Kanamycin resistance of germinating pollen of transgenic plants

The influence of kanamycin on the percentage of pollen germination and on tube growth of pollen from non-transformed and transformed plants of various species containing a chimaeric kanamycin resistance gene (NPTII) was investigated. Pollen grains isolated from kanamycin resistant plants expressed resistance when germinating in vitro, whereas kanamycin impaired tube growth of pollen from non-transformed plants. Pollen grains from transgenic plants were less sensitive and produced significantly longer tubes. mRNAs of the chimaeric gene are probably presynthesized concurrently with the other mRNAs during microsporogenesis, and kanamycin resistance is expressed by mRNA translation during pollen tube elongation. Received: 24 August 1999 / Revision accepted: 20 October 1999     

Expression of Kanamycin resistance introduced byAgrobacteriutn binary vector intoNicotiana tabacum andAtropa belladonna

Kanamycin resistance gene was introduced into tobacco and Atropa belladonna cells by binary vectors, based on Agrobacterium, by means of inoculation of seedlings. The plasmid pGA472, which carries chimaeric kanamycin resistance gene expressed in plants was introduced by transformation into A. tumefaciens Bo542, harbouring pTiBo542 plasmid and A. rhizogenes 8196, carrying pRi8196 plasmids and the resulting two strains were used as binary vectors. Tobacco tumors induced by A. tumefaciens Bo542(pGA472) grew as undifferentiated, kanamycin resistant tissues. Those induced by A. rhizogenes 8196(pGA472) differentiated into transformed plants. When cultivated in vitro on 200 μg ml^-1 kanamycin medium, they showed yellow green sectoring, which was not selected out during vegetative propagation. Atropa belladonna tissues transformed by both A. tumefaciens Bo542(pGA472) and A. rhizogenes 8196(pGA472) differentiated plants which grew well on 200 μg ml^-1 kanamycin as green, non-sectoring plants; sensitive cells obviously did not divide at all. Selection of Atropa belladonna transformed tissues on kanamycin medium is much more efficient than selection of transformed tobacco tissues with introduced kanamycin resistance gene.     

Transformation of Populus tomentosa with Insecticidal Cowpea Proteinase Inhibitor Gene

Cowpea trypsin inhibitor (CpTI) gene, an insecticidal gene, was introduced into poplar ( Populus tomentosa Carr. ) by gene transformation mediated by Agrobacterium tumefac/ens (Smith et Townsend) Conn. The influences on regeneration and transformation frequency of poplar by the concentration and addition of kanamycin were compared. Kanamycin resistant (Kmr) plantlets were obtained by 3 -4 cycles screening in selective condition. The ability of leaf regeneration and shoot subculture and rooting from the transformed and non-transformed plants in the presence of 50 mg/L kanamycin was examined. The presence of CpTI gene in the transgenic plants were confirmed by PCR and PCR-Southern blot. Assay on proteinase inhibition activity demonstrated that leaf protein extracts of the transgenic poplar showed higher inhibition activity against trypsin than that of control plants.     

Transgenic antibiotic resistance may be differentially silenced in germinating pollen grains

Transgenic tobacco (Nicotiana tabacum L.) plants, carrying the neomycin phosphotransferase (NPT II) gene from E. coli, are resistant to kanamycin when grown from seeds on kanamycin containing medium. Tissue and cell cultures derived from those transformants also express resistance and regenerate complete plantlets in the presence of the antibiotic. This unspecific response to the selective condition has led to the belief that the foreign gene is continuously active or uniformly inducible in all cells of the transgenic plant. However, our experiments show that this view is not true for pollen grains during in vitro germination. Pollen grains isolated from kanamycin resistant tobacco plants carry and transmit the foreign gene but do not express resistance when germinating in vitro. This data presents evidence for differential silencing of a foreign gene in a mature gamete. On the other hand, immature pollen grains (microspores) appear to express resistance. The point of the downregulation of the neomycin transferase gene during pollen maturation is discussed.Abbreviations kan kanamycin sulfate - NPT II neomycin phosphotransferase II - sr streptomycin sulfate     

