0766-B1
Shyamal K. Roy[1]
Natural regeneration of forest trees is poor because of sporadic seed production and because most of the species produce recalcitrant seeds that are intolerant to desiccation, so that their genetic diversity cannot be preserved in conventional seed banks. Concerted efforts must therefore be made to evolve methods for mass multiplication of forest trees, their conservation and cloning of superior genotypes for use in tree improvement programmes.
For clonal mass propagation, through in vitro culture, from shoot tip and nodal explants of mature trees, we have established protocols for some tropical trees e.g. Artocarpus heterophyllus, Azadirachta indica, Dipterocarpus turbinatus, Syzygium cuminii, Gmelina arborea and Elaeocarpus robustus. Small twigs were collected in spring season from coppiced branches of selected trees and protocols have been established for their mass clonal propagation. The techniques consisted of four major steps. 1. Establishment of in vitro cultures from rejuvenated tissues, 2. Induction of multiple shoots from individual explants, 3. Rooting and 4. Acclimatization.
For culture establishment, multiplication and rooting Murashige and Skoog's (MS) nutrient medium was used. Explants of different species showed different responses when cultured on MS medium supplemented with various concentrations and combinations of cytokinin, auxins, casein hydrolysate and coconut milk. Polyvinylpyrrolidone (PVP) was required to overcome the effects of phenolic compounds secreted from the explants. Depending on species, 65-85% cultures produced shoots. For multiplication, newly formed shoots were subcultured to fresh medium designed for each plant species, and 15-20 shoots per subculture were obtained through several subcultures. Well-developed shoots were rooted by implanting them on root induction medium. For acclimatization four to six-week-old regenerants were transplanted into sterilized soil and compost (1:1) and covered with transparent polyethylene sheets misted three times per day. After 3-4 weeks the plantlets were transplanted singly in polyethylene bags containing sand, soil and compost (1:1:1) and kept under indirect sunlight. After 8-10 months they were transplanted in the open field where 70-85% plants survived. The technique is feasible as viable alternative to the continuous supply of planting material for recalcitrant trees, as well as for the conservation of forest genetic resources.
Excessive logging of trees in the forests of southeast Asia has gravely threatened its ecosystem as areas have been cleared of countless plant and animal species. If this situation continues, it will pose a great threat ultimately, to mankind. Natural regeneration of forest trees is poor because of sporadic seed production and most of the species produce recalcitrant seeds that are intolerant to desiccation, so that genetic diversity cannot be preserved in conventional seed banks. Concerted efforts must therefore be made to evolve methods for mass multiplication of forest trees, their conservation and cloning of genotypes for use in tree improvement programmes.
During the last decades, significant progress has been made in the propagation of fruit and forest plants through tissue culture technology (Bajaj, 1986; Bonga and Durzan, 1987; Hanover and Keathly, 1988). Some important tree species, especially hardwood trees for which in vitro propagation has been achieved are Eucalyptus citriodora (Gupta et al., 1981) E. grandis (D. Fossard et. al., 1977; Lakshmi Sita and Vaidyanathan, 1979), Tectona grandis (Gupta et. al., 1980) etc. Litz et al.(1985) and James (1988) demonstrated the application of tissue culture methods for the improvement and mass propagation of fruit trees.
For clonal mass propagation through in vitro culture, from shoot tip and nodal explants of mature trees we have established protocols for Artocarpus heterophyllus, Azadirachta indica, Syzygium cuminii, Dipter acarpus turbinatus, Gmelina arborea and Elaeocarpus robustus. Azadirachta indica A. Juss. (Meliaceae) and Gmelina arborea Roxb. (Verbenaceae) are hardwood species valued for its timber. The species adapted to withstand drought strees and are good potential for afforestation programmes. Artocarpus heterophyllus Lam. (Moraceae) or jackfruit and S. cuminii (Myrtaceae) are large, tropical evergreen fruit trees. The trees are of considerable economic importance for their timber and fruits. Elaeocarpus robustus Roxb. (Elaeocarpaceae) is also an important tree for timber, fuel wood and fruits. Dipteterocarpus turbinatus (Dipterocarpaceae) is a major dipterocarp species of rain forests in the south and south-east Asia. As tissue culture technologies may provide methods for large-scale propagation of tree species (Bajaj, 1986; Dunstan and Thorpe, 1986; Boulay, 1987), this work was undertaken to establish efficient and reproducible method for in vitro mass propagation of these species from mature trees. Regeneration from adult material will be useful, since the pheno-type and desirability of the selected plants are known.
Shoot tips and/or nodal segments of all the six species were collected from copicced branches of 15-20-year-old trees growing in the Jahangirnagar University Campus.
After collection the explant materials were kept in water and brought to the laboratory. They were washed thoroughly under running tap water. Surface sterilization was done with 0.2 per cent HgCl2 for 5 min. followed by washing with sterile double-distilled water changed three to five times.
