根癌农杆菌介导转化诸葛菜获得转基因植株

以诸葛菜下胚轴和子叶为材料,在附加BA和NAA的MS培养基上诱导芽再生,在1／2MS培养基上诱导生根,获得完整再生植株,建立了诸葛菜组织培养高频再生体系,再用很癌农杆菌介导转化诸葛菜下胚轴和子叶,在附加一定量的氨苄青霉素、头孢霉素和卡那霉素的相应培养基上进行筛选,并培养再生成苗,获得完整抗性再生植株,移植到盛有土壤的花盆中均可存活,生长正常。将再生植株叶片,进行GUS、NPTⅡ酶活性测定和Southernblot分子杂交,证实外源基因已稳定整合到植物基因组中,并高效表达。     

番茄ACC合酶反义基因对河套蜜瓜的转化

河套蜜瓜（CucumismeloLcvHetau）的子叶经预培养。芽诱导和生根培养,获得再生小植株,诱导率达58％。取带有番茄ACC合酶反义基因的双元载体pMQ6／JM109与农杆菌（Agrobacteriumtumefaciens）LBA4404经三亲融合后,与在MS0上萌发5d、并在MS＋1mg／LNAA培养基上预培养3d的子叶共培养48h,然后转入含50mg／L卡那霉素的MS＋6mg／LZT的芽诱导培养基中,1l个月后诱导生芽,待芽长1．5－2cm时转入生根培养基中,1－2周后可诱导产生大量的根,形成完整的转基因小植株。经PCR和分子杂交检测证明,目的基因已整合入河套蜜瓜的基因组中。     

药用植物金荞麦愈伤组织诱导与植株再生

目前转基因技术已成为植物定向遗传改良的重要手段,而建立稳定高频的离体再生系统是实现遗传转化的基础和前提.本试验以25 ～30 d苗龄的金养麦(Fagopyrum dibotrys)无菌苗叶片、茎节间、叶柄为外植体进行愈伤组织诱导与植株再生研究.结果表明:叶片在MS +2,4-D 4.0 mg/L +6-BA 1.0 mg/L培养基上愈伤组织诱导率达到89％.茎节间在MS +2,4-D 2.0 mg/L +6-BA 2.0 mg/L培养基上愈伤组织诱导率为87％.叶柄在MS +2,4-D 4.0 mg/L +6-BA 2.0 mg/L+ IBA 0.2 mg/L培养基上的最高诱导率仅为54％.愈伤组织分化不定芽的适宜培养基为MS +6- BA2.0 mg/L +TDZ0.2 mg/L +NAA0.2 mg/L;金荞麦不定芽在1/2 MS +NAA 0.5 mg/L的培养基上生根效果最好.组培再生植株经炼苗后移栽到田间成活率达80％以上,且生长表现正常.高频完整再生体系的建立,为金荞麦进一步遗传操作和扩大药材资源奠定了基础.     

荞麦组织培养及高频植株再生体系的建立

通过对荞麦(Fagopyrum esculentum Moench)不同外植体、不同激素配比的比较研究,建立了荞麦离体培养高效植株再生体系。荞麦子叶切段在含2．0 mg/L 2,4-D和1．0 mg/L 6-BA的MS培养基上愈伤组织诱导率为89．6％,而下胚轴切段在含2．0 mg/L 2,4-D和1．0-2．0 mg/L 6-BA MS培养基上愈伤组织诱导率高达100％。在2．0 mg／L 6-BA、0．1 mg,L IAA和1 mg／L KT的MS培养基上通过愈伤组织间接分化或外植体直接分化形成不定芽。来自子叶和下胚轴的愈伤组织的分化率分别为42．5％和73．6％,下胚轴的分化率明显高于子叶。将生长状态良好的不定芽转至含1．0 mg/L IBA和0．5mg/L NAA的1/2 MS培养基上生根,生根率达到100％。再生植株移栽到盆土中,成活率达91．6％,并且生长状态和特征均表现正常。     

根癌农杆菌对健康和患丛枝病泡桐的遗传转化

选取健康及患丛枝病泡桐（Paulownia spp.)为材料,建立组织培养和植株再生系统,以茎段作为转化受体,诱导分化和生根的最佳激素组合分别是MS＋BA4mg/L NAA0.2mg/L和1／2MS＋KT0.5mg/L IBA0.25mg/L。芽分化频率可达22．8％。健康和患病泡桐的茎段经农杆菌共培养3d后,在附加50mg/Lm的选择分化培养基上培养20d左右再生出抗性芽,经培养、诱导生根,获得了转基因再生植株,建立了泡桐的遗传转化体系。PCR和Southern杂交检测证明外源基因已整合到泡桐的基因组,标记基因ITPⅡ在再生植株中也得到表达,同时对影响转化的一因素进行了探讨。     

高效诱导甜菜再生植株的研究

研究了栽培甜菜(Beta vulgaris L.)4倍体品系405叶柄外植体的离体培养。成功地建立了一套高频率诱导再生芽的程序。外植体取自生长在改良MS(MSB)附加BA和NAA或者单加BA的培养基中。经过30d以上预培养后的幼苗叶柄,在MS附加BA 1．0mg／L或NAA0．3mg／L,Bal．0mg／L培养基上直接诱导再生芽,并发育成苗．诱导频率最高可达51．3％。在1／2MS(MS培养基大量元素减半)附加NAA0．5～1．0mg／L的培养基上诱导生根．这一程序为甜菜扩大繁殖和遗传转化提供了一个良好的试验系统。     

影响农杆菌介导的大豆子叶节遗传转化的因素

利用携带pCAMBIA1301质粒（含hpt和gus基因）的超毒根癌农杆菌菌株EHA105对大豆子叶节外植体进行遗传转化,研究了影响农杆菌介导的大豆子叶节遗传转化的因素。研究结果表明．农杆菌侵染液和共培养培养基中添加200μmok/L乙酰丁香酮和50mg／L抗坏血酸可以有效促进农杆菌对大豆子叶节的转化。农杆菌与子叶节共培养后羧苄青霉素（250mr／L）和头孢霉素（100mg／L）结合使用能有效抑制农杆菌过度繁殖并提高转化芽诱导频率;在转化细胞的分化和转化芽伸长过程中,改进的筛选策略可以明显改善对转化芽的筛选效果,从而提高转化频率。应用优化后的转化体系．获得了3个国内大豆主栽品种的转基因植株,PCR阳性植株频率为3．8％～7．6％。转化植株叶片总DNA的PCR和Southern blot实验表明,T-DNA上的外源基因已经整合到大豆基因组中。     

根癌农杆菌介导转化诸葛莱获得转基因植株

以诸葛莱下胚轴和子叶为材料,在附加ＢＡ和ＮＡＡ的ＭＳ培养在上诱导芽于生,在１／２ＭＳ培养基上诱导生根,获得完整再生植株,建立了诸葛菜组织培养高频再生体系,再用根癌农杆菌介地转化炒下胚了叶,在附加一定量的氨邪说青霉素,头孢霉和卡那霉素的相应增减基上进行筛选,并增减再生成苗,获得完整抗性再生植株,移植到盛有土壤的花盆中均可存活,生长正常,将再生植株叶片,进行ＧＵＳ、ＮＰＴⅡ酶活性性测定和Ｓｏｕｔｂｅｒ     

荞麦高频离体再生及发根农杆菌转化体系的建立

荞麦无菌苗下胚轴切段在不同激素配比的MS培养基上诱导愈伤组织,出愈率均为100％。在2．0mg/L2,4-D和1．5mg/L 6-BA组合下诱导产生的愈伤组织;转入2．0mg/L 6-BA和1．0mg/L KT的MS培养基,再生苗分化率在80％以上。根尖色体分析表明再生植株具一定的遗传稳定性。发根农杆菌A4转化荞麦下胚轴和子叶获得发状根,纸电泳检测所有随机取样测定的发状根均有相应冠瘿碱的存在。     

茎用芥菜细胞质雄性不育系子叶原生质体培养和高效植株再生

利用茎用芥工细胞质雄性不育系原生质体培养获得了再生植株,并研究了影响原生质体培养的因素。结果表明,子叶是茎用芥菜原生质体培养最佳的外植体,10d苗龄的子叶原生质体在改良MS培养基上培养3d后发生第1次细胞分裂,6d后发生第2次分裂,3周后形成细胞团,5周后形成肉眼可见的小愈伤,培养基中减少NAA或2,4－D都会降低愈伤组织的再生能力,在含一定浓度的NAA（0．25mg/L)和2,4－D（0．25mg/L)培养基上诱导的愈伤组织地致密且有光泽,芽的分化能力高;在MS＋BA 1mg/L NAA0.2mg/L的培养基上芽分化频率高达近29％,再生芽1／2MS＋NAA0．1mg/L培养基上生根,形成完整植株。     

Transformation of Orychophragmus violaceus Using Agrobacterium tumefaciens And Regeneration of Transgenic Plants

ＴｒａｎｓｆｏｒｍａｔｉｏｎｏｆＯｒｙｃｈｏｐｈｒａｇｍｕｓｖｉｏｌａｃｅｕｓＵｓｉｎｇＡｇｒｏｂａｃｔｅｒｉｕｍｔｕｍｅｆａｃｉｅｎｓＡｎｄＲｅｇｅｎｅｒａｔｉｏｎｏｆＴｒａｎｓｇｅｎｉｃＰｌａｎｔｓａ￥ＺＨＯＵＪｉ－ｍｉｎｇ（周冀明）;ＷＥＩＺｈ...     

Preliminary studies on tissue culture and agrobacterium-medicated transformation of Brassica campestris ssp. chinensis]

The hypocotyls and cotyledons of the asepetic seedling of Brassica campestris ssp. chinensis L cv. Pudongaijiecai) were used as explants for tissue culture. Adventitious buds were differentiated on modified MS medium supplemented with TDZ 1-2 mg/L, NAA 0.2-1 mg/L and AgNO3 7.5 mg/L. The percentage of explants which formed buds of cotyledons was about 56%, and that of hypocotyls was about 37%. When the regenerated explants were transferred onto MS medium with 2 i.p. 5 mg/L and NAA 0.1 mg/L for two weeks, whole plantlets were obtained by culturing the regenerated shoots on 1/2 MS medium with NAA 0.1 mg/L. Agrobacterium tumefaciens strain (LBA 4404/PBI 121) carrying the GUS gene and Npt II gene was used for transformation. After 2 days of coculture, the hypocotyls and cotyledons were transferred onto regenerated medium containing CP 300 mg/L for bud formation. After 4-5 weeks, the differentiated buds were transferred onto selection medium with CP 200 mg/L and Km 10 mg/L for 1 month, then the green shoots were transferred onto the rooting medium containing Cef 100 mg/L and Km 20 mg/L. 4-5 weeks later, plantlets with Km resistance were obtained and some of them showed higher enzymatic activities of beta-glucuronidase than control ones.     

Transformation of Brassica napus Using Agrobacterium tumefaciens and Regeneration of Transgenic Plants

Seeds of Brassica napus L. cv. "Yunbei 2" were surface-sterilized and germinated on hormone-free MS medium. After 4—5 days the cotyledons were excised in such a way that each has a 1—2 mm petiole was remained at its base. These cotyledons were used as the explants for tissue culture and genetic transformation. This paper first deals with the improvement of the medium for shoot regeneration. Of the elements tested, AgNO3 and carbenicillin enhanced shoot regeneration. The highest frequency (52 %) was obtained on MS medium containing 4.5 mg/L BAP, 20 μmol/L AgNOa and 500 mg/L earbenicillin. An efficient gene transfer system based on the regeneration procedure was established. After 2 days of cocultivation with Agrobacterium tumefaciens strain A208SE (pTi T37-SE, pROA93), the explants were transferred onto selection medium containing 25 mg/L kanamycin. After 1.5 months shoots emerged from 27% of the explants inoculated. They were excised and transferred onto rooting medium containing 25 mg/L kanamycin and 200 mg/L cefotaxime which is better than carbenicillin for root induction. Whole plants were transplanted into pots, and grew well in the phytotron. Transformation was confirmed by β-glueuronidase assay and Southern blotting analysis.     

农杆菌转化的小冠花发状根的诱导及其植株再生

利用野生型发根农杆菌15834菌株感染小冠花15日龄无菌苗子叶和下胚轴切段,建立了高效的发状根培养及其体细胞胚胎发生再生体系。发状根可直接从受伤的外植体表面产生,也能在外植体诱导的愈伤组织上发生,在无外源激素的MS固体和液体培养基上,转化根能自主生长,表现出典型的发根特征。用适宜浓度的乙酰丁香酮处理对数生长期的农杆菌菌液2h,感染预培养2d的子叶获得了最高的转化频率(87.4%)。在附加0.2mgL2,4_D,0.5mgLNAA和0.5mgLKT的MS培养基上,发状根能100%形成胚性愈伤组织,并于含0.5mgLKT,0.2mgLIBA和300mgL脯氨酸的MS培养基上顺序经过体细胞胚胎发育的各个典型时期,转换成完整植株。再生植株除具有发达的侧根外,其它形态特征与未转化植株未见明显的差异,但在获得的5个转化克隆中,其中1个的发状根及其再生植株叶片中有毒物质3_硝基丙酸的含量显著下降,分别为未转化对照的57.68%和58.17%。冠瘿碱纸电泳检测和rolB基因PCR扩增检测均证明农杆菌Ri质粒上的T_DNA已经整合到小冠花转化细胞的基因组中。     

Callus Induction and Plant Regeneration of Leaf Explants in Orychophragmus violaccus(L.) O. E.Schulz

Explants of Orychophragmus violaceus (L.) O. E. Schulz were acquired from young leaves which lower epidermis was stripped, Differentiation of calli in high frequency is in the case of that calli grown on B5 culture medium supplemented with 1 mg/l 2, 4-D should be transferred onto MS culture medium supplemented with 0.2 mg/l NAA. Effect of basic culture medium on the differentiation was discussed. In addition, protoplasts derived from calli of Orychophragmus violaceus were cultured, and small calli consisted of more than hundred cells had been obtained.     

龙牙匆木幼芽体细胞胚胎发生和植株再生（简报）

Explants excised from the young shoots of Aralia elata (Miq.) Seem. were cultured on MS media. Calli were induced from the explants on MS medium supplemented with 0.5 mg/L 2, 4-D, 0.5 mg/L BA and 0.5 mg/L NAA. Then these calli were transferred onto the MS medium containing 2.0 mg/L 2,4-D + 0.5 mg/L BA + 0.5 mg/L NAA and 0.2% activated charcoal. Under these conditions the somatic embryoids were observed and regenerated plants were obtained from somatic embryogenesis. Then, a experimental system with stability and high regenerating efficiency has been set up for the propagation of the young plants, the cell breeding technology and the control of somatic embryogenesis of Aralia elata (Miq.).     

龙牙楤木幼芽体细胞胚胎发生和植株再生(简报)

龙牙楤木[Aralia elate(Miq.)Seem]是五加科楤木属的多年生乔本药用植物,又称刺老鸦,具有补气、活血、祛风、利湿、止痛等功效。主要分布于我国东北地区、朝鲜、日本和俄罗斯的西伯利亚地区。其幼嫩茎叶是有名的山菜,在我国及东南亚一些国家很受欢迎。但是,近些年来由于人为恶性采伐,使     

Plant Regeneration and Transformation of Sweet Papper (Capsicum frutescens)

Three strains of sweet pepper, (Capsicum frutescens) “Shuang Feng”, “Zhong Jiao No. 2” and “Zhong Jiao No.3” were screened out of six Chinese cuhivars for their high capacity of regeneration. The normal flowering and fertile regenerated plants have been obtained from cotyledons of seedlings from 10 to 16 days old by a four-step culture procedure; short induction, shoot elongation, rooting of excised shoot and transplanting into soil. MS was the basal medium in all steps, supplemented with different kind and different concentration Of phytohomores. Optimal shoot ioduction medium is MS +4–6mg/LBA+0.5mg/LIAA which gives rise a shoot regeneration frequency of 100%. 35% of the induced shoots elongated on the medium of MS+2mg/L Zeatin or 2mg/L. BA+l–3mg/L GA, and subsequently rooted on MS medium or in addition of 0.1–0.5mg/LNAA. The regenerants were transplanted into soil and developed into normal plants. In the transformation of sweet pepper using the leaf disc method, two kinds of wild type Agrobacterium tumefaciens, C58 and GV3111, have been screened out in regard to their high infection capacity. The transient expression of GUS gene was detected and Kanamycin-resistant shoots from infected cotyledons have been obtained. Further assay and transfering the TMV-resistant and CMV-resistant genes into sweet pepper are in progress.     

提高西瓜离体培养植株再生效率的研究

本文以“京欣1号”母本和“伊选”西瓜4天苗龄子叶为外植体,研究离体培养植株高频率再生体系。结果表明：“伊选”子叶远轴端外植体的再生频率仅为10％,子叶近轴端外植体在5mg／LBA 0．1mg／L IAA的激素组合下植株再生频率为100％,平均每个外植体的丛生芽数在所有组合中最多,为10.3个;“京欣1号”母本子叶近轴端外植体在2mg／LBA 0．5mg／L IAA激素组合下植株再生频率为100％,平均每个外植体的丛生芽数在所有组合中最多,达6．9个。本试验条件下,子叶近轴端外植体接种4天即分化出不定芽,至再生苗的移栽仅需40天,在MS 0．1mg／L NAA的生根培养基上的生根率为97．3％,移栽成活率达98．5％。