Studies on in vitro propagation of Himalayan cedar (Cedrus deodara) using zygotic embryos and stem segments

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1 Indian Journal of Biotechnology Vol. 3, April 2004, pp Studies on in vitro propagation of Himalayan cedar (Cedrus deodara) using zygotic embryos and stem segments Sushma Tamta* and L M S Palni G B Pant Institute of Himalayan Environment and Development, Kosi-Katarmal, Almora , India Adventitious bud formation was examined in zygotic embryos (decoated seeds) of Cedrus deodara, planted in the normal position, using four different media variously supplemented with or without plant growth regulators (PGRs). Following incubation for 7, 14, 21 and 28 days on PGR containing media, the embryos were transferred to respective PGRfree basal media. While embryo swelling was observed in all cases, normal germination was observed only in embryos cultured initially in PGR-free media. Best response, in terms of bud formation (93%), was observed in embryos first cultured on LP medium containing BA and Kinetin (1.0 μm each) for 28 days and then transferred to LP medium without any PGR. While the mean number of shoots formed per cultured embryo was also highest (34%) in this combination, these shoots eventually dried when excised and sub-cultured for further multiplication. Success was, however, obtained when the embryos were initially planted in an inverted position on LP medium containing 20.0 μm BA for 10 days, and then shifted, again in the inverted position, on the same medium without BA. Although the mean number of shoots formed was low (only 2.5 shoots per embryo), these could be multiplied and elongated. The microshoots ( cm) thus obtained could be rooted (100%) using ½ strength, PGR-free, LP medium, hardened and established in pots. Besides excised embryos, stem segments were also used as explants. While only a quarter of explants could be established in culture, in contamination-free condition, good sprouting response (3-4 buds per segment) was obtained in certain PGR combinations. The sprouted buds developed into shoots on LP medium containing 5.0 μm BA; further subculturing of excised shoots for multiplication, however, resulted in drying up of shoots. Further work is required to overcome this difficulty. Keywords: Cedrus deodara, conifer, deodar, micropropagation, inverted embryo technique, stem segments, rooting IPC Code: Int Cl. 7 A 01 H 4/00, 7/00 Introduction Himalayan cedar [Cedrus deodara (Roxb. ex Lamb.) G. Don; deodar], an important conifer found between 1,200 and 3,030 m, is a prominent member of the Himalayan moist temperate forests 1. Cedrus is the main source of commercial timber in the Indian subcontinent. Although managed under Regular Shelter Wood system with fixed periodic blocks in the past plan, the forests are understocked, almost evenaged and deficient in regeneration 2. Deodar is propagated by direct sowing of seeds ; however, seed production is irregular and seeds are in short supply. While plantations have been successfully raised from cuttings in South Africa, the method has not been practiced in India 3. Although some success has been reported recently 4, this needs to be standardized for large-scale multiplication. Tissue culture techniques can also be applied for propagation of deodar. Tissue culture of conifers has concentrated *Author for correspondence: Fax: , sushma_tamta@yahoo.com on multiplication via embryonic tissues. This process has utility in multiplying small amount of genetically improved material. The regeneration of adventitious buds, shoots and roots has been reported in a few gymnosperms e.g., C. deodara and Pinus roxburghii 5 ; P. roxburghii and P. wallichiana 6 ; P. caribaea and P. kesiya 7 ; P. caribaea 8 ; P.gerardiana 9 ; P. elliottii 10 and Taxus brevifolia 11,12. Somatic embryogenesis has also been achieved in P. radiata 13 ; P. patula 