Explants were cultured on a 0.7 per cent agar-gelled medium with 0.7 per cent aqueous polyvinylpyrrolidone. MS medium (Murashige and Skoog, 1962) with 3 per cent sucrose and different combinations of auxin and cytokinin were used for shoot proliferation and half-strength MS for the induction of root. Different concentrations and combinations of auxins were also used in the case of root induction. Effects of casein hydrolysate (CH) and coconut milk (CM) on shoot multiplication and growth were also determined. The pH of the media was adjusted to 5.8 before autoclaving. The cultures were incubated at 24 ± 1°C under 16 hour photoperiod with a light intensity of 2000 lux under white fluorescent tubes. The cultures were regularly subcultured on fresh media at 4 weeks intervals. All treatments consisted of 15 replicates and the experiment was repeated three times. The plantlets regenerated in vitro were removed from the culture flasks, washed to remove the nutrient agar medium from the roots and transplanted into small polyethylene bags containing sterile soil and compost (3 : 1 v/v), plantlets were subsequently transferred to open place and gradually acclimatized to out door conditions.
Gmelina arborea: Explants showed different responses when cultured on MS medium with various concentrations and combinations of cytokinin and auxin. Among the two cytokinin used, BA (benzyladenine) was found superior for shoot regeneration. Explants responded best for shoot regeneration on MS medium, containing 1.5 mg/1 BA and 0.2 mg/l NAA (naphthaleneacetic acid). About 82 and 79 per cent cultures of shoot tip and nodal explants, respectively regenerated into multiple shoots. For subculture, individual shoots grown on MS containing 1.5 mg/l BA+0.2 mg/l NAA were excised and reinoculated into fresh medium with the same combination of hormones. In each subculture, axillary and adventitious shoots grew from each shoot and lateral branches. A clump of 10-15 shoots was produced after three to four subcultures. For further multiplication the regenerated microshoots were excised several times and cultured on the same medium. But the growth of the regenerated shoots on these media was not enough. For inducing axial growth in regenerated shoots the concentration of BA and NAA were lowered and CH (50-150 mg/l) and CM (5-20 percent v/v) were added to the medium. The best combination of the medium for axial growth was found to be MS containing 0.5 mg/l BA+0.05 mg/l NAA + 100 mg/l CH + 10 per cent (v/v) CM, in which the length of shoots was 4-7.5 cm.
For rooting, the shoots were excised and implanted on half strength MS medium. Shout roots with laterals emerged in 100 per cent shoots when the medium contained 2.0 mg/l each of IBA (indole-3-butyric acid) and NAA.
Artocarpus heterophyllus: The explants were found to become swollen; they produced three to four shoots within 3-4 weeks after inoculation on MS medium containing BA alone but the percentage of proliferated cultures was only 12-23. BA-NAA combinations were found to be comparatively better for proliferation of shoot buds and the optimal doses of BA and NAA were found to be 2.5 mg/l and 0.5 mg/l, respectively; in these the culture percentage of induced shoot buds was 92. The number of shoot buds per culture was 12. These shoot buds were not found to grow axially, so the medium was further amended. When 15 per cent (v/v) coconut milk (CM) was added to the medium with 2.5 mg/l BA, in 100 per cent cultures shoot buds were induced; the number of shoot buds per culture was 20 and their axial growth was satisfactory. The medium selected was MS+2.5 mg/l BA + 15 per cent (v/v) CM in which shoots continued to proliferate through 36 subcultures with an average of 20 shoots per transfer.
For rooting, well developed shoots were excised form the culture flask and implanted individually on root induction medium containing half-strength MS salts with different concentrations and combinations of IBA and NAA. One mg/l each of IBA and NAA was found to be the best combination of auxins for proper rooting in which 85 per cent shoots rooted.
Elaeocarpus robustus: The explants were cultured directly on MS medium with 0.5 mg/l BA + 0.5 mg/l Kn (kinetin) in which multiple shoot buds were formed. The rate of shoot multiplication was enhanced at the time of subculturing. Incorporation of 10 per cent CM and 100 mg/l CH into the medium resulted in satisfactory shoot growth and development. These shoots continued to proliferate through 10 or more subcultures with an average of 20 shoots per transfer.
Rooting was achieved at 100 per cent when the excised shoots were cultured individually on root induction medium consisting of half-strength MS salts with 1.0 mg/l IBA and 0.5 mg/l IAA (indole-3-acetic acid).
