In vitro assay for 2,4-D resistance in transgenic cotton

Summary 2,4-Dichlorophenoxyacetic acid (2,4-D) resistant plants of transgenic cotton (Gossypium hirsutum L.) were produced using Agrobacterium tumefaciens containing a plasmid carrying the neomycin phosphotransferase II (npt II) and 2,4-D monooxygenase (tfd A) genes. An in vitro assay was performed to determine the sensitivity of seed germination, and the growth of seedlings of transgenic and non-transgenic cotton to various concentrations of kanamycin and 2,4-D. The results indicated that kanamycin caused the cotyledons of non-transgenic plants to turn white, but transgenic plants grew normally. Seed germination and seedling growth of non-transgenic plants were strongly inhibited by 2,4-D, but only slightly for transgenic plants. Transgenic plants and non-transgenic plants can be clearly distinguished by the use of 2 mg l⁻¹ 2,4-D in seed germination medium. There was a high correlation between the response of seed germination and the growth of seedlings to kanamycin or 2,4-D, based on the germination ration, albino ratio, dry weight or fresh weight. On this basis, we development a rapid method for identifying transgenic plants that has been verified in the field. These findings will allow identification of cotton transformants at an early stage of plant development, saving time and improving cultivars containing the 2,4-D resistance trait.     

Measuring gene flow in the cultivation of transgenic cotton (Gossypium hirsutum L.)

Transgenic Bt cotton NewCott 33B and transgenic tfd A cotton TFD were chosen to evaluate pollen dispersal frequency and distance of transgenic cotton (Gossypium hirsutum L.) in the Huanghe Valley Cotton-producing Zone, China. The objective was to evaluate the efficacy of biosafety procedures used to reduce pollen movement. A field test plot of transgenic cotton (6×6 m) was planted in the middle of a nontransgenic field measuring 210×210 m. The results indicated that the pollen of Bt cotton or tfd A cotton could be dispersed into the environment. Out-crossing was highest within the central test plot where progeny from nontransgenic plants, immediately adjacent to transgenic plants, had resistant plant progeny at frequencies up to 10.48%. Dispersal frequency decreased significantly and exponentially as dispersal distance increased. The flow frequency and distance of tfd A and Bt genes were similar, but the pollen-mediated gene flow of tfd A cotton was higher and further to the transgenic block than that of Bt cotton (χ² = 11.712, 1 degree of freedom, p<0.001). For the tfd A gene, out-crossing ranged from 10.13% at 1 m to 0.04% at 50 m from the transgenic plants. For the Bt gene, out-crossing ranged from 8.16% at 1 m to 0.08% at 20 m from the transgenic plants. These data were fit to a power curve model: y=10.1321x ^−1.4133 with a correlation coefficient of 0.999, and y=8.0031x ^−1.483 with a correlation coefficient of 0.998, respectively. In this experiment, the farthest distance of pollen dispersal from transgenic cotton was 50 m. These results indicate that a 60-m buffer zone would serve to limit dispersal of transgenic pollen from small-scale field tests.     

Cotton plants transformed with a bacterial degradation gene are protected from accidental spray drift damage by the herbicide 2,4-dichlorophenoxyacetic acid

The agronomic performance of broad leaved crop plants such as cotton would be greatly improved if genetically-engineered resistance to broadleaf herbicides could both protect the plants from accidental spray drift damage and allow the suppression of problem broadleaf weeds by chemical means. Followingin vitro modification and the addition of plant expression signals, the gene for 2,4-D monooxygenase, a bacterial enzyme that degrades the broadleaf herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), was introduced into cotton plants byAgrobacterium-mediated transformation. First generation homozygous progeny of regenerated transgenic cotton plants carrying this gene exhibited up to a 50–100 fold increase in tolerance to 2,4-D compared with untransformed controls, and glasshouse trials suggest that the genetically-engineered plants would be completely protected from spray drift of 2,4-D, at least up to the normal field application rates commonly used on neighbouring cereal crops.     

Expression of a bacterial gene in transgenic tobacco plants confers resistance to the herbicide 2,4-dichlorophenoxyacetic acid

Plants resistant to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) were produced through the genetic engineering of a novel detoxification pathway into the cells of a species normally sensitive to 2,4-D. We cloned the gene for 2,4-D monooxygenase, the first enzyme in the plasmid-encoded 2,4-D degradative pathway of the bacterium Alcaligenes eutrophus, into a cauliflower mosaic virus 35S promoter expression vector and introduced it into tobacco plants by Agrobacterium-mediated transformation. Transgenic tobacco plants expressing the highest levels of the monooxygenase enzyme exhibited increased tolerance to 2,4-D in leaf disc and seed germination assays, and young plants survived spraying with levels of herbicide up to eight times the usual field application rate. The introduction of the gene for 2,4-D monooxygenase into broad-leaved crop plants, such as cotton, should eventually allow 2,4-D to be used as an inexpensive post-emergence herbicide on economically important dicot crops.     

Mutation analysis of the different<Emphasis Type="Italic"> tfd</Emphasis> genes for degradation of chloroaromatic compounds in<Emphasis Type="Italic"> Ralstonia eutropha</Emphasis> JMP134

Ralstonia eutropha JMP134 possesses two sets of similar genes for degradation of chloroaromatic compounds, tfdCDEFB (in short: tfd _I cluster) and tfdD _II C _II E _II F _II B _II (tfd _II cluster). The significance of two sets of tfd genes for the organism has long been elusive. Here, each of the tfd genes in the two clusters on the original plasmid pJP4 was replaced by double recombination with a gene fragment in which a kanamycin resistance gene was inserted into the respective tfd genes reading frame. The insertion mutants were all tested for growth on 2,4-dichlorophenoxyacetic acid (2,4-D), 2-methyl-4-chlorophenoxyacetic acid (MCPA), and 3-chlorobenzoate (3-CBA). None of the tfdD _II C _II E _II F _II B _II genes appeared to be essential for growth on 2,4-D or on 3-CBA. Mutations in tfdC, tfdD and tfdF also did not abolish but only retarded growth on 2,4-D, indicating that they were redundant to some extent as well. Of all tfd genes tested, only tfdE and tfdB were absolutely essential, and interruption of those two reading frames abolished growth on 2,4-D, 3-CBA (tfdE only), and MCPA completely. Interestingly, strains with insertion mutations in the tfd _I cluster and those in tfdD _II , tfdC _II , tfdE _II and tfdB _II were severely effected in their growth on MCPA, compared to the wild-type. This indicated that not only the tfd _I cluster but also the tfd _II cluster has an essential function for R. eutropha during growth on MCPA. In contrast, insertion mutation of tfdD _II resulted in better growth of R. eutropha JMP134 on 3-CBA, which is most likely due to the prevention of toxic metabolite production in the absence of TfdD_II activity.     

Increased glycine betaine synthesis and salinity tolerance in AhCMO transgenic cotton lines

Glycine betaine is an osmoprotectant that plays an important role and accumulates rapidly in many plants during salinity or drought stress. Choline monooxygenase (CMO) is a major catalyst in the synthesis of glycine betaine. In our previous study, a CMO gene (AhCMO) cloned from Atriplex hortensis was introduced into cotton (Gossypium hirsutum L.) via Agrobacterium mediation to enhance resistance to salinity stress. However, there is little or no knowledge of the salinity tolerance of the transgenic plants, particularly under saline-field conditions. In the present study, two transgenic AhCMO cotton lines of the T₃ generation were used to study the AhCMO gene expression, and to determine their salinity tolerance in both greenhouse and field under salinity stress. Molecular analysis confirmed that the transgenic plants expressed the AhCMO gene. Greenhouse study showed that on average, seedlings of the transgenic lines accumulated 26 and 131% more glycine betaine than those of non-transgenic plants (SM3) under normal and salt-stress (150 mmol l⁻¹ NaCl) conditions, respectively. The osmotic potential, electrolyte leakage and malondialdehyde (MDA) accumulation were significantly lower in leaves of the transgenic lines than in those of SM3 after salt stress. The net photosynthesis rate and Fv/Fm in transgenic cotton leaves were less affected by salinity than in non-transgenic cotton leaves. Therefore, transgenic cotton over-expressing AhCMO was more tolerant to salt stress due to elevated accumulation of glycine betaine, which provided greater protection of the cell membrane and photosynthetic capacity than in non-transgenic cotton. The seed cotton yield of the transgenic plants was lower under normal conditions, but was significantly higher than that of non-transgenic plants under salt-stressed field conditions. The results indicate that over-expression of AhCMO in cotton enhanced salt stress tolerance, which is of great value in cotton production in the saline fields.     

A partially disarmed<Emphasis Type="Italic"> vir</Emphasis> helper plasmid,pKYRT1, in conjunction with 2,4-dichlorophenoxyactic acid promotes emergence of regenerable transgenic somatic embryos from immature cotyledons of soybean

Agrobacterium tumefaciens strain KYRT1 harboring the virulence helper plasmid pKYRT1 induces transgenic somatic embryos (SEs) at high frequency from infected immature soybean cotyledons. KYRT1 is derived from the highly oncogenic strain Chry5. However, pKYRT1 is not completely disarmed and still contains an entire T-right (T_R) and a portion of T-left (T_L). In this report, binary strains, each carrying fully disarmed vir helper plasmids including pKPSF2, which is a fully disarmed version of pKYRT1, were compared to strain KYRT1 for their ability to induce transgenic SEs on immature cotyledons of soybean. Six weeks following cocultivation, histochemical GUS assays of cultured explants indicated that all fully disarmed vir helper plasmids transferred their binary T-DNA, containing a GUS-intron gene, into soybean tissues. However, none of these transformed tissues developed SEs on medium with or without 2,4-dichlorophenoxyactic acid (2,4-D). On the other hand, immature cotyledons cocultivated with strain KYRT1 exhibited high induction of transgenic SEs, but only on medium supplemented with 2,4-D. Derivatives of strain Chry5 harboring other vir helper plasmids did not induce transgenic SEs under any conditions tested, thus suggesting that the chromosomal background of KYRT1 alone was not sufficient to promote somatic embryogenesis. PCR analysis indicated that 55% of transgenic embryogenic cultures and 29% of transgenic T₀ soybean plants derived by transformation using strain KYRT1 contained T_R from pKYRT1 in addition to the uidA gene from the binary construct. None of the transgenic tissues or T₀ plants contained T_L DNA. These results suggest that some function coded for by T_R of pKYRT1 influences somatic embryogenesis in conjunction with exposure of the plant tissues to 2,4-D. Since the co-transformation frequency of the undesirable T-DNA sequences from the vir helper plasmid was relatively low, the partially disarmed strain KYRT1 will likely be very useful for the production of normal transgenic plants of diverse soybean cultivars.Abbreviations 2,4-D 2,4-Dichlorophenoxyactic acid - GUS -Glucuronidase - hpt Hygromycin phosphotransferase gene - SE Somatic embryo - uidA -Glucuronidase gene     

Overexpression of Maize IAGLU in Arabidopsis thaliana Alters Plant Growth and Sensitivity to IAA but not IBA and 2,4-D

