Agrobacterium Mediated Transformation Of Lycopersicon Esculentum And Achyranthes Bidentata And In Vitro Plant Regeneration Of Zygophyllum Xanthoxylon

Ⅰ Transformed hairy roots were produced from the stem and leaf explants of Achyranthes bidentata BL. after infect by Agrobacterium rhizogenes strains A4, 9402Binl9 and ATCC15834 respectively.The best effective strains were screened.The transformation frequencies of three strains were quite different.The highest frequency, i.e 60%~80%,was obtained from ATCC15834;the following one(40%~55%) from A4,and 9402Binl9(30%~40%).The transformation frequency from leaf explants was higher than that from stem explants.The hairy roots transformed by ATCC15834 grew rapidly on MS medium without any phytohormones, and were characterized by strongly branching,clustering and non-geotropism,etc. The highest growth rate of hairy roots was observed on MS liquid medium (shaked at 110r/min at 25 ℃ in darkness) ,and their fresh weight was increased by 8.4 times after culturing for 25 days.Determined by HPLC,the content of oleanolic acid in hairy root clone w02 was found to be 25.59mg/g, higher than that in 1-year-old cultivated Achyranthes bidentata(14.73 mg/g). Mannopine was detected in the hairy roots of Achyranthes bidentata by the high-voltage paper electrophoresis.The reaction of PCR proved that the hairy roots were transformed By the strain ATCC15834 and the T-DNA of Ri plasmid had been integrated into the DNA of hairy roots.When the hairy roots transformed by ATCC 15834 were cultured for two weeks on MS medium supplemented with 1.5mgL~-1 2,4-D and 0.5 mgL~-1 6-BA , calli were induced. After transferred on the MS-medium added with 1.0mgL~-1 6-BA and 0.5mgL~-1 NAA, a large number of buds were induced from calli. All of these buds could grow further.The buds were rooted on the MS-medium containing l.0mgL~-1IBA.and then developed into plantlets.II In vitro plant regeneration system was established from the leaf and stem segments of Lycopersicon esculentum Mill, and was used for transformation. Lycopersicon esculentum was transformed via Agrobacterium twnefaciens(LBA4404)harboring plasmids carrying genes of arresten.Transformants were obtained in the experiment. It was demonstrated that arresten gene had been inserted into Lycopersicon esculentum genome by PCR and RT-PCR analysis.Factors influencing transformation were studied. Genetic transformation system of Lycopersicon esculentum mediated by Agrobacterium tumefaciens was built. Ill An efficient regeneration system of Zygophyllum xanthoxylon Maxim was established from leaf base segments via tissue culture. MS medium supplemented with 2mgL"' 2,4-D and 0.2mgL~! 6BA was suitable for calli induction. A large number of shoots were induced from calli on the MS-medium added with lmgL'1 6BA and 0.5mgL"! NAA. Roots were effectively induced on the MS-medium containing lmgL"1 KT. The stem segments of regenerated shoots directly rooted on the MS-medium containing lmgL'1 KT. The regenerated plantlets were transplanted into soil, and over 90 % of them were survival. The number of chromosome of the regenerated plantlets checked was the same as the normal plant,i.e 2n=22; However RAPD analysis indicated that one special DNA fragment about 800bp was amplified in regenerated shoots.This showed that exogenous phytohormone might influence the genetic structure of Zygophyllum xanthoxylon in the course of plant tissuse culture. The effects of main elements, including concentration of Mg2+, dNTPs , DNA, primer and Taq polymerase dosage on RAPD amplification were studied through single factor experiment. A following efficient protocol for optimal reaction system of RAPD analysis in Zygophyllum xanthoxylon was established , i.e , the 25 ul reaction mixture contained 1.5—2.5 mmolL"1 Mg2+, 0.2—0.3 mmolL'1 of each dNTPs, 1.2 umolL"1 arbitrary primer , 2.0U Taq polymerase, 50ng template DNA.