Effects of kanamycin on tissue culture and somatic embryogenesis in cotton

The aminoglycoside antibiotic kanamycin was evaluated for its effects on callus initiation from hypocotyl and cotyledon explants, proliferation of non-embryogenic and embryogenic calli, initiation and development of somatic embryos in cotton (Gossypium hirsutum L.). On this basis, the potential use of kanamycin as a selective agent in genetic transformation with the neomycin phosphotransferase II gene as the selective marker gene was evaluated. Cotton cotyledon and hypocotyl explants, and embryogenic calluses were highly sensitive to kanamycin. Kanamycin at 10 mg/L or higher concentrations reduced callus formation, with complete inhibition at 60 mg/L. Kanamycin inhibited embryogenic callus growth and proliferation, as well as the initiation and development of cotton somatic embryos. The sensitivity of embryogenic callus and somatic embryos to kanamycin was different during the initiation and development stages. Kanamycin was considered as a suitable selective agent for transformed callus formation and growth of non-embryogenic callus. Forty to sixty mg/L was the optimal kanamycin concentration for the induction and proliferation of transformed callus. The concentration of kanamycin must be increased (from 50 to 200 mg/L) for the selection of transformation embryogenic callus and somatic embryos. A scheme for selection of transgenic cotton plants when kanamycin is used as the selection agent is discussed.     

Transgenic Brassica napus plants obtained by cocultivation of protoplasts with Agrobacterium tumefaciens

Hypocotyl protoplasts of German winter oilseed, rape (Brassica napus) lines of double-low quality were transformed using Agrobacterium tumefaciens harbouring pGV 38501103 neo (dimer) containing chimaeric kanamycin resistance reporter genes. Transformed protoplasts were regenerated to fertile and phenotypically normal plants. Transformation was confirmed by kanamycin resistance, nopaline production, neomycinphosphotransferase II activity, and Southern blot hybridization. Seed progeny from self-pollinated transformants expressed the introduced kanamycin resistance as a Mendelian trait.Abbreviations BAP 6-benzylaminopurine - Cf Claforan^R - 2.4D 2,4-dichlorophenoxy acetic acid - Km kanamycin - MS Murashige and Skoog (1962) - NAA -naphthalene acetic acid - NPT II neomycinphosphotransferase - npt II neomycinphosphotransferase II gene - NOS nopaline synthase - nos nopaline synthase gene - ocs octopine synthase gene - IAA indole-3-acetic acid     

表达核糖核酸酶基因的雄性不育油菜的获得

从细菌Ｂａｃｉｌｌｕｓａｍｙｌｏｌｉｑｕｅｆａｃｉｅｎｓ染色体ＤＮＡ中克隆了ＲＮａｓｅ（ｂａｒｎａｓｅ）基因,构建了ＴＡ－２９基因５＇调控区（－１３００－＋３）与ｂａｒｎａｓｅ基因的嵌合基因,通过农杆菌介导的遗传转化,获得了“双低”甘蓝型油菜“中双８２１”的转基因植株。转化植株与末转化植株在高度、生长速度、花器形态、花色等方面基本相同,但转化植株花丝短小、花药干瘪、没有花粉;自花授粉或以其为父本进行的异花授粉均不能结实,表现为完全的雄性不育。花药的横向解剖结构表明：转基因油菜雄性不育与绒毡层细胞的破坏有关     

Analysis of the T-DNA structure in a large number of transgenic petunias generated by Agrobacterium-mediated transformation

Southern hybridisation was performed on ninety-six transgenic petunias that had been selected for resistance to kanamycin. Just over half of the plants contained intact copies of the T-DNA. The most common rearrangements (at least 24 plants out of 96) were simple deleted derivatives that had lost one or both ends of the T-DNA. T-DNAs lacking the left border occurred at a frequency of 20%, and estimates of the frequency of T-DNAs lacking the right border were at least this high. Three plants contained grossly rearranged T-DNAs, of which all expressed the kanamycin resistance gene but only one transmitted the gene to progeny. Two plants lacked T-DNA homology altogether and did not express kanamycin resistance in their leaves or their progeny. Circumstantial evidence suggests that plants containing a chimaeric kanamycin resistance gene driven by the ocs promoter do not root efficiently in the presence of kanamycin. There was no correlation between intactness of the T-DNA and Mendelian inheritance of the kanamycin-resistance phenotype. However, a disproportionate number of plants showing non-Mendelian inheritance had a high copy number of their T-DNA.     

Celery transformation by Agrobacterium tumefaciens: cytological and genetic analysis of transgenic plants

Transgenic celery plants were obtained following co-cultivation of petiole explants with Agrobacterlum tumefaciens containing pMON200, a cointegrate vector carrying genes for kanamycin resistance and nopaline synthase. Transformants were selected by ability of callus to grow in the presence of 50mg/l kanamycin. Transformation was confirmed either by the presence of nopaline or by Southern blots. Cytological analysis of 14 transformed plants revealed chromosomal aberrations, both in structure and number. Only 20% of the regenerated plants had the normal karyotype. Kanamycin resistance behaved as a monogenic, dominant trait, segregating in a 3:1 ratio in three families derived from plants with normal karyotypes.Abbreviations KB Kilobases - 2-4D 2,4-diphenoxyacetic acid     

Transfer of a 4.3-kb fragment of the TL-DNA of Agrobacterium rhizogenes strain A4 confers the pRi transformed phenotype to regenerated tobacco plants

Two strategies were used to transfer into tobacco a 4.3-kb fragment of the TL-DNA of the Ri plasmid of Agrobacterium rhizogenes strain A4. In the liposome-mediated procedure a plasmid containing a neomycin phosphotransferase II (NPT II) gene conferring kanamycin resistance and another plasmid containing the 4.3-kb Eco RI fragment (pRiA4 Eco RI-15) were co-transferred into the tobacco genome. In the Agrobacterium transformation procedure, a micro-Ri vector containing a kanamycin resistance gene and the same pRiA4 fragment was used to transform tobacco leaf fragments. Kanamycin resistant plants were regenerated in both cases. They present a phenotype similar to that of plants regenerated from hairy roots induced by A. rhizogenes, that is wrinkled leaves, reduced apical dominance and ability to form hairy root on leaf fragments. In one plant (Ka158), the organization, expression and transmission to the progency of the inserted foreign DNA were analyzed more precisely.     

基因枪轰击成熟花粉粒转化玉米的研究

利用基因枪轰击花粉粒再授粉的基因转化途径,将豇豆胰蛋白酶抑制剂基因（CpTI）成功导入玉米受体中。经卡那霉素筛选结果表明,非转化植株经1000ppm卡那霉素溶液处理后白化、死亡,余下大量健壮、可育的抗性植株,转化率约1．59％。通过对抗性植株进行PCR和PCR—Southern检测,初步确定CpTI基因已导入玉米基因组。饲虫实验结果表明转化植株具有较强的抗虫性。     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of embryogenic cultures and plant regeneration in <Emphasis Type="Italic">Vitis rotundifolia</Emphasis> Michx. (muscadine grape)

A method to produce transgenic plants of Vitis rotundifolia was developed. Embryogenic cultures were initiated from leaves of in vitro grown shoot cultures and used as target tissues for Agrobacterium-mediated genetic transformation. A green fluorescent protein/neomycin phosphotransferase II (gfp/nptII) fusion gene that allowed for simultaneous selection of transgenic cells based on GFP fluorescence and kanamycin resistance was used to optimize parameters influencing genetic transformation. It was determined that both proembryonal masses (PEM) and mid-cotyledonary stage somatic embryos (SE) were suitable target tissues for co-cultivation with Agrobacterium as evidenced by transient GFP expression. Kanamycin at 100 mg l⁻¹ in the culture medium was effective in suppression of non-transformed tissue and permitting the growth and development of transgenic cells, compared to 50 or 75 mg l⁻¹, which permitted the proliferation of more non-transformed cells. Transgenic plants of “Alachua” and “Carlos” were recovered after secondary somatic embryogenesis from primary SE explants co-cultivated with Agrobacterium. The presence and stable integration of transgenes in transgenic plants was confirmed by PCR and Southern blot hybridization. Transgenic plants exhibited uniform GFP expression in cells of all plant tissues and organs including leaves, stems, roots, inflorescences and the embryo and endosperm of developing berries.     

Uptake, integration, expression and genetic transmission of a selectable chimaeric gene by plant protoplasts

Summary Genetic transformation of Nicotiana tabacum protoplasts was achieved by incubation of protoplasts with a plasmid DNA-calcium phosphate coprecipitate, followed by fusion of the protoplasts in the presence of polyvinyl alcohol and subsequent exposure to high pH. A derivative of the plasmid pBR322 containing a chimaeric gene, consisting of the nopaline synthase promoter, the coding region of the aminoglycoside phosphotransferase gene of Tn5 and the polyadenylation signal region of the octopine synthase gene, was used for these transformation experiments. This chimaeric gene confers resistance of transformed plant cells to kanamycin. This novel transformation procedure reproducibly yielded transformants at frequencies of approximately 0.01%. Aminoglycoside phosphotransferase II activity was detected in both transformed calli and in regenerated plants. DNA from some of the transformed clones was analyzed by Southern blot hybridization. The input DNA appears to be integrated into high molecular weight cellular DNA. Genetic analysis of one of the kanamycin resistant plants shows that the chimaeric gene is transmitted to the progeny as a single dominant trait in a Mendelian fashion. As a comparison the input DNA was also introduced into tobacco protoplasts using Agrobacterium tumefaciens and Ti-plasmid derived gene vectors.Dedicated to Professor Georg Melchers to celebrate his 50-year association with the journal     

Kanamycin selection in temporary immersion bioreactors allows visual selection of transgenic citrus shoots

In mature citrus transformation, the nptII gene is most commonly used for selection and it is confounded by the high number of non-transformed, escaped shoots that develop on semi-solid kanamycin selection medium, even at high concentrations. Selection in liquid medium with kanamycin in temporary immersion bioreactors might provide a better means of distinguishing between transformed and non-transformed shoots. A dose-response curve was constructed for wild-type Carrizo rootstock in liquid medium to evaluate the effects of kanamycin concentration on the number and the length of microshoots. Kanamycin at 200 mg/l was chosen as the optimal concentration for selection of transgenic mature citrus shoots in bioreactors. At this dose, most non-transgenic microshoots turned yellow and their lengths and numbers were significantly reduced in comparison to the no kanamycin controls. Selection of transgenic shoots in bioreactors was tested after Agrobacterium transformations of mature Carrizo and Valencia using three different binary vectors containing nptII as the selectable marker. Shoots developed on semi-solid medium and were transferred to temporary immersion bioreactors containing liquid MS medium with 200 mg/l kanamycin. After two weeks of culture in bioreactors, 21 dark green shoots were visually selected on the basis of color from a total of 6882 microshoots, and 17 of them (81%) were confirmed as transgenic with either the GUS histochemical assay, GFP fluorescence or PCR. Yellow shoots (5675) to be discarded from pTLAB21 and pCAMBIA2301 transformations were also tested for GUS or GFP expression and only one (0.01%) was positive. Kanamycin selection of mature transgenic shoots in temporary immersion bioreactors permitted transgenics to be visually distinguished on the basis of color, and reduced labor and consumable costs for PCR screening, particularly when reporter genes were not used.     

Free auxin level and inheritance of introduced markers in tobacco transformed by binary vector eased on A4 Ri plasmid

Tobacco cv. Mamont was transformed by leaf disc method with A.tumefaciens C58C1 (pRiA4b) (pGA472). Transformed leaf segments were cultivated on medium with kanamycin as a selection agent, but without phytohormones. Transformed tissues grew as roots which later regenerated plants. Kanamycin in the medium ensured that only doubly transformed tissues, which contain in their genome both pRi T-DNA as well as pGA472 chimeric kanamycin resistance gene were recovered. Regenerated plants showed clear-cut morphological deviations, but only a slight increase of the auxin content. Kanamycin resistance in the progeny segregated in the Mendelian ratio 3: 1 or 2: 1.     