Azadirachta indica: The explants responded positively to direct regeneration when cultured in MS medium suplemented with BA and Kinetin alone or in combination with NAA and IBA in different concentrations. Best shooting response was observed in MS + 1 mg/l BA + 0.1 mg/l NAA medium. Eighty five percent of the cultures were found to regenerate four to five shoots in this medium. The percentage of cultures regenerating shoots in BA and Kinetin alone was 57 per cent followed by 48 per cent cultured in media MS + 1.5 mg/l BA and MS + 2.0 mg/l Kinetin, respectively. Among the Kinetin - NAA combinations the best shoot regeneration was 68 per cent cultured in MS + 2.5 mg/l Kinetin + 0.2 mg/l NAA. So, the primary experiment determined the suitable medium for shoot multiplication, which was MS + 1.0 mg/l BA + 0.1 mg/l NAA. Number of shoots per culture increased with the subculture. The medium further amended with addition of CM (95-10% v/v) and CH (50-200 mg/l). Addition of 10 per cent CM and 150 mg/l CH with MS + 1.0 mg/l BA + 0.1 mg/l NAA was competent to regenerate multiple shoots (25) and their average height was 6 + 1.56 cm.
For rooting, the shoots were excised and implanted individually in rooting medium. Within three weeks of transfer, 100 percent rooting was achieved on a root induction medium consisting of half-strength MS salts supplemented with 1.0 mg/l each of IBA and IAA.
Dipterocarpus turbinatus: The cotyledonary node of a germinated seed was found to be the best explant for shoot multiplication of D. tubinatus. The explants become swollen and produced 4-5 shoots within 3-4 weeks after inoculation in half strength MS medium supplemented with 2.25 mg/l BA. The number of shoots increased with the number of subculture and after 5 subcultures number of shoots were found to be 10-15 per culture. For further development of the medium CH (50-150 mg/l) and CM (5-20%) were added individually and found that 10% CM was effective for satisfactory growth. Thus the medium was ½ MS + 2.25 mg/l BA + 10% CM for shoot multiplication (18 per culture) and growth.
Rooting was achieved on half strength MS medium in combination with 1.0 mg/l each of IBA (Indole-3-butyric acid) and NAA. Stout roots with laterals emerged in 90% of regenerated shoots within 4 weeks of culture.
Syzygium cuminii: Full strength MS medium was found suitable for shoot regeneration in the shoot tip and nodal explants of S. cuminii. In MS + 2.5 mg/l Kn medium shoots were induced in 65-85% of the culture. At the time of subculturing newly formed shoots were separated and implanted onto fresh medium with the necessary growth regulators and thus shoots continued to proliferate through several subculture with an average of 10-15 shoots per transfer. For further development of the medium, urea (50-200 mg/l), CH (50-200 mg/l) and CM (15-20%) were added either individually or in combination. Addition of 100 mg/l urea enhanced the number of shoots up to 60, where as incorporation of CH (100 mg/l) and CM (15%) to the medium increased the length of shoots.
Rooting was achieved when well developed shoots were excised form the culture flask and implanted individually on half strength MS basal medium with 0.5 mg/l each of IBA, IAA and IAA.
The plantlets regenerated in vitro were removed from the culture flasks wahed thoroughly of the nutrient medium and transplanted into small pots containing sterile soil and compost. The plantlets were reared under semicontrolled temperature (25-32°C) and light (2000 lux) in a chamber. After 18-20 days, plants were transferred to an open room and gradually acclimatized to outdoor conditions. About 20 per cent mortality occurred following transplantation of the plantlets.
Mature trees demonstrating superior genotypes are ideal for mass propagation. However, it is usually more difficult to establish shoot cultures from mature trees than from juvenile (Bonga, 1987; Hackett, 1987). The results of the present study show that shoot tip explants from mature trees of G. arborea, A. indica, A. heterophyllus, S. cuminii and E. robustus could be induced to produce multiple shoots in vitro, with the adjustment of hormonal concentration. The results reported here are in agreement with those on Tectona grandis (Gupta et al., 1980). Albizia lebbek (Gharyal and Maheshwari, 1982). Mitrogya parvifolia (Roy et al.,1988) and other trees. According to Bonga (1982), multiplication of shoot bud-explants of both tropical and temperate regions is easier if explants are derived from germinated seedlings. But multiplication of trees through seed is not a satisfactory means of conserving the characteristics of a desired clone (Das and Mitra, 1990) because of strong heterozygosity of trees. To avoid such a situation explants were collected from new shoots of copicced area of mature plants. Collection and inoculation of explants were tried round the year and the period from June to August was found to be the best season for excision for enhanced multiplication.
Among the six species the culture of A. heterophyllus and A. indica were established in 1990 and rootable shoots are still being regenerated from this 10-year-old culture. This result reveals that shoot tips of mature selected trees could be used as an alternative source of seeds for the genetic conservation and propagation of tropical forest trees.
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| [1] Department of Botany, Jahangirnagar
University, Savar, Dhaka, Bangladesh. Tel: 880-2-7708478 Ext. 436; Fax:
880-2-7708383; Email: [email protected] |