Presentation on,

POLYEMBRYONY

Shivanand. M.R
UHS11PGM143 Jr. MSc. (Hort.) Dept. of PMA
1 KITTUR RANI CHANNAMMA COLLEGE OF HORTICULTURE, ARABHAVI 591 310

POLYEMBRYONY
Polyembryony can be defined as the occurrence of more than one embryo in a seed . The additional embryos-do not always mature. They may become arrested at very early stages or may degenerate during the course of seed development. Therefore, if mature seeds are taken into account the percentage of polyembryony in a species would be far less than its actual frequency. polyembryony includes all those instances in which there is a clear indication of the actual occurrence of two or more proembryos or embryos in a developing ovule. Except for a few taxa (Citrus, Mangifera), polyembryony occurs only as an abnormal feature.

Picture of germinated bi-embryonate seed of Osyris Wightiana & Citrus (after Bhojwani, 1968)

 The first case of polyembryony was reported in certain orange seeds by Antonvan Leeuwenhoek (1719). Braun (1859) gave a survey of 58 cases of polyembryony recorded in the botanical literature at that time and referred them to four categories on the basis of the origin of the additional embryos. Polyembryony in angiosperms may arise by: (1) Cleavage of proembryo, (2) Formation of embryos by cells of the embryo sac other than the egg, (3) Development of more than one embryo sac within the same ovule, (4) Activation of some sporophytic cells of the ovule.

Cleavage Polyembryony
Cleavage and proliferation of zygote or its derivatives leading to the establishment of separate embryonal primordia is widespread among gymnosperms. In angiosperms this feature is less frequent. Among angiosperms cleavage polyembryony is quite common in orchids. In Eulophia epidendraea, Swamy (1943) recorded three different modes of supernumerary embryo formation:

I. The zygote divides irregularly to form a mass of cells of which those lying toward the chalazal end grow simultaneously and give rise to many embryos.
Three embryos arisen by proliferation of the zygote.

II. The proembryo

forms small buds or

outgrowths which
may themselves

function as embryos.

Budding of the embryo

III. The filamentous

embryo becomes

branched, and each

branch forms an

embryo

Two embryos arisen by splitting of a single embryo. Large, vacuolated cells belong to the suspensor.

Embryos from Cells of the Embryo Sac Other than the Egg
In this category the most common source of additional embryo is the synergid. Depending on whether it arises from fertilized synergid or unfertilized synergid, the embryo may be diploid or haploid. In Aristalachia bracteata, Poa alpina and Sagittaria graminea besides the egg and the polars, one or both the synergids may get fertilized. This can be brought about either by the entry of more than one pollen tube into the embryo sac or by the presence of additional sperms in the same pollen tube. In such a situation the zygotic as well as the synergid embryos are diploid. Embryos arising from unfertilized synergids are known in Argemane mexicana and Phaseolus vulgaris. In such cases the zygotic embryo can be distinguished from the synergid embryo (haploid) by its diploid nature.

Formation of embryos from antipodals is rather rare. It has been observed in Paspalum scrobiculatum, Ulmus americana and U. glabra. The antipodal cells may divide a few times to form proembryo-like structures (side Fig.). However, they fail to grow into an adult embryo and there is no suggestion of antipodals forming germinable embryos.

More Than One Embryo Sac in the Same Ovule

Multiple embryo sacs in an ovule may arise from: (1) Derivatives of the same megaspore mother cell, (2) Derivatives of two or more megaspore mother cells,
OR

(3) Nucellar cells. Formation of twin embryo sacs within an ovule is seen in Casuarina equisetifalia, Citrus and Paa pratensis. In Pemiisetum ciliare 22 percent seeds contain twin embryos. The normal embryo sac develops only up to the 4-nucleate stage, and the multiple embryos are formed by aposporous embryo sacs.