Expression of mouse metallothionein-I gene confers cadmium resistance in transgenic tobacco plants

Transgenic tobacco plants containing a mouse metallothionein-I (MT-I) gene fused to the cauliflower mosaic virus 35S (CaMV 35S) promoter and nopaline synthase (nos) polyadenylation site were obtained by transforming tobacco leaf discs with an Agrobacterium tumefaciens strain carrying the chimaeric gene. Transformants were directly selected and rooted on medium containing cadmium and kanamycin. A total of 49 individual transgenic tobacco plants were regenerated. Among them 20% showed a very high expression level and their growth was unaffected by up to 200 M cadmium, whereas the growth of control plants was severely affected leaf chlorosis occurred on medium containing only 10 M cadmium. The concentration of MT-I in leaves of control and transgenic tobacco was determined with Cd/haemoglobin saturation assay, a polarographic method and western blotting. In addition, seeds from self-fertilized transgenic plants were germinated on medium containing toxic levels of cadmium and scored for tolerance/susceptibility to this heavy metal. The ratio of tolerant to susceptible plants was 3:1 indicating that the metallothionein gene is inherited as a single locus.     

POLLEN TUBE COMPETITION AND SELECTION FOR METAL TOLERANCE IN SILENE DIOICA (CARYOPHYLLACEAE) AND MIMULUS GUTTATUS (SCROPHULARIACEAE)

An experiment was conducted to test whether metal tolerance expressed in pollen would provide a competitive advantage during pollen tube growth and fertilization. Copper or zinc was introduced into the pistil by growing metal tolerant plants of Silene dioica or Mimulus guttatus in a nutrient solution to which metals were added. Flowers from treated and control plants were pollinated with either metal tolerant or metal sensitive pollen. The rate of pollen tube growth, the number of seeds set, and the number of viable seeds produced were measured. In general, no effects were found on the rates of pollen tube growth. However, the number of fertilizations and viable seeds were affected. When toxic metals were present, the relative success of pollen from the nontolerant parent was reduced.     

Comparison of the effects of kanamycin and geneticin on regeneration of papaya from root tissue

Kanamycin and geneticin are commonly used for the selection of neomycin phosphotransferase II (npt II) transformed plants. Since papaya tissue is sensitive to both antibiotics, it is difficult to explore their effects on the regeneration process solely based on using non-transformed tissues. Adventitious roots derived from npt II-transgenic and non-transgenic papaya shoots in vitro were used as explants in this investigation. The effects of kanamycin and geneticin on callus formation, embryogenesis, and conversion of somatic embryos to shoots were compared. Callus growth derived from npt II-transformed root explants was apparently enhanced on kanmycin within 50–200 mg l^–1 or on geneticin within 12.5–50 mg l^–1 as compared to those on antibiotic-free controls. The percentages of npt II-transformed somatic embryo-forming callus were not significantly different (16.3–18.3%) on geneticin less than 6.25 mg l^–1 and only slightly reduced (11.2–15.7%) on geneticin within 12.5–50 mg l^–1, whereas, formation of somatic embryos was strongly suppressed on kanamycin media. Conversion rates of npt II-transformed somatic embryos to shoots were not significantly different among all kanamycin or geneticin treatments. Percentages of the callus derived from non-transformed root explants were greatly reduced on the medium containing more than 25 mg l^–1 kanamycin or geneticin, and no somatic embryos formed from untransformed callus on any kanamycin or geneticin media. Our results indicated that somatic embryogenesis of callus derived from npt II-transformed root explants of papaya was strongly inhibited by kanamycin. Thus, to regenerate npt II-transformed cells from papaya root tissue, we recommend using the lower concentration geneticin (12.5–25 mg l^–1) to avoid the adverse effects of kanamycin on embryogenesis.