Micropropagation of superior clones of teak for the Western Ghats of Kerala (Final Report of Project No. KFRI 433/06)

Transcription

1 KFRI Research Report 457 Micropropagation of superior clones of teak for the Western Ghats of Kerala (Final Report of Project No. KFRI 433/06) E.M. Muralidharan Kerala Forest Research Institute Peechi, Thrissur, October 2012

2 Abstract of Project Proposal 1. Title of the Project : Micropropagation of selected teak clones for improvement of planting stock 2. Principal Investigators : E.M.Muralidharan 3. Date of commencement : April Date of completion : March Funding agency : Western Ghats Development Programme, Planning and Economic Affairs, Government of Kerala. 6. Objectives : i. Rapid large scale micropropagation of teak clones selected for faster growth ii. Attempt cost-reduction through innovations in the culture system and equipment. iii. Develop methods for long-term storage of shoot cultures 1

3 Contents Item Pages 1. Abstract : 3 2. Introduction : 4 3. Materials and Methods : 6 4. Results and Discussion : 9 5. References : 13 2

4 ABSTRACT Micropropagation of selected six clones derived from plus trees teak plantations of Kerala was carried out successfully. Shoot cultures were induced to form by culture of shoot tips of epicormic shoots that sprouted from branch cuttings maintained in the mist chamber. Surface sterilized were inoculated on a modified MS medium with various levels of Benzyl aminopurine ( BA) and Kinetin (Kn) in test tubes. The best combination of hormones was found to be 1.5 mg/l BAP and 1.5 mg/l Kin. Multiple shoot formation was obtained in third subculture onwards on the same medium. Shoot cultures were maintained through subculture carried out at 4-6 week intervals. The best media was with 0.5 mg/l BAP and 0.2 mg/l Kin. Ex vitro rooting was successful in microshoots treated with 250 mg/l IBA and transferred to the hardening chamber. Plantlets were transferred to soil after 4 weeks of hardening. 95 % of the plantlets were hardened. Upto 500 plantlets each of the selected clones were produced through the above method. Shoot cultures did not perform well in liquid media even when PU supports were provided or when the cultures were agitated on shaker. The shoots appeared to be vitrified and the leaves deformed when compared to shoots on the solid medium. Shoot cultures were maintained in PP bags modified the size of 10 cm (l) X 5 cm (w) X 10 cm (h) with a heat sealer and in PP food containers of 300 and 500 ml capacity without any difference in multiplication rates and change in quality of shoots. Attempts to maintain teak shoot cultures for long period without subculture did not give promising results. Cultures on minimal growth media could be sustained only for two months whereas with a mineral oil overlay cultures could last only upto 5 weeks. Only shoot cultures maintained in the dark at 4 ºC in the refrigerator could be maintained for upto 6 months and rejuvenated to give normal shoot cultures. 3

