根癌农杆菌介导转化Lesquerella fendleri L．的研究

以Lesquerella fendleri L.叶片和叶柄为外植体,研究了根癌农杆菌介导的β-葡萄糖醛酸苷酶基因(β-glucuronidase,Gus)的遗传转化技术,将再生植株叶片进行PCR分子鉴定和Gus组织染色,证实外源基因已稳定整合到植物基因组中,并得到表达。     

Effects of the Introduction of Agrobacterium tumefaciens T-DNA ipt Gene in Nicotiana tabacum L. cv. Petit Havana SR1 Plant Cells

Transformation of tobacco leaf discs with the ‘cytokinin’ipt gene yielded several transgenic callus tissue lines, respectiveto the kind of ipt construction present in the A. tumefacienscointegrates. Those calli containing an active ipt gene wereable to grow hormone-autotrophically and showed an increasedendogenous cytokinin level in comparison with controls. Analysisof endogenous IAA level did not allow any quantitative correlationwith the cytokinin content. However, a minimal level of auxinseems to be necessary to obtain hormone-autotrophic growth.Exogenously supplied NAA significantly reduced the endogenouscytokinin content without modifying growth characteristics. The varying chlorophyll content in the different callus lineselicited the study of the ultrastructure of the plastids. Thecontrols contained small plastids, often filled with starchor accumulated vesicles that did not allow observation of theinternal membrane system. The ‘Pssu-ipt’ line, havinga higher cytokinin content, showed plastids with an internalmembrane system consisting of stroma and grana thylakoids, butthis structure was lost during subculture. Swollen thylakoidsappeared, the amount of starch was reduced and vesicles wereaccumulating. (Received November 15, 1990; Accepted March 4, 1991)     

Transformation of azuki bean by Agrobacterium tumefaciens

Stable transformation and regeneration was developed for a grain legume, azuki bean (Vigna angularis Willd. Ohwi & Ohashi). Two constructs containing the neomycin phosphotransferase II gene (nptII) and either the -glucuronidase (GUS) gene or the modified green fluorescent protein [sGFP(S65T)] gene were introduced independently via Agrobacterium tumefaciens-mediated transformation. After 2 days of co-cultivation on MS medium supplemented with 100 M acetosyringone and 10 mg l^–1 6-benzyladenine, seedling epicotyl explants were placed on regeneration medium containing 100 mg l^–1 kanamycin. Adventitious shoots developing from explant calli were excised onto rooting medium containing 100 mg l^–1 kanamycin. Rooted shoots were excised and repeatedly selected on the same medium containing kanamycin. Surviving plants were transferred to soil and grown in a green house to produce viable seeds. This process took 5 to 7 months after co-cultivation. Molecular analysis confirmed the stable integration and expression of foreign genes.     

Transformation of Corynespora cassiicola by Agrobacterium tumefaciens

The fungus causing target spot disease, Corynespora cassiicola (Berk. & M. A. Curtis) C. T. Wei, poses an increasing threat to watermelon (Citrullus lanatus), muskmelon (Cucumis melo), and cucumber (Cucumis sativus); the most economically important cucurbit crops grown in China. An understanding of the molecular mechanisms underlying the pathogenicity of C. cassiicola is essential for the development of new strategies to control this disease-causing fungus. Agrobacterium tumefaciens-mediated transformation (ATMT) might be useful to obtain transformants of C. cassiicola, for the ultimate identification of genes involved in pathogenicity. In the present work, we established and optimized an ATMT protocol using A. tumefaciens strain AGL-1 carrying the vector pATMT1 for C. cassiicola. Efficiency of ATMT was 102–148 transformants per 10⁶ conidia and successive subculturing of transformants on non-selective and selective media demonstrated that the integrated transfer (T)-DNA was stably inherited in C. cassiicola transformants. The integration of the hygromycin B phosphotransferase (hph) gene into C. cassiicola was validated by PCR and Southern blot analyses, which revealed that nearly 90 % of the transformants contained single-copy T-DNA. The transformants with altered phenotypes were characterized. Three of these transformants completely lost pathogenicity and other three showed strongly impaired pathogenicity relative to the Cc-GX strain on muskmelon leaves. These results strongly suggest that ATMT may be used as a molecular tool for identifying genes relevant to pathogenicity in the fungus C. cassiicola, an emerging threat to several agronomically important plants in China.     

