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Studies on Transformation of Arabidopsis Thaliana rd29A Gene of Medicago Sativa by Agrobacterium Tumefaciens and Explore of Regeneration Technology of Salix Viminalis L

Author: ZhangTianYu
Tutor: ZhangJunLian；XiaYang
School: Gansu Agricultural University
Course: Olericulture
Keywords: Alfalfa Agrobacterium-mediated transformation system willow tissue culture regeneration system
CLC: S792.12
Type: Master's thesis
Year: 2007
Downloads: 235
Quote: 1
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Abstract


Saline soil is wildspread in china. the development of saline soil is of significant with the increasing population and decreasing arable land.The development and application of gene engineering creates a new path for crop salt-resistant breeding and becomes a mean of economy and effective for improving saline soil.Cultivated alfalfa,perennial legume crop,is wildly planted in the world.It is a very good forage,more over people like it .Willow is excellent tree for garden virescence and zoology,meanwhile there is important use in other ways.people perpetrated many work in routine breeding of crop、forest and hasty and obtaid many fruit.But its cycle is longer and the load is hard ,so the development and application of gene engineering creates a new path for alfalfa and willow salt-resistant breeding.In this research, Studies on Transformation of Gene rd29A of Medicago.sativa Zhongmuyihao by Agrobacterium tumefaciens and achived a new transgenic plant;Studies on Establishment of organise culture Regeneration System of asepsis willow Q106 by choice of every effective factor for transformation willow. The main results were as following:1.Optimization of medicago’s genetic transformation and the transgenic plants medicago were obtained Agrobacterium culture stay over in YEB medium containing 50mg/L Kan.A liquid culture of A.tumefaciens was centrifuged and the pellet was resuspended in liquid MC medium contained AS10 mg/L to the OD600 of 0.3～0.5.The leaves cultivated 15 days make wound in the A.tumefaciens prompt(exsect stipe and transversely two bistoury in the lamina).The injured leaves are cultured and shaked 30 minutes in the A.tumefaciens. The excrescent A.tumefaciens on the lamina is blotted by the asepsis filter paper. The laminas were transferred and cultured on MC medium(pH=6.0, AS10 mg/L).After co-cultured 3 days, the laminas were bathed by the asepsis water and transferred onto the MC.They were choice cultured on the MC(PPT2 mg/L, Cef 400 mg/L).After 50～60 days,PPT was removed and reduced Cef concentration to 200 mg/L.The fastness embryogenesis cullus were transferred and cultured onto the MSO medium.In the 1 month ,the fastness plants were obtaided.After 3～4 era,principium proved,eight PPT-resistant plants are the transformation rd29A gene plant by PCR analysis.2 Regeneration tissue culture system establishment of willow Q106 The effect of callus inducing is fine that the willow stem is cultured on MS +6-BA 1.0 mg /L + NAA0.5 mg/L +sucrose30 g/L in the dark,after 20 days ,cultured in the light. The effect of cluster shoot differentiation is fine that the willow armpit bud stem is cultured MS+KT3.0mg/L+NAA0.2 mg/L +sugrose20 % medium. It is fine to cultivate haleness seeding on MS+6-BA 0.1 mg/L+NAA 0.2 mg/L+sucrose30% medium. It is fine to rhizogenesis inducement on MS+BA0.01 mg/L+NAA0.2 mg/L + +sucrose30% medium. Selected haleness seeding transplant after domestication seeding 15～20 days ,its survival frequency reach over 98%.

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CLC: > Agricultural Sciences > Forestry > Forest tree species > Broad-leaved trees > Willow
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