ABSTRACT
Objective: The physiological regulation mechanism of seedlings germinated from Jiangxi local yam (Ruichang yam, Nancheng yam, Yongfeng yam, and Guangfeng yam) microtubers under salt stress was studied and the salt tolerance of seedlings germinated from microtubers of four kinds of local yams in Jiangxi was compared, which provided some references for salt tolerance mechanism research and salt tolerance breeding of four local yams in Jiangxi. Methods: Some physiological and biochemical index detection of seedlings germinated from Jiangxi local yam microtubers under salt stress were studied by spectrophotometer method. The subordinate function, principal component analysis (PCA), and clustering analysis of their salt tolerance were accomplished by fuzzy mathematics subordinate function formula and SPSS 19.0 software, respectively. Results: The total chlorophyll content and root activity of seedlings germinated from Jiangxi local yam microtubers under 0-300 mmol/L salt stress decreased significantly. The soluble protein content of seedlings germinated from Ruichang yam, Nancheng yam, and Guangfeng yam microtubers under 0-300 mmol/L salt stress increased firstly and then decreased, while the soluble protein content of seedlings germinated from Yongfeng yam microtubers under 0-300 mmol/L salt stress decreased significantly. The total soluble sugar content, proline content, MDA content and membrane permeability of seedlings germinated from Jiangxi local yam microtubers under 0-300 mmol/L salt stress significantly increased. The POD, SOD, and CAT activities of seedlings germinated from Jiangxi local yam microtubers under 0-300 mmol/L salt stress firstly increased and then decreased. Based on the subordinate function and PCA of SPSS 19.0 software, three principal components of 10 physiological and biochemical indexes of seedlings germinated from Jiangxi local yam microtubers under salt stress were induced. The salt tolerance order of seedlings germinated from Jiangxi local yam microtubers was Guangfeng yam > Yongfeng yam > Ruichang yam > Nancheng yam. Through the cluster analysis by SPSS 19.0 software, the salt resistance of four kinds of Jiangxi local yam is divided into two types, the salt tolerance of Guangfeng yam and Yongfeng yam was strong, and of Ruichang yam and Nancheng yam were sensitive to salt stress. Conclusion: The regulation mechanism of salt tolerance of seedlings germinated from four kinds of Jiangxi local yam microtubers is revealed and the objective evaluation of salt tolerance of four kinds of Jiangxi local yam is made in the paper, which will provide a theoretical basis for sowing in the field of four kinds of Jiangxi local yam microtubers.
ABSTRACT
Objective: In order to provide a theoretical basis for clarifying the mechanism of synthesis of saponins during different induction formation period of Dioscorea bulbifera microtubers, squalene synthase (SQS) gene expression of D. bulbifera microtubers was analyzed in this paper. Methods: Using Actin gene as a reference gene, Real-time quantitative PCR (qRT-PCR) analysis technology was applied. The amplification curve and solubility curve of SQS (target gene) and Actin (reference gene) were successfully constructed by SYBR Green I after RNA was extracted from different formation period of D. bulbifera microtuber and cDNA was obtained by reverse transcription. Result: Quantitative results showed that: During the initial stage (18-36 d) of D. bulbifera microtuber formation, the expression level of SQS gene increased significantly. During the mid-term (36-72 d) of D. bulbifera microtuber formation, the expression level of SQS gene decreased significantly, and tended to be stable. During the later stage (72-90 d) of D. bulbifera microtuber formation, the expression level of SQS gene decreased significantly again. Conclusion: In general, in the process of D. bulbifera microtuber formation, the expression level of SQS gene shows the trend of "low-high-low-constant-low", which indicates that SQS is a key enzyme of saponin biosynthesis during the different induction formation period of D. bulbifera microtubers.
