TITLE: SOMATIC EMBRYOGENESIS AND PLANT REGENERATION OF

TECTONA GRANDIS LINN (TEAK)

1. ABSTRACT

1.1 Abstract

The main objective of the research is to study the production of Teak (Tectona
Grandis L.) from zygotic embryo. Teak is highly demanded because of its high
quality compared to other timbers. Zygotic embryos are being used to increase the
propagation of the teak. To regenerate teak, direct somatic embryogenesis is being
used. Direct somatic embryogenesis is a method an asexual form of plant propagation
in nature that mimics many of the events of sexual reproduction. This process may be
reproduced artificially by the manipulation of tissues and cells in vitro. The expected
result is the teak will regenerate from the culturing of zygotes in specific medium.
Thus, it can reduce the breeding cycles and may harvest quickly. Direct somatic
embryogenesis may also used to culture other species such as Kacip fatimah (Labisa
pumila) and other herbs for commercial purposes.

2. LITERATURE REVIEW

2.1 Zygotic Embryo

Early attempts to develop somatic embryogenesis for sandalwood (one of

wood plant) propagation focussed on indirect embryogenesis using hypocotyl, nodal
and endosperm explants. However, when somatic embryos were obtained through
callus, the conversion or germination of these embryos into plantlets was problematic
(Rai & McComb, 2002). Despite the use of hypocotyl, Rain and McComb (2002)
reported successful induction of somatic embryogenesis from mature zygotic embryos
of sandalwood without involving a callus phase and a large number of plants
regenerated in this way from the somatic embryos have been established in the soil in
a greenhouse.

In addition, it is also reported that the explants of zygotic embryos of a timber

yielding leguminous tree Hardwickia binata has been reported by the work of Chand
 and Singh in 2001, for high frequency somatic embryogenesis and subsequent plant
conversion (Giri et al., 2004).

2.2 Direct Somatic Embryogenesis

Direct somatic embryogenesis is preferable due to the somatic embryo

obtained through callus which is known as indirect SE has a problem at the
conversion or germination of these embryos into plantlets (Rai & McComb, 2002).
Somatic embryogenesis has many potential advantages for mass propagation and
genetic improvement of hardwood forest trees (Merkle, 1995). The method offers fast
multiplication of high value clones for reforestation and provides a means of gene
transfer and the production of new plants from transformed cells for the mass
production of transgenic trees (Rai & McComb, 2002).

Although, potential application of somatic embryos for a large scale

production of plants via direct somatic embryogenesis in teak is still under studies that
need to be focused to obtain a high rate of somatic embryo induction, maturation and
germination. At the present, the result of SE already reaches small scale production of
a few hundred plants. Further experimentation will be required to enhance
embryogenic frequency in cell cultures and to use such cultures for scale up studies.
In addition, the other benefits offer by somatic embryogenic system can be in
applying a number of genetic manipulation (Giri et al., 2004).

2.3 Media Composition

The formation of embryogenic tissues from somatic cells can be stimulated by

altering either the levels of exogenously applied auxins or the ratio of auxins to
cytokinins (Murthy, Murch, & Saxena, 1998)

The induction of somatic embryogenesis has been experimented in different

media such as MS, B5, WPM, mWPM, AE (Arnold and Erikksson 1981), LM (Litvay
et al. 1985), and MCM (Bornman and Jansson 1981). As well as different plant
growth regulators, such as 2,4-D (2, 4-dichloro phenoxyacetic acid) NAA, IAA, IBA,
IPA (indole propanoic acid) BA, KN, TDZ, 2iP, Zeatin, CPPU, TIBA (tri-
iodobenzoic acid) GA3, and ABA (abscisic acid) were used for callus induction,
 somatic embryo formation, shoot organogenesis and for the conversion of somatic
embryos into plantlets. In some cases somatic embryo formation occurred directly on
the callus induction medium whereas in some cases different media and different
hormones were needed for callus induction, somatic embryo formation, shoot
organogenesis and plant conversion. Immature zygotic embryos were cultured on MS
medium supplemented with B5 vitamins and 2, 4-D produced embryogenic cultures.
Somatic embryos were also produced on MS medium containing NAA, KN, and
glutamine in Litchi (Murthy et al., 1998).

Generally, auxins are included in cell culture media for the induction of cell
proliferation and callus growth. Synthetic auxins [naphthaleneacetic acid (NAA) and
others] and growth regulators with auxin-like activity [2,4-dichlorophenoxyacetic acid
(2,4-D)] have been extensively used for this purpose (Murthy et al., 1998).

3. INTRODUCTION

3.1 Project Background

Tectona grandis Linn which is also known as Teak is a family of Lamiaceae

and native to the tropical forests of Southeast Asia in which grows well in a fairly
moist warm and tropical climate. Under favorable conditions T. grandis can grow into
a large tree with clean cylindrical bole and height up to 30 meters with 1.5 meter
diameter. Its bark is grayish pale. The strong, durable timber from teak has fine
quality and is valued for furniture and construction which is also recognized as high
commercial commodity (Merkle & Nairn, 2005).

In addition, its leathery elliptic or ovate leaves have rough upper surface and
the lower surface is covered with veins and vein lets. Its leaves can measure up to 60
cm long and 30 cm wide. It has tiny white flowers, which are found in clusters in
about a meter long stalk at the terminal end of the branches. The fruit of the tree is
subglobos and four-lobed with dense stellate hairs (Pradhan, 2004). The seeds can be
found in the fruit which each fruit contains one to four of seeds from which the teak is
naturally regenerated. However, seed germination is often difficult due to various
physiological, physical and morphological barriers. Alternatively teak has been
 propagated via cuttings as well as budding and grafting but these methods have severe
limitations, including rooting and incompatibility problems, and only provide a few
propagules from selected individuals (Lim, 2008).