14 ; P. sylvestris 15 and P. monticola 16. P. maximartinezii and P. pinceana, the two endemic pines from Mexico, were successfully regenerated by in vitro organogenesis, starting from mature embryos 17. In P. banksiana (Jack pine), little or no shoot elongation was observed in cultured embryos 18 and limited success was reported in C. deodara 19. Materials and Methods Seed Collection Mature seeds of C. deodara were collected from a forest in Jageshwar, Almora ( N and E, m; about 45 km from the Institute) during the month of October and brought to the laboratory. After washing with running tap water,

2 210 INDIAN J BIOTECHNOL, APRIL 2004 seeds were air-dried under shade and stored in a refrigerator at 4 C. Media Preparation In most experiments, LP medium 20 was used; various plant growth regulators (PGRs; Table 1) and sucrose (3.0%, w/v) were added to the basal medium. The other media used were MS 21, SH 22 and WP 23. In all cases ph of the medium was adjusted to 5.8 before autoclaving and the medium was gelled either with phytagel (0.25%, w/v; Sigma Chemical Co., St. Louis, USA) or agar (0.8%, w/v; Himedia Chemicals, Mumbai). 100 ml of medium was dispensed in 250 ml Erlenmeyer flasks and the flasks were plugged with non-absorbent cotton. The medium was sterilized by autoclaving at 1.05 kg/cm 2, C for 20 min. Various PGRs used in this study were from Sigma Chemical Co., St. Louis, USA. Surface Disinfection of Seeds and Culture Establishment The seeds were wetted in 100 ml of detergent solution (labolene, 0.1%, v/v; 10 min), and then washed under running tap water for 5 min. Subsequently, the seeds were rinsed in distilled water (x4), sequentially treated in solutions containing a systemic fungicide (Bavistin, 0.2%, w/v; 30 min; from BASF India Ltd, Mumbai), an antioxidant (ascorbic acid, 0.02%, w/v; 30 min), and finally surface disinfested with an aqueous solution of mercuric chloride (0.05%, w/v; 10 min); each treatment was followed by repeated washings (x4) with sterile distilled water under aseptic conditions. After surface disinfection the embryos were gently taken out from the endospermic mass and inoculated on various media. The flasks were kept in the culture room at 25 ±1 C under 16 hrs light and 8 hrs dark cycle, with irradiance (42.0 μ mol m -2 s -1 ; inside the flasks) by cool fluorescent tubes (Philips; 40 W). Shoot Multiplication via Short Duration Exposure of Embryos to PGRs Excised embryos, used as explants, were cultured on MS, LP, SH and WP media either as such or supplemented with different concentrations of PGRs, i.e., 2,4-D (10.0 and 20.0 µm ), IBA +BA (5.0 µm each), BA (5.0 µm ), BA+2,4-D ( µm), BA+Kin (Kinetin; 1.0 µm each). These were cultured for 7, 14, 21 and 28 days and then transferred to respective PGR-free meda. A minimum of 15 embryos were incubated for each treatment (5 embryos per flask). Subculturing was carried out at 2-3 weeks interval. Shoot Multiplication via Inverted Embryo Technique The disinfected seeds, after removal of the testa and operculum, were inoculated on water agar (0.8%, w/v) medium for 3 days for germination. After germination, the embryos were excised from the decoated seeds. Excised embryos were carefully implanted in an inverted orientation, with the cotyledon portion in LP medium, supplemented with BA (10.0 or 20.0 µm). After 10 days the BA treated embryos were reimplanted in the same inverted position in LP medium containing activated charcoal (1.0%, w/v) and incubated for 3 weeks. They were then implanted in the normal orientation, in ½ strength LP medium without PGRs. After a week, individual cotyledons and hypocotyl were dissected and again cultured on ½ strength LP medium. 