5 INTRODUCTION Teak (Tectona grandis L. f ) is the most important of the plantation trees in many of the states of India including Kerala. Most of these plantations have been established using propagules raised from seeds collected from seed stands located in different areas. The mother trees are selected only based on the phenotypic traits and therefore these seeds are genetically untested. It is desirable that genetically superior propagules are used for establishing plantations to maximize the gains from the land area available for such use. At the Kerala Forest Research Institute, Peechi, the technology for vegetative propagation of teak through rooting of stem cuttings was developed and standardized (Surendran and Sharma, 2005). Propagation of selected trees from different locations in Kerala was carried out. Several clones have been identified that showed consistently good growth characteristics when fieldtested in different locations. The growth was exceptionally good in some of the selections. Although vegetative propagation, especially through rooting of stem cuttings, is a simple and cheap method for large-scale clonal propagation, the serious constraint is that the availability of buds from the mother tree is limited in number. Micropropagation or the use of tissue culture techniques for rapid clonal propagation has in recent times been recognized as a very useful tool for mass multiplication of trees. The distinct advantage that micropropagation offers in comparison with conventional vegetative propagation is that much higher rates of multiplication are possible from a few buds and plantlets can be produced all year round. Successful tissue culture of teak has been one of the earliest breakthroughs in this area from India (Gupta, 1980, Mascarenhas et al., 1987, 1993). Since then the method and its modifications have been reported to be used for propagation of teak from several parts of the world. Monteuuis (1994) was also successful in micropropagation of mature teak trees using a modified procedure. Tiwari and Pandey (1995) also reported successful micropropagation from mature trees. Results of field trials of micropropagated teak reveal that early and faster growth and uniformity can be obtained (Gupta et al., 1991). Several commercial facilities now produce micropropagated teak but as shown by Muralidharan and Pandalai (2000) the nursery management of the plantlets needs to be carefully done to ensure the quality of the propagules and good survival in the field. As in any other species, genotypic differences in the response in culture and in multiplication or rooting rates is to be expected and fine tuning of the protocols will be required for the individual clones. Cost of plant production is one of the greatest hurdles in the wider application of tissue culture in forestry. It has been estimated that a 50% reduction in cost of micropropagated plants would increase the market demand by 10 times (Kozai, 1991).The potential for cost reduction through modifications in the equipment and media components is to be explored so as to make micropropagation more acceptable. Some of measures standardised at KFRI for cost reduction in different species of plants (Muralidharan, 1995, 1998) could be tested for their suitability for teak shoot cultures. Besides standardizing the protocol for individual clones, it is necessary that material for initiating cultures be available on a continuing basis since long term multiplication through subculture is 4

6 is not desirable due to problems such a poor rooting response, vitrification and build up of latent microbes. Since Plus trees themselves cannot be expected to be the source of explants due to problems of logistic the setting up of clonal gradens /clonal hedges should be encouraged. Another alternative is to have long term storage of cultures without subculture so that cultures are available at the end of several months of years to be revives and used for multiplication. It is in this background that micropropagation of selected clones of outstanding performance among those already identified at KFRI was undertaken in this study. The objectives of the study was : i. Rapid large scale micropropagation of teak clones selected for faster growth. ii. Attempt cost-reduction through innovations in the culture system and equipment. iii. Develop methods for long-term storage of shoot cultures 5

7 MATERIALS AND METHODS A. Plant material: Shoot tips for initiation of cultures were obtained either from dormant buds collected from branches of plus trees of teak in plantations or from epicormic shoots induced on branch cuttings maintained in soil filled bags placed under mist. The teak clones used for this study were T1, T9, T10, T26, T36, and T47 B. Basal Media: Culture media used in this study was based on the basal medium consisting of the mineral salts and vitamins as described by Murashige and Skoog ( 1962). C. Preparation of media All chemicals used in this study were of analytical grade and double glass distilled water was used for preparation of all media. Stock solutions of the mineral salts and vitamins in distilled water were stored in the refrigerator or freezer respectively. After addition of sucrose, necessary additions of growth regulators and other additives were made from stock solutions stored below 5 o C and the ph adjusted to 5.7 with 1 N NaOH or HC1. For preparation of solid medium, agar (Hi-Media, Mumbai) was added at 0.5% w/v and melted in a microwave oven before dispensing into the culture vessels. Aliquots of 20 ml were dispensed into each test tube. Culture media were autoclaved at 1.5 kg -1 cm -2 at 12l o C for 15 min. Sucrose was added at 2 % w/v and the media solidified wherever required by addition of agar at 0.7 % w/v. ph of the media were adjusted to 5.6 before autoclaving the media for 15 min. Media were dispersed into borosilicate test tubes of the size 150 mm x 25 mm or 100 mm x 25 mm (20 ml media /tube) or 600 ml glass bottles with PP lids ( 30 ml/bottle ) or higher volumes in the larger PP containers. D. Culture conditions: All cultures were maintained in the culture room where a temperature of 25 C + 2 C was maintained through airconditioning The cultures were illuminated with cool daylight fluorescent tubes and the photosynthetically active radiation (PAR] at the level of the cultures was µe m -2 s -l. A photoperiod of 8 hr light and 16 hr darkness was provided to the cultures. Cultures were normally maintained for 4 weeks of growth before subculture onto fresh media. Depending on the growth response, the culture period was sometimes increased upto 8 weeks. Cultures were usually evaluated after three passages on a particular media. 6

