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Studies on Somatic Embryogenesis and in Vitro Propagation Systems in Myrica Rubra Sieb & Zucc

Syed Asghar Tori  
【摘要】:a) Direct induction of somatic embryogenesis and plant regeneration from cotyledon explants ofMyrica rubra Sieb Zucc.Somatic embryogenesis was induced on BW medium supplemented with TDZ alone or in combination with 2,4-D from mature cotyledon explants of Myrica rubra. All concentrations of TDZ except 1.0 mg L~-1 induced somatic embryos and adventitious shoots simultaneously within two months of culture. Addition of 2,4-D in the medium significantly improved induction of somatic embryos. Frequency of embryogenesis was only 3.34% with 7.00 embryos per explants when TDZ was fortified as a single growth regulator which was improved to 22.00 % with the addition of 0.1 mg L~-1 2,4-D in the media. Repetitive embryogenesis was induced on optimized concentrations of BA and TDZ (0.5 mg L~-1 BA and 0.05 mg L~-1 TDZ) in combinations with various concentrations of 2, 4-D. Continuous culture of the explants with cluster of embryos on the induction media did not induce repetitive embryogenesis. On repetitive embryogenesis induction media, most of the embryos induced were smaller than those of the primary embryos during their induction stage. Effect of BA in combination with 2,4-D was more remarkable in the induction of repetitive embryogenesis than TDZ. Maturation and rhizogenesis of somatic embryos was achieved on the media containing IBA. Mature green embryos were used to induce adventitious shoots. TDZ in combination with IBA induced multiple shoots on the surface of somatic embryos explants. TDZ (0.2 mg L~-1 ) plus IBA (1.0 mg L~-1) was the most effective combination with maximum number (8.5) of shoots per explant. Shoot elongation was achieved on the media supplemented with optimized BA concentration (0.5 mg L~-1) plus 0.1 mg L~-1 NAA. Full strength WPM media supplemented with IBA concentrations did not induce roots in M. rubra micro-shoots. Microshoots rooted on BW medium fortified with 0.5 mg L~-1 IBA with highest rooting efficiency. Rooted plants were successfully hardened and grown in the greenhouse. The protocol reported here for Myrica rubra is efficient, reproducible and could be used for genetic transformation experiments. b) Micropropagation ofMyrica rubra sieb zucc. using shoot tips and nodal explantsMicropropagation of Myrica rubra, an important horticultural, forestry and ornamental tree was achieved using shoot tip and nodal explants from two commercial cultivars "Biji and Dongkui" on BW medium supplemented with Thidiazuron (TDZ). TDZ alone induced multiple but stunted shoots on the explants especially on those buds adjacent to the surface of the medium. Maximum number of shoots (5.75) was obtained for both the cultivars with 0.6 mg L"1 TDZ. Shoot length was significantly improved with various concentrations of 2,4-D in combination of lower (0.012 mg L"1) TDZ concentration. BW medium was found as suitable medium for micropropagation of two cultivars with maximum and longer shoots. WPM and MS media ranked second in multiple shoot regeneration of both the cultivars. Micro-shoots were continuously harvested (3-6 harvests) in the presence of TDZ in combination with 2,4-D. All the explants lost their efficiency to nurture micro-shoots for the second harvest in the absence of TDZ. Micro-shoots cultured on plant growth regulator-free medium for 2-3 weeks and subsequent transfer to the % and Vi strength media containing 0.1- 0.6 mg L"1 IB A initiated roots within 2-3 weeks of culture. Roots were induced with a high frequency of 95% in Biji and 87% in Dongkui with 0.3 mg I/1 IBA respectively. No callus was observed on shoots in rooting media. All concentrations of IAA either on reduced or full strength media did not initiate roots. Rooted plants were successfully acclimatized and grown in the greenhouse.c) In vitro adventitious bud formation and organogenesis from cotyledon explants of Myrica rubra Sieb. Zucc.This study involves in vitro adventitious bud formation from mature cotyledon explants of Myrica rubra on WPM medium with various cytokinins either used alone and/or in combination with 2,4-D. Zeatin induced adventitious buds when used in combination with 2,4-D while KN did not induce adventitious buds. In the induction of adventitious buds, TDZ and BA were more effective than Zeatin. Cytokinins combined with 2,4-D significantly promoted shoot length. Maximum number of adventitious shoots (26.60) was induced by 0.3 mg L'1 TDZ plus 0.1 mg L"1 2,4-D. Cytokinins especially TDZ suppressed the growth of microshoots and resulted in a rosette of shoots when continued to be cultured on same medium. Prolonged culture in the higher concentrations of TDZ either