Plantlet regeneration of Pinus kesiya Royle ex Gord. from mature embryos

Transcription

1 Indian Journal of Biotechnology Vol 6, April 2007, pp 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 , 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 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 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 seedderived plants. Keywords: growth characteristics, micropropagation, shoot buds induction, zygotic embryos, Pinus kesiya IPC Code: Int. Cl. 8 A01H4/00, 7/00 Introduction Pinus kesiya Royle ex Gord. (Khasi pine) is widely distributed in the eastern Himalayan region of the Indian subcontinent and extends up to Myanmar, Philippines and Vietnam 1. Extensive extraction of timber and unplanned developmental activities had denuded pine forests in the region. Regeneration of P. kesiya through seeds and vegetative cuttings is quite poor and regenerants show heterogeneous quality. Therefore, there is an urgent need for production of a large number of improved and fast growing trees of this species. Successful plantlet regeneration in conifers through shoot bud formation has been achieved in many cases 2-7. We have earlier reported successful regeneration of P. kesiya using needles 8, axillary buds 9 and shoot tips 10 as explant sources. Here we report a protocol for in vitro plantlet regeneration from excised mature zygotic embryos of P. kesiya. Comparison of growth characteristics of micropropagated plants with that of seed-derived plants has also been presented. Materials and Methods Plant Materials Mature seeds of selected genotype of P. kesiya were obtained from the Forest Department, Government of Meghalaya, Shillong, India. Seeds *Author for correspondence: Tel: / / ; Fax: tandon1@sancharnet.in were surface-sterilized with 1% (w/v) mercuric chloride for 2-3 min, washed 3-4 times with sterilized pure water and then treated with 6% (v/v) hydrogen peroxide for 10 min and stratified at 4 C for 24 h. Shoot Bud Induction MS (Murashige and Skoog) 11, LP (Quoirin and Lepoivre) 12 and SH (Schenk and Hildebrandt) 13 media with varying concentrations ( µm) of benzylaminopurine (BAP) or kinetin (Kn) were used. The ph of the medium was adjusted to 5.8, solidified with 0.8% Difco bacto-agar and then autoclaved at 125 kpa (121 C) for 20 min. The stratified seeds were germinated on ½ strength MS basal medium in the dark at 25±2 C and 3-4 d embryo were dissected out. Three embryos per petri plate (35 mm 15 mm) were inoculated in the above media and the cultures were maintained at 25±2 C under 12 h photoperiod of light intensity of 150 µmole m -2 s -1 provided by fluorescent lamps. After 4 wk, the embryos were transferred to respective media devoid of cytokinins but containing reduced sucrose (2%) for shoot buds induction. The bud-forming capacity (BFC) was calculated as follows based on the average number of buds and percentage of response 5,7. BFC = (average number of buds per explant) (% of explants forming buds) 100 Shoot Multiplication and Elongation The shoot buds were multiplied and elongated in 1/2 LP medium supplemented with reduced growth

2 TANDON et al: IN VITRO REGENERATION OF P. KESIYA 263 regulators [4.5 µm BAP/Kn with varying concentrations ( µm) of IBA] and activated charcoal (0.3%). Shoot buds were subcultured after every 21 d on fresh medium for further growth and multiplication. Rooting of Shoots The isolated shoots measuring 2-3 cm were cultured in ½ LP medium containing different concentrations ( µm) of α-naphthaleneacetic acid (NAA) and indolebutyric acid (IBA), separately. In another experiment, the basal ends of the isolated shoots were dipped in NAA or IBA solutions of varying concentrations ( µm) for different duration (12-48 h) and then cultured in ½ LP basal salts medium free of growth regulators or on semi-solid water-agar (0.6%). Hardening of Plantlets The rooted plantlets were transferred to plastic pots containing different potting mixture, namely peat:vermiculite:pumice (1:1:1), peat:pumice (1:1), vermiculite:pumice (1:1), pumice alone and nonsterile soil (upper layer cm depth) from pine forests. The potted plantlets were hardened at 25±2 o C, under 12 h photoperiod of light intensity of 150 µmole m -2 s -1 and 80-85% RH. Plants were fed with 1/10 th of the MS nutrient salt solution twice a wk for 2 wk. The plants were hardened in about 4-5 wk and transferred first to the net-house and then to the natural habitat. Growth Analysis Both, tissue culture and seed-derived plantlets measuring around 2.5 cm were established in the glasshouse. The growth analyses of plants were done after 10 and 22 months by measuring the shoot and root lengths and dry mass accumulation. Ten replicates for each of the above experiments were taken and the experiments were repeated thrice. Results and Discussion Soaking the seeds of P. kesiya softened the hard seed coat and helped in elimination of the nonviable/empty seeds, which floated on the surface. Also, preculturing the seeds in basal medium for 3-4 d eliminated non-responsive embryos, which turned yellow. The bud induction was not observed in embryos cultured in SH and MS media. In case of the control, each embryo germinated directly into a plantlet. A maximum of buds/embryo were formed in about 6 wk s time on 90% embryos, which were initially cultured for 4 wk in LP medium supplemented with µm Kn and then transferred to basal medium devoid of growth regulators, but containing 2% sucrose (Table 1; Fig. 1). On the other hand, with µm of BAP pretreatment, buds/embryo were formed in 75% embryos. Highest BFC index of was recorded with Kn pretreatment, which was 1.2 times higher than with BAP (12.38). The BFC index obtained in the present study is 4.59 times higher than those reported for blue pine (BFC 4.53) 7. The BFC index is an efficient indicator of bud induction as it takes into consideration both the number of explants showing bud induction as well as the number of buds per explant. The morphological changes that accompanied shoot development started with nodular structures emerging on the surface of the embryo of P. kesiya. Such findings are similar to earlier reports on pines 14,15. In the present study, full strength medium was suited for bud induction and ½ strength for subsequent stages of growth. The transfer from a high salt medium to low salt medium was found beneficial for preconditioning, shoot and root initiation and subsequent development before transferring to ex vivo conditions 16. Transferring them along with the Table 1 Shoot bud formation on embryos of P. kesiya cultured in growth regulator-free LP medium LP medium + PGR (µm)# % response No. of shoot buds formed/embryo BFC Control 96.5±2.53* Embryo germinated - BAP ± ± ± ± Kn ± ± ± ± PGR: Plant Growth Regulator *Data±S.D., data scored after 2 wk of culture # The embryos were cultured for 4 weeks before transfer to PGR free medium ANOVA test shows that the number of shoots formed, as the result of treatments is highly significant at 5% level of significance.

3 264 INDIAN J BIOTECHNOL, APRIL 2007 explants to fresh medium increased the survivability of the developing shoot buds in P. kesiya. Multiplication and elongation of shoot buds (Fig. 2) were highest in the medium containing 0.5 µm IBA along with reduced concentration of BAP or Kn at 4.5 µm, which is in line with earlier findings of Schwarz et al 13 in case of P. strobus. Half-strength basal nutrient medium with 0.2% activated charcoal enhanced the shoot proliferation in P. kesiya. The use of activated charcoal probably adsorbs excess hormones or inhibitory substances produced during culture conditions 17. In all, about shoots per embryo were formed in a period of about wk. In ½ LP medium containing 1.61 µm NAA, optimum rooting response of 64.2% was observed after 4 wk of culture (Table 2); 2-3 healthy roots emerged at the base of the isolated shoots (Fig. 3). However, a lower rooting of 43.6% shoots was observed using 1.47 µm IBA in the medium. In the present study, the best rooting response of 72.3% was observed in shoots treated with µm NAA for 24 h, followed by those cultured in water-agar medium. This is in contrast to the report of Mathur and Nadgauda 7 who observed IBA to be preferential growth regulator for induction of rooting in P. wallichiana shoots. Addition of charcoal to the rooting medium seemed to be beneficial as it either inhibits light at the shoot base or adsorbs rooting inhibitors 18. After taking adequate care, 70% plantlets were hardened in about 4-5 wk in soil collected from pine forests. The growth and survivability of the plantlets was much lower ( %) in other substrata tried. The mycorrhizae available in the soil from pine forest may have helped in better establishment of in vitro grown plantlets. The growth analyses of tissue culture raised and seed-derived plants showed better performance of the former. After 22-months of transfer, tissue culture raised plants exhibited 3.2 times higher biomass yield than those of the seedderived plants (Table 3). Also, the shoot and root length of the micropropagated plants is expected to Table 2 Effect of NAA and IBA on root formation of P. kesiya shoots Treatment Concentration (µm) % rooting response of shoots Pulse NAA, 12h ±2.58 NAA, 24h 72.3±1.83 NAA, 48h 59.2±1.82 NAA, 12h ±2.83 NAA, 24h 45.3±1.56 NAA, 48h - NAA, 12h ±1.98 NAA, 24h - NAA, 48h - IBA, 12h IBA, 24h 35.3±2.98 IBA, 12h ±1.58 IBA, 24h - IBA, 12h IBA, 24h - Semi-solid medium (LP ½, sucrose 3%, activated charcoal 0.2%) + NAA ± ± ± ± IBA ± ± ± *Data scored after 4 wk of culture ANOVA test shows that the number of roots formed is highly significant in shoots treated with NAA but not significant in those treated with IBA at 5% level of significance Table 3 Growth analyses of seed-derived and in tissue culture-raised plantlets Plantlet type Shoot length (cm) Root length (cm) Biomass (mg plantlet -1 ) 10 month 22 month 10 month 22 month 10 month 22 month Seed-derived 4.80±1.86* 15.6± ± ± ± ±0.65 Tissue culture-raised 7.30± ± ± ± ± ±0.84 *Data±S.D. m=months

