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Pinus Kesiya Royle Ex Gord Indian Journal of Biotechnology Vol 6, April 2007, pp 262-266 Plantlet regeneration of Pinus kesiya Royle ex Gord. from mature embryos Pramod Tandon*, Suman Kumaria and Hiranjit Choudhury Plant Biotechnology Laboratory, Department of Botany, North-Eastern Hill University, Shillong 793 022, India Received 7 September 2005; revised 8 May 2006; accepted 10 August 2006 A high frequency (90.5%) of shoot bud induction was observed in mature zygotic embryos of Pinus kesiya Royle ex Gord. that were pre-cultured in Quoirin and Lepoivre (LP) medium containing 23.15 µM Kn for 4 wk and then transferred to growth regulator-free medium. Multiplication and elongation of the shoot buds resulted in 1/2 LP medium containing 0.5 µM IBA and 4.5 µM BAP or Kn. Rooting was 72.3% in isolated shoots that were treated with 53.76 µΜ ΝΑΑ for 24 h and then cultured on water-agar medium. Plantlets were hardened and successfully established on soil collected from pine forests with 70% survival. Biomass of the micropropagated plants was 3.2 times higher than seed- derived plants. Keywords : growth characteristics, micropropagation, shoot buds induction, zygotic embryos, Pinus kesiya IPC Code: Int. Cl. 8 A01H4/00, 7/00 Introduction were surface-sterilized with 1% (w/v) mercuric Pinus kesiya Royle ex Gord. (Khasi pine) is chloride for 2-3 min, washed 3-4 times with sterilized widely distributed in the eastern Himalayan region of pure water and then treated with 6% (v/v) hydrogen the Indian subcontinent and extends up to Myanmar, peroxide for 10 min and stratified at 4°C for 24 h. Philippines and Vietnam 1. Extensive extraction of timber and unplanned developmental activities had Shoot Bud Induction MS (Murashige and Skoog) 11 , LP (Quoirin and denuded pine forests in the region. Regeneration of P. 12 13 kesiya through seeds and vegetative cuttings is quite Lepoivre) and SH (Schenk and Hildebrandt) media poor and regenerants show heterogeneous quality. with varying concentrations (11.06-69.45 µM) of Therefore, there is an urgent need for production of a benzylaminopurine (BAP) or kinetin (Kn) were used. large number of improved and fast growing trees of The pH of the medium was adjusted to 5.8, solidified this species. Successful plantlet regeneration in with 0.8% Difco bacto-agar and then autoclaved at conifers through shoot bud formation has been 125 kPA (121°C) for 20 min. The stratified seeds achieved in many cases 2-7. We have earlier reported were germinated on ½ strength MS basal medium in successful regeneration of P. kesiya using needles 8, the dark at 25 ±2°C and 3-4 d embryo were dissected axillary buds 9 and shoot tips 10 as explant sources. out. Three embryos per petri plate (35 mm × 15 mm) Here we report a protocol for in vitro plantlet were inoculated in the above media and the cultures regeneration from excised mature zygotic embryos of were maintained at 25 ±2°C under 12 h photoperiod of P. kesiya . Comparison of growth characteristics of light intensity of 150 µmole m -2 s -1 provided by micropropagated plants with that of seed-derived fluorescent lamps. After 4 wk, the embryos were plants has also been presented. transferred to respective media devoid of cytokinins but containing reduced sucrose (2%) for shoot buds Materials and Methods induction. The bud-forming capacity (BFC) was calculated as follows based on the average number of Plant Materials 5,7 Mature seeds of selected genotype of P. kesiya buds and percentage of response . were obtained from the Forest Department, BFC = (average number of buds per explant) × Government of Meghalaya, Shillong, India. Seeds (% of explants forming buds) ÷ 100 —————— Shoot Multiplication and Elongation *Author for correspondence: Tel: 91-364-272 2244/272 2214/255 0150; Fax: 91-364-255 0300 The shoot buds were multiplied and elongated in E-mail: [email protected] 1/2 LP medium supplemented with reduced growth TANDON et al : IN VITRO REGENERATION OF P. KESIYA 263 regulators [4.5 µM BAP/Kn with varying were initially cultured for 4 wk in LP medium concentrations (0.05-5.00 µM) of IBA] and activated supplemented with 23.15 µM Kn and then transferred charcoal (0.3%). Shoot buds were subcultured after to basal medium devoid of growth regulators, but every 21 d on fresh medium for further growth and containing 2% sucrose (Table 1; Fig. 1). On the other multiplication. hand, with 22.10 µM of BAP pretreatment, 15-18 buds/embryo were formed in 75% embryos. Highest Rooting of Shoots BFC index of 20.82 was recorded