Studies On Regeneration Of Three Litchi(Litchi Chinensis Sonn.)Cultivars In Vitro

It is vital to establish regeneration system in vitro for litchi bioengineering breeding and rootstock propagation.It is reported that litchi can be regenerated in vitro; however, the regeneration is highly genotype-dependent. The frequency of both normal somatic embryogenesis and regenerated plants were all relatively low. Three cultivars,"Feizixiao","Ziniangxi" and "Wuhe" were superior litchi cultivars. The tissue culture of these three cultivars can provide with important information for further studies of genetic improvement for promoting litchi production through establishing in vivo regeneration technologies.The objectives of the study were to examine tissue cultural development of above three litchi cultivars and to establish in vitro regeneration technology system that can provide a technique basis for further genetic improvement of litchi trees. The tissue culturing of "Feizixiao","Ziniangxi"and "Wuhe" were systematically studied in the present studies and the embryonic callus of the three cultivars were induced using the young anther as explant. The regeneration system was successfully established for "Feizixiao" via the somatic embryogenesis pathway. A series of histological observation was performed through the process of the "Feizixiao" somatic embryogenesis and development. The genetic analysis and characteristics of the cell morphology changes and the causes of the formation of malformation embryo were done through the process of somatic embryogenesis, which have established a foundation for future study of litchi cell engineering and genetic transformation. Meanwhile, the study can provide new approaches and conditions for further creating new germplasm resources. The important results of the study are as follows:1. The anther callus induction technique system was established up. Through anther induction in the late uninucleate stage of the three litchi varieties,"Feizixiao","Ziniangxi" and "Wuhe", the callus of different forms were obtained. The highest induction rate reached to92.9%,87.5%and87.5%for "Feizixiao","Ziniangxi" and "Wuhe", respectively. It is suggested that this induction technique system can be used for anther induction of embryonic callus of different varieties of litchi.2. The selection of embryonic callus and transgenerational technology systems were set up. The embryonic callus of color Kelly, granular, grown in good conditions from early generation of callus induction to transgenerational the best combination medium were chosen. Solid-liquid cross training method was adopted in the selection process, then it was subcultured25days interval and transgenered over for3-4times. Thus, a stable line of embryonic callus was obtained. 3. The mature technology system for cv."Feizixiao" was established. The anther fragile embryonic callus was transferred to somatic embryo induction medium; then high frequency somatic embryo induction culture medium formula was screened.4. The systems of embryo cell germination and plant regeneration of cv "Feizixiao" were established. The anther mature somatic embryos were transferred to plant regeneration medium, and then singled out plant regeneration culture medium formula. Plant regeneration rate was up to7.0%of transferred mature somatic embryos.5. The tissue culture seedling transplanting technology system was established. After7days of outdoor hardening, properly developed plantlets were transferred into the pots containing mixtures of peat soils, perlites and coconut chaffs (ratios1:1:1). The seedlings were then transferred into the field when they were growing well.6. The secondary somatic embryo callus induction system was studied. The embryoid, cotyledon, stems and leaves of regenerated plants were used to induce callus. It was demonstrated that the rates of callus induction were strongly different with the organization. While callus forms exhibited obvious difference, the embryoid induction callus worked well. The best embryoid induction culture medium formula was treated with MS,2.4-D2.0mg/L and KT1.0mg/L, adding30g/L Sucrose and7g/L Agar. The induction rate was93.4%.7. The genesis process of somatic embryogenesis from embryogenic callus was observed. The main development process including the globular embryo and various malformations embryo stages of the fragile embryogenic callus were observed using paraffin slice. Nuclear mass ratio of the embryonic callus was low. Embryonic callus cells are relatively small, so its nuclear mass ratio was high. Somatic embryos at each developmental stage were visible in the shape of their procambium. Procambium cell arrangement was close, cytoplasm was thick, dyeing was deep; and nucleuses were big except the cell of other parts. Malformation embryo generally didn’t have the procambium or incomplete development of procambium.8. The embryogenic callus with ploidy was examined. The identification was carried out with flow cytometer. The friable embryogenic callus of "Feizixiao","Zininagxi" and "Wuhe" cultivars litchi were2X、2X and8X. It is concluded that the current studies contribute to understanding litchi callu induction and somatic embryogenesis in plant regeneration and genetic improvement. Further examinations using these in-vivo regeneration technologies are still needed for further genetic improvement of litchi cultivars.