Overexpression of the IAGLU gene from maize (ZmIAAGLU) in Arabidopsis thaliana, under the control of the CaMV 35S promoter, inhibited root but not hypocotyl growth of seedlings in four different transgenic lines. Although hypocotyl growth of seedlings and inflorescence growth of mature plants was not affected, the leaves of mature plants were smaller and more curled as compared to wild-type and empty vector transformed plants. The rosette diameter in transgenic lines with higher ZmIAGLU expression was also smaller compared to the wild type. Free indole-3-acetic acid (IAA) levels in the transgenic plants were comparable to the wild type, even though a decrease in free IAA levels might be expected from overexpression of an IAA-conjugate–forming enzyme. IAA-glucose levels, however, were increased in transgenic lines compared to the wild type, indicating that the ZmIAGLU gene product is active in these plants. In addition, three different 35SZmIAGLU lines showed less inhibition of root growth when cultivated on increasing concentrations of IAA but not indole-3-butyric acid (IBA) and 2,4-dichlorophenoxyacetic acid (2,4-D). Feeding IAA to transgenic lines resulted in increased IAA-glucose synthesis, whereas the levels of IAA-aspartate and IAA-glutamine formed were reduced compared to the wild type. Our results show that IAA homeostasis can be altered by heterologous overexpression of a conjugate-forming gene from maize.     

Inheritance and segregation of exogenous genes in transgenic cotton

Three transgenic cotton varieties (lines) were chosen for the study of inheritance and segregation of foreign Bt (Bacillus thuringiensis toxin) andtfdA genes in cotton. The transformed cotton varieties CCRI 30 and NewCott 33B expressing the BtcryIA gene, and cotton line TFD expressing thetfdA gene were crossed with CCRI 19, CCRI 12 and Lumian 6. The results confirm inheritance and segregation of (i) the exogenous Bt gene in transgenic CCRI 30 and NewCott 33B, governing resistance to bollworm, and (ii) the exogenoustfdA gene in transgenic TFD, governing resistance to the herbicide 2,4-D. Both resistance characters were governed by a single dominant nuclear gene, and were not affected by cytoplasm. Our data support the conclusion that foreign traits encoded by single genes are inherited and expressed in Mendelian fashion in cotton. Our results also indicate that a practical backcross breeding program could be used to develop cotton cultivars combining one or more resistance traits from foreign and native gene sources.     

过表达或沉默棉花GhPCBER基因株系的获得及其茎叶木质素和木脂素含量研究

编码苯基香豆满苄基醚还原酶(phenylcoumaran benzylic ether reductase,PCBER)的基因PCBER属于PIP亚家族,是苯丙烷代谢途径中参与木脂素合成的关键基因。该研究构建了棉花GhPCBER基因的植物过表达载体并转化拟南芥,同时构建了VIGS(virus induced gene silencing,病毒诱导的基因沉默)载体转化棉花,采用实时荧光定量PCR技术对GhPCBER基因在不同组织中的表达进行分析;对野生型和转基因植株茎叶组织中的木质素和木脂素含量进行测定分析。结果表明:(1)成功构建了GhPCBER植物过表达载体pGWB17-GhPCBRE以及基因沉默重组载体pTRV2-GhPCBER;经遗传转化获得6株转棉花GhPCBER基因抗性拟南芥植株,同时获得15株GhPCBER基因沉默棉花植株(5株为一组)。(2)PCR检测表明,6株转基因拟南芥均为过表达株系,其中株系1、2、3相对表达量更高,且在茎、叶组织中的表达量分别较野生型提高了7～14倍和6～16倍,表明GhPCBER基因成功在拟南芥中过表达;GhPCBER基因沉默棉花植株的茎、叶组织中的表达量分别比野生型棉株约下降12%和26%,表明烟草脆裂病毒(TRV)体系(pTRV2-GhPCBER)成功抑制了GhPCBER基因的表达。(3)转GhPCBER基因拟南芥茎、叶中木质素和木脂素含量较野生型均显著降低;GhPCBER基因沉默棉花植株茎、叶中木质素和木脂素含量较野生型均极显著降低;组织化学染色观察发现GhPCBER基因沉默棉花植株茎秆颜色明显比野生型染色浅,也证明沉默基因棉花植株茎秆中的木质素含量减少。(4)苯丙烷代谢通路中8个相关基因的实时荧光定量PCR分析发现,过表达或抑制GhPCBRE基因均会导致苯丙烷代谢途径发生重新定向。     

Inheritance of a bacterial hygromycin phosphotransferase gene in the progeny of primary transgenic pea plants