with Kn The isolated shoots measuring 2-3 cm were pretreatment, which was 1.2 times higher than with cultured in ½ LP medium containing different BAP (12.38). The BFC index obtained in the present concentrations (0.98-2.67 µM) of α-naphthaleneacetic study is 4.59 times higher than those reported for blue acid (NAA) and indolebutyric acid (IBA), separately. pine (BFC 4.53) 7. The BFC index is an efficient In another experiment, the basal ends of the isolated indicator of bud induction as it takes into shoots were dipped in NAA or IBA solutions of consideration both the number of explants showing varying concentrations (53.76-161.29 µM) for bud induction as well as the number of buds per different duration (12-48 h) and then cultured in ½ LP explant. basal salts medium free of growth regulators or on The morphological changes that accompanied semi-solid water-agar (0.6%). shoot development started with nodular structures Hardening of Plantlets emerging on the surface of the embryo of P. kesiya . The rooted plantlets were transferred to plastic pots Such findings are similar to earlier reports on 14,15 containing different potting mixture, namely pines . In the present study, full strength medium peat:vermiculite:pumice (1:1:1), peat:pumice (1:1), was suited for bud induction and ½ strength for vermiculite:pumice (1:1), pumice alone and non- subsequent stages of growth. The transfer from a high sterile soil (upper layer 10-12 cm depth) from pine salt medium to low salt medium was found beneficial forests. The potted plantlets were hardened at 25 ±2oC, for preconditioning, shoot and root initiation and under 12 h photoperiod of light intensity of 150 subsequent development before transferring to ex vivo 16 µmole m -2 s -1 and 80-85% RH. Plants were fed with conditions . Transferring them along with the 1/10 th of the MS nutrient salt solution twice a wk for 2 Table 1—Shoot bud formation on embryos of P. kesiya cultured wk. The plants were hardened in about 4-5 wk and in growth regulator-free LP medium transferred first to the net-house and then to the natural habitat. LP medium % response No. of shoot buds BFC + PGR formed/embryo Growth Analysis (µM)# Both, tissue culture and seed-derived plantlets measuring around 2.5 cm were established in the Control 96.5 ±2.53* Embryo germinated - BAP glasshouse. The growth analyses of plants were done 11.06 62.0 ±1.87 4-6 3.10 after 10 and 22 months by measuring the shoot and 22.10 75.0 ±1.58 15-18 12.38 root lengths and dry mass accumulation. 44.20 7-8 3.75 Ten replicates for each of the above experiments 50.0 ±2.77 were taken and the experiments were repeated thrice. 66.30 48.2 ±1.20 2-4 1.45 Kn Results and Discussion 11.57 61.2 ±1.58 6-8 4.28 Soaking the seeds of P. kesiya softened the hard 23.15 90.5 ±1.49 20-25 20.82 seed coat and helped in elimination of the non- 46.30 46.0 ±2.91 8-10 4.14 viable/empty seeds, which floated on the surface. 69.45 35.0 ±2.43 8-10 3.15 Also, preculturing the seeds in basal medium for 3-4 d eliminated non-responsive embryos, which turned PGR: Plant Growth Regulator yellow. The bud induction was not observed in *Data ±S.D., data scored after 2 wk of culture # The embryos were cultured for 4 weeks before transfer to PGR embryos cultured in SH and MS media. In case of the free medium control, each embryo germinated directly into a ANOVA test shows that the number of shoots formed, as the plantlet. A maximum of 20-25 buds/embryo were result of treatments is highly significant at 5% level of formed in about 6 wk’s time on 90% embryos, which significance. 264 INDIAN J BIOTECHNOL, APRIL 2007 explants to fresh medium increased the survivability Table 2—Effect of NAA and IBA on root formation of of the developing shoot buds in P. kesiya . P. kesiya shoots Multiplication and elongation of shoot buds (Fig. 2) Treatment Concentration % rooting response were highest in the medium containing 0.5 µM IBA (µM) of shoots along with reduced concentration of BAP or Kn at 4.5 Pulse µM, which is in line with earlier findings of Schwarz NAA, 12h 53.76 45.1 ±2.58 13 et al in case of P. strobus . Half-strength basal NAA, 24h 72.3 ±1.83 nutrient medium with 0.2% activated charcoal NAA, 48h 59.2 ±1.82 enhanced the shoot proliferation in P. kesiya . The use of activated charcoal probably adsorbs excess NAA, 12h 107.53 48.4 ±2.83 hormones or inhibitory substances produced during NAA, 24h 45.3 ±1.56 culture conditions 17 . In all, about 80-100 shoots per NAA, 48h - embryo were formed in a period of about 12-15 wk.
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