ABSTRACT
Objective: To discuss the influence of several factors on the cryopreservation of embryogenic calli induced from Dioscorea bulbifera microtuber by droplet-vitrification and to test the genetic stability of the regenerated plantlets after freezing from the aspects of morphology, physiology, DNA content, as well as the photosynthetic characteristics and chlorophyll fluorescence parameters in this paper. Methods: Plant tissue culture (including microtuber induction and embryogenic callus induction), plant physiology index detection (including total chlorophyll, soluble protein, soluble sugar and superoxide dismutase enzyme and peroxide enzyme activity), and cell flow cytometry were applied. Results: The best cryopreservation conditions of embryogenic callus of D. bulbifera microtuber were as following: Embryogenic calli were precultured in liquid media of MS+KT 2 mg/L+NAA 0.5 mg/L+2, 4-D 0.5 mg/L+0.3 mol/L sucrose for 1 d and then treated in loading liquid (MS+2 mol/L glycerol+0.4 mol/L sucrose, pH 5.8) for 20 min. In order to dehydrate, embryogenic calli were transferred in 100% PVS2 at 0℃ for 40 min. After dehydration, the embryogenic calli were inoculated to PVS2 small drops in the aluminum foil strips and then dipped in liquid nitrogen (LN). Finally the aluminum foil strips were quickly transferred to freezing tube that filled with LN and then put into LN tank. After conserving for 1 d in LN, the aluminum foil strips were removed and the embryogenic calli were immersed into liquid washing media (MS+KT 2 mg/L+NAA 0.5 mg/L+2, 4-D 0.5 mg/L+1.2 mol/L sucrose, pH 5.8) preheated in 37℃ warm water. After separated from the aluminum foil strips, the embryogenic calli were washed with fresh liquid washing media at room temperature for there times, 10 min each time. After washing, the embryogenic calli were transferred onto differentiation medium (MS+KT 2 mg/L+NAA 0.5 mg/L+30 g/L sucrose+5 g/L agar), and cultured in dark for 2 d and then cultured in 12 h/d photoperiod, the cell survival rate reaches above 89%. The morphological and physiological indexes and the content of DNA of two kinds of plantlets, which regenerated from cryopreserved and non-cryopreserved embryogenic calli induced from D. bulbifera microtuber by droplet-vitrification, showed no significant difference (P>0.05). Conclusion: Cryopreservation technology system of embryogenic calli induced from D. bulbifera microtuber by droplet-vitrification is established and the regeneration plants have no genetic variation, which provides the theoretical basis and technical basis for the long-term preservation of germplasm resources in the plants of Dioscorea L.
ABSTRACT
Objective: In order to find the suitable concentration and combination of plant growth regulators, the effects of plant growth regulators (NAA, 2, 4-D, 6-BA, KT, and PP333) on in vitro induction formation for the plantlet microtuber of Dioscorea bulbifera was studied. Methods: Through plant tissue culture technique, single factor test, and orthogonal test, taking the stems with a bud of D. bulbifera plantlets as explants, the effects of plant growth regulators on the in vitro induction formation for the microtubers of D. bulbifera were investigated. Results: Auxin using alone was conducive to the induction formation for the microtuber of D. bulbifera. The suitable concentration of both NAA and 2, 4-D inducing the microtuber formation was 0.5 mg/L, but the inducing effects of NAA and 2, 4-D had no significant difference. Cytokinin using alone was not conducive to the induction formation for the microtuber of D. bulbifera. The suitable concentration of both KT and 6-BA inducing microtuber formation was 2 mg/L, but the inducing effect of KT is better than that of 6-BA. The combination of auxin, cytokinin, and PP333 could significantly promote the in vitro induction formation for the microtuber of D. bulbifera, the better combination was MS+NAA 0.5 mg/L+6-BA 2.0 mg/L+PP333 0.5 mg/L. Conclusion: Based on these experimental results, the paper selects the suitable concentration of plant growth regulators conducive to the in vitro induction formation for the microtuber of D. bulbifera, which has laid the technical foundation for their in vitro induction formation of microtuber and factory production.