Embryo sac of Ulmus glabra showing zygotic and antipodal embryos. (after Ekdahl, 1941)

Activation of Some Sporophytic Cells of the Ovule

The embryos arising from the maternal sporophytic tissues (outside the embryo sac) are called adventive embryos. The only maternal tissues which are known to form adventive embryos are the nucellus and the integuments. Besides the more popular examples of Citrus and Mangifera, nucellar polyembryony occurs in Opuntia dillenii and Trillium undulatum.

 Some species of Citrus are monoembryonate (C grandis, C. limon) while others are polyembryonate (C. microcarpa, C. reticulata).
Seeds with as many as 40embryos have been recorded in C. unshiu.

In polyembryonate species the adventive embryos arise by the proliferation of the nucellar cells. With rare exceptions (Trillium undulatum) nucellar embryos arise from the micropylar half of the nucellus.

 In Mangifera the nucellar cells destined to form adventive embryos can be distinguished from other cells of the nucellus by their dense cytoplasm and starchy contents. The inception of nucellar embryos takes place outside the embryo sac but they are gradually pushed into the embryo sac cavity where they divide and differentiate into mature embryos (Fig: 13.6). The adventive embryos do not show synchronous development. A single seed may show embryos at various stages of development. Nucellar embryos can be distinguished from the zygotic embryo by their lateral position in the embryo sac, irregular shape, and lack of suspensor(Fig: A).

Fig. A: Longisection of a seed of Citrus sinensis, 80 days after anthesis, showing adventive embryos located in the micropylar and three lateral islands (arrowhead). There is a clear difference in embryo development in four islands. In the chalazal half the adventive embryogenesis is suppressed. (after Wakana and Uemoto, 1988)

CLASSIFICATION OF POLYEMBRYONY
Broadly speaking, polyembryony is of two types: (1) Spontaneous: Includes instances of naturally occurring polyembryony. (2) Induced: Includes instances of experimentally induced polyembryony. Spontaneous polyembryony has been subdivided by Ernst (1901, 1910; also adopted by other leading embryologists) into two categories: (1) True polyembryony: Two or more embryos arising in the same embryo sac from; zygote or embryo (Eulophia, Vanda), synergid (Sagittaria), antipodal cell (Ulmus), or nucellus or integument (Citrus).

(2) False polyembryony: Development of embryos in more than one embryo sac in the same ovule (Fragaria) or placenta (Loranthaceae). Yakovlev (1967) proposed a classification of polyembryony on genetic basis. He distinguished two types of spontaneous polyembryony: (1) Gametophytic: Arising from any gametic cell of the embryo sac after or without fertilization. (2) Sporophytic: Arising from the zygote, proembryo or the initial sporophytic cells of the ovule (nucellus, integuments).

Camaroon and Soost (1979) classified polyembryony on the basis of frequency of polyembryony into 3 types. i. Strictly polyembryonic: Plant species in which the frequency of multiple embryos is lesser than 7%. ii. Nearly monoembryonic: Here the frequency of polyembryony varies 6-10 %. iii. Polyembryonic : If the percentage of multiple seed is more than 10, is called polyembryony & plants are called polyembryonate.

CAUSES OF POLYEMBRYONY
Many theories have been advanced to explain the occurrence of polyembryony but none is sufficiently validated. In different theories polyembryony has been considered to be caused by to hybridization, necrohormones, the effect of recessive genes, etc. Haberlandt (1921, 1922) proposed the "necrohormone theory". He regarded the degenerating cells of the nucellus as source of stimulus for the adjacent cells to divide and form adventive embryos.

 Leroy (1947) thought that polyembryony in mango was caused by one or more recessive genes. According to Frusato et al. (1957), the embryo number in Citrus seeds may be influenced by the following factors: (1) Age of the tree; It increasing in older trees. (2) Fruit-set; being higher in years of higher fruit- set. (3) Nutritional status of the plant; It decreasing with reduced food supply. (4) Orientation of the branch of the tree; being higher on northern than on southern branches.