Transformation of rice mediated by Agrobacterium tumefaciens

Agrobacterium tumefaciens has been routinely utilized in gene transfer to dicotyledonous plants, but monocotyledonous plants including important cereals were thought to be recalcitrant to this technology as they were outside the host range of crown gall. Various challenges to infect monocotyledons including rice with Agrobacterium had been made in many laboratories, but the results were not conclusive until recently. Efficient transformation protocols mediated by Agrobacterium were reported for rice in 1994 and 1996. A key point in the protocols was the fact that tissues consisting of actively dividing, embryonic cells, such as immature embryos and calli induced from scutella, were co-cultivated with Agrobacterium in the presence of acetosyringonc, which is a potent inducer of the virulence genes. It is now clear that Agrobacterium is capable of transferring DNA to monocotyledons if tissues containing competent cells are infected. The studies of transformation of rice suggested that numerous factors including genotype of plants, types and ages of tissues inoculated, kind of vectors, strains of Agrobacterium, selection marker genes and selective agents, and various conditions of tissue culture, are of critical importance. Advantages of the Agrobacterium-mediated transformation in rice, like on dicotyledons, include the transfer of pieces of DNA with defined ends with minimal rearrangements, the transfer of relatively large segments of DNA, the integration of small numbers of copies of genes into plant chromosomes, and high quality and fertility of transgenic plants. Delivery of foreign DNA to rice plants via A. tumefaciens is a routine technique in a growing number of laboratories. This technique will allow the genetic improvement of diverse varieties of rice, as well as studies of many aspects of the molecular biology of rice.     

Rapid flowering Nicotiana tabacum L.

 Selected Nicotiana tabacum Tobacco Introductions from the Nicotiana Germplasm Collection were crossed, back-crossed, and then selfed. When compared to parents, the new genotypes produce smaller plants with a shortened life-cycle that go from seed to seed in under 11 weeks. Received: 11 December 1998 / Revision accepted: 8 January 1999     

Transformation of arctic bramble (Rubus arcticus L.) by Agrobacterium tumefaciens

Genetic transformation of arctic bramble (Rubus arcticus L.) was achieved utilizing a Ti-plasmid vector system of Agrobacterium tumefaciens. Internodal stem segments were inoculated with Agrobacterium strain EHA101 carrying a T-DNA with the CaMV 35 S promoter-gus-int marker gene from which β-glucuronidase (GUS) is expressed only in plants. Regenerants were produced on Murashige and Skoog medium. Growth of Agrobacterium was inhibited with cefotaxime. Kanamycin was used as the selective agent for the transformants. Regenerants were assayed by histochemical GUS staining, and by Southern analysis using a gus-int probe. Transgenic arctic bramble plants containing gus-int and expressing GUS were recovered. Expression has been stable for 3 years in micropropagation. Received: 22 October 1997 / Revision received: 7 January 1998 / Accepted: 2 February 1998     

Transformation of Metarhizium anisopliae mediated by Agrobacterium tumefaciens

A simple, highly efficient, and reliable Agrobacterium tumefaciens-mediated transformation method was developed for the insect pathogenic fungus Metarhizium anisopliae. Expression of the green fluorescent protein gene, egfp, and the benomyl resistance gene, benA3, were used as markers in transformed M. anisopliae. Transformation efficiencies were dependent on the strain of A. tumefaciens used. With strain AGL-1, 17.0 +/- 1.4 transformants per plate could be obtained using conidial concentrations of 10(6) conidia/mL and a 2 day co-cultivation in the presence of 200 micromol/L acetosyringone. On the other hand, transformations using strain LBA4404 were unsuccessful. Ten transformants were tested by Southern analysis and found to contain a single copy T-DNA. Twenty transformants were subcultured for five generations on nonselective media, and 95% of the transformants were mitotically stable. Agrobacterium tumefaciens-mediated transformation of M. anisopliae can serve as a useful tool to investigate genes involved in insect pathogenicity.     