8 E. Stages in culture a. Initiation: Shoot tips were surface sterilized according to the methods described earlier by Muralidharan (1997) after treatment to minimize browning due to phenolic exudation and oxidation. Explants were inoculated in test tubes on a solid initiation medium consisting of MS basal media supplemented with BAP (1.5) and Kn ( 1.5 mg/l) and sucrose 2 %. b. Multiplication: Sprouted shoots from shoot tip explants were subcultured on to fresh media and when multiple shoot formation was observed after 2-4 subcultures, shoots were split into clusters of 3-4 shoots for transfer to fresh media. Various cytokinin combinations ( BAP and Kinetin ) were tested to find the best media for shoot multiplication c. Rooting: Shoots of 2-4 cm length with at least 3-4 well formed leaves were excised from shoot clusters and treated with an auxin solution for rooting. The base of the shoot was dipped in IBA ( 250 mg/l) for 1-2 minutes and planted ina 1: 1 mixture of vermiculite and sand in trays and transferred to the mist chamber. d. Hardening: Rooted plantlets after 4 weeks in the mist chamber were transferred to soil in polybags and placed in shade for a week before shifting to the nursery. F. Cost reduction measures a. Use of liquid media with or without supports: A comparison was made between the solid multiplication media and a liquid media of the same composition. Cultures were carried out on liquid media in bottles and conical flasks with and without support in the form of polyurethane (PU) foam blocks. Cultures without supports were maintained either stationary or placed on an orbital shaker set to 60 rpm. b. Use of PP bags/pp food container as culture vesses: Teak multiple shoots were maintained in polypropylene (PP) bags modified with a heat sealer to form a vessel of size. 10 cm (l) X 5 cm (w) X 10 cm (h). The mouth of the bag was folded down and secured in place with paper clips. Clear transparent PP food containers of 300 ml and 500 ml were procured form the supermarket andused as culture vessels. Cultures maintained in glass bottles were the controls for comparison. 7

9 G. Long term storage of cultures a. Minimal growth media A simple culture medium with low levels of sucrose ( 0.5 and 1 % w/v) and mineral salts at half strength of that in MS media was used for subculture of shoots. Cultures were subcultured at intervals of 8 and 12 weeks to test the feasibility of maintaining cultures for long term without subcultures. Control cultures were maintained on the regular multiplication media with subculture at 4-6 week intervals. b. Low temperature storage Shoot cultures which were freshly subcultured to fresh media were allowed to grow for a week and transferred to low temperature ( 4 C in the refrigerator) and dark after sealing the test tubes with cling film to prevent desiccation. Cultures were subcultured after 8 weeks Shoots maintained under growth room conditions were control. c. Mineral Oil overlay: Shoot cultures were subcultured to fresh media and allowed to grow for 2 weeks and mineral oil (autoclaved paraffin oil) overlaid on the shoots. Cultures were observed for ability to survive and revive after various periods of time. 8

