An efficient system for the regeneration of adventitious shoots from in vitro cultured leaf sections of Lycium chinense Miller was developed and silent somaclones from the regenerants detected with RAPD method. Among the eight media tested (B5, SH, N&N, 1/2MS, MS, 3/2MS, GD and WPM), and four cytoki...
An efficient system for the regeneration of adventitious shoots from in vitro cultured leaf sections of Lycium chinense Miller was developed and silent somaclones from the regenerants detected with RAPD method. Among the eight media tested (B5, SH, N&N, 1/2MS, MS, 3/2MS, GD and WPM), and four cytokinins (BA, kinetin, 2ip and zeatin) with different concentrations (1, 5, 10, 20, 30 and 40 $\mu{M}$), 1/2 MS medium supplemented with 20 and 30 $\mu{M}$ zeatin showed the best regeneration frequency (100% and 93.7%) and higher average number of shoots (9.0 and 9.4). All regenerants easily elongated after subculturing on 1/4MS without growth stimulants and produced spontaneous adventitious roots from their basal parts. With phenotypically normal 40 regenerants, RAPD analysis with 15 different random primers was performed to examine the cryptic somaclonal variants. No substantial differences in banding patterns were found in the amplified polymorphic DNAs implying no DNA changes during dedifferentiation into adventitious shoots. However, one (OPF-4) of the 15 primers detected silent somaclonal variation in one regenerant in which two different polymorphic bands did not appear when compared with the rest regenerants. The results indicate that regenerantion via intervening callus phase can be used to establish true-to-type planting stocks for homogeneous population.
An efficient system for the regeneration of adventitious shoots from in vitro cultured leaf sections of Lycium chinense Miller was developed and silent somaclones from the regenerants detected with RAPD method. Among the eight media tested (B5, SH, N&N, 1/2MS, MS, 3/2MS, GD and WPM), and four cytokinins (BA, kinetin, 2ip and zeatin) with different concentrations (1, 5, 10, 20, 30 and 40 $\mu{M}$), 1/2 MS medium supplemented with 20 and 30 $\mu{M}$ zeatin showed the best regeneration frequency (100% and 93.7%) and higher average number of shoots (9.0 and 9.4). All regenerants easily elongated after subculturing on 1/4MS without growth stimulants and produced spontaneous adventitious roots from their basal parts. With phenotypically normal 40 regenerants, RAPD analysis with 15 different random primers was performed to examine the cryptic somaclonal variants. No substantial differences in banding patterns were found in the amplified polymorphic DNAs implying no DNA changes during dedifferentiation into adventitious shoots. However, one (OPF-4) of the 15 primers detected silent somaclonal variation in one regenerant in which two different polymorphic bands did not appear when compared with the rest regenerants. The results indicate that regenerantion via intervening callus phase can be used to establish true-to-type planting stocks for homogeneous population.
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제안 방법
Based on the results of the first experiment, the best meduim was selected and used to determine the best cytokinin and its concentration. The four cytokinins (2ip: 6- 7-r-dimethylallylamino purine, BA: benzyladenine, kinetin and zeatin) at six concentrations (1, 5, 10, 10, 30 and 40 μM) with control were examined for this experiment.
Our objectives in this research were to optimize adventitious shoot regeneration protocols from leaf sections of L. chinense and examine silent somaclones with RAPD methods. This would provide information about the frequency of spontaneous mutation in vitro and assess the level of background genetic changes resulting from adventitious pathway.
Each reaction was overlaid with three drops of mineral oil to prevent evaporation. PCR reactions were carried out in Techne in a GENUS the「m기 cycler (England) under the following pro gramme conditions: 94 °C for 5 min at initial step, 35 cycles at 92°C for 45 sec, 36°C for 45 sec and 72°C for 1 min, followed by one cycle at 72°C for 5 min. Random 10-mer primers (F-01 to F-15) were obtained from OPERON (Almeda, California).
Based on the results of the first experiment, the best meduim was selected and used to determine the best cytokinin and its concentration. The four cytokinins (2ip: 6- 7-r-dimethylallylamino purine, BA: benzyladenine, kinetin and zeatin) at six concentrations (1, 5, 10, 10, 30 and 40 μM) with control were examined for this experiment. All cyotkinins except zeatin were added before autoclaving.
대상 데이터
A cultivar of L. chinense was obtained from the Forest Research Institution (FRI), South Korea and used for pro liferating in vitro stocks. Stems from plants grown in the pots were cut into 2 cm each and sterilized with commercial이。!"ox (5.
Each plate was regarded as individual replicate and used for statistical analysis. For all experiments, data were collected on number of adventitious shootsafter eight weeks of in vitro culture. For statistical analysis, per cent data were transformed into arcsine values and anlayzed by ANOVA; means were subjected to LSD and ANOVA tests using SAS statistical package (SAS Institute Inc.
데이터처리
For all experiments, data were collected on number of adventitious shootsafter eight weeks of in vitro culture. For statistical analysis, per cent data were transformed into arcsine values and anlayzed by ANOVA; means were subjected to LSD and ANOVA tests using SAS statistical package (SAS Institute Inc. 1985).
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