Genetic Transformation of Wheat Mediated by Agrobacterium tumefaciens

A rapid Agrobacterium tumefaciens-mediated transformation system for wheat was developed using freshly isolated immature embryos, precultured immature embryos, and embryogenic calli as explants. The explants were inoculated with a disarmed A. tumefaciens strain C58 (ABI) harboring the binary vector pMON18365 containing the [beta]-glucuronidase gene with an intron, and a selectable marker, the neomycin phosphotransferase II gene. Various factors were found to influence the transfer-DNA delivery efficiency, such as explant tissue and surfactants present in the inoculation medium. The inoculated immature embryos or embryogenic calli were selected on G418-containing media. Transgenic plants were regenerated from all three types of explants. The total time required from inoculation to the establishment of plants in soil was 2.5 to 3 months. So far, more than 100 transgenic events have been produced. Almost all transformants were morphologically normal. Stable integration, expression, and inheritance of the transgenes were confirmed by molecular and genetic analysis. One to five copies of the transgene were integrated into the wheat genome without rearrangement. Approximately 35% of the transgenic plants received a single copy of the transgenes based on Southern analysis of 26 events. Transgenes in T1 progeny segregated in a Mendelian fashion in most of the transgenic plants.     

In vitro Transformation of Ovules into Rudimentary Pistils in Nicotiana tabacum L.

In our experiments with in vitro cultured young ovaries of Nicotianatabacum, var. havanensis we observed greening and extensivegrowth of the ovular primordia. Four to 6 weeks after inoculationa mass of rudimentary pistils bearing secondary ovules was tobe seen on the protruding placenta. Histological investigationproved these pistilloids not to be adventive organs arisingfrom callusing tissue, but the result of altered growth of theoriginal ovu1ar prirnordia. Though comparatively rare, reportson spontaneous ovular pistillody, including two Nicotiana species,exist in the teratological literature. However, whereas thesespon taneous ovular transformations are described as carpel-likestructures with marginally inserted secondary ovules, the invitro proliferating ovules develop into rudimentary pistilswith an early differentiating and very prominent central placenta.An interpretation of these differences is proposed.     

Transformation of Indica Rice (Oryza sativa L.) Mediated by Agrobacterium tumefaciens

A system has been developed for the transfer of genes by Agrobacteriumtumefaciens to indica rice (Oryza sativa L. cv. Taichung Native1), and optimal conditions for such Agrobacterium-mediated transformationhave been determined. Excised stems, leaves, and roots from3- to 4-day-old seedlings were infected with strains of Agrobacteriumthat carried plasmids with chimeric derivatives of genes forß-glucuronidase (GUS) and neomycin phosphotransferase(NPTII). After selection with the antibiotic G418, only rootssurvived and formed calli. The integration of chimeric genesin the rice genome was demonstrated by Southern analysis andassays of enzymatic activity. We obtained a transformation frequencyof 37.5% for transgenic callus. In addition, the absence ofhybridization with vir probes indicated that transformants werenot contaminated with Agrobacterium. The complete failure oftransformation in the absence of medium from potato suspensioncultures (PSC) indicates that PSC is essential for Ti-mediatedtransformation in indica rice. A high efficiency of transformationwas observed upon infection with the cointegrate vector pGV2260::NG1. (Received August 13, 1991; Accepted April 28, 1992)     

Sperm dimorphism in Nicotiana tabacum L.

Sperm cells of tobacco have been intensively studied as examples of isomorphic gametes in which major cellular and organellar parameters remain statistically indistinguishable in the two sperm cells. An examination of sperm cells late in maturation, however, displays that the sperm cell associated with the vegetative nucleus becomes statistically significantly smaller than the other sperm cell in tobacco. If late divergence occurs in the two sperms of other angiosperms, sperm dimorphism may be more prevalent than has previously been assumed and dimorphism may have a major influence on the pattern of double fertilization. Received: 15 December 2000 / Accepted: 4 May 2001     

根癌农杆菌介导的高粱遗传转化体系的研究

采用根癌农杆菌介导法将杀虫晶体蛋白基因cryIA(b)转入高粱胚性愈伤组织中,建立农杆菌转化高粱的遗传转化体系,获得70棵再生植株。经GUS及PCR检测,结果表明,cryIA(b)基因确实转移到高粱恢复系0-30中,报告基因GUS在再生植株中也得到表达。转化植株的抗病性鉴定正在进行中。     