The monoembryonate condition in some species of Citrus has been ascribed to the synthesis and release of certain volatile and non-volatile embryogenic inhibitors in their ovules which do not occur in the ovules of polyembryonate species. Ethanol is one of the volatile inhibitors produced by the ovules of C. medica. When applied at a concentration equal to that produced by the ovules of C. medica, ethanol markedly inhibited embryogenesis in carrot tissue cultures. The non-volatile component of the inhibitors has been identified with IAA, ABA and GA3.

EXPERIMENTAL INDUCTION OF POLYEMBRYONY

In Eranthis hiemalis, a member of the family Ranunculaceae, a seed at shedding encloses an undifferentiated embryo which is pear-shaped and possesses a long suspensor (Fig.1). The radicle as well as the cotyledons differentiate several weeks later while the seed is in the soil. Treatment of the freshly harvested seeds with substances such as 2,4-D, 2,4,5-T or NAA at a concentration of 0.1% induces many abnormalities. Some of the seeds (up to 8%) develop twin embryos (Fig.2 AC; Haccius, 1955). These treatments damage the plumule, and the cells which are destined to produce cotyledons give rise to additional plumules.

Fig:1 Pear-shaped embryo excised from a freshly harvested seed of Eranthis hiemalis. (after Haccius, 1957)

Fig:2 Induction of polyembryony in seeds of Eranthis hiemalis treated with auxins. A, B. Formation of twin embryos. C. Twin seedlings.(after Haccius, 1955)

The potential to reconstitute additional embryos is also retained by mature embryos.

When the older embryos are treated with acidic buffer (pH 4) the embryonal body is selectively killed and the surviving suspensor cells develop into a new adventive embryo (Fig.3. A,B).
If the treatment is now repeated, the adventive embryo-1 is killed and a second adventive embryo arises (Fig.3.C).

Fig: 3.Eranthis hiemalis. Formation of adventive embryos from suspensor cells of the mature embryos treated with acidic buffer solution. (after Haccius, 1965)

 In nature the formation of embryos is restricted to the ovular tissues. For long it was assumed that, for their development, the embryos require a special physical and chemical environment which is available only inside the "magic bath" of the embryo sac.
However, it is now well established that by providing suitable nutritional and environmental conditions inside culture vials, any cell of the plant body can be stimulated to give rise to viable embryos. Embryos formed in tissue cultures are called adventive embryos.

 Depending on their origin these embryos are termed somatic embryos (arising from any somatic tissue) or pollen embryos. In vitro adventive embryogenesis has been reported in the cultures of zygotic embryos (bamboo, cotton, oil palm, soybean, wheat), nucellus (mono- and polyembryonate species of citrus and mango), stem segments (tobacco), root segments (carrot), fruit tissue (pumpkin), and anthers/pollen (grape, mustard, rice).

Somatic embryogenesis has been observed in over 100 species, which include many important crop plants, such as alfalfa, apple, coffee, cotton, grape, rice, soybean and wheat (Rangaswamy, 1986).
Ranunculus sceleratus (buttercup) and Daucus carota (carrot) have proved especially suitable for somatic embryogenesis. Any part of these two plants will form embryos in tissue culture. The other plants in which this phenomenon has been studied in detail are Citrus sp., Coffea sp. and Macleaya cordata.

 The work on buttercup was originally done at the Department of Botany, University of Delhi by Konar and Nataraja. Very young flower buds of Ranuneulus seeleratus were excised and planted on White's nutrient medium (which contains mainly inorganic salts of major and minor elements, vitamins, and sucrose, and is jelled with 0.8% agar). The buds proliferated to form an amorphous mass of tissue, called callus (Fig.4A). After six weeks numerous embryos differentiated from the callus (Fig.4B).

