| Zoysia matrella [L.] Merr., namely manilagrass, from Southeast Asia, is an important turfgrass species in warm climatic regions and transitional zones. Zoysia matrella produce asexually by stolons in nature, so traditional breeding methods such as hybridization can not be used for its genetic improvement. Hence, biological technologies such as genetic transformation and somaclonal variation selection play a key role in its breeding and improving its resistance and ornamental characters.In this experiment, eight-year old embryogenic callus induced from the stolons of manilagrass were taken as materials. Effects of different combinations of different growth regulators (BA and ZR) with various concentrations (0.00,0.05,0.10,0.20and0.40mg/L), callus sizes(1,2,3and4mm), amino acids (L-Pro, L-Arg and L-Glu), gelling agents (Phytagel、Gelzan, Agar、Carrageenan and Agargellan), vessels (Bottle, Erlenmeyer flask and glass Petri dish) and doses of60Co γ-irradiation (0and20Gy) on plant regeneration were studied. Furthermore, we conducted ultrastructure observation (SEM and TEM) in the process of its regeneration to get a better understanding of its regeneration.The primary results were summarized as follows:1. Percentage of regeneration varied significantly depending on plant regulators. As concentrations of BA and ZR increased from0.00to0.40mg/L, regeneration rates ranged from2.5%to70.0%, the addition of BA with lower concentration (0.05mg/L) stimulated regeneration. However, increased concentrations of BA resulted in a significant decrease of regeneration rate. On the other hand, in the media absence of BA, no significant difference was observed with increasing concentrations of ZR. Results showed that the addition of ZR and BA at0.05mg/L in the regeneration media gave the best overall regeneration response with the highest regeneration rate (70.0%) and highest average number of shoots (109.5shoots) per20clumps.2. No significant difference was shown between the regeneration rate or capacity in medium with or without L-Glu (18.0%and30.0%). Addition of L-Arg and L-Pro resulted in a significantly lower regeneration (p<0.05). Media with L-Arg exhibited the lowest regeneration rate (8.0%) and regeneration capacity (74.5%).3. No significant difference of regeneration capacity was detected among the six gelling agents. Agargellan showed highest regeneration rate (39.0%), Agar (32.0%) and Carrageenan (27.0%) showed no significant variation from Agargellan. Phytagel (19.0%), GelzanTM-Kelco (21.0%) and GelzanTM-Sigma (12.0%) all showed significant lower regeneration rates than Agargellan, Agar and Carrangeenan.4. No significant difference among regeneration rates inoculated in1mm (21.0%),2mm (19.0%) and4mm (18.0%), but the3mm callus showed significantly higher regeneration rate (68.0%) and had the highest number of shoots (data not shown).5. Two weeks after inoculation, the highest frequencies of regeneration rate and regeneration capacity,64.0and91.0%respectively, were both observed in bottles. Callus in Erlenmeyer flasks and glass Petri dishes showed significant lower regeneration rates (7.0%and13.0%respectively).6. During the subculture, we found that callus treated with radiation had a higher level of cell division than those of callus without any treatment. However, a significant decline was observed in regeneration rate (20.0%) of the radiated callus (p<0.05) compared to regeneration rate of the non-radiated callus (71.0%).7. The results of ultrastructural observation showed the regeneration course belongs to the somatic embryogenesis way and its regeneration showed a similar in vitro developmental pathway of warm-season turfgrasses. Embryogenic callus varied in different states during regeneration with active metabolism. Besides, a very compact, nodular, organized structure were observed, organs such as mitochondria, chloroplast and vacuole varied during regeneration. |
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