4 TANDON et al: IN VITRO REGENERATION OF P. KESIYA 265 Figs Initiation of multiple shoot buds from embryos in LP medium free of cytokinins but containing 2% sucrose after 6 wk; 2. Elongation and multiplication of the buds in ½ LP medium containing 4.5 µm Kn and 0.5 µm IBA after 12 wk; 3. Rooting of the isolated shoot treated with µm NAA for 24 h and then cultured in water-agar medium after 4 wk; and 4. A 3-year-old tissue culture derived plant in the field. show exponential growth effect over the years. The present protocol of multiplication from zygotic embryos will be more desirable for the production of large numbers of regenerants for mass propagation and establishment of Khasi pine. Acknowledgement This study was supported by a research grant no. BT/TF/08/01/90 to PT from the Department of Biotechnology, Government of India, New Delhi, India. References 1 Singh K P & Mugal V, Gymnosperm, in Floristic diversity and conservation strategies in India, edited by V Mudgal & P K Hajra (Botanical Survey of India Publication, Calcutta) 1997, 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) Harry I S & Thorpe T A, Regeneration of plantlets through organogenesis from mature embryos of jack pine, Plant Cell Tissue Organ Cult, 37 (1994) Goldfarb B, Howe G M, Hackett W P & Monteuuis O, Survival and growth of eastern white pine shoot apical meristems in vitro, Plant Cell Tissue Organ Cult, 46 (1996) Saborio F, Dvorak W S, Donahue J K & Thorpe T A, In vitro regeneration of plantlets from mature embryos of Pinus ayacayuite, Tree Physiol, 17 (1997) Gonzalez M V, Rey M, Tavazza R, La-Malfa S, Cuozzo L et al, In vitro adventitious shoot formation on cotyledons of Pinus pinea, Hortic Sci, 33 (1998) Mathur G & Nadgauda R, In vitro plantlet regeneration from mature zygotic embryos of Pinus wallichiana A B Jacks, Plant Cell Rep, 19 (1999) Kumar A & Tandon P, Caulogenesis in cultures needles of Pinus kesiya Royle ex Gord, Proc Natl Acad Sci India, 62 (1995) Kumar A & Tandon P, In vitro propagation of Pinus kesiya Royle ex Gord, in Advances in plant tissue culture in India,

5 266 INDIAN J BIOTECHNOL, APRIL 2007 edited by P Tandon (Pragati Prakashan, Meerut, India) 1994, Nandwani N, Kumaria S & Tandon P, Micropropagation of Pinus kesiya Royle ex Gord, Gartenbauwissenschaft, 66 (2001) Murashige T & Skoog F, A revised medium for rapid growth and bioassays with tobacco tissue cultures, Physiol Plant, 15 (1962) Quoirin M & Lepoivre P, Etudes de milieux ad qstes aux cultures in vitro de prunus, Acta Hortic, 78 (1977) Schenk R U & Hildebrandt A C, Medium and technique for induction and growth of monocotyledonous and dicotyledonous plant cell cultures, Can J Bot, 50 (1972) Schwarz O J, Schlarbaum S E & Beaty R M, Plantlet regeneration from mature zygotic embryos of eastern white pine (Pinus strobus L), Plant Cell Rep, 7 (1988) Halos S C & Go N E, Micropropagation of Pinus caribaea Morlet, Plant Cell Tissue Organ Cult, 32 (1993) Horgan K & Aitken J, Reliable plantlet formation from embryos and seedling shoot tips of radiata pine, Physiol Plant, 53 (1981) Fridborg G, Pedersem M, Landstrom L & Eriksson T, The effect of activated charcoal on tissue cultures: Adsorption of metabolites inhibiting morphogenesis, Physiol Plant, 43 (1978) Dumas E & Monteuuis O, In vitro rooting of micropropagated shoots from juvenile and mature Pinus pinaster explants: Influence of activated charcoal, Plant Cell Tissue Organ Cult, 40 (1995)