3 weeks after this, the cotyledons bearing shoot buds were dissected into pieces. The pieces as well as the hypocotyl portion containing the main shoot were further incubated in ½ strength LP medium for another 3 weeks. Shoot buds were allowed to elongate in ½ strength LP medium to an average height of cm by subculturing onto fresh medium every four weeks. In vitro Rooting For rooting, the microshoots were implanted in (i) ½ strength LP medium supplemented with or without different concentrations of IBA (5.0, 10.0, 15.0 and 20.0 µm) or NAA (5.0, 10.0, 15.0 and 20.0 µm)- Experiment I, or (ii) in LP medium supplemented with IBA (25.0, 50.0, 75.0 and µm) for 24 and 48 hrs only and then transferred to LP basal medium- Experiment II. Sprouting of Buds from Stem Segments and Shoot Multiplication Stem segments ( cm long), taken from fouryear-old seed raised plants, were used as explants. They were cultured on LP medium after surface disinfection as mentioned earlier. After one week, uncontaminated explants were transferred to the multiplication medium (LP medium supplemented with µm BA) and further incubated for 3 weeks. The sprouted buds were then excised from the stem explants and again cultured on LP medium supplemented with 5.0 µm BA for multiplication. Results Shoot Multiplication via Short Duration Exposure of Embryos to PGRs Excised embryos were cultured on MS, LP, SH and WP media supplemented with or without different

3 TAMTA & PALNI: IN VITRO PROPAGATION OF CEDRUS DEODARA 211 concentrations of PGRs for 7, 14, 21 and 28 days for induction of adventitious buds. White embryos swelled and became green within 3-6 days. Embryos grown on PGR-free media developed into normal seedlings whereas those grown in PGR supplemented media also swelled, but their root growth was found to be inhibited (Fig. 1A). Shoot bud induction was not observed in any of the treatments involving SH medium. However, small needles with nodular structures developed in MS and WP media, without further development. Most of the embryos did not show any growth when taken out from the induction medium (containing PGRs) after 7, 14 and 21 days and transferred to respective medium without any PGRs. Some response was, however, obtained from the embryos which were transferred to PGR-free medium (LP) after 28 days exposure to different PGR treatments (Table 1). The morphological change that accompained shoot development could be noticed after one week with the appearance of nodular structures on the surface of the explant, most of which were concentrated on the cotyledons. They developed into adventitious buds and needle rosettes (Fig. 1B); the adventitious buds elongated into shoots. All the treatments were capable of inducing adventitious buds on embryos; adventitious bud formation was found to be highest (93.33%) in those embryos; following transfer to the PGR-free medium (LP), which were earlier exposed to BA and Kin (1.0 µm each) for 28 days, followed by embryos earlier exposed to media containing BA+IBA (73.33%) and BA (66.67%) (Table 1). The buds further proliferated following subculture on the LP medium for a period of further one month. Mean number of shoots formed per embryo was also highest (33.75 ±1.05) in BA and Kin (1.0 µm each) treatment. After a month of subculture, shoots ( cm height) were separated from the original explant and again transferred to LP medium supplemented with BA (2.3 µm) for further proliferation and elongation. During the period of proliferation and elongation, shoots dried after 4 weeks in most cases. Only a few shoots (around 20.0%) survived beyond this period, and were subjected to rooting experiment. However, no success was obtained and the microshoots finally dried. Shoot Multiplication via Inverted Embryo Technique While the number of shoots formed was high, in some treatments, when the excised embryos were placed in the normal position in the medium (Table 1); the shoots formed, by and large, failed to elongate and survive beyond a certain period. Therefore, to avoid this problem embryos were placed in an inverted position. In the shoot induction medium supplemented with BA (10.0, 20.0 µm), inverted embryos turned dark green in 2-3 days. The cotyledon and hypocotyl portions were found to be swollen by the 7 th day, but shoot buds were not visible. Fig. 1C shows the germination of embryos in an inverted position. When the BA treated (for 10 days) embryos were transferred to LP medium containing 1.0% activated charcoal, the cotyledons and hypocotyl portion elongated. These embryos were then (after 3 weeks) implanted in the normal position in ½ strength LP basal medium, and shoot buds appeared at the cotyledon tips after two months (Fig. 1D). Inverted embryos incubated in LP medium supplemented with 10.0 µm BA did not develop shoots at all. Embryos treated with 20.0 µm BA developed shoots at the cotyledon tips only (1-3 shoots per embryo; Table 1). After 3 weeks the shoots were subcultured again in ½ stergth LP medium (Fig.1E-F). The new shoots were dissected and subcultured for further shoot multiplication in the same medium, i.e., on ½ strength LP medium. Although the mean no. of shoots formed per embryo was low (only 2.5 shoots per embryo, Table 1), these microshoots could be successfully rooted when transferred to the rooting medium. In vitro Rooting The microshoots ( cm height) when transferred to the rooting medium, became yellow and died after 2 weeks in the experiment I, except those which were in ½ strength LP medium without any PGR. After one month roots were found to emerge from the basal portion and the rooting was cent percent in microshoots kept on PGR-free ½ strength LP medium (Fig. 1G-H). These rooted shoots were transferred to small plastic cups (6.1 cm diam; 8.5 cm height) containing non-sterile soil, sand and FYM (equal parts) for hardening in a mist chamber (25 C, 80% RH; Fig. 1I). In the experiment II the shoots were found to develop basal callus, but no roots and gradually died. Sprouting of Buds from Stem Segments and Shoot Multiplication Twenty-five per cent of stem explants were found to be free from contamination when cultured. Heavy contamination, mostly of bacterial origin, was frequently observed. Though different concentrations of two surface disinfectants, namely mercuric chloride

4 212 INDIAN J BIOTECHNOL, APRIL 2004 Fig. 1 In vitro propagation of Cedrus deodara: A, Swollen embryo (normal orientation) with inhibited root growth, one week after incubation in LP medium containing BA and Kin (1.0 μm each); B, Multiple, elongated shoots developing from an embryo, 3 weeks following transfer to PGR-free LP medium after initial exposure to BA and Kin (1.0 μm each) containing LP medium for 28 days; C, Germinating embryos planted in an inverted position in LP medium containing BA (20.0 μm; tubes 1-3; tube1: one day after transfer; tube 2-3: 6 and 10 days after transfer, respectively) or activated charcoal (1.0%; tube 4; initial culture on 20.0 μm BA containing medium for 10 days); D, New shoots developing from the cotyledon tip following transfer of embryos in the normal position, after initial exposure to BA (20.0 μm; 10 days) and activated charcoal containing medium (3 weeks) in an inverted position; E-F, Excised shoots developing from the cotyledon tip, as in D above, obtained using the inverted embryo technique being elongated in ½ strength LP medium without PGRs; G, Rooting of a microshoot following elongation, as in F above, on ½ strength PGR-free LP medium (viewed from the bottom of the flask); H, A well rooted microshoot ready for transplanting; I, Micropropagated plants of deodar after hardening in the mist chamber (25 C, 80% RH) for 10 weeks; J, Sprouted buds on a stem segment cultured on shoot multiplication medium (LP medium supplemented with 5.0 μm BA); K, Elongating shoots developed from excised buds, as in J above, on LP medium containing 5.0 μm BA; & L, Drying up of shoots following prolonged culture on shoot multiplication medium.

5 TAMTA & PALNI: IN VITRO PROPAGATION OF CEDRUS DEODARA 213 ( %, w/v; 10 min) and sodium hypochlorite ( %, w/v; 10 min) were also tried to minimize contamination, these were found to be not very effective. Axillary buds on the contamination-free stem explants sprouted within days of culture on the shoot multiplication medium (Fig. 1J). After 3 weeks, the sprouted buds of cm length were excised and cultured on the same medium supplemented with 5.0 µm BA. Further, shoot multiplication was observed after 15 days of inoculation. Best shoot multiplication was found when the LP medium was supplemented with 5.0 µm BA. The number of shoots formed per stem segment was 3-4 in this treatment (Fig. 1K). No multiplication was observed with a higher concentration of BA (10.0 µm). With lower concentration of BA, i.e., 0.5 and 2.0 µm, the sprouted buds could not survive and dried after a month. After one month, multiple shoots were found to sprout and these were again excised and cultured on BA (5.0 µm) containing LP medium for further shoot multiplication. However, they did not survive and gradually became brown and died (Fig. 1L). Discussion The nodular structures present on the surface of the explants (decoated embryos) of C. deodara were similar to the developing buds described previously 24. Control treatment (medium without PGRs) did not show any bud formation and the embryos inoculated in this treatment simply developed into normal seedlings. Cytokinins are essential for the induction of adventitious bud primordia in conifers. The duration of exposure to different PGRs also showed a significant effect on the frequency of embryos producing adventitious buds. In the present study, embryos exposed to different PGRs for 7, 14 and 21 days did not show any growth except for a few (1 or 2) adventitious buds which later developed into shoots. If the embryos were maintained on PGR containing medium for a long time, they were overgrown by callus. Exposure to PGR containing medium for 28 days, before transfer to PGR-free LP medium was estimated to be most effective for optimal bud production. The duration of exposure to different PGRs showed a significant effect on the frequency of embryos producing adventitious buds. The per cent embryos forming adventitious buds was maximum in BA and Kin (treatment no. 7, Table 1) supplemented LP medium, followed treatments no. 4 and 5, respectively. Ellis and Bilderback 25 also observed bud formation on cotyledonary leaves in the presence of BA, IAA and/or IBA. Similar results with BA and NAA application in MS medium have been reported 9. Shoot elongation was observed to be relatively slower at higher concentrations of BA, and in most explants the shoot growth was inhibited following prolonged exposure. This inhibitory effect of very high BA concentration on shoot development has also been observed in P. sylvestris 26. Table 1 Effect on shoot formation in excised embryos of deodar previously exposed to different PGRs in LP medium No. PGR Treatments (μm) % embryos forming adventitious buds ±SE Number of shoots formed/embryo Mean number of shoots/ embryo ±SE Embryos * placed in the normal position 1. Control 2. 2,4-D (10.0) ± ± ,4-D (20.0) ± ± BA+IBA ( ) ± ± BA (5.0) ± ± BA+2,4-D ( ) ± ± BA+ Kin ( ) ± ± 1.05 LSD(p=0.05) Embryos ** placed in an inverted position 8. Control 9. BA (10.0) 10. BA (20.0) 25.0 ± ± 0.25 *Embryos transferred to basal medium (LP) after 28 days exposure to different PGRs, control embryos were cultured on PGR-free medium throughout; ** Embryos were exposed to BA for 10 days followed by transfer to LP medium containing activated charcoal for 3 weeks; - denotes no response; SE= Standard error; n= 15; 5 embryos per flask