The loss of the world’s tropical forests has been accelerating at an alarming
rate. While much of this deforestation is due to conversion of rain forest to farm and
pasture land, the harvesting of tropical hardwoods for forest products, most strikingly
for fuelwood, also contributes substantially to these losses. For example, according to
recent statistics from the Food and Agriculture Organization (FAO) of the United
Nations, over 545 million m3 of wood is consumed for fuel annually in Africa, which
is over six times the amount of wood consumed for all other purposes (roundwood,
sawnwood, pulp, etc.) combined (Merkle & Nairn, 2005).

In conjunction, due to its vast economic value, several commercial

organizations have launched massive teak plantation schemes throughout India.
Natural regeneration is mainly through seeds and is widely employed in several
nurseries. Other methods like grafting, rooted stumps and buding are also practiced
for propagation (Rao, Suprassana, Ganapathi & Bapat, 2000). Whilst, the
conventional breeding is rather slow and less productive and cannot be used
efficiently for the genetic improvement of trees (Giri, Shyamkumar & Anjaneyulu,
2004). Other attempts are required to acquire massive, fast regeneration of this tree
with large quantities of good planting stock.

3.2 Objectives

3.2.1 To study the regeneration of teak from zygotic embryo

3.2.2 To generate production of teak by direct somatic embryogenesis

3.2.3 To produce teak plantlets in large scale

3.2.4 To determine suitable plant growth regulator as well as medium formulation

on regeneration of somatic embryo
 3.3 Research Question

Does production of Teak increase by the use of direct somatic embryogenesis?

3.4 Hypothesis

The regeneration of teak plantlets might be increased by the use of direct somatic
embryogenesis.

4. METHOD

4.1 Materials

4.1.1 Immature teak seeds

4.1.2 Somatic embryogenesis induction media

4.1.3 Somatic embryo proliferation media

4.1.4 Hardening media

4.2 Methods

4.2.1 Sample preparation

Immature seeds will be obtained from Forest Research Institute Malaysia

(FRIM) Kepong. The seeds will be sterilized using standard sterilization
method. Zygotic embryos can be obtained by dissecting the seeds of Tectona
grandis.

4.2.2 Induction and proliferation of somatic embryos

The zygotic embryos will be pre-cultured on MS medium with high

concentration of 2,4-D for four weeks. After that, the zygotic embryos will be
cultured on MS medium with varied low concentration of 2,4-D and high
concentration of sucrose. The somatic embryo obtained will be isolated and
proliferated.
 4.2.3 Maturation and germination of somatic embryos

Isolated embryo will then be cultured on MS medium with 0.5-1.0 mg/l BAP.
The embryo will develop into plantlets.

4.2.4 Hardening Media

The plantlets are transferred to sand for hardening process. This process is
very important so as to prepare the plantlets before it is transfer to the natural
environment.

4.2.5 Other factors

Other factors such as the physical factors that should be taken into
considerations include the nitrogen sources, light intensity and other
constituents such as amount of dissolved oxygen and many more.

4.3 Expected Result

4.3.1 Embryoids/ Embryo-like structure

4.3.2 Regeneration of plantlets

4.3.3 Mass production of Tectona grandis plantlets

4.3.4 Cryopreservation of somatic embryos

5. CONCLUSION

The ever-increasing demand for teak timber has resulted in large-scale plantations.
Nevertheless teak growers often faced difficulties in obtaining sufficient planting
materials. Thus, many studies and approaches have been taken to accommodate the high
demand for these species. Hence, this study is very necessary as an integral component of
tree improvement and may indeed be useful to overcome the handicaps associated with
teak production.
7. MILESTONES AND GANTT CHART

7.1 Milestones and Dates

1. Obtain seed for the sample preparation (November 2010)

2. Regenerate the teak from the zygotic embryo. ( February 2011)
3. Maturation and germination of somatic embryos for large scale. (March 2011)
4. Transfer of plantlets into hardening medium. (April 2011)
5. Transfer of plantlets to the plantation. (May 2011)

7.2 Gantt Chart

7. REFERENCES

Giri, C. C., Shyamkumar, B. & Anjaneyulu C. (2004). Progress in tissue culture, genetic
transformation and applications of biotechnology to trees: an overview. DOI:
10.1007/s00468-003-0287-6

Merkle, S. A., Nairn, C. J. Hardwood tree biotechnology. DOI: 10. 1079/IVP2005687

Murthy, B. N. S., Murch, S. J. & Saxena, P. K. (1998). Review Thidiazuron: A Potent

Regulator Of In Vitro Plant Morphogenesis. In VitroC ell. Dev. Biol.--Plant3. DOI
: 10.1007/BF02822732

Lim, Sooi Ping. (2008). Somatic Embryogenesis And Organogenesis In Teak (Tectona

Grandis L.). Masters thesis, Universiti Putra Malaysia.  From http://
http://psasir.upm.edu.my/5519/1/FP_2008_5.pdf

Pradhan, H. K. (2004). Gorkha And Other Ethnic Herbal Medicines (E-Book). Florida:
Universal Publishers.

Rai, V. R. & McComb, J. (2002). Direct somatic embryogenesis from mature embryos of
sandalwood. Plant Cell, Tissue and Organ Culture. DOI:
10.1023/A:1015037920529

Rao, P.S., Suprassana, P., Ganapathi, T. R. & Bapat, V. A. (2000). Status of Somatic
Embryogenesis in Indian Forest Tree. Somatic Embryogenesis in Woody Plants,
Volume 6. Netherlands: Kluwer Academic Publishers