10 RESULTS AND DISCUSSION a. Initiation: On the initiation medium consisting of MS basal media supplemented with BAP (0.5 mg/l and and Kinetin (0.2mg/l) and sucrose 2 %, shoot tip explants sprouted to give rise to shoots ( Figure 2 and 3). Severe browning of the media immediately below many of the explants ( Figure 1) occurred within a few hours in spite of the antioxidant treatment and was countered by shifting the position of explants in the same test tube or fresh media if needed. Rinsing of the explants immediately after collection was found to reduce the intensity of phenolic exudation. When that was not possible the plantmeterial was brought to the lab and placed in running tap water for 30 min to an hour before further processing and surface sterilization. b. Multiplication: Sprouted shoots from shoot tip explants were subcultured on to fresh media and were maintained through subcultures. Growth was initially slow for the first two months during which subcultures to fresh media was carried out every three weeks. When multiple shoot formation ( Figure 4) was observed after 2-4 subcultures, the shoots were split into clusters of 3-4 shoots for transfer to fresh media. Single shoots as well as bases of shoots ( Figure 5) were also transferred, but compared to explants consisting of shoot clusters their growth was slower. Figure 1. Browning in shoot tips inoculated on initiation medium Figure 2: Sprouted shoot tip explants Figure 3. Development of shoot from shoot tip explants 9

11 Figure 4. Multiple shoots of teak on multiplication medium Figure 5. Sprouting of axillary buds of in vitro shoots Shoot cultures derived from buds of mature teak were subcultured on the cytokinin series to test the effect of BAP and Kin, singly and in combinations,on shoot multiplication rates. After three passages the best shoot multiplication was obtained on 0.5 mg/l BAP and 0.2 mg/l Kin. Elongation and growth of healthy shoots (Fig. 3) was obtained on lower levels of cytokinins. The best results were obtained on media with both BAP and Kin where the shoots had bigger leaves. The shoot cultures developed a mass of callus at the base during the passage. In some of the clones, size of the callus growth threatened to disrupt the growth of shoots and had to be removed and the shoots transferred to fresh media. c. Rooting: After multiple shoot formation had proceeded for 6-10 subcultures and sufficient shoots were available for each clone, rooting of the microshoots were attempted. Ex vitro rooting was very successful and in most attempts all the shoots rooted within two weeks ( Figure 6) d. Hardening: Rooted plantlets after hardening for 4 weeks in the mist chamber were transferred to soil in polybags ( Figure 7) and placed in shade for a week before shifting to the nursery. Newer leaves of the plantlets increased in size as the plantlets established but for the first few months had not attained the stiff and leathery nature of rooted teak stem cutting and were more like teak seedlings. About 500 plantlets of the selected clones of teak were produced and hardened and transferred to the nursery using the above method. Plantlets were observed to have normal morphology and growth habit in the nursery. 10

12 Figure 6. Microshoot with root induction after two weeks (left) and plantlet after hardening ( right) Figure 7. Hardened plantlets of teak H. Cost reduction measures i. Use of liquid media with or without supports: Teak shoot cultures were not amenable to culture in liquid media. Shoot clusters placed on liquid media with or without PU foam supports showed slower growth and multiplication rates than the cultures maintained in culture bottles on solid media. Shoots showed sign of vitrification and were easily damaged when transfers were made. The use of temporary immersion systems for improved shoot multiplication of shoot cultures in species like pineapple ( Escalona et al. 1999) has been demonstrated and it will be worth testing for teak since savings in cost of solidifying agents and cost of cleaning of vessels will be be an advantage in large scale muliplication ii. Use of PP containers The use of PP bags (Figure 8) as well as food containers for culture of shoots was found to be successful and no difference in the growth rates or in quality of the cultures were observed. Difficulty in handling the PP bag during transfers is a disadvantage since the wall of the bags are flexible and opening and closing of the mouth of the vessels requires some skill to be developed. Increased chances of contamination might be one of the problems that could be encountered in routine use of such containers. The advantages are however in the light penetration afforded by the clear transparent wall and the possibility of accommodating more vessels on culture room shelves with out blocking light availability. Such vessels are inexpensive when compared to the glass bottles. PP food containers also gave satisfactory results when compared to cultures maintained in glass bottles and no differences in quality of cultures or in multiplication rates was observed. Precaution while sterilizing the relatively thin walled vessels was however required to avoid deformation. Stacking of vessels was therefore avoided during sterilization. 11