 Addition of coconut water from young nuts to the nutrient medium enhanced the formation of adventive embryos. The differentiation of somatic embryos in cultures is not synchronous. Squashes of a differentiated callus show various stages of embryo development. It has not been possible to compare very early stages of embryo development with their parallel stages in the development of zygotic embryo because of the difficulty in recognizing the embryo initials. However, the post-globular stages in somatic embryo development showed normal histogenic differentiation. The somatic embryos in buttercup were attached to the callus by a distinct suspensor and were virtually indistinguishable from the seed embryos.

 They possessed a radicle-plumule axis and 2 (rarely 3

or 4) well developed cotyledons. If left attached to the callus, the embryos germinated in situ to form complete plantlets (Fig.4C). A striking feature of the plantlets thus formed was the

activation of the intact epidermal cells of their

hypocotyl to form embryos.

In a young seedling one could see numerous exposed

embryos hanging from the hypocotyl (Fig. 4C,D).

Fig.4: Somatic embryogenesis in floral bud cultures of Ranunculus sceleratus. A. Callused floral bud. B. Six-week-old culture, showing numerous embryos arising from the callus. C. Five-week-old plantlets formed by the somatic embryos; note numerous embryos at the hypocotyledonary region. D. Portion of the hypocotyl enlarged from C to show the somatic embryos. (after Nataraja, 1968)

PRACTICAL VALUE OF POLYEMBRYONY

Nucellar adventive polyembryony is of great significance in horticulture. The adventive embryos provide uniform seedlings of the parental type, as obtained through vegetative propagation. However, nucellar seedlings of Citrus furnish better clones of orchard stock than cuttings. This is because of the following reasons: (1) The nucellar seedlings have a tap root and, therefore, develop a better root system than do the cuttings. The latter have only a lateral root system. (2) The nucellar seedlings show a restoration of the vigour lost after repeated vegetative propagation. (3) Polyembryony helps in large scale propagation of desired genotype.

(4) Avoids the complications associated with sexual reproduction like pollinators and cross incompatibility.
(5) The nucellar embryos are free from diseases. So far nucellar polyembryony is the only practical approach to raise virus-free clones of polyembryonate Citrus varieties in nature. For monoembryonate cultivars of Citrus there is no in vivo method for raising virus-free clones. However, it can be achieved by culturing their nucellus and inducing somatic embryogenesis (Button, 1977).

 In vitro embryogenesis is looked upon as a practical approach to large scale propagation of selected genotypes. In tissue cultures plant regeneration occurs via organogenesis or somatic embryogenesis. The latter process offers the possibility of using bioreactors to automate large scale production of somatic embryos. Moreover, somatic embryos directly germinate to form plantlets because of the presence of root and shoot primordia. Somatic embryos can also be converted into "artificial seeds" suitable for field planting using mechanical devices similar to those used for normal seeds. Artificial seeds are produced by encapsulating individual somatic embryos with a hydrogel, such as Ca-alginate (Redenbaugh, 1993).

Callus cultures often show spontaneous changes in their genetic material so that the plants regenerated from them exhibit variability which can be exploited in crop improvement programme. Unlike shoot buds, somatic embryos arise from single cells and, therefore, the new strains of plants obtained through embryogenesis would be solid mutants. Very little is known about the molecular biology of embryogenesis. One of the difficulties has been that the zygotic embryo is embedded in the seed and is thus, not easily accessible to experimental manipulations. Somatic embryogenesis provides an excellent opportunity for such studies.

Disadvantages
It constitutes problem in crop improvement because it makes

the identification of hybrids, particularly in crosses involving

taxonomically closely related parents. Loss of vigour of zygote embryo due to suppression by the

nucellar embryos during seed development.

Germination of monoembryonic seed is low due to the competition between the embyos in a single seed. It reduces the variability since nucellar seed will have the same genotype as the parental genotypes.

REFERENCES
Bhojwani, S. S. and Bhatnagar, S. P., 2000, The embryology of Angiosperms. PP: 236-253. Peter, K. V., (ed.) 2011, The science of Horticulture. Vol.(1): 145-163