used alone or in combination with 2,4-D induced callus. Shoots were tremendously elongated with proper regulation of BA levels in combination with NAA and 2,4-D. WPM was found to be a suitable medium for adventitious shoot regeneration followed by MS and BW. Roots were induced with a high frequency of 88-100% in WPM medium supplemented with various concentrations of IBA as compared to the lowest 3-9% by IAA. Plants were successfully acclimatized to the green house conditions.d) In vitro adventitious shoot formation and organogenesis from embryonic axes explants of Myrica rubra Sieb Zucc.An efficient in vitro adventitious shoot formation and organogenesis protocol was developed for Myrica rubra using embryonic axes explants. Embryonic axes were excised and cultured on MS medium fortified with 0.6 mg L"1 TDZ for different time intervals. Explants not exposed to TDZ concentration did not germinate. Exposure of explants for 2-3 weeks was found the optimal time interval for induction of adventitious shoots. Shoot regeneration was suppressed by longer exposures. Highest shoot induction frequency (99.80%) was observed when explants were exposed for 21 days followed by 14 days (97.00%) exposure. Maximum number of shoots (16.66) were recorded when explants were exposed to media containing TDZ for 21 days. Shoots were tremendously elongated with proper regulation of BA concentrations alone or in combination with NAA. Longer shoots (4.60 cm) were harvested with 0.5 mg L"1 BA in combination with 0.9 mg L"1 NAA. Roots were induced with a high frequency of 100% in MS medium supplemented with 0.6 mg L"1 IBA. Plants were successfully acclimatized to the green house conditions.e) Adventitious shoots regeneration and organogenesis from leaf explants of Myrica rubra Seib Zucc.Adventitious shoots were regenerated from leaf explants of Myrica rubra plants with a scion cultivar "Biji" on WPM medium supplemented with thidiazuron (TDZ) alone or in combination with 2,4-D. All concentrations of TDZ tested in this experiment induced adventitious shoots, however, higher concentrations were associated with callus and heavy exudation of phenolic compounds. TDZ concentrations at (0.1 and 0.2 mg L"1) were found to be optimum for adventitious shoot regeneration without intervening callus phase. Highest regeneration response (92.6 and 90.6%) to produce more adventitious shoots (3.4 and 4.4) without intervening callus phase was observed in 0.1 and 0.2 mg L"1 TDZ. Combined effect of TDZ with 2, 4-D on induction of adventitious shoots was more prominent than TDZ alone. TDZ (0.2 mg L'1) plus 2,4-D (0.05 mg L'1) was the most effective combination with maximum number (5.24) of shoots per explant. Shoot elongation was achieved on the media supplemented with low BA concentration (0.05 mg L"1) plus NAA (0.6 mg L"1). Root induction on micro-shoots was directly related to the media strength. Full strength WPM media supplemented with IBA concentrations did not induce roots in M. rubra micro-shoots. Roots were only initiated on % strength WPM medium containing IBA. Media supplemented with 0.3 mg L"1 IBA with 81.4 % rooting efficiency was found to be the best concentration. Rooted plants were successfully hardened and grown in the greenhouse. The protocol reported here for Myrica rubra is efficient, reproducible and could be used for genetic transformation experiments.f) Factors affecting the in vitro germination of Myrica rubra Sieb Zucc. seedAn in vitro germination protocol for Myrica rubra Sieb Zucc. seeds is described. A hard endocarp and internal seed dormancy prevent freshly harvested Myrica rubra Sieb Zucc. seeds from germinating. To soften the seed coats, facilitate the scarification procedure and improve germination, seeds were treated with concentrated H2SO4 for different time intervals. To break the internal seed dormancy, seeds were stratified at -2 and 5°C for chilling requirements. Intact endocarp, intact testa, naked seeds, halved seeds and pieces of cotyledons were used as explants and incubated on MS basic medium for germination. Chilling plus H2SO4 treatment increased germination. Seeds with endocarp did not germinate even after chilling. Seeds chilled but not treated with H2SO4 exhibited poor germination, while seeds with intact testa germinated poorly (3.4%). As compared to the untreated seeds, germination was improved to 77.0, 83.4 and 28.0% in the naked, halved and pieces of cotyledons respectively. Comparing two chilling temperature regimes, germination was improved by 28.6 % in the seeds chilled at -2°C than 5°C. Treating seeds with 1000 ppm GA3 for 15 h adversely affected their germination. More seeds were germinated in the light than in the dark. TDZ and BA had positive effects on


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