13 Figure 8. Shoot cultures of Teak maintained in PP bag The use of PP containers and bags on a routine basis is expected to result in substantial savings in cost of production of micropropagation. Proper selection of vessel shape and size including custom manufacturing of the optimum sizes is also feasible. iii. Long term storage of cultures Shoots on the maintenance media developed callus and roots when subculture periods were extended to beyond 5 weeks. The lower leaves also turned senescent and brown gradually and soon there was a mass of dead tissue at the base of the shoot clusters. Shoots maintained on the media free of growth regulators as well as those maintained on sucrose free media showed a slowing of growth but it was difficult to recover such cultures to normal growth rates after maintaining for beyond two months. Cultures overlaid with paraffin oil remained green for more than 5 weeks but subsequently turned senescent. Shoot cultures on the shoot multiplication medium that were maintained in the dark at 4 C in the refrigerator survived for six months and could be recovered to normal growth rates after repeated subcultures. Further experimentation is required to standardise the conditions including pretreatments before storage at low temperature and culture in light throughout the storage period. Acknowledgements The financial support given through a research and Development project under the centrally sponsored Western Ghats Development Programme by the Planning and Economic Affairs (WGC) Department, Government of Kerala is gratefully acknowledged. 12

14 References Escalona, M. Lorenzo, J. C., González B., Daquinta, M., González,,J. L,. Desjardins, Y. C. Borroto, G. (1999) Pineapple (Ananas comosus L. Merr) micropropagation in temporary immersion systems. Plant Cell Reports (1999) 18: Gupta, P.K., Nadgir, A.L., Mascarenhas, A.F. and Jagannathan, V. (1980) Tissue culture of forest trees - Clonal multiplication of Tectona grandis (Teak) by tissue culture. Plant Sci. Lett., 17: Gupta, P.K, Timmis, R. and Mascarenhas, A.F. (1991) Field performance of micropropagated forestry species. In Vitro Cell Dev. Biol. 27P: Kozai, T. (1991) Micropropagation under photoautotrophic conditions. In: Debergh, P.C. and Zimmerman. R.H (eds.) Micropropagation. Technology and Application, Kluwer Monteuuis, O.. Bon, M. and Goh, K.S. (19981 Teak propagation by in vitro culture. Bois Et Forets des Tropiques, 256(2): Mascarenhas, A.F., Kendurkar, S.V., Gupta, P.K., Khuspe, S.S and Agrawal, D.C. (1987) Teak. In: Bonga, J.M. and Durzan, D.J. (eds.), Cell and Tissue Culture in Forestry, Vol:3, Martinus Nijhoff, Dordrecht. pp Mascarenhas, A.F. and E. M. Muralidharan (1993) Clonal Forestry with Tropical Hardwoods. In: Ahuja, M.R. and W.J. Libby (Eds.), Clonal Forestry II: Conservation and Application. Springer Verlag. pp Muralidharan, E.M. (1997) Micropropagation of Teak, Rosewood and Sandal. KFRI Research Report No 119: 20p Muralidharan, E.M. (1995) An evaluation of cost reduction measures in micropropagation. Proc. of Seventh Kerala Science Congress, Palakkad, January 27-29: Muralidharan, E.M. (1998) Development and dissemination of Low cost techniques for micropropagation of Kaempferia galanga ( Kacholam) KFRI Research Report No. 159: 26p Muralidharan,. E.M. and Pandalai, R.C. (2000) Assessment of Field Performance of Micropropagated Teak and Eucalypt, KFRI Research Report No. 182, 2000 Surendran. T and Sharma.J.K (2005) Clonal propagation of plus trees of teak and field performance of ramets: In Proceedings of the International Conference on Quality Timber Products of Teak fromsustainable Forest Management, Peechi, India, 2-5 December 2003 pp Tiwari. S.K. and Pandey. R.K. (1995) A preliminary observation on in vitro propagation of teak (Tectona grandis) through tissue culture from excised apical and seedling explants. J. Trop. For. 11: