PEG法介导转化诸葛菜下胚轴原生质体获得转基因植株

采用诸葛菜无菌苗的下胚轴组织为材料,分离原生质体,在原生质体培养基中作液体浅层暗培养,植板率为５％,植株再生频率为１００％。作者进而开展了遗传转化研究。为研究ＰＥＧ介导转化诸葛菜原生质体的影响因素,通过瞬间表达,实验了ＰＥＧ法转化子叶原生质体的过程,在此基础上将分离纯化后的原生质体与带ＨＰＴ基因的质粒ＤＮＡ（ｐＢＩ２２２）混合,ＨＰＴ基因作选择标记,ＰＥＧ介导转化;重新收集转化后的原生质体,以５×１０４／ｍｌ的密度在原生质体培养基中作浅层培养;培养１０—１５天后用２５ｍｇ／Ｌ的潮霉素（ｈｙｇｒｏｍｙｃｉｎ）进行筛选,一月后出现少量细胞团,转入含潮霉素５０ｍｇ／Ｌ的扩增培养基扩增愈伤组织,进而转入含５０—１００ｍｇ／Ｌ潮霉素的分化培养基诱导分化成苗,分化率为１００％,转入生根培养基中生根成完整植株。抗性植株再生率为４×１０（－５）。在获得再生转基因植株后,以再生植株叶片为材料,进行Ｓｏｕｔｈｅｒｎｂｌｏｔ分子杂交,证实外源基因已稳定整合到植物基因组中并表达,再生转基因植株频率为１０（－５）。国内外首次转化诸葛菜属植物原生质体获得成功。     

亚麻下胚轴离体培养和转化的研究

报道了亚麻（Ｌｉｎｕｍ　ｕｓｉｔａｔｉｓｓｉｍｕｍ）的高频率植株再生和细胞转化。亚麻下胚轴切段培养在ＭＳＢ分化培养基上,诱导分化出不定芽。最佳的激素组合是ＢＡ２ｍｇ／Ｌ＋ＩＡＡ０．５ｍｇ／Ｌ,分化频率可达９７％。在附加ＢＡ１ｍｇ／Ｌ和ＮＡＡ０．０２ｍｇ／Ｌ的ＭＳＢ培养基上,亚麻下胚轴外植体的不定芽分化频率也可达９０％以上。用根癌农杆菌（Ａｇｒｏｈａｃｔｅｒｉｕｍ　ｔｕｍｅｆａｃｉｅｎｓ）感染亚麻下胚轴外植体,在含卡那霉素５０ｍｇ／Ｌ的选择培养基上筛选获得卡那霉素抗性苗,并检测到ＧＵＳ基因活性表达。表明它们是转化植株,转化频率约为２％。     

导入β-1,3-葡聚糖酶及几丁质酶基因的转基因可育油菜及其抗菌核病的研究

利用农杆菌转化法,将组成型表达β-1,3葡聚糖酶及几丁质酶基因的双价植物表达载体pBLGC转化优质甘蓝型油菜品种H165,并得到了抗卡那霉素(Kanamycin,Kan)的再生植株。我们对所得到的抗Kan的再生苗进行了初步分子生物学检测,结果表明,在K15(Kan15mg/L)培养基上的绿苗中有30%为PCR阳性植株,而在K25(Kan10mg/L)培养基上的绿苗有53%的阳性率。对部分PCR阳性的转基因植株进行了点杂交分析,结果均表现出较强的杂交信号,这初步说明外源基因已整合到油菜基因组中。转基因植株的活体接种油菜菌核病菌(Sclerotiniasclerotiorium)试验表明,部分转基因植株比对照显示较强抗病性。     

抗生素对谷子外植体的影响

通过抗生素潮耆素（Ｈｍ）和卡拉霉素（Ｋｍ）进行谷子外植体抗体性浓度的筛选,结果说明,不同外植体抗性浓度不同,谷子愈伤分化再生苗阶段,潮霉素与卡拉霉素的选浓度范围为５ｍｇ／Ｌ－１０ｍｇ／Ｌ和１５ｍｇ／ｌ－２０ｍｇ／Ｌ,米粒长苗时其浓度为１５ｍｇ／Ｌ、３０ｍｇ／Ｌ。     

影响木薯次生胚状体发生及植株再生因素的研究

研究了在外植体的不同发育阶段中,碳源以及不同的生长激素配比对木薯次生胚状体诱导及植株再生的影响。结果表明：以固体成熟培养基上生长１５ｄ的胚状体子叶为外植体,次生胚状体的产量最高,达２９．３个成熟胚状体／１个外植体。在次生胚状体的诱导阶段,以麦芽糖（４０ｇ／Ｌ）代替蔗糖作碳源,能同时提高次生胚状体的产量（３２．５个胚次体／１个外植体）及植株再生频率（７４．３％）。２,４－Ｄ与ＰＰ３３３;（０．１ｍｇ／Ｌ）配合能提高植株再生频率到７７．６％。２,４－Ｄ与ＢＡＰ（２ｍｇ／Ｌ）或激动素（２．０ｍｇ／Ｌ）配合则大大降低了胚状体诱导及植株再生频率。     

南苜蓿组织和原生质体培养及转化试验

主要探讨南苜宿子叶和下胚轴外植体,子叶原生岳体培养中的器官发生及遗传转化。南苜蓿子叶和下胚细外植体培养在附加１ＡＡ０．５－１ｍｇ／Ｌ和细胞分裂素（ＢＡ或ＺＴ）０．５－２ｍｇ／Ｌ的ＭＳ培养基上,在有当照的条件下诱导形成不定芽,进而再生成完整植株。子叶原生质体培养在附加２,４－Ｄ０．５ｍｇ／Ｌ和ＫＴ０．２ｍｇ／Ｌ的Ｂ５液体培养基中,细胞分裂频率可达３０－４１％,原生质全来源的愈伤组织在ＭＳＢ培养基（Ｍ     

辣椒转基因植株再生

用改建的农杆菌转化辣椒,已建立有效的转化两再生系统。辣椒无菌苗（１２天苗龄）子叶在再生培养基（ＭＳ＋３ｍｇ／Ｌ ＢＡ）上预培养２天,再用农杆菌感染,共培养２天,再转入含Ｋｍ（１００μｇ／ｍｌ）的再生培养基中培养２－３周后,子叶基部有多丛芽形成。分切多丛芽,将芽转选择性的芽伸长培养基（ＭＳ基本成分＋Ｂ５维生素类＋２ｍｇ／Ｌ ＢＡ＋７５μｇ／ｍｌ）上,待芽长至２－３ｃｍ后,切下并转入生根培养基（ＭＳ＋     

杯山药零余子愈伤组织诱导及植株再生的研究

对怀山药（Ｄｉｏｓｃｏｒｅａ ｏｐｐｏｓｉｔａ）零余子愈伤组织的诱导、分化、再生苗的生根和移栽进行了研究。结果表明：⑴在不同激素组合的培养基上怀山药零余子均能产生愈作组织,而且具有一次成苗的能力。ＢＡ２ｍｇ／Ｌ＋ＮＡＡ２ｍｇ／Ｌ的培养基对诱导愈伤组织最有利,其出愈率达１００％;⑵在愈伤组织的分化中,ＢＡ１ｍｇ／Ｌ＋ＮＡＡ１ｍｇ／Ｌ的激素组合是最佳的,其分化率为６３．６％,且多形成丛生芽;⑶再生植株     

高寒藏药—抱茎獐牙菜组织培养研究及其愈伤组织有效成分的测定

从抱茎獐牙菜的胚轴、幼叶及未成熟种子诱导出愈伤组织并再生植株,试验选用ＭＳ、Ｂ５和Ｎ６三种培养基,其中以附加２．４－Ｄ３．０ｍｇ／Ｌ＋６－ＢＡ０．５ｍｇ／Ｌ的ＭＳ培养基诱导率最高;以附加６－ＢＡ０．５ｍｇ／Ｌ＋ＮＡＡ０．２ｍｇ／Ｌ的ＭＳ培养基分化苗频率最高;以附加２．４－Ｄ２．０ｍｇ／Ｌ＋６－ＢＡ０．５ｍｇ／Ｌ的ＭＳ培养基愈伤组织的生长最好。结果表明,外植体,培养基,激素等对愈伤组织诱导、继代和分化均有明显影响。采用高压液相色谱法（ＨＰＬＣ）测定抱茎獐牙菜愈伤组织中齐墩果酸含量的结果表明,愈伤组织中齐果墩酸含量因培养基、继代培养时间的不同而有所差异     

月光花素对彩叶芋愈伤组织细胞生长和分化的影响

在含有ＮＡＡ０．２ｍｇ／Ｌ,２．４－Ｄ０．２ｍｇ／Ｌ的ＭＳ基础培养基中添加月光花素培养彩叶芋愈伤组织,结果显示,低浓度的月光花素能促进愈伤组织生长,高浓度则有明显的抑制作用,最适浓度为１．０￣２．０ｍｇ／Ｌ;培养两个月后,添加适宜浓度的月光花素（０．２￣２．０ｍｇ／Ｌ）的培养基能促进体细胞分化和植株再生,而高浓度处理和对照均未能分化出绿苗,表明适宜浓度的月光花素在合适的各行其事纱剂量的协同作用下,     

Insect-resistant transgenic cabbage plants and their progenies

An insecticidal crystal protein gene of Bacillus thuringiensis was transferred into cabbage genome with the method of Agrobacterium infection. Cotyledons with petioles as explants were cocultivated with Agrobacterial suspension. Calli generated at the basis of petiole were subjected to selection on the MS medium containing 15-30 mg/L kanamycin (Km). About 5% explants produced calli growing continuously on the selective medium. Green shoots appeared on these calli when they were transplanted onto medium with Km and 6-BA for plant differentiation. The shoots were separated and cultivated on medium with kanamycin. About 80% shoots were rooted. Non-transformed control calli could not give normal shoots and roots and brownized and died gradually. Larvae of Pieris rapae showed poisonous symptoms: growth inhibition and mortality when fed with the leaf of the transgenic plants. About 80% of regenerated plants showed positive hybridization bands when their DNA were probed with crystal protein sequence of Bacillu     

甘蓝型油菜抗虫转基因植株及其抗性分析

通过油菜子叶外植体－农杆菌共培养法将苏云金杆菌杀虫蛋白基因导入甘蓝型油菜,获得抗虫的转基因植株。带有１￣２ｍｍ子叶柄的油菜子叶经农杆菌感染后,共培养２￣３天,然后转移到附加１５ｍｇ／Ｌ卡那霉素的ＭＳ选择培养基上筛选转化愈伤组织及不定芽。卡那霉素抗性苗相继在含２０￣５０ｍｇ／Ｌ卡那霉素的选择培养基上继代培养,再转移到含２５ｍｇ／Ｌ卡那霉素的生根培养基上诱导生根。以苏云金杆菌杀虫蛋白基因为探针,进行１     

油菜的遗传转化及抗溴苯腈转基因油菜的获得

以油菜(Brassica napus L.)的下胚轴和子叶为转化受体,建立了油菜的高效转化系统。在此基础上,将抗除草剂溴苯腈基因(bxn基因)导入油菜,获得了抗溴苯腈转基因油菜。分子检测实验证明,转基因油菜中含有bxn基因。转基因油菜可抗高达10~(-3)mol/L的溴苯腈。     

Genetic manipulation in cultivars of oilseed rape (Brassica napus) using Agrobacterium

Summary The response of oilseed rape cultivars to infection with Agrobacterium tumefaciens and A. rhizogenes and the possibility of regenerating genetically transformed oilseed rape plants were examined. The frequency at which Agrobacterium induced galls or hairy-roots on in vitro cultured plants ranged from 10% to 70%, depending on the cultivar. From galls induced by the tumorigenic strain T37, known to be strongly shoot inducing on tobacco, roots developed frequently. Occasionally, shoots formed and some of these produced tumour cell specific nopaline. Attempts to grow the transformed shoots into plants have so far been unsuccessful. Whole plants transformed with Ri-T-DNA, however, were regenerated. These had crinkled leaves and abundant, frequently branching roots that showed reduced geotropism, similar to previously isolated Ri T-DNA transformed tobacco and potato plants. The transformed oilseed rape plants flowered, but failed to form seeds.     

水稻几丁质酶基因导入芥菜型油菜的研究

以芥菜型油菜 (BrassicajunceaL .)下胚轴为转化材料 ,通过根癌农杆菌 (Agrobacteriumtumefaciens)介导将水稻的几丁质酶基因 (Ricechitinasegene)导入“泸洲四陵”油菜品种中 ,获得抗潮霉素的再生转基因植株 ,并讨论了影响油菜再生及转化效率的几个因素。对部分经潮霉素筛选得到的再生植株进行了多次重复PCR检测 ,发现其中 40 %以上的潮霉素抗性植株均表现出较强的阳性反应 ,初步证明几丁质酶基因已整合到油菜细胞核基因组中。     