Summary An analysis of the progeny of primary transgenic pea plants in terms of transmission of the transferred DNA, fertility and morphology is presented. A transformation system developed for pea that allows the regeneration of fertile transgenic pea plants from calli selected for antibiotic resistance was used. Expiants from axenic shoot cultures were co-cultivated with a nononcogenic Agrobacterium tumefaciens strain carrying a gene encoding hygromycin phosphotransferase as selectable marker, and transformed callus could be selected on callus-inducing media containing 15 mg/l hygromycin. After several passages on regeneration medium, shoot organogenesis could be reproducibly induced on the hygromycin resistant calli, and the regenerated shoots could subsequently be rooted and transferred to the greenhouse, where they proceeded to flower and set seed. The transmission of the introduced gene into the progeny of the regenerated transgenic plants was studied over two generations, and stable transmission was shown to take place. The transgenic nature of the calli and regenerated plants and their progeny was confirmed by DNA and RNA analysis. The DNA and ploidy levels of the progeny plants and primary regenerants were studied by chromosome analysis, and the offspring of the primary transformants were evaluated morphologically.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - BA 6-ben-zyladenine - hpt hygromycin phosphotransferase gene - IAA indole acetic acid, kin, kinetin - NAA -naphtalene acetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid     

Genetic tranformation of cotyledon explants of cowpea (Vigna unguiculata L. Walp) using Agrobacterium tumefaciens

Mature de-embryonated cotyledons with intact proximal end of Vigna unguiculata were cultured on B5 basal medium containing varying concentrations of BAP. Thirty-six percent of the explants produced shoots on B5 medium supplemented with 8× 10^–6 M BAP. Cotyledon explants were pre-incubated for 24 h, inoculated with A. tumefaciens pUCD2614 carrying pUCD2340, co-cultivated for 48 h and transferred to hygromycin-B (25 mg/l) containing shoot induction medium. Approximately 15–19% of the explants produced shoots on the selection medium. The elongated shoots were subsequently rooted on B5 basal medium containing hygromycin. The transgenic plants were later established in pots. The presence of hpt gene in the transgenic plants was confirmed by Southern blot hybridization.Abbreviations BAP 6-Benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - hpt hygromycin phosphotransferase - IAA Indole-3-acetic acid - NAA 1-naphthaleneacetic acid     

海岛棉GbHCT13基因的功能验证

该研究利用海岛棉‘新海21’和陆地棉ND203以及模式植物拟南芥,通过转基因及荧光定量检测等方法探究海岛棉GbHCT13基因(GenBank 登录号MW048849)在纤维发育中的功能。结果显示：(1)成功构建重组载体pCAMBIA3301 GbHCT13,经农杆菌介导法转化、除草剂抗性基因筛选、荧光定量检测方法鉴定获得转GbHCT13基因拟南芥T₃代植株4株;qRT PCR检测表明,转基因植株中GbHCT13基因表达量较野生型极显著增加。(2)转基因拟南芥过表达GbHCT13基因使植株同一时期的生长较野生型旺盛,株形、叶片数、抽薹数和茎秆表皮毛数量均与野生型存在差异;组织化学分析发现,转GbHCT13基因的拟南芥较野生型茎秆初生木质部生长活跃,导管增粗,次生木质部导管细胞壁横截面积变大,但髓质细胞无明显变化;过表达GbHCT13使拟南芥中木质素合成途径基因发生不同程度改变,其中CAD、CCoAOMT、PAL和4CL与GbHCT13基因的表达呈正相关。(3)经大田筛选、分子鉴定,成功获得转GbHCT13基因棉花植株3株;转GbHCT13基因棉花的棉纤维伸长率增加,纤维强度增大;沉默GbHCT13基因使棉花植株木质素含量降低,茎秆表皮毛数量减少,木质部导管细胞数量减少,导管细胞壁中木质素沉积量降低,而棉株并未发生株高上的明显矮化现象,且木质素合成通路中的CAD、CCoAOMT、CCR、PAL 4个基因的表达均呈降低