Establishing And Optimization Of Genetic Transformation System In Crambe Abyssinica

Crambe abyssinica belongs to genus Crambe of the Cruciferae family. It has an oil content of34.48%in the seeds and an erucic acid content of55%～62.50%in the seed oil. Crambe has the potential to be developed into an industrial oil crop through genetic engineering since it is self-fertilizing and does not belong to genus Brassica, and thus does not cross with the edible Brassica crops. In this thesis the regeneration and transformation systems of Crambe hypocotyls and cotyledons were established and factors affecting the regeneration and transformation systems were exploited.First the influence of kanamycin on un-transformed Crambe shoots was exploited. More and more untransformed Crambe shoots turned white when the kanamycin concentration was increased. The difference among different kanamycin concentrations were significant after both15days and30days of inoculation. When the untransformed Crambe shoots were cultured on medium with the same kanamycin concentration, more and more shoots turned white when the culture time was extended with exceptions of5mg/L and10mg/L kanamycin. The explanation is that probably these two kanamycin concentrations were too low and some shoots got used to these concentrations. Therefore we suggest that the selection kanamycin should be higher than lOmg/L.Our results indicated that NAA signifficantly affected the hypocotyl regeneration frequency after transformation. The regeneration was the highest when NAA was0.2mg/L, then it decreased when NAA was further increased. The hypocotyl regeneration frequency was increased when ZT was increased from0mg/1to3mg/1and then it decreased when ZT was further increased.8mg/L TDZ gave the highest hypocotyl regeneration frequency. The hypocotyl regeneration frequency was slightly increased when6-BA was increased, but too high6-BA decreased the regeneration dramatically. Statistic analyses indicated that none of ZT, TDZ and6-BA affected the hypocotyl regeneration signifficantly.The length of hypocotyls affected the regeneration frequency signifficantly. The shorter was the hypocotyls, the higher was the regeneration frequency after transformation. Kanamycin signifficantly affected the hypocotyl regeneration frequency. The higher was the kanamycin, the lower was the hypocotyl regeneration frequency after transformation.16mg/L kanamycin was chosen for selection after considering the regeneation frequency and selection efficiency. All the rcgencrants turned to be PCR-positivc after repeated selections The hypocotyl regeneration frequency after transformation was highest when0.5mg/1of AgNO3was used, but statistic analysis indicated that neither AgNO3nor the AS added to the Agrobacteria solution signifficantly affected the regeneration frequency after transformation.The time length of hypocotyl pre-culture signifficantly affected the regeneration frequency after transformation. A pre-culture of2days gave the highest regeneration frequency after transformation. Too long or too short pre-culture decreased the hypocotyl regeneration frequency after transformation. The time length of Agrobacteria infection signifficantly affected the regeneration frequency after transformation. The longer was the infection time, the lower was the regeneration frequency after transformation. The hypocotyl regeneration frequency after transformation was increased when the concentration of AS was increased in the co-cultivation medium. Statistic analyses indicated that none of AS concentration, Agrobacteria concentration, the Cef/Car ratio and pH value signifficantly affected the hypocotyl regeneration frequency after transformation.6-BA signifficantly affected the cotyledon regeneration frequency after transformation. The higher was the6-BA concentration, the lower was the cotyledon regeneration frequency after transformation. The cotyledon regeneration frequency after transformation reached the peak when NAA was increased to1.5mg/l, then it began to decrease when NAA was further increased. Higher TDZ gave a higher cotyledon regeneration frequency after transformation. Statistic analyses indicated that none of NAA, TDZ and ZT signifficantly affected the cotyledon regeneration frequency after transformation.Kanamycin signifficantly affected the cotyledon regeneration frequency after transformation. The cotyledon regeneration frequency after transformation was the highest when nanamycin was8mg/l, then it began to decrease when kanamycin was increased.10mg/L of kanamycin was used for selection after considering the regeneration frequency and selection efficiency.The selection presure was gradually increased after the shoot regeneration. AgNO3signifficantly affected the cotyledon regeneration frequency after transformation. The cotyledon regeneration frequency after transformation reached the peak when AgNO3was increased to lmg/L, then it decreased when AgNO3was further increased. Statistic analyses indicated that none of infection time, the time length of pre-culture and co-cultivation signifficantly affected the cotyledon regeneration frequency after transformation.Most of the kanamycin-tolerant TO Crambe plantlets turned out to be PCR-positive. Southern blot also confirmed that we have got transgenic Crambe.Constructs were introduced into Crambe to increase crude acid and wax content in Crambe seed oil. These materials will be very useful for future Crambe utilization.