6 214 INDIAN J BIOTECHNOL, APRIL 2004 Many reports exist on the importance of the type of basal media used for bud induction. Bastola et al 27 used 4 different media and found that LP medium yielded most favourable results. However, Bhatnagar et al 5 have used MS medium for organ differentiation in C. deodara and P. roxburghii. In our investigations, MS, WP, SH and LP media were tried. Out of these 4 media, LP was found to be the best. There are also other reports where LP medium has been used as a basal medium for culture of higher plants. LP medium was used for black spruce 28, Norway spruce 29, and for P. caribaea 30. Exposure to BA (10.0 or 20.0 µm) did not promote the growth of shoot buds even when the inverted embryo technique was used. However, shoot buds in explants cultured on media containing higher level of BA (20.0 µm) exhibited much better growth after transfer to LP medium (PGR-free) containing 1.0% activated charcoal. Since low concentration (10.0 µm) of BA did not induce shoot buds, 20.0 µm BA was used subsequently for shoot induction using the inverted embryo technique. The positive effect of activated charcoal on shoot bud proliferation is consistent with the reported observations in P. roxburghii 31, and in P. wallichiana 27. Mature embryos when cultured on LP medium supplemented with 2,4-D, NAA and/or BA resulted in callus formation (details not given). Such type of response was also observed in C. deodara, P. roxburghii and P. wallichiana 27. Bavistin (Carbendazim), a systemic fungicide showing toxicity towards ascomycetes and basidiomycetes is effectively used for the decontamination of moulds. When the stem segments were taken as explants, heavy contamination, mostly of bacterial origin, was frequently observed. Gupta and Durzan 32 also reported difficulties in obtaining uncontaminated cultures from explants of mature conifers. While HgCl 2 has been effectively used for the surface sterilization of several hard woody species 33, it was not very effective in the present investigation. At present, in vitro regeneration of C. deodara, using stem explants, has proven to be very difficult. However, Piola and Rohr 34 reported in vitro propagation of C. lebani through axillary bud production. Multiple shoots were also formed from terminal and axillary buds taken from mature trees of P. caribaea and P. kesiya 7. Much more work is required to develop a workable protocol for in vitro propagation of C. deodara using stem explants. While some success has been obtained in in vitro propagation of deodar using the inverted embryo technique, the leads obtained are expected to provide valuable insight for doing goal oriented work in future. Acknowledgement The authors thank Department of Biotechnology, Govt. of India, New Delhi for financial assistance and Drs S K Nandi, A Kumar, N Bag and V K Purohit for their help. References 1 Champion H G & Seth S K, A revised survey of the forest types of India (Manager of Publications, Delhi) Srivastava M B & Siaguru P, Silvicultural system and sustainable management of Cedar (C. deodara) and Blue pine (Pinus wallichiana) forests in western Himalaya, Indian J For, 20 (1997) Troup R S, The silviculture of Indian trees, vol 3 (Clarendon Press, Oxford) 1921, Nandi S K, Tamta S & Palni L M S, Adventitious root formation in young shoots of Cedrus deodara L., Biol Plant, 45 (2002) Bhatnagar S P, Singh M N & Kapur N, Preliminary investigations on organ differentiation in tissue culture of C. deodara and P. roxburghii, Indian J Exp Biol, 21 (1983) Agrawal V P, Shakya S, Bastola D R & Joshee N, Inverted embryo technique for in vitro propagation of Himalayan pines P. roxburghii and P. wallichiana, Biotechnol Lett, 1 (1991) Nadgauda R S, Nagarwala N N, Parasharami V A & Mascarenhas A F, Bud break and multiple shoot formation from tissues of mature trees of Pinus caribaea and P. kesiya, In vitro Cell Dev Biol, 29 (1993) Halos S C & Go N E, Micropropagation of Pinus caribaea Morelet, Plant Cell Tissue Organ Cult, 32 (1993) Gupta D, Purohit M & Srivastava P S, Adventitious buds from cotyledonary leaves of Pinus gerandiana Wall. The Chilgoza pine, Beitr Biol Flanzen, 68 (1994) Burns J A, Schwarz O J & Schlarbaum S E, Multiple shoot production from seedling explants of slash pine (Pinus elliottii, Engelm.), Plant Cell Rep, 10 (1991) Chee P P, In vitro culture of zygotic embryos of Taxus species, Hort Sci, 29 (1994) Chee P P, Organogenesis in Taxus brevifolia tissue cultures, Plant Cell Rep, 14 (1995) Smith D R, The role of in vitro methods in pine plantation establishment: The lesson from New Zealand, Plant Tissue Cult Biotechnol, 3 (1997) Jones N B, Staden J V & Bayley A D, Somatic embryogenesis in Pinus patula, J Plant Physiol, 142 (1993) Lelu M A, Bastien C, Drugeault A, Gouez M L & Klimaszeweska K, Somatic embryogenesis and plantlet development in Pinus sylvestris on medium with and without growth regulators, Physiol Plant, 105 (1999)

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