农杆菌介导的拟南芥rd29A基因转化苜蓿的研究及柳树组织培养再生技术初探
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摘要
我国盐渍化土壤分布广泛,大量土地荒芜,盐碱地资源的开发利用是我国增加农业可利用土地资源的需要,也是改善生态环境、增加绿色植被、提高森林覆盖率的需要。现代生物技术的发展为培育耐盐的作物品种提供了新方法和途径,已经成为改良盐碱地经济而有效的手段。紫花苜蓿(Medicago sativa L.)是世界上种植面积最大,应用最广泛的多年生豆科作物,不仅作为很好饲料而且现在人们将它作为美味的菜肴使用。柳树(Salix viminalis L.)是优良的园林绿化和生态树种,同时在很多方面有重要的用途。目前,人们在农作物和林草常规育种方面做了大量工作,取得了很多成果,但周期长,工作量大,因此基因工程的发展和应用为苜蓿和林木抗盐育种开辟了一条新的途径。本研究以中苜一号为材料,进行农杆菌介导rd29A基因转化体系的优化研究,获得了转基因新株系;以柳树Q106无菌苗为材料,通过对各种影响因素的筛选,初步建立了组培再生体系,为柳树基因转化奠定基础。目前取得的主要研究结果如下:
1、优化了苜蓿转基因体系,获得了转rd29A基因植株
农杆菌用含有50mg/L Kan的YEB培养基培养过夜,离心后用MC液体培养基重悬,同时在MC液体培养基中加入AS10 mg/L,调整菌液OD600值为0.3～0.5,作为受体侵染菌液;取继代培养15天的叶片,在菌液中迅速制造伤口(切去叶柄,然后在叶面上横切两刀),将致伤的叶片在菌液中摇动培养,侵染30分钟,无菌滤纸吸干叶片上多余菌液,转到pH值为6.0、并加有10mg/L AS的共培培养基MC上;共培养3天至叶片周围有淡而透亮的菌斑出现,用无菌水洗去叶片上的农杆菌,接到MC选择培养基上进行筛选(MC培养基加入PPT 2mg/L+Cef 400 mg/L),选择50～60天后撤掉PPT,同时降低Cef浓度至200 mg/L,将抗性胚性愈伤组织转到MSO培养基上进行分化诱导,一个月后获得抗性植株。继代3～4代后,经过PCR检测,初步证明有8株PPT抗性再生植株为转rd29A基因植株。
2、柳树Q106组织培养再生体系初探
以柳树Q106茎段作为外植体, MS + 6-BA 1.0 mg /L + NAA0.5 mg/L +蔗糖30 g/L为基本培养基,暗培养20天,然后转到光下培养,诱导愈伤组织效果好。以带腋芽茎段为外植体,在MS+KT 3.0mg/L+NAA 0.2 mg/L +蔗糖20 g/L培养基上,丛生芽分化效果好。采用MS+6-BA 0.1 mg/L+NAA 0.2 mg/L+蔗糖30 g/L培养基培养健壮苗较好,最佳生根培养基为MS+6-BA 0.01 mg/L+NAA 0.2 mg/L +蔗糖30g/L。选取健壮苗移栽,经驯化炼苗15-20天后,大田成活率可达98%。
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%.
1、优化了苜蓿转基因体系,获得了转rd29A基因植株
农杆菌用含有50mg/L Kan的YEB培养基培养过夜,离心后用MC液体培养基重悬,同时在MC液体培养基中加入AS10 mg/L,调整菌液OD600值为0.3～0.5,作为受体侵染菌液;取继代培养15天的叶片,在菌液中迅速制造伤口(切去叶柄,然后在叶面上横切两刀),将致伤的叶片在菌液中摇动培养,侵染30分钟,无菌滤纸吸干叶片上多余菌液,转到pH值为6.0、并加有10mg/L AS的共培培养基MC上;共培养3天至叶片周围有淡而透亮的菌斑出现,用无菌水洗去叶片上的农杆菌,接到MC选择培养基上进行筛选(MC培养基加入PPT 2mg/L+Cef 400 mg/L),选择50～60天后撤掉PPT,同时降低Cef浓度至200 mg/L,将抗性胚性愈伤组织转到MSO培养基上进行分化诱导,一个月后获得抗性植株。继代3～4代后,经过PCR检测,初步证明有8株PPT抗性再生植株为转rd29A基因植株。
2、柳树Q106组织培养再生体系初探
以柳树Q106茎段作为外植体, MS + 6-BA 1.0 mg /L + NAA0.5 mg/L +蔗糖30 g/L为基本培养基,暗培养20天,然后转到光下培养,诱导愈伤组织效果好。以带腋芽茎段为外植体,在MS+KT 3.0mg/L+NAA 0.2 mg/L +蔗糖20 g/L培养基上,丛生芽分化效果好。采用MS+6-BA 0.1 mg/L+NAA 0.2 mg/L+蔗糖30 g/L培养基培养健壮苗较好,最佳生根培养基为MS+6-BA 0.01 mg/L+NAA 0.2 mg/L +蔗糖30g/L。选取健壮苗移栽,经驯化炼苗15-20天后,大田成活率可达98%。
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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