Establishment Of Vitro Regeneration System And Transformation Of HAL1 Gene On Ningxia Japonica Rice

Japonica rice in Northwest is one important type of rice in China with the characteric of high yield and elite quility,because of abundant sunshine and large diurnal temperature amplitude,which was benefit for nutrient accumulation.Inspite of small planting area,it is named as arable ecological zone for high quality rice production.For example,rice production in Ningxia ranked top or second among rice production areas in China according average yield/unit of years.In Ningxia,improving the varieties was one of important ways to promote local food production through continuously improving yield and quality.Plant genetic transformation, developed from molecular biology,was a new technologies using directional crops modified and breeding on molecular level.HAL1 gene,acting on balance of Na~+/K~+,was a key gen on salt tolerance from Saccharomyces cerevisiae.Despite it was not a kind of gene transferering protein,it can keep lower Na~+/K~+ with other gene and transferering sistem.So HAL1 gene have great potentiality in the gene engineering of salt tolance.In theoretical,it played important role on using HAL1 gene to transform Ningxia Japonica rice for rice salt-tolerant.Embryo and anther tissues culture are main explants to establish the vitro regeneration system in cereal crops genetic modified.Some reports on using rice mature embryo and anther organizations to establish high frequency rice regeneration system and exogenous genetic transformation can be found on rice,but few on Ningxia Japonica rice.In this study,experiments were conducted on Ningxia Japonica rice material through technology optimization.Firstly,highly efficient and stable mature embryos and anther organizations vitro regeneration system was established.Secondly,agrobacterium tumefaciens-mediated genetic transformation of conditions and the factors affecting were explored and optimized.Thirdly,using of Agrobacterium-mediated transformed HAL1 into the Ningxia Japonica rice,and finally HAL1 Transgenic rice was identified with molecular biology.The result showed as following.1.Mature embryos vitro culture regeneration technology system was established and optimized using Ningxia Japonica rice,and genotype of better culture effects were selected. The resulted showed that effects of mature embryo culture was vastly differenence among 34 varieties of Ningxia Japonica rice.Callus induction rate was between 16.0%to 87.3%,green plantlet differentiation rate was between 0%to 68.9%.Mature embryos of Youyu28 performed the best effect culture,with callus induction rate and green plantlet differentiation rate were 87.3%and 65.6%,separately.Effect culture of Ningjing24 and Ningjing34 were also good.Different genotypes had different culture abilities in N6 culture medium and NB culture medium.Using N6 culture,the addition of 2mg/L 2,4-D to induction medium was favorable for effect of culture.Adding 0.2mg/L-0.5mg/L 6-BA can improve mature embryo culture ability.Sucrose and Maltose were higher than Glucose to induce and differentiate callus.And 30-40g/L was the best for proportion of sucrose to induce and differentiate callus.Using Ms culture medium,adding KT2mg/L+NAA1mg/L+IAA0.5mg/L was better for green plantlet differentiation of Ningxia Japonia rice mature embryo callus.Subculture 1-2 times,green plantlet differentiation of callus was no significant impact.Subculture more than 3 times,callus become gloomy,dull,and growing slow down,green plantlet differentiation of callus was significantly deteriorate.Overall,in Ningxia Japonica rice mature embryos vitro regeneration,best genotype was Youyu28,best induction culture medium is N6+2,4-D 2mg/L+6-BA 0.2-0.5mg/L+hydrolyzed casein protein 0.5g/L+Proline 2.8g/L+ sucrose 30g/L+Agarose powder 6g/L(pH5.8),best green plantlet differentiation is Ms+KT 2mg/L+IAA 1mg/L+NAA 0.5mg/L+hydrolyzed casein protein 0.5g/L+sucrose 30g/L+ Agarose powder 6g/L(pH5.8).2.Established and optimized anther vitro regenetation technology system of Ningxia Japonica rice.The resulted showed that anther of 3279 and Hua107 were high culture ability among 22 Ningxia Japonica rice cultivars.Effects of culture medium showed that A4 culture medium was better than N6 culture medium.Different genotypes had different culture abilities for concentration of 2,4-D,the extent was 1-3mg/L.Sucrose was better to induce callus using anther,and 60 g/L was the best proportion.Effects of anther culture in greenhouse cultivation is better than in field cultivation.Low-temperature pretreated about 8d had the best effect culture.So in anther vitro regeneration system of Ningxia Japonica rice, plant 3279 and Hual07 had better culture effect in greenhouse,and put anther in 4℃refrigerator pretreated 8d.Induction medium is N6+2,4-D 1-3mg/L+KT 0.5mg/L+ hydrolyzed casein protein 0.5g/L+Proline 0.5g/L+sucrose 60g/L+gluose powder 6g/L (pH5.8).Green plantlet differentiation medium was MS+KT 2.0mg/L+NAA 1mg/L+ IAA 0.5mg/L+sucrose 30g/L+gluose powder 6g/L(pH5.8).3.The Agrobacterium-mediated genetic transformation technology system was established and optimized using mature embryo callus of Ningxiat Japonica rice.Using Youyu28 mature embryo callus as transformed receptor,the effects of agrobacterium-mediated genetic transformation were studied on influence of Kan-resistant concentration,Cb concentration,co-culture time,washing agrobacterium,drying treatment etc.The results showed that 250mg/L was Kan-resistant concentration.Concentration of Cb more than 600mg/L inhibited the growth of the callus significantly.Co-culture 3d can obtain the most resistant callus.Using sterile water added Cb and Cef 500mg/L separately can inhibit agrobacterium perfectly,the resistant callus rate was the highest.Before inoculating filtration medium,drying treatment can inhibit the growth and reproduction of Agrobacterium tumefaciens effectively,and improve resistant rate of callus.4.Through the establishment of vitro regeneration system and research on HAL1 genetic transformation,genetic transformation system was established firstly using Ningxia Japonica rice.Using Youyu28 mature embryo callus as transformed receptor,by Agrobacterium tumefaciens and Kan resistance filtrating,69 plants resistant to Kan were obtained.The results of PCR amplification showed that 3 transformants obtained with the special bands at 879bp.Initial proof that HAL1 gene had being integrated into the genome of northwest Japonica rice Youyu28.