铁棍山药微型块茎遗传转化体系的建立

怀山药(Dioscorea opposita)遗传转化是对其进行基因功能分析和遗传改良的基础, 但目前国内外尚未见相关报道。以怀山药优良品种铁棍山药(D. opposita cv. ‘Tiegun’)的微型块茎为受体材料, 对影响遗传转化的因素进行优化, 建立了由根癌农杆菌介导的山药遗传转化体系。过表达质粒载体pCAMBIA1301-DoSERK2含GUS标记基因和潮霉素(Hyg)抗性筛选基因, 沉默质粒载体pART27-DoSERK2含卡那霉素(Kan)抗性筛选基因。根癌农杆菌抑制剂特美汀(Tim)的最佳浓度为500 mg·L ^-1; 再生芽和生根时, Hyg的最佳浓度分别为15和20 mg·L ^-1, Kan的最佳浓度分别为120和160 mg·L ^-1。对转化植株进行PCR和GUS组织化学检测, 结果显示外源基因已整合到铁棍山药转基因株系的基因组中并在细胞中表达。该研究建立了一套取材便利的铁棍山药遗传转化方法, 对其它品种山药的转化也具有参考价值。     

改良菜心离体培养植株再生体系的研究(简报)

菜心(Brassica campestris L.subsp.chinensis Makino var.parachinensis Tsen et Lee)为十字花科芸薹属芸薹种中国白菜亚种中的一个变种,又名“菜薹”。它是我国南方特产蔬菜之一,在蔬菜的周年供应上有重要地位。目前迫切需要培育抗病虫、抗逆和具有其他优良农艺性状的新品种,以提高菜心的     

中国木薯栽培种通过器官发生再生植株研究

本文研究了中国木薯栽培种四种外植体通过器官发生再生植株的条件。结果表明:在MS附加0.05mg/L TIBA,1mg/L BA的培养基上“NZ 188”初步的萌发胚状体“切头”后切口处可直接产生丛芽,出芽率为43%。“SC201”胚状体子叶块在MS附加0.5 mg/L NAA,0.5mg/L BA的培养基上可直接出芽,出芽率为42%,在MS附加0.5mg/L IBA,1.5mg/L BA培养基上·出芽率为31%,AgNO_3和ABA单独使用或配合使用均不利于芽的再生。“NZ188”胚状体下胚轴在MS附加0.5mg/LNAA,0.5mg/L BA的培养基上形成的愈伤组织转入MS附加1mg/L NAA,2mg/L BA的培养基上,3周后大多数愈伤组织有绿点出现、仅4.4%外植体分化出芽。“HZ188”无菌苗茎段接种在MS附加0.05mg/L TIBA,2mg/LBA的固体培养基上,2周后形成大量愈伤组织,4周后仅见一块愈伤组织分化出芽。     

Oilseed Rape Transformation and the Establishment of a Bromoxynil Resistant Transgenic Oilseed Rape

Using hypocotyls and cotyledons as transformed materials, an efficient transformation system of oilseed rape (Brassica napus L.) was established. Bxn gene (bromoxynil-resistant gene)was introduced into these plants, and bromoxynil-resistant transgenic oilseed rape was obtained. Themolecular monitoring experiments showed that these transgenic plants contained bxn gene. The herbicide experiments showed that these transgenic plants had resistance to 10-3 moL/Lbromoxynil.     

Transformation of a grain legume (Lupinus angustifolius L.) via Agrobacterium tumefaciens-mediated gene transfer to shoot apices

Transgenic plants of Lupinus angustifolius L. (cvs. Unicrop and Merrit) were routinely generated using Agrobacterium-mediated gene transfer to shoot apices. The bar gene for resistance to phosphinothricin (PPT, the active ingredient of the herbicide Basta) was used as the selectable marker. After co-cultivation, the shoot apex explants were transferred onto a PPT-free regeneration medium and their tops were thoroughly wetted with PPT solution (2 mg/ml). The multiple axillary shoots developing from the shoot apices were excised onto a medium containing 20 mg/l PPT. The surviving shoots were transferred every second week onto fresh medium containing 20 mg/l PPT. At each transfer, the number of surviving shoots decreased, until it stabilized. Indeed, some of these chimeric shoots surviving the PPT selection, eventually produced new green healthier axillary shoots which could be transferred to soil. This whole process took from 5 to 9 months after co-cultivation. Average transformation frequencies of 2.8% for cv. Unicrop and of 0.4% for the commercial cultivar Merrit were achieved. Molecular analysis of T₀, T₁, and T₂ generations demonstrated stable integration of the foreign gene into the plant genome and expression of the integrated gene. Transformed plants of the T₁ and T₂ generations were resistant in glasshouse trials where the herbicide Basta (0.1 mg/ml) was sprayed onto whole plants. These results demonstrate that Agrobacterium-mediated gene transfer to preorganised meristematic tissue combined with axillary regeneration can form the basis of a routine transformation system for legume crop species which are difficult to regenerate from other explants.