Studies On Chromosomal Karyotype And Transformation Of Sunflower

Carotenoids,a kind of nutrition elements have various health benefits such as improving human's immunity, retarding the ageing process of organs, preventing cancer, reducing the risks of nyctalopia and infant amblyopia for lack of vitamin A.Human blood contains lycopene, xanthophyl,β-cryptoxanthin,α-carotene and other carotenoids although human body can not synthesize those, and take them from food. Fruit and vegetable, is the main source of carotenoids. Carotenoids are lipophilic soluble, which can't be absorbed lack of lipid.β- carotene in albumen of"the golden rice"have gained a higher content, whereas, how much can be taken need a further research. Additionally, it is hard and poisonous to synthesize by chemical method, by which only a few kinds of carotenoids obtained and commercialized, such asβ- carotene, astaxanthin and canthaxanthin etc. In another way, we gotβ- carotene and astaxanthin by fermentation.Karyotype analysis is a basic method for investigating the cell genetics, chromosome engineering, gene location, cytotaxonomy and modern evolution theory. It also provides cytology proof for cytotaxonomy breeding, selecting parents of hybrid Combination and cytology test of trans-genes sunflower. Carotenoids synthase genes were applied to transformation some plants such as tobacco, cole, tomato and rice. Although it is usually called as golden rice and golden cole for higher carotenoid content by genetic transformation, no related research and report about sunflower was found. Sunflower, a kind of annual herbaceous plant belongs to composite family, which bears character of leanness, salt and drought resistance. Seed of Sunflower, the main source of protein and plant lipid, is one of the important biomass energy. Sunflower plays an important role in supplying lipid and protein and improving food structure as well as promoting development of animal husbandry and processing industry. Scientists aim to breed new breeds with high yield and good quality by modern biotechnology, whereas, sunflower is a generally acknowledged recalcitrant crop for transformation. So far no efficient and stable transformation system was set up because of frequency of plant regeneration.Researchers have attached importance to utilizing tissue culture and gene engineering to meliorate quality of sunflower since the 1980s, but most research focus on improving the method of plant regeneration and transformation as well as on promoting transformation efficiency. No satisfying result was reported, except single or multi-genes transformed sunflower and very few characters were altered.There will be significant economy and society benefit for health promotion and promoting the quality of sunflower through transformation of carotenoids synthase gene to enhance the carotenoids content.Karyotype analysis of eight genotypes of sunflower showed that chromosome numbers in the eight sunflowers were 2n = 2x = 34. All the chromosomes were median and submedian centromeric. Their karyotype formulas were F53: 2n=2x=34=24m+10sm,2603: 2n=2x=34=24m+10sm,2736: 2n=2x=34=26m+8sm,89B1: 2n=2x=34=22m+12sm,HA300: 2n=2x=34=28m+6sm,B7: 2n=2x=34=22m+ 12sm,RHA266: 2n=2x=34=30m+2sm+2st,PR29: 2n=2x=34=30m+4sm;The eight genotypes of sunflowers shared two karyotypes (1B and 2B), which indicated there were some karyotypes diversity within different genotypes of sunflowers.The regeneration frequency of induced bud with different explants in different culture medium was further researched by taking different genotypes sunflower as material. The result showed that, it is easy to form the callus that explants of different genotype sunflower in the culture medium which contain suitable endocrine such as IAA,6-BA and so on, but the possibility that callus forms bud is slim. The frequencies of induced bud which explants of different genotype sunflower are in turn as followed: cotyledenary node > hypocotyls> cotyledon> euphylla; the suitable concentration of 6-BA to induce the cotyledenary node to generate the bud is 1.2mg/l, hypocotyls is 1.8mg/l, cotyledon is 0.6mg/l, euphylla hasn't been induced to form the bud. The capability of induced adventitious bud is very different with different genotypes of sunflower. The regenerated frequency of PR29 showed to be the highest.The young embryos of different genotype sunflower were also used as material. The rule of somatic embryogenesis was studied preliminarily, including the size of young embryo, the concentration of sucrose, carbohydrate, endocrine (2,4-D and 6-BA). The results showed that somatic embryogenesis was induced on callus or between or marginal of two cotyledons, but the most be on one side of the cotyledon in line. The somatic embryogenesis frequency showed the highest as sucrose (120 g/L) added into the medium. When the size of young embryo is less than 2mm, the frequency of the embryo to embryogenesis was suitable (45.5%). The optimum culture medium for somatic embryogenesis is Ms+0.4mg/l+2,4-D+120 mg/l sucrose. Except that, the somatic embryogenesis frequency varied with genotypes, and RHA266 gained the highest (38.4%), on the contrary 2736 got the lowest(10.1%).Cotyledonary nodes of three different genotypes of sunflowers (RHA266, PR29 and 2603) were transformed with Agrobracterium tumefaciens EHA101 carrying the PSY gene. Meanwhile the factors affecting transformation were studied. Transgenic sunflower's plants were examined by PCR and Southern hybridiazation analysis. The results as follows: The Optimum concentration of Hyg is 8mg/L. The time of co-culture in 3 days might be suitable. Eight minutes is suitable for infection. The optimum range of OD600 is 0.6. PCR and PCR-Southern blot showed that the target LycB gene integrated into the genome of sunflower.Carotenoid biosynthesis enzyme gene (LycB) was transformed to 3 genotypes of sunflowers via pollen tube pathway technique. The results showed that stigma method affecting seed operated in 2~4 hrs pollinated, and 4hr later, no effect was observed. Meanwhile, times of operation had slim effect on seed. Ovary injection method gave more effect on seed, which brought a very low seed ratio in 2hrs, after that gained height in another 2hrs, whereas, seed ratio got no strong promotion in 8hrs. 40 mg/L kanamycin is the best density to select the positive via pollen tube pathway technique. Additionally, different induction methods gained distinguished positive ratio, that is to say, ovary injection method gave higher setting rate than stigma. The LycB gene has integrated into genome of sunflower testified by the analysis of Southern blot.