Transformation of Stylosanthes spp. using Agrobacterium tumefaciens

Tumours were incited on leaf sections of Stylosanthes humilis, S. hamata, S. guianensis and S. scalra following infection by Agrobacterium tumefaciens. The suitability of 2 binary vectors (pGA472, BIN6) for gene transfer in S. humilis was tested and kanamycin-resistant tumour tissue was obtained from infected leaf pieces. The presence and expression of the neomycin phosphotransferase (NPT II) gene in the plant cells was demonstrated by hybridization of the coding region of the NPT II gene of the transposon Tn5 to DNA and RNA of kanamycin resistant tumours and by detection of significant NPT II activity in tissue extracts. Tumours also produced teratomatous shoots expressing the NPT II gene, but these could not be rooted.     

根癌农杆菌介导丝状真菌遗传转化的研究进展

根癌农杆菌介导的丝状真菌遗传转化是近年建立起来的一种新方法,该方法和以往的真菌转化体系相比具有转化方法简单、材料易得、效率高以及转化子中T-DNA单拷贝插入比例高等特点。就根癌农杆菌转化的丝状真菌种类、转化的具体过程以及影响转化效率的因素等方面进行了综述,并展望了该方法的应用前景。     

根癌农杆菌介导的苜蓿体胚转化

以苜蓿体细胞胚胎作为根癌农杆菌介导转化的受体,通过对GUS基因瞬时表达率的分析,研究该转化体系的最佳实验参数。实验结果显示,负压处理10min和共培养5d时表达率最高(可达17．4％)。以这一转化方法分别对带有3种不同启动于的表达载体进行比较,发现由CMV35S启动于驱动的GUS基因的瞬时表达率可达82．7％,Ubil启动于驱动的可达57．8％,而Actl启动于驱动的则未见表达。     

根癌农杆菌介导的水稻遗传转化

就根癌农杆菌介导水稻遗传转化的研究历程和影响农杆菌转化水稻的几个关键因素以及这一问题的前景作了评述和展望     

Characterization of the Nicotiana tabacum L. genome by molecular cytogenetics

Nicotiana tabacum (2n=48) is a natural amphidiploid with component genomes S and T. We used non-radioactive in situ hybridization to provide physical chromosome markers for N. tabacum, and to determine the extant species most similar to the S and T genomes. Chromosomes of the S genome hybridized strongly to biotinylated total DNA from N. sylvestris, and showed the same physical localization of a tandemly repeated DNA sequence, HRS 60.1, confirming the close relationship between the S genome and N. sylvesfris. Results of dot blot and in situ hybridizations of N. tabacum DNA to biotinylated total genomic DNA from N. tomentosiformis and N. otophora suggested that the T genome may derive from an introgressive hybrid between these two species. Moreover, a comparison of nucleolus-organizing chromosomes revealed that the nucleolus organizer region (NOR) most strongly expressed in N. tabacum had a very similar counterpart in N. otophora. Three different N. tabacum genotypes each had up to 9 homozygous translocations between chromosomes of the S and T genomes. Such translocations, which were either unilateral or reciprocal, demonstrate that intergenomic transfer of DNA has occurred in the amphidiploid, possibly accounting for some results of previous genetic and molecular analyses. Molecular cytogenetics of N. tabacum has identified new chromosome markers, providing a basis for physical gene mapping and showing that the amphidiploid genome has diverged structurally from its ancestral components.     

根癌农杆菌介导的高羊茅遗传转化研究

采用携带卡那霉素抗性基因nptⅡ和Na^ ／H^ 反向运输AtNHX1基因的表达载体pROK2／AtNHX1(带有35S启动子)和pROK2U／AtNHX1(带有ubi1启动子)的根癌农杆菌AGL1和GV3101,对4个品种高羊茅下胚轴来源的胚性愈伤组织进行了遗传转化。胚性愈伤组织经根癌农杆菌感染和共培养后,用50～150mg／L巴龙霉素筛选抗性愈伤组织,获得1126棵再生植株,用10～20mg／L卡那霉素进一步筛选再生植株,总共得到525棵绿色抗性植株。抗性植株的总DNA用AtNHX1基因的特异引物进行PCR检测,其中21棵为PCR阳性,最高转化频率为1．77％。Southern杂交结果证实,外源基因以低拷贝整合到高羊茅的基因组中,实验发现,在不同品种之间转化效率有所差异。