Kalopanax septemlobus (Thunb. ex Murray) Koidz, belonging to the family Araliaceae is a woody perennial species that reaches up to 25m in height. The tree has been used for timbers as well as folk medicine. Traditionally, stem bark including cortical tissue has been the source of oriental medicine. ...
Kalopanax septemlobus (Thunb. ex Murray) Koidz, belonging to the family Araliaceae is a woody perennial species that reaches up to 25m in height. The tree has been used for timbers as well as folk medicine. Traditionally, stem bark including cortical tissue has been the source of oriental medicine. Conventional propagation of the tree has been achieved via seeds or stem cuttings with very low efficiency. Seeds require 2 years to germinate under natural conditions. Moreover, zygotic embryos at harvest time are in an immature stage showing extreme dormancy. Micropropagation techniques have been applied to develop an efficient propagation method for the species. Among various in vitro techniques, somatic embryogenesis is an applicable technique in practical point of view if an optimized cultural conditions are established. In the present study attempts were made to determine the factors affecting somatic embryogenesis of the species. First, induction of embryogenic calli (EC) from leaf segments was investigated with pre-treatments as 100μM 2,4-D and 1M sucrose. Second, comparison of the embryogenic competency was made using different embryogenic cell lines. Third, the effects of several additives and plant growth regulators on induction of somatic embryos was studided. Finally plant growth regulators influencing development of somatic embryos and germination were examined. 1. Induction of embryogenic calli To induce embryogenic calli (EC) from leaf segments of in vitro plant, pre-treatments such as high 2,4-D (100μM), high sucrose (1M), cold pre-and starvation were evaluated. Without pre-treatments, no calli were induced even when the cultures were conducted on MS medium with 2,4-D. In contrast, EC could be induced by the pre-treatment of 100μM 2,4-D or 1M sucrose, respectively. The highest EC formation (23.3%) was obtained by 1M sucrose treatment for 6 hours, but the explants turned brown and died when the treatment time was over 6hrs. 100μM 2,4-D resulted in 16.7% of EC induction for 1day exposure, 10% EC for 3 days and 5% EC for 5 days, respectively. Longer treatments resulted in necrosis and browning of the explants. However, neither starvation nor low temperature pre-treatment produced EC in the present study. 2. Comparison of embryogenic competency by cell lines Embryogenic competency was compared using 4 different embryogenic cell lines maintained for 2 and half years. Significant differences among the cell lines were observed in callus proliferation, SE induction and plantlet conversion. It was revealed that short term cultured embryogenic calli formed more somatic embryos compared to long-term cultured calli. As for cellular hormone, short-term cultured EC had a higher value in a ratio of endogenous cytokinin/ABA. As the culture period was extended, the rate of somatic embryo induction decreased. 3. Comparison of somatic embryo induction rate Generally, ABA treatment appeared to be an essential step to induce SEs in Kalopanax septemlobus. It also requires some additives like sugars, activated charcoal and higher concentration of gelling agent. In conclusion, the highest somatic embryo induction was accomplished on 1/2MS medium containing 0.37μM ABA, 5% PEG, 3% sucrose, 0.2% AC and 0.3% gelrite. 4. development somatic embryos and plant conversion by plant growth regulators To optimize embryo development and its conversion, ABA, zeatin and GA3 were treated at various development stages of somatic embryos. Generally, more synchronized embryo proliferation and plant conversion were obtained when the PGRs were applied in an earlier stage (globular) somatic embryos, whereas more uniform SEs were developed when ABA was treated on globular stages. Relatively large number of normally converted plants was developed when GA3 was treated on heart stages. Above results suggested that specific PGR treatments were necessary for multiplication of normal somatic embryo multiplication and then plant conversion plant in Kalopanax septemlobus.
Kalopanax septemlobus (Thunb. ex Murray) Koidz, belonging to the family Araliaceae is a woody perennial species that reaches up to 25m in height. The tree has been used for timbers as well as folk medicine. Traditionally, stem bark including cortical tissue has been the source of oriental medicine. Conventional propagation of the tree has been achieved via seeds or stem cuttings with very low efficiency. Seeds require 2 years to germinate under natural conditions. Moreover, zygotic embryos at harvest time are in an immature stage showing extreme dormancy. Micropropagation techniques have been applied to develop an efficient propagation method for the species. Among various in vitro techniques, somatic embryogenesis is an applicable technique in practical point of view if an optimized cultural conditions are established. In the present study attempts were made to determine the factors affecting somatic embryogenesis of the species. First, induction of embryogenic calli (EC) from leaf segments was investigated with pre-treatments as 100μM 2,4-D and 1M sucrose. Second, comparison of the embryogenic competency was made using different embryogenic cell lines. Third, the effects of several additives and plant growth regulators on induction of somatic embryos was studided. Finally plant growth regulators influencing development of somatic embryos and germination were examined. 1. Induction of embryogenic calli To induce embryogenic calli (EC) from leaf segments of in vitro plant, pre-treatments such as high 2,4-D (100μM), high sucrose (1M), cold pre-and starvation were evaluated. Without pre-treatments, no calli were induced even when the cultures were conducted on MS medium with 2,4-D. In contrast, EC could be induced by the pre-treatment of 100μM 2,4-D or 1M sucrose, respectively. The highest EC formation (23.3%) was obtained by 1M sucrose treatment for 6 hours, but the explants turned brown and died when the treatment time was over 6hrs. 100μM 2,4-D resulted in 16.7% of EC induction for 1day exposure, 10% EC for 3 days and 5% EC for 5 days, respectively. Longer treatments resulted in necrosis and browning of the explants. However, neither starvation nor low temperature pre-treatment produced EC in the present study. 2. Comparison of embryogenic competency by cell lines Embryogenic competency was compared using 4 different embryogenic cell lines maintained for 2 and half years. Significant differences among the cell lines were observed in callus proliferation, SE induction and plantlet conversion. It was revealed that short term cultured embryogenic calli formed more somatic embryos compared to long-term cultured calli. As for cellular hormone, short-term cultured EC had a higher value in a ratio of endogenous cytokinin/ABA. As the culture period was extended, the rate of somatic embryo induction decreased. 3. Comparison of somatic embryo induction rate Generally, ABA treatment appeared to be an essential step to induce SEs in Kalopanax septemlobus. It also requires some additives like sugars, activated charcoal and higher concentration of gelling agent. In conclusion, the highest somatic embryo induction was accomplished on 1/2MS medium containing 0.37μM ABA, 5% PEG, 3% sucrose, 0.2% AC and 0.3% gelrite. 4. development somatic embryos and plant conversion by plant growth regulators To optimize embryo development and its conversion, ABA, zeatin and GA3 were treated at various development stages of somatic embryos. Generally, more synchronized embryo proliferation and plant conversion were obtained when the PGRs were applied in an earlier stage (globular) somatic embryos, whereas more uniform SEs were developed when ABA was treated on globular stages. Relatively large number of normally converted plants was developed when GA3 was treated on heart stages. Above results suggested that specific PGR treatments were necessary for multiplication of normal somatic embryo multiplication and then plant conversion plant in Kalopanax septemlobus.
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