Somatic Embryogenesis And Genetic Fidelity And Tetraploid Regeneration In Vitis Vinifera L.

Grapevine (Vitis vinifera L.) is the most widely cultivated fruit crop. Due to the complicated genetic background and the long breeding cycles of hybrid breeding in grapevine, the conventional breeding was extremely restricted in the improvement of grapevine varieties. But with the development of biotechnology, somaclonal variation breeding and genetic transformation using somatic embryos make feasible in grapevine varieties improvement and rapid germplasm propagation. Therefore, it is a prerequisite to establish a stable high-frequency system of somatic embryogenesis and plant regeneration by using somatic cell, tissues, and organ as explants. At present, successful somatic embryogenesis and plant regeneration system has been ducumented in several grapevine species and cultivars. However, low production efficiency of regeneated plants and long-term cycles of somatic embryogenesis are often associated with these methods. Information about factors affecting somatic embryogenesis, morphological and histological aspects of somatic embryos and genetic stability of plants regenerated from somatic embryos is rather scarce. And only few examples of somaclonal variation breeding have been reported in grapevines. Therefore, it is very important to establish a stable high-frequency system of somatic embryogenesis in grapevine varieties improvement using somaclonal variation such as polyploidy production and genetic transformation via somatic embryos, in vitro propagation and preservation of germplasm etc.In this thesis, based on our research results and the published reports, the development in some very important aspects in plant somatic embryogenesis such as concept, characteristics, uses for crop, factors crucial for induction of somatic embryos, conversion into plant, physiological and biochemical changes, chimerism, genetic stability, embryo gene expression and molecular markers for somaclonal variation in the process of somatic embryogenesis was reviewed. Based on previous reports in somatic embryogenesis with particular reference to Vitis vinifera L., this study carries on research from three respects including establishment of somatic embryogenesis and plant regeneration system, analysis of genetic fidelity in somatic embryogenesis and tetraploidy production from diploid somatic embyos. The study's main findings and conclusions are as follows:(1) Establishment of somatic embryogenesis and morphological and histological studies on somatic embryosIn order to obtain a reproducible plant regeneration system that allows the application of biotechnological tools to grapevine breeding, somatic embryogenesis and plant regeneration were induced and successfully established on Nitsch and Nitsch (NN) medium from immature zygotic embryos of of grapevine (Vitis vinifera L.) by controlling PGRs, light, temperature and other factors affecting embryos production. The optimum hormone combinations were 1.0mg/L 2,4-D for primary callus induction at 28°C in the dark and 1.0mg/L NAA+0.5mg/L BA for embryos production and 0.03mg/L NAA + 0.5mg/L BA for embryos conversion and plant regeneration at a temperature of 25±2°C with an irradiance of 50μmol/m~2s provided by cool white fluorescent lamps for 16-h photoperiod. The efficiency on induction of embryogenic callus ranged from 65.8% to 85.4%, and the frequency of somatic embryogenesis varied from 10.5% to 37.5% among six genotypes. 15.5-42.1% of somatic embryos converted into normal plantlets. 5°C and 500Lx conditions will be suitable for the preservation of somatic embryos.The observation of morphology and ultrastructure of somatic embryo showed that two different callus, embryogenic and non-embryogenic, can be obtained from immature zygotic embryos. Compared to non-embryogenic callus, some changes such as regular cell wall, big nuclear, concentrated cytoplasm, cytoplasmic membrane close to cell wall, plasmodesmata and lots of starch grains in somatic cells were observed. Somatic embryos originated from mononuclear cells of embryogenic callus surface. Histological observations reveal that the somatic embryos successively differentiate through spherical embryo, pear-shaped embryo, heart-shaped embryo and cotyledon five typical developmental stages, and gradually developed into normal plants. At same time, there are several abnormal embryos including joined embryo, abnormal-bud embryo and hyperhydric embryo, etc. in the induction stage of somatic embryos. The studies on somatic embryo conversion and plant regeneration showed that the formation of chlorophyll and cotyledons is signs of somatic embryo conversion. And, too big or small embryos are not converted well to normal plants. In parallel with embryo germination, Secondary embryogenesis and process of embryo development progress-not synchronized were also frequently observed. Therefore further studies are needed to optimize the condition in somatic embryogenesis and plant regeneration for increasing percentage of somatic embryo conversion into plantlets and avoiding to abnormal embryos.(2) Monitoring of the genetic fidelity of regenerated plantsThe analysis of DNA content determined by flow cytometry and chromosome counting of the plantlets regenerated from somatic embryos derived from a zygotic embryo clearly indicated that no ploidy level changes were induced during somatic embryogenesis and plant regeneration established on Nitsch and Nitsch (NN) medium from immature zygotic embryos, the nuclear DNA content and ploidy levels of the regenerated plants were stable and homogeneous to those of the donor plants. No significant differences (P<0.05) were detected between diploid and parental field plants. The rate of normal diploid cells with 2C DNA of donor parent and regenerated plants accounted for 78.8% and 83.5%, of the total number of monitoring cells. The ratio of the number of tetraploid cells to diploid cells is 1.960 and 1.963 respectively, i.e. about 2. And, 12.9% and 16.5% tetraploid cells with 4C DNA were found in donor and regenerated plants. However, chromosome doubling can not be attributed to genetic variation, but to the cell division cycle.In order to further confirm genetic fidelity of plants regenerated from somatic embryos at molecular level, the quality of 14 regenerated plants (cv. Sinsaut) was screened with the 38 random RAPD primers. The results were scored as patterns of bands obtained from the regenerated plants and compared with the donor plants maintained in the field. Out of 38 random primers tested, eight primers that produced distinct amplification profiles, displayed same banding pattern in all plants as the DNA sample from donor plant. The total number of amplification products generated by polymerase chain reaction was 46 bands (5.8 bands per primer). The size of polymorphic fragments with eight primers varied from 150 in S20 to 1200 bp in S37 and the number of amplified products ranged from 3 in S20 to 7 in S37. These results confirmed the genetic fidelity of the grapevine plants regenerated from somatic embryos. We conclude that somaclonal variation is almost absent in our grapevine plant regeneration system. Of course, it is not enough to determine genetic stability of regenerated plants only from chromosome number, DNA content and molecular level. In the future, further studies are needed to analysis genetic stability of obtained plants from phenotype, physiological and biochemical, and agronomic traits in field.(3) Tetraploid regeneration from diploid somatic embyosTetraploids often generate variants that may contain favorable horticultural characteristics, such as a large fruit size, sturdiness and high productivity. Most breeders focus on tetraploids production in grapevine varieties improvement. Traditionally, induced tetraploid plant was performed on tissue, organs or whole plants in a colchicine solution. However, low production efficiency of polyploid plants and a high frequency of chimeras are often associated with this method. A protocol for in vitro induction of tetraploids via colchicine-treated somatic embryos from immature zygotic embryos of diploid grapevine (Vitis vinifera L.) is reported. A factorial experiment, with a control and two concentrations of colchicine, 0, 10, 20mg/L, and three durations of treatment, 1, 2, or 3 days, was conducted. The explants selected for colchicine treatment were vigorously growing somatic embryos of small size (about 2mm) taken from embryogenic callus lines known to have retained their regenerable capacity and from which shoots are routinely regenerated into whole plantlets. Colchicine dissolved in 1% dimethylsulphoxide was filter-sterilized and added to autoclaved liquid NN medium supplemented with 0.03mg/L NAA and 0.5mg/L BA, and shaken (60rpm) at 25°C in darkness.The survival and germination of the somatic embryos after colchicine treatment were dependent on colchicine concentration and the treatment duration. The effects of colchicine on embryo germination were similar to those observed in embryos survival. As for embryos survival, the number of germinated embryos decreased with increasing colchicine concentration. At 0mg/L (the control), 10mg/L and 20mg/L colchicine concentrations, the total percent of embryo germination were 18.7%, 12.0%, and 7.3%, respectively. Shoot formation following colchicine treatment was in general slow. Among 29 randomly investigated plantlets regenerated from colchicine-treated somatic embryos, five solid tetraploids (2n=4x=76) were identified by flow cytometry and chromosome counting analysis; all others were diploid (2n=2x=38). No chimeras with both 2C and 4C nuclei was produced. The best induced results that four tetraploids were obtained at a frequency of 2.7% by using 20mg/L colchicine for 1, 2, and 3d, and one tetraploid obtained at a frequency of 0.7% by using 10mg/L colchicines for 3d.Significant differences in leaf stomata parameters were observed between diploid and induced tetraploid plantlets. The stomata sizes of tetraploid plants were significantly larger than those of diploid plants, while the frequency of stomata was reduced significantly. Similarly, the chloroplast number of guard cells of tetraploid plants increased significantly. The average size of stomata in diploid plantlets was 26.2±0.3×18.4±0.4μm (length×width), and that of tetraploid plantlet was 36.6±0.5×26.6±0.7μm. The average density of stomata in tetraploid plantlets was significantly lower than that of diploid plantlets. Simple correlation coefficients were computed between stomata size, stomata density and ploidy level. Significantly positive correlation was observed between stomata size and ploidy level, indicating that the increase in stomata size may relate directly to the increase in ploidy level. Furthermore, a significant negative correlation was recorded between stomata density and ploidy level, indicating that the decrease in stomata density may result from increased ploidy level. Highly significant correlations between ploidy level and stomata parameters were confirmed. These stomata parameters information could be strong indicators of change in ploidy level.In summary, a protocol for reproducible and stable somatic embryogenesis and plant regeneration in grapevine (Vitis vinifera L.) is established. Fundamental differences on ultrastructure of the cells between embryo and non-embryogenic callus in somatic embryogenesis were confirmed. Somatic embryos, originated from mononuclear cell from epidermis of embryogenic callus, differentiated and developed step by step through five typical developmental stages similarly to zygotic embryos. The analysis of DNA content determined by flow cytometry and chromosome counting, and RAPD markers clearly indicated that no significant differences were detected between the plantlets regenerated from somatic embryos and the donor field plants. Our results also showed that it is feasible to produce artificial solid tetraploids in grapevine by colchicine treated somatic embryos from immature zygotic embryo callus. Undoubtedly, the results and conclusion obtained in this study are very important in technology platform for further more studies in theoretical and applied research including genetic transformation, germplasm improvement and preservation etc. in grapevine.