growth
and development in vitpo indicates that these (or their degrada-
tion products) must be taken up into the cells, converted to a
metabolizable form and integrated into cellular metabolism.
In the intact plant approximately 70% of the fixed C02 is util-
ized in cell wall biosynthesis. There is no evidence to suggest
that in culture most of the carbohydrate is not similarly in-
volved in cell wall biosynthesis. However, it will become
apparent that more information has been obtained on other as-
pects of metabolism using cell cultures. Although most of the
326
2. NUTRITIONAL ASPECTS
Since sucrose is the sugar of transport in the intact plant
it is not surprising that this disaccharide and its constituent
hexoses, glucose and fructose generally best serve the carbon
nutritional requirements of cells in culture, including woody
tissues (6, 7). However a variety of other carbon sources have
supported some growth in culture (see 110, 145). The capacity
of tissues to utilize other carbohydrates varies with the species
from which the explant was selected (72). Furthermore, cultures
derived from different cultivars, e.g.,of Malus (22), different
organs of the same plant, e.g., root- vs. stem-derived callus of
sugar maple (113), and even single cell isolates from the sane
clone, e.g., of tobacco, have all been shown to differ greatly
in their response to a particular carbohydrate. Finally, if a
carbohydrate will support growth of a tissue in culture, it will
apparently also support its diff e rentiation (158, 170, 179).
Some selected examples of carbohydrate use, mainly in culture~
species (e. g., 116, 178, 183) is unable to serve as the sole
carbon source. On the other hand, sorbitol supports excellent
growth of Malus tissue culture (23), as well as cultures of
several genera in the Roseae (25). However, it must be pointed
out that sorbitol is an important carbohydrate in the metabolism
of apple and related species in vivo. Mannitol supports callus
growth in Fraxinus (178). Glycerol can also be used by some
tissues (20, 56, 80, 136).
A specific morphogenetic role for carbohydrate has been
suggested by experiments carried out on the induction of vascular
elements in vitro. At a constant auxin concentration, low con -
centrations of sucrose (1.5-3.0%) favoured xylem formation in
lilac callus, while at higher concentrations (4.5-5.0%) ph lo em
was produced (174). Similar results were obtained with Phaseolus
callus (78). In liquid cultures of Parthenocissus callus the
number of xylary cells formed increased with sugar concentration
(up to 8%) (132). Sucrose also affected the quality of xylem
by altering the pattern of wall thickening (11). Low sucrose
concentrations (0.5%) induced elements with annular and scalari-
form thickening, whereas higher concentrations (1.5-3.5%) favour-
ed scalariform and reticulate elements. In Helianthus tuberosus,
sucrose and g lucos e were equa lly effective in differentiation
when used separately, but inhibitory when used together (114).
Althou gh it was once suggested that a -glucosyl disaccharides
possess special xylogenetic properties (78), it appears that
any carbohydrate which permits rapid cell pro liferation in a
particular tissue will effectively support xylogenesis (127).
From the above it is clear that tissues in culture can
utilize a variety of carbon sources for growth and organized
development . IIowever, rarely are any of these superior to
sucrose and its hexose constituents. For most cultures there-
fore sucrose at a level of 2-4% (w/v of medium) is the carbo-
hydrate of choice (50). It has been shown that the time at which
maximum yield is obtained coincides with the time at which carbo-
hydrate is exhausted from the medium (138, 167). However, all
the carbohydrate need not be added at the start of culture; if
some is added during log phase growth , it supports the same
328
3. CARBOHYDRATE UPTAKE
In studying the uptake of exogenously supplied carbohydrates
a distinction should be made between those fueling primarily
exergonic pathways and those accumulated and utilized exclusively
for the endergonic synthesis of structural cell components (110).
Thus, most cells have been found to be freely perneable to glyco-
lytic intermediates, as well as sugars such as arabinose, xylose
and fucose, which do not promote growth but are components of the
cell wall. The major question which arises is whether the carbo-
hydrate uptake into cells is an active process, a passive dif-
fusional one or some combination.
Studies on sugar uptake into tobacco (122) and carrot callus
(125) and Acer c e ll suspensions (43) led to the idea that uptake
was passive. In contrast studies with sugar cane cells in sus-
pension indicated that the uptake of sugars was active because
it occurred against a steep concentration gradient, normal efflux
was not greater than 10 % of the influx rate, and uptake was in-
hibited by substances such as iodoacetate and dinitrophenol (106,
107). It was further concluded that specific transport sites
for glucose uptake were present on the cell membrane. Further
the almost instantaneous appea ranc e of sugar phosphates with
glucose uptak e suggested that the formation of phosphorylated
intermediates was an important part in the active uptake process,
which required a permease and a kinase in the plasmalemma (43).
Similarly, active uptake of maltose into variant strains of soy-
bean grown in cell suspension has been observed (101). The up-
take was inhibited by NaN 3 and dinitrophenol.
Whether uptake is passive or active, or a combination is
still not clear. However, it would appear that a distinction
has to be made between uptake into a tissue, i.e., into the
apoplastic portions (intercellular spaces, apparent free space,
etc.) and entry into cell. Improved techniques for the isolation
of membrane vesicles, vacuoles, etc., will allow this question
to be answered definitively. Indeed, some progress has been
329
4. CARBOHYDRATE METABOLISM
Studies on carbohydrate metabolism in vitro have been carried
out with callus cultures, cell suspensions and more recently with
continuous (chemostat) cultures.
In addition, to the metabolism of exogenous carbohydrates,
cultures grown in the light will contribute some photosynthate
to the endogenous carbon pool. For example, it was shown that
there was a greater increase in dry weight in a green strain of
rue over a colourless strain, as a consequence of higher absorp-
tion rates of sucrose from the medium and the refixation of res-
piratory C02 in the photoheterotrophic cells (139). Furthermore
dark or non-autotrophic C02 fixation occurred in culture (135,
154, 169). In spite of the presence of exogenous and endogenous
carbohydrates, it has been shown that CO 2 is essential for the
initiation of growth in cultured sycamore cells (52). The CO 2
requirement was not related to any effect on pH. The cells fixed
the C02 into organic and amino acids. This non-photosynthetic
330
This finding indicates the probable demand for reducing power for
N03 reduction.
In a study on the effect of oxygen supply on the growth of
tobacco cells in suspension under aerobic-dark batch culture
conditions it was found that a very low value for the volumetric
02-transfer coefficient was sufficient to produce 14.9 g bio~ass
aweight % composition
343
HO - o C H 2 -CH(NH 2 )
Tyrosine
- COOH o CH 2 -CH(NH 2 )
Phenylalanine
- COOH
-0-
CH 3 0
+ t t
L - -~ P - Coumaryl alcohol Coniferyl alcohol Sinapyl alcohol
I I :
I I I
:L ________________ ~'
I
LIGNIN 4-------------------
~
: I
the steps outlined above. Thus work with parsley cell suspension
cultures have shown that the coordinated activation of the enzymes
involved in phenylpropanoid metabolism is preceded by RNA and
protein synthesis (67).
Lignin synthesis in tobacco cell cultures has been studied in
cell aggregates (97). Larger aggregates produced more tracheary
elements and had higher activities of shikimate dehydrogenase,
cinnamic acid 4-hydroxylase, caffeic acid-O-methyltransferase, and
5-hydroxyferulic acid-O-methyltransferase. The peak of activity
occurred just prior to tracheid formation. Similar changes have
been observed during xylogenesis in bean callus (65).
The activity of the pentose phosphate pathway was elevated
during lignification in Jerusalem artichoke and Coleus blumei
tissues relative to glycolysis (131). The enhanced activity of
the pentose phosphate pathway indicated that it was probably
providing the reducing power needed for lignification, in addition
to erythrose-4-P (see Section 4.5.). The development of an
active lignifying system in Zinnia elegans (47, 48) will allow,
348
5. OSMOTIC ROLE
The major component of a tissue and cell culture medium is
usually the carbohydrate. For most species sucrose best serves
this purpose (see section 2.). As indicated earlier (section
4.1.) sucrose can be taken up without hydrolysis or some
hydrolysis may occur extracellularly and the products taken into
the cell. In any case the intracellularly hydrolyzed sucrose
increases the osmotic potential in the cell, unless the products
of hydrolysis are rapidly removed by metabolic utilization.
Furthermore, the optimum level of sucrose (or other carbon source)
in the medium, is in excess of the requirements of the tissue
for growth, and relatively large quantities of sucrose and
reducing sugars accumulate eventually (151, 159).
Osmotic potential is regulated in plant cells by both in-
organic ions and organic molecules, including organic acids,
sugars and sugar alcohols (184). It has been suggested that
because the cytoplasmic inorganic ion concentration remains
fairly constant, osmotic adaptation of the cytoplasm is mainly
achieved by the accumulation of non-toxic organic molecules (180).
350
6. CONCLUDING THOUGHTS
In this chapter I have discussed various aspects of carbo-
hydrate utilization and metabolism relative to in vitro cultures.
Studies on the nutritional capacity of different carbon sourc es
can better be carried out with cell, tissu e and organ cultures,
than with intact plants since most cultures are nutritionally
heterotrophic. These studies tend to indicate that plant cells
in culture are very plastic and possess the capacity to utilize
a variety of carbon sources, in addition to fixing C02 non-
photosynthetically. Use of protoplasts and cell suspension
cultures have gone a long way towards resolving the problem of
active versus passive uptake of carbohydrates. While uptake into
the tissue is indeed passive, it now appears fairly certain that
uptake into the cell, i.e., across the plasmalemma is an active
process, which is relatively unspecific.
The relatively recent indication of an osmotic component to
shoot initiation should stimulate conc e rted efforts to evaluate
the role of other physical phenomena in differentiation. Work
done in the 1960's (e.g., see 140) has certainly shown a con-
tributory role for pressure in organized development. In
addition, the recent acceptance of the membrane as a focus for
the regulation of a large segment of a cell's b e haviour, lends
importance to studies in this area.
As an advance on the use of tissue slices, cell and tissue
cultures have proven extremely useful tools in the study of
356
REFERENCES
1. AITKEN J, KJ HORGAN, TA THORPE 1981 Influence of explant
selection on the shoot-forming capacity of juvenile tissue
of Pinus r adiata . Can J For Res 11: 112-117
2. ALBERSHEIM P 1976 The primary cell wall I n J Bonner,
JE Varner, eds, Plant Biochemistry, 3rd edition. Academic
Press, New York, pp 226-277
3. ALSOP WR, RD LOCY, WR OKLE 1981 Selection and partial
characterization of tomato ( Lycopers i con e s cule n t um Mill.)
cell lines for ability to grow on ribose. Plant Physiol
Suppl 67: 117
4. A~~IRATO PV, FC STEWARD 1971 Some effects of environment
on the development of embryos from cultured free cells.
Bot Gaz 132: 149-158
5. ASPINALL GO 1980 Chemistry of cell wall polysaccharides
In J Preiss, ed, The Biochemistry of Plants, Vol 3
Carbohydrates: Structure and Function. Academic Press, New
York, pp 473-500
6. BALL E 1953 Hydrolysis of sucrose by autoclaving media, a
neglected aspect in the technique of culture of plant
tissues. Bull Torrey Botan Club 80: 409-411
358
12. THE USE OF IN VITRO TECHNIQUES FOR GENETIC MODIFICATION OF FOREST TREES
E. G. KIRBY
1. INTRODUCTION
Encouraging reports of the development of techniques for in vi t r o mutant
selection (56) and for fusion, culture and regeneration of somatic hybrid
plants from protoplasts of herbaceous species (79) suggest applications of
new techniques in genetic improvement programs for forest trees. In
addition, through a more thorough understanding of gene expression in
higher organisms (9), genetic engineering of plants, including forest
species, is likely to become a reality in the near future (47). Previous
reviews have addressed the topic of somatic cell genetic manipulation of
plants (26, 28, 56, 72, 78, 79, 83). The present paper will discuss the
use of in vitro systems for genetic modification of forest trees. It must
be noted at the outset that recent contributions are few in number. Con-
s iderable additional effort must be expended before use of such techniques
becomes an integral part of tree breeding programs.
2. IN VITRO SELECTION
Alterations in established culture procedures for selection of specific
variants in a population of microorganisms is a standard procedure (14).
Considerable interest has centered on the application of similar procedures
to plant cells. The ideal s ystem for such application consists of tech-
niques enabling indefinite culture of friable callus, complete regeneration
of whol e plants from cell suspensions or callus cultures resulting from
s uspensions , production of haploid cell lines from pollen culture or other
t echnique and stab i liz ation of the chromosome karyotype (72). This review
will discuss significant progress which has been made with regard to sev-
eral of these prerequisites of a model system for forest species, although
considerable work remains for full rea lization of in vitro procedures.
370
2.5. Applications
There are many applications for variant cell lines in tree breeding
programs, including establishment of resistance clones, such as those
resistant to low temperatures, fungal pathogens including blister rust,
Dutch elm disease, fusiform rust and Melampsora rust (94) and also those
resistant to various insect pathogens. To date, however, little headway has
been made in this potentially significant area. In terms of screening
genotypes for rapid growth, several laboratories have found a good correlation
between in vitro and in vivo performance. Examples include correlations
between field performance and callus growth in aspen callus cultures (59) and
frequency of adventitious bud formation in cotyledon cultures of conifers
(1, 64). Tissue cultures may be utilized for screening a variety of tree
species for effects of environmental stresses including low temperatures in
spruce (87) and susceptibility to fungal pathogens, such as Dutch elm disease
in ulmus (86). Advantages of in vitro genotype testing include the short
time required to obtain results; tests may be able to be performed on
seedling-derived cultures; in vitro screening tests generally require only
minimal space; tests can be performed at any time of year. In our laboratory
investigations are currently in progress determining the usefulness of
several biochemical markers of cell cultures as a means of early genotype
374
assessment.
A few words of caution, however, are needed. Under most circumstances
conditions bn vitro do not simulate conditions under which plant cells
normally grow bn vivo . The mere act of placing plant mat eria l in culture
exposes cells to continuous selection pressure resulting in an increased
population of cells that are adapted to specific culture conditions (72).
In addition, differences in performance in vitro vs. in vivo may not be a
consequence of genetic differences, but rather a result of one or several
epigenetic factors controlling phenotype, as discussed earlier.
3. SOMATIC HYBRIDIZATION
Breeding programs for trees and crops are often faced with the major
problems of interspecific and intraspecific incompatibility which prevent
genetic recombination between unlike plants. Consequently, the first report
of the use of cell wall hydrolyzing enzymes for the isolation of large
numbers of naked protoplast s of higher plants (18) coupled with availability
of a procedure enabling high frequency of protoplast fusion (48) and culti-
vation procedures (11) have sparked considerable interest in production of
somatic hybrid plants. Several recent reviews (11, 48, 89) have focused on
principles and applications of protoplast techniques to crop improvement.
The discussion here centers on protoplast research with regard to potential
applications in tree breeding.
Another factor which has aided our ability to regenerate dividing cells
from conifer protoplasts is use of a non-woven polyester fabric support, as
described earlier. fabric supports and similar such procedures may aid in
gas exchange and diffusion of inhibitory substances from actively growing
cells.
377
been performed (13, 63, 82). Considerable work rema in s and some doubt has
been expressed as to whether isolated chloroplasts, when incorporated into
foreign cytoplasm, are capable of surviving and expressing the chloroplast
genome (27).
One aspect of protoplast work that may have application to t r ee species
is the use of in vitro systems for the e stablishment of nitrogen f ixation
(19 , 29). Progre ss ha s been made with r e gard to the establishment of the
symbiotic association in vi t ro (4, 5, 10,42). Giles and Whitehead (30, 31)
report successful transfer of both cells and nitrogen fixin g ability of
Azotobacter vinelandii to protoplasts of the fungu s Rhizopogon . Thi s
specific fungus forms mycorrhiz a l associations with the roots of radiata
pine (Pinus r adi ata). When the nitrogen-fixing variants of Rhizopogon wer e
associated with roots of nitrogen starved seedlings of radiata pine, an in-
crease in growth and nitrogen levels in seedling material was observed.
Such studies demonstrate potential usefulness of functional symbiotic gene
transfer.
4. GENETIC TRANSFORMATION
4.1. Principles
Isolation, culture and plant regeneration from protoplasts have enabled
the combination of genes from diverse sources. On the other hand, genetic
transformation involves uptake of selected DNA molecules by competent cells,
integration of the DNA into the genome of those cells and ultima te expres-
sion of integrated foreign genetic information (84) . Studies of genetic
379
4.2. Procedures
4.2.1. DNA uptake. Since the original demonstration, almost thirty
years ago, that bacteria could be genetically transformed by exogenously
supplied DNA (2), considerable work in plant systems has focused on simple
uptake studies. Some controversy has developed in terms of the results
that have been obtained. Work from Ledoux's laboratory (54, 55), claiming
correction of a thiamine-less mutant in Arabidopsis as a result of incuba-
tion with bacterial DNA has been hotly contested (52, 53). In spite of
considerable effort in this area, including the direct uptake of exogenous
DNA by cell cultures, callus cells, and protoplasts (52, 66, 67, 68, 88)
there remains little convincing evidence that uptake of exogenously supplied
DNA offers potential usefulness for genetic transformation. An exception
is the recent report that plasmids isolated from a bacterium, Agrobacterium
tumefaciens, can directly transform Petunia protoplasts (20).
4.2.2. Transformation using biological vectors. There is some prom-
ising evidence which suggests that biological vectors may be utilized for
successful transfer of bacterial genes to plant cells. The most thoroughly
investigated vector is the crown gall bacterium, Agrobacterium tumefaciens.
It is well established that tumor-inducing strains of A. tumefaciens contain
large (100-150 megadalton) extrachromosomal, covalently closed, circular
DNA plasmids (76). The direct association of tumor-inducing properties of
A. tumefaciens with such plasmids (Ti plasmids) has been clearly demonstra-
ted. Strains of A. tumefaciens which have lost the Ti plasmid have also
lost the ability to induce tumors; introduction of a Ti plasmid into a non-
tumor-inducing acceptor strain by conjugation confers the capacity to induce
tumors in plant cells; deletion mutants of Ti plasmids are not able to in-
duce tumors (84). Recently it has been shown that a portion of the Ti
plasmid, termed T-DNA, is stably incorporated into chromosomal DNA of plant
tumor cells (17, 24, 95). Transformed plant cells produce a novel class of
amino acids referred to as opines. Naturally-occurring strains of A.
tumefaciens are unusual in that they can utilize opines as sole sources of
380
5. CONCLUSIONS
This review has presented a brief overview of recent sophisticated and
theoretical advances in genetic modification of plants using in vitro sys-
tems. It is readily apparent that very few reports have centered on appli-
cation to forest trees. Applications of such procedures, including in vitro
selection and somatic hybridization, await development of techniques for
production of plantlets from single cells or callus cultures of economically
important species. This must be our immediate research goal.
For the long term, it is felt that in vitro procedures will have appli-
cation in the development of genetically improved forest trees. Production
of intra- and interspecific hybrid lines will likely be achieved through
protoplast procedures. An understanding of fundamental aspects of naturally-
occurring genetic transformations, such as crown gall disease, may lead to
the harnessing of such systems for genetic improvement of trees. Simple
transformations may involve maintenance and expression of genes for single
peptides, such as those conferring characters as diverse as insect resistance
and attachment of nitrogen-fixing bacteria. Application is seen in terms
of expanding the ranges of certain forest species as well as increasing
yield.
382
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8. BRIGHT SWJ, PB NORBURY, BJ MIFLIN 1979 Isolation of a recessive
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9. BROWN DD 1981 Gene expression in eukaryotes. Science 211:667-674
10. BRUGOON AC, PJ BOTTINO 1976 Uptake of the nitrogen-fixing blue-green
algae GZeocapsa into protoplasts of tobacco and maize. J Hered 67:
233-236
11. BUTENKO RG 1979 Cultivation of isolated protoplasts and hybridization
of somatic plant cells. IntI Rev Cytol 59:323-373
12. CARLSON PS 1970 Induction and isolation of auxotrophic mutants in
somatic cell cultures of Nico tiana tabacum . Science 168:487-489
13. CARLSON PS 1973 The use of protoplasts for genetic research. Proc
Natl Acad Sci USA 70:598-602
14 . CHALEFF RS, PS CARLSON 1974 In vitpo selection for mutants of higher
plants. In L Ledoux, ed, Genetic Manipulations with Plant Material,
Plenum Press, New York, pp 351-363
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Ann Rev Genet 8:267-278
16. CHALEFF RS, J POLACCO 1977 In H Smith, ed, Molecular Biology of
Plant Cells, University of California Press, Berkeley, pp 429-441
17. CHILTON MD, MN DRU~4MOND, DJ MERLO, D SCIAKY, AL MONTOYA, MP GORDON,
EW NESTER 1977 Stable incorporation of plasmid DNA into higher plant
cells : The molecular basis of crown gall tumorigenesis. Cell 11:263-
271
18. COCKING EC 1960 A method for the isolation of plant protoplasts and
vacuoles. Nature 187:927-929
19. DAVEY MR 1977 Bacterial uptake and nitrogen fixation. In J Reinert,
YPS Bajaj, eds, Applied and Fundamental Aspects of Plant Cell, Tissue
and Organ Culture, Springer, Berlin, pp 551-561
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of Petunia protoplasts by isolation Agpobactepium plasmids. Plant Sci
Lett 18:307-313
21. DAVID A, H DAVID 1979 Isolation and callus formation from cotyledon
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23. DIX PJ, HE STREET 1976 Selection of plant cell lines with enhanced
chilling resistance. Ann Bot 40:903-910
24. DRUMMOND MH, MP GORDON, EW NESTER, MD CHILTON 1977 Foreign DNA of
bacterial plasmid origin is transcribed in crown gall tumors.
Nature 269:535-536
25 . DUHOUX E 1980 Protoplast isolation of gymno sperm pollen.
Z Pflanzenphysiol 99:207-214
26. GAMBORG OL, K OHYAMA, LE PELCHER, LC FOWKE, K KARTHA, F CONSTABEL,
K KAO 1979 Genetic modification of plants. I n WR Sharp, PO Larsen,
EF Paddock V Raghavan, eds, Plant Cell and Tissue Culture: Principles
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27. GILES KL 1977 Chloroplast uptake and genetic complementation. In
J Reinert, YPS Bajaj, eds, Applied and Fundamental Aspects of Plant
Cell, Tissue and Organ Culture, Springer, Berlin, pp 536 -550
28. GILES KL 1978 The uptake of organelles and microorganisms by plant
protoplasts: Old ideas, but new horizons. In TA Thorpe, ed, Frontiers
of Plant Tissue Culture, International Association for Plant Tissue
Culture, Calgary, pp 67-74
29. GILES KL, IK VASIL 1980 Nitrogen fixation and plant tissue culture.
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30. GILES KL, H WHITEHEAD 1976 Uptake and continued metabolic activity
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31. GILES KL, HCM WHITEHEAD 1977 The localization of introduced
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36. HESS 0 1978 Genetic effects in Petunia hybri da induced by pollination
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37. HESS 0 1979 Genetic effects in Petuni a hybrida induced by pollination
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Principles and Methods, Plenum Press, New York, pp 49-55
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385
J.M. Bonga
1. INTRODUCTION
For forest tree improvement it is essential to develop methods of vegeta-
tive propagation of mature trees, because the potential for obtaining better
forests through sexual breeding is limited. The major problem with sexual
breeding, besides the long life cycle of trees, is that many woody species,
especially those in temperate climate zones, are highly heterozygous (1, 64).
This limits the use of inbreeding and controlled hybridization as a means of
obtaining genetic gain because inbreeding of heterozygous plants often leads
to severe growth depression in the offspring (64, 93, 120) . Nevertheless,
with continuous breeding and selection over several generat i ons some tree
improvement can be expected (93), but the gain is likely to be less than if
selected trees are propagated vegetatively (Fig. 1). Another advantage of
mass vegetative propagation is that increased productivity is immediate, whereas
with sexual propagation it requires several generations. Furthermore, vegeta-
tive propagation allows mass propagation of hybrids and polyploids with low
sexual fertility (12).
Vegetative propagation of woody plants of a size large enough to have demon-
strated their potential is often difficult or impossible with the traditional
methods of rooting of cuttings and grafting. Cloning by means of tissue cul-
ture is an alternative to the traditional methods , and has been successfully
employed to obtain propagules from mature plants of some tree species (see
earlier chapters on vegetative propagation). However, most mature hardwoods
and conifers are still difficult or impossible to propagate by tissue culture.
In fact, tissue culture techniques often offer no advantage over traditional
rooting of cutting techniques because the capacity of a plant to propagate
vegetatively in tissue culture is often lost at an age when the plant can still
be propagated by rooting its cuttings (Fig. 2). For example, in many conifers
the capacity for embryogenesis and morphogenesis diminishes shortly after
388
germination (10), and in some species even before germination (75). There-
fore, to achieve vegetative propagation of hitherto "recalcitrant" plants,
some of the presently used tissue culture methods may have to be drastically
changed. To develop such new methods, factors such as juvenility, maturation,
determination in meristems, clonal deterioration, genetic stability, and
rejuvenation will have to be considered. Each of these factors must be
controlled before uniform, true-to-type, long-term cloning of trees can be
achieved.
ISMALL'
17 LARGE .1
GENETIC GAIN
2. JUVENILITY - MATURITY
2.1. Definitions
The capacity to vegetatively propagate trees is associated with juve-
nility. Generally, the more juvenile the specimen, the easier it is to prop-
agate vegetatively. There is no clearly defined transition from the juvenile
to the mature phase in most plants. Often some parts of the tree may be
mature, or senescent, while other portions still display juvenile charac-
teristics. Furthermore, in some trees major morphological characteristics
389
may change abruptly (heteroblastic) when maturing, while in others, the changes
are more gradual (homoblastic) (98).
*.!-~
1--1/!~~
I D
c\ 414-
~~
~1'i)/
E A
Juvenile Mature
Apical meristems Small dome (43, 49, 100) High dome with increased
Large cell s (43) RNA (49)
b This obviously does not apply to the zygote, which has a large
nucleus.
c Reference number.
391
result of environmental limitations, but others may have arisen from cells in
which some maturity was still retained, i.e., rejuvenation was almost, but not
entirely complete. 5) "Dedifferentiation" is often used in the tissue culture
literature to describe events similar to those described above under partial
and complete rejuvenation. However, in other literature the term dedifferenti-
ation often has other meanings than reprogramming of cells to a state in which
they are capable of adventitious organ or embryo formation. Therefore, the
term rejuvenation is preferred to describe increasing morphogenetic ability of
cells, but the term dedifferentiation may be used when discussing a literature
reference in which it is used.
2.2. Determination in meristems
Meristematic apices, the centers of growth and organization in plants,
undergo changes when the plant matures. Therefore, the tissues derived from
these apices behave differently in young and old plants (13, 43, 64, 100). One
consequence of this is that shoot cuttings from older trees often root poorly
or not at all. Furthermore, if rooting occurs, the propagules may not behave
true-to-type, but show undesired characteristics such as plagiotropic growth,
reduced growth rate, changed taper, etc. (51, 94). Differences between rooted
cuttings and seed-derived specimens are often maintained throughout the life of
the trees. For example, mature trees grown from rooted Pinus radiata cuttings
are different in several respects from trees grown to maturity from seed (65).
In some cases, specific growth charac teristics are very firmly "determined" 1
in the meristems. The classical example is Araucaria, where only cuttings fron:
vertical stems will form normal plants after rooting. Rooted cuttings from
primary and secondary branches, instead of growing vertically, have been ob-
served to maintain a horizontal growth habit for over 50 years, with the rooted
primary branch cuttings forming laterals, and with the rooted secondary branch
cuttings failing to do so (40, 44, 73, 100). Similar, but less extreme and
persistent trends have been observed in Larix, Picea, Pseudotsuga (40, 118) and
some hardwoods (40), while in Douglas fir plagiotropism is already noticeable
in rooted cuttings from seedlings only 5-7 months old (97).
It is obvious from these examples that true-to-type vegetative propagation
is often possible only with very young, juvenile material. However, as was
pointed out earlier, the objective is to find methods to propagate trees old
enough to have expressed their genetic potential. This indicates the need of a
close study of juvenility and of the possibility of experimentally obtaining at
least partial, but preferably complete, rejuvenation. Therefore, first it should
be determined which cells and tissues in the plant retain juvenility the long-
est, and use these tissues and cells as explants for in vitro propagation.
Next, those species which have propagated vegetatively naturally, sometimes over
thousands of years, should be investigated. How did these species maintain
their meristems in a sufficiently juvenile condition for true-to-type cloning
over many generations of propagules; how did they maintain their genetic
stability during those years? Lastly, it should be determined if partial or
complete rejuvenation occurs naturally, and if this process can be duplicated
experimentally.
Sometimes there are reasons other than vegetative propagation why main-
tenance or reestablishment of juvenility is of interest. The juvenile phase
of tree growth has some attributes which are important in a practical forestry
context. Firstly, when trees mature their growth rates often drop. Secondly,
in many hardwoods the trunk is single as long as the tree is juvenile, but
starts to fork when the tree reaches maturity (Fig. 3). Thirdly, juvenile
trunks have more radial growth, and juvenile bark is more resistant to dis-
ease and stress (84, 100, 112). However, juvenile characteristics are not
always the ones desired. For example, fibre length is short in juvenile wood
(33) and in conifers the mature growth form is sometimes better than the juve-
nile one (112, 113). Therefore, in some vegetative propagation programs cut-
tings of older trees are preferred over those of younger trees, because when
cuttings of older plants are rooted, the juvenile growth phase can be bypassed
resulting in straighter, less tapered stems, although, alas, with less volume
growth (112, 113).
2.3. Juvenile zones
Most trees have zones that retain a degree of juvenility longer than other
areas of the tree. For example, roots often retain juvenility and thus a capa-
city for vegetative propagation and juvenile type of growth, for a long time
(16, 82, 90, 117). In the above-ground part of the tree, the less distance a
shoot apical meristem is located from the base of the trunk, the more juvenile
it generally is. In a regularly shaped tree, like a conifer, this means that
the meristem at the apex of the leader is the least juvenile; the apical meri-
stem at the end of a first-order branch in the upper part of the tree is more
394
juveni1e; the apical bud of a first-order branch in the lower part of the tree
is more juvenile again, and the apical bud on a second order branch of this
lower branch is more juvenile still (in Fig. 3, AB>AC>AD>AE>AF) (61, 84). In
the tree, this zonation of juvenility is reflected in cuttings from lower
branches rooting easier than those from higher branches, and cuttings from a
higher branching order rooting easier than those from a lower branching order
(40) . Zonal differences in juvenility are also demonstrated by differences in
winter leaf retention, in apical meristem shape and cytochemistry (49, 100,
101), and in many other characteristics (Table I, Fig. 3). This increase in
juvenility towards the center and lower parts of the crown is exploited in
vegetative propagation practices. The most common practice is heavy and re-
peated pruning, or cutting down of the tree, thus forcing new shoot formation
from the lower and innermost branches or from the lower parts of the trunk
(Fig. 3). Most of these juvenile sprouts do not arise de novo from adventi-
tious buds, but from buds laid down early in the life cycle of the tree that
have remained strongly suppressed and thus juvenile (arrested juvenility) up to
the time of pruning or cutting (34, 39).
It has been suggested that the differences in degree of juvenility between
different shoot apical meristems in the tree are related to the number of cell
divisions that separate each meristem from the original embryo shoot apex. To
assume some loss in juvenility in each successive division (84, 95), in analogy
to what has been observed in lower organisms (78) and several animal cell cul-
tures (47, 109), is probably reasonable, but only up to a point, as will be
discussed later.
Differences in maturity in the above-ground parts of a tree are not con-
fined to apical meristems, but are evident also in the cambium. The cambium
near the base of the trunk is more juvenile than that in the upper part of
the trunk, and may retain some juvenile characteristics for many years (84).
This juvenility is expressed in the cambial derivatives by bark type, tracheid-
vessel ratio, and tracheid length (84, 88, 98).
2.4. Clonal aging
On the basis of the earlier discussed maintenance of mature characteris-
tics in rooted cuttings or grafts if the cuttings or scions are taken from
the mature part of the tree, one would expect that in successive generations
of cloning, including repeated in vitro cloning, mature characteristics would
accumulate progressively. In older literature it is often stated that repeated
vegetative propagation eventually leads to clone senility, especially in plants
395
that in nature reproduce only sexually, and that this senility can only be
alleviated by going through a sexual cycle (40, 69, 88). However, this con-
cept of clonal degeneration is generally no longer accepted, most clonal de-
generation is currently ascribed to pathogen accumulation or adverse environ-
mental conditions (40, 46, 63). It appears therefore, that even though mature
characteristics may be transmitted to the first generation of propagules,
later generations do not progressively become more mature when propagated
regularly. In fact, many tree species, including those that predominantly
propagate sexually, have been cloned for hundreds or some even a few thousand
years without any signs of clonal degradation (28, 76, 116). Furthermore, in a
few clones instead of degradation, a degree of rejuvenation has been achieved
by application of special techniques. For example, a Vitis clone, which for
centuries has been propagated only in its mature form, reverted to the juvenile
form after several generations of in vitro repropagation of propagules when
these were still very small (76). Early repropagation, by rooting cuttings,
has resulted in arrested maturation in some species (70, 74), probably because
this procedure keeps the shoot apex close to roots (70, 82). However, the
opposite has also been observed. Robinson and Wareing (95) found that if black
currant was regularly cloned before reaching mature height, flower induction
became easier after a few generations of cloning, i.e., the clone had become
more mature. Clonal maturation and senility are distinct features in proto-
zoans, fungi, and other lower organisms. Growth slows down and can only be
restored to the original rate by a passage through the sexual cycle (42, 106).
Clonal senility also occurs in several animal cell cultures, but, in these,
unlike in most plant clones, senility is not caused by transmissible viruses or
other pathogens (109).
Many plant cell or tissue clones can be maintained in vitro well beyond the
normal life span of the plants that provided the cells or tissue for culture.
For example, callus cultures from tomato plants have maintained vigorous growth
continuously since 1945 (62), although it could be claimed that these cells are
transformed (habituated) cells, i.e., they are cells that are different from
the cells in the original explant and have lost the capacity to mature. Even
though seemingly immortal, long-term in vitro plant cell clones often deteri-
orate; chromosome numbers change and the competence for embryogenesis or orga-
nogenesis generally diminishes with subculturing (72).
396
In the past, the prevailing view was that genes are gradually activated
during embryo initiation and later growth. Currently it is more generally
accepted that progressive gene repression dominates selective gene activa-
tion during development (24, 55). One mechanism of gene inactivation appears
to be methylation of chromosomal DNA (50, 51, 92); possibly others are euchro-
matization (101), or to the contrary heterochromatization and histone forma-
tion (52, 55, 107). However, maturation is not solely determined by the nucleus,
but also by the accumulation of self-replicating determinants in the cytoplasm,
which can be passed on through many cell generations (13, 15, 105, 107, 119).
These self-replicating determinants presumably are cytoplasmic organelles which
contain DNA and thus genetic information (4, 15, 42, 107), or they are free
cytoplasmic DNA released by disintegrating organelles (38). This organellar DNA
is circular and is structurally much simpler than chromosomal DNA, and although
sometimes present in large amounts, its genetic information is always limited in
kind (42, 57). However, organellar DNA may be a principal factor in determining
the level of maturation of a cell. For example, in fungi transfer of cytoplas-
mic DNA (probably of mitochondrial origin) from a mature mycelium into a juve-
nile one causes maturation of the latter (37, 38).
Even though maturation of a cell may, to a degree, be determined by the num-
ber and distribution of these DNA-containing organelles, or by DNA released by
these organelles into the cytoplasm, there are other factors that could be
important. For example, in time, organelles tend to differentiate structurally
to more complex forms (60,), which may affect the expression of their DNA, and
their DNA often becomes more polyploid or amplified (4, 38, 48).
The effects of the cytoplasm on the nucleus, and vice versa, during matura-
tion have been clearly shown in several nuclear transplant studies. For exam-
ple, in experiments with Acetabularia, mature cytoplasm induced rapid maturation
of immature nuclei (8, 15, 89). Conversely, if nuclei of mature cells were
implanted in the enucleated cytoplasm of young cells, the mature nuclei rejuve-
nated (8, 89). However, nuclear rejuvenation in a juvenile cytoplasm is not a
universal phenomenon; in nuclear transplant experiments with Paramecium, mature
nuclei did not rejuvenate in juvenile cytoplasm (17). Mature frogs have been
cloned by transplanting nuclei from skin cells into enucleated egg or embryo
cells (19, 24), although the success rate decreases as the maturation level of
the transplanted nucleus increases (19, 71). Therefore, nuclear rejuvenation is
possible, but only if maturation has not progressed too far. Conceivably, in
many mature plants and animals only those somatic cells that will eventually
398
enter meiosis may have nuclei at a maturation level low enough to be rejuvena-
ted by juvenile cytoplasm.
2.7. Mechanisms of juvenility retention
From earlier comments (section 2.3.) one would be inclined to conclude
that the degree of maturity of a cell or meristem of a tree is proportional
to the number of cell divisions that separate the cell or meristem from the
zygote. With respect to the general principle of maturation, this statement
is probably correct, but with regard to different cells in different loca-
tions within the plant or meristem it is an oversimplification, because: 1)
In some cell lines, the cells may mature more in each division than those in
other cell lines. 2) If maturity of a cell is partly determined by cyto-
plasmic determinants, unequal divisions would result in differences in matur-
ity of the two daughter cells because of unequal distribution of the cyto-
plasm between the two cells (13). 3) In some cell lines, a mechanism may be
present for occasional or regular, partial rejuvenation of the cells (see
later).
Nevertheless, limiting the number of cell divisions probably favors juve-
nility retention. On that basis, the following mechanism is suggested to
explain retention of juvenility in some tissues, especially in long-lived
clones. In all major plant meristems there are tissues with a low mitotic
activity, and the function of this low mitotic activity may well be to main-
tain a pool of relatively juvenile cells within the meristems. In the more
active areas of the root and shoot apical meristems, the cells divide and
organize lateral root, shoot, or leaf primordia. In this process, the cells
presumably become slightly more mature in each cell division, and eventually
reach a level of maturity where cell division is still possible but the
capacity to form primordia is lost. When this stage is reached, some cells
may be removed from the juvenile low activity portions of the meristem to
replenish the active tissue with relatively juvenile cells (3, lOS, 114).
The low activity zones are not completely inert; their cells are dividing
occasionally at a low rate, and thus one would expect a slow gradual matura-
tion even in these low activity areas. However, to explain how very long-
lived trees such as sequoia and bristlecone pine (69), and long-lived clones
such as poplar (2S) and creosote bush (116), manage to produce functional new
shoot and root primordia after centuries of active growth, we probably have to
assume, that a counteracting, rejuvenating mechanism is present in at least a
few cells in the low activity areas. Following this concept further, species
399
that are difficult to propagate vegetatively probably differ from easily prop-
agated species in that the maturation - rejuvenation balance in the low activ-
ity zones in the latter is such that the cells are maintained at a more juve-
nile level.
Relatively inactive zones are found in root tips, shoot tips, and the cam-
bium. Of these, the quiescent center in the roots is generally the least
active . It possesses great regenerative capacity; if the root apex is damag-
ed, a new root will arise from surviving quiescent cells (3, 114). In the
vegeta ti ve shoot apex, the central zone, sometimes called the "meris tern d' a t-
tente", is relatively inactive (6B, 96, lOB) as long as the shoot remains veg-
etative. In developing flowers the apices behave differently. Flowers are
"determinate" structures (lOB), I.e., they die after they have performed their
flowering function. Therefore, there is no need to maintain a pool of rela-
tively inactive cells in the central zone of the flower apex, and the division
rate in this zone increases (21, lOB), as some of the cells are prepared for
meiosis.
The central zone of the shoot apex has been traced back to a few specific
cells of the embryo, and remains relatively inactive as it is carried along
inside the shoot apex from the time of germination to the onset of flowering
(110). Thus, the central zone in the apex of a tall, old tree has undergone
only a limited number of divisions since its conception in the germinating
embryo.
Another major meristem with a relatively inactive zone is the cambium,
where the rate of division is much lower in the cambial initials than in adja-
cent cells (lOB, Ill). Long-term retention of function in the cambium also
may depend on the relative inactivity of some of its cells, i.e., of the cam-
bial initials.
2.B. Mechanisms of genetic stability
Genetic stability is a prerequisite for successful cloning, especially if
the cloning is repeated over several successive generations. Therefore, it is
important to discover how this stability is maintained in meristems of long-
lived trees and natural clones. Again, low mitotic activity may be crucial,
because gene mutation rates are proportional to the rate of division (23), and
therefore, good genetic stability can be expected in relatively inactive areas.
Evidence of genetic stability of inactive areas in meristems has been pro-
vided by experiments with root tips. The quiescent center of the root tip is
very stable in adverse environmental conditions. For example, it is relatively
400
species. This warrants a closer look at the one stage in the life cycle of
every tree where complete rejuvenation always occurs, namely during sexual
reproduction. If we could duplicate this meiotic rejuvenation mechanism in
somatic cells without reducing the number of chromosomes, asexual propagation
from somatic tissues of mature trees would likely become possible.
2.10. Sexual rejuvenation
Since the subject of sexual rejuvenation has been reviewed earlier (13),
the subsequent discussion will be confined mostly to literature not considered
at that time.
It has been claimed, that some cellular dedifferentiation occurs in the
prefloral (21), and premeiotic (82) stages. However, the most dramatic reju-
venation probably occurs during the meiotic prophase (13). During the meiotic
prophase, there is a drastic reduction in numbers of cytoplasmic ribosomes and
plastids, and a simplification (dedifferentiation) of mitochondria (30, 67,
102). The apparent function of this process is to remove residual, long-lived,
ribosome-associated mRNA from the cytoplasm during the transition from the
mature sporophyte to gametophyte and male and female gametes (30, 31), and
results, after fertilization, in a completely juvenile zygote. The reduction
in complexity of organelles, or their partial removal, is accomplished by lys-
ing enzymes, primarily acid phosphatase and ribonuclease (30). The most like-
ly sources of these enzymes are vacuoles, cell walls, and membranes (86, 87),
although the lysosomal function of the vacuole has been questioned (53).
Lysing during meiosis is not restricted to the cytoplasm. A somewhat simi-
lar process occurs within the nucleus when vacuoles and other structural com-
ponents of the sporophyte nucleus are reconditioned to those of a gametophyte
nucleus (103). Whether the genetic information of the nucleus is reprogrammed
at the same time for the juvenile phase of growth is not known; it could be
reprogrammed later, either shortly before, during, or even after fertilization
(50).
Plastid and ribosome destruction is not total during the meiotic prophase.
A small number of these organelles is sequestered from the rest by membranes
and thus is partially protected from lysing (30). Initially, the sequestered
organelles undergo little degradation and presumably remain sufficiently func-
tional to properly maintain cell metabolism while the rest of the cytoplasm is
devoid of functional organelles. During the later phases of meiosis, the
cytoplasm is rapidly repopulated with new (presumably juvenile) organelles.
402
New mitochondria are formed from the partially lysed, structurally simplified
ones (67, 102), and new ribosomes are released by the nucleus (30, 102).
It is not clear what happens to organellar DNA during meiosis. Earlier
(section 2.6.) it was pointed out that DNA in the cytoplasmic organelles prob-
ably is directly involved in part of the maturation process in the cell.
Therefore, it is important to establish what happens to the cytoplasmic DNA
during meiosis. Does it survive during organelle dedifferentiation or elimi-
nation, what happens to its ploidy, and does it become reprogrammed during
meiosis? These are just a few of the many still unanswered questions.
It has been found that tumor-inducing plasmid DNA disappears from the cyto-
plasm during meiosis (18, 122). At present, it is not known if the DNA from
the regular organelles is similarly eliminated, but if so, where would the new
DNA for new organelles come from? During meiosis there is an abundance of
circular extrachromosomal DNA in the nucleolus organizer in the nucleus of
some organisms (52). It is not known if this is a source for new cytoplasmic
organellar DNA. Observations suggesting release of de novo organelles with
extra-chromosomal DNA from the nucleus have been described (6, 7), but have
also been questioned (4, 32).
In conclusion, it appears that old information is being removed from the
cytoplasm during meiosis. However, it is not yet clear how the cytoplasm is
subsequently provided with new information, and how and when the nuclear
genetic information is reprogrammed. A better understanding of the mechanisms
of cytoplasm and nucleus rejuvenation during meiosis is important because it
may suggest means of removing the mature determination from cells of mature
trees, thus reestablishing their capacity for somatic embryogenesis and true-
to-type vegetative propagation.
3. SIGNIFICANCE FOR PROPAGATION BY TISSUE CULTURE
If we are faced with a recalcitrant in vitro culture from which true-to-
type vegetative propagation does not materialize, the following general ap-
proach should be considered.
It probably is wise to first develop methods for vegetative propagation
from highly juvenile material, i.e., from sections of embryos or very young
seedlings. Once propagation from juvenile material has been achieved, and the
basic environmental parameters for culture of the species have been establish-
ed, the following procedures are suggested for mature material. 1) It may be
necessary to select explants containing cells that will eventually enter meio-
sis, because these may be the only cells in the plant body in which the nuclei
403
have not matured past the point where reprogramming of the nucleus for embryo-
genesis has become impossible. However, it is not currently known how far the
nuclei in non-meiotic tissues have matured, and it is possible that the nuclei
of at least some tissues other than those entering meiosis could be completely
rejuvenated for use in somatic embryogenesis. 2) Since genetic stability and
juvenility are probably best retained in cell lines that are separated from
the embryo by low numbers of mitosis (section 2.7.), it is recommended that
tissues containing such relatively inactive cell lines be cultured. It is
also advisable to limit cell division between tissue excision and induction of
morphogenesis, i.e., an intervening callus stage should be avoided as much as
possible. 3) Based on the discussion on rejuvenation during meiosis, it is
recommended that tissues be selected for culture which have recently undergone
a reduction in number or complexity of cytoplasmic organelles.
Obviously, the choice of explant is important, and therefore, a survey is
presented below of which parts of the tree, excluding the embryo, would be
most suitable for excision and culture.
3.1. Choice of explants
3.1.1. Flower parts. Somatic tissues of flowers of many plants have a high
capacity for vegetative reproduction, possibly because of their proximity to
the rejuvenating sexual cells (81, 82, 83). With regard to trees, morphogene-
sis has been obtained in cultures of somatic tissues of sexual shoots of sev-
eral species (12, 14, 35). According to some authors (21, 82), a dedifferent-
iation of cells occurs just before or shortly after flower induction. There-
fore, explants should probably be taken from flowers at an early developmental
stage. One part of later stage flowers that is of considerable interest is
the nucellus. In some citrus varieties, adventitious embryos arise naturally
from the nucellus (20, 58, 61), but the propagules raised from these do not
always develop true-to-type (58). Adventitious embryos have also been obtain-
ed in vitro from nucellar tissues of citrus (22) and apple (36). In view of
earlier comments regarding rejuvenation of the cytoplasm during meiosis, it is
of interest to note that during development of the nucellus the cytoplasmic
organelles in its cells are reduced in number and become disorganized (79, 80).
3.1.2. Vegetative buds. Vegetative buds, or parts thereof, have frequently
been used as explants in experiments designed to obtain vegetative propagation
of trees (see chapters on vegetative propagation). The central part of the
apex is probably the most responsive. Its cells have low mitotic rates and
404
low numbers of ribosomes (21), both of which, as was pointed out earlier, may
be significant in relation to the morphogenetic capacity of the tissue.
In cultures of shoots dissected from buds of mature conifers, small, shoot-
like structures were obtained, mostly at the base of young needles (14). Many
of these structures may have arisen from the lining of the resin ducts in the
needles, which again is a tissue with structurally simple plastids and, in the
later stages of its development, a low number of ribosomes (25, 121).
3.1.3. Roots. Shoot buds will form naturally near the apex of roots of
some species and have been induced in root cultures of others (90). They will
form only rarely from the root apical meristem itself, i.e., the root apical
meristem, like the shoot apical meristem, is firmly determined, and the two
meristems normally are not interconvertible.
3.1.4. Root-shoot junction. This area may contain arrested juvenile buds
(section 2.3.) which will flush into juvenile sprouts if the tree is cut down
or severely pruned. In vitro culture of such juvenile sprouts has resulted in
clonal propagation of, for example, sequoia (2). However, in many tree spec-
ies such sprouts are not formed, and thus are not available for cloning pur-
poses.
As was emphasized throughout this section on explants, many of the tissues
suitable for vegetative propagation have low numbers of organelles, or struc-
turally simple organelles. Of course, one may not automatically assume that
this condition is always causally related to morphogenesis. For example, a
reduction in numbers and complexity of organelles is a common feature in sen-
escing tissues (5, 38). Senescence generally does not favor morphogenesis,
although both are not always mutually exclusive. For example, in citrus cul-
tures, aging of the callus resulted in increased embryogenesis (54).
3.2. Chemical and physical methods of reducing organelles
It is still far from clear, whether the mechanisms of reprogramming of
cells for morphogenesis are indeed similar to the mechanisms of rejuvenation
during sexual reproduction. Nevertheless, it may be worthwhile to determine
if some of the processes occurring during sexual rejuvenation can be duplicat-
ed in somatic cells by chemical or physical means, and if this is subsequently
followed by morphogenesis. Since the first step, and possibly the most impor-
tant one, in sexual rejuvenation appears to be a reduction in number and com-
plexity of organelles, possibly accompanied by a removal of organellar DNA,
this process should receive some attention. An alternative approach would be
405
5. ACKNOWLEDGEMENTS
I wish to thank Dr. W.K. Coleman, Canada Department of Agriculture, and Dr.
J.E.A. Seabrook, University of New Brunswick, Fredericton, Canada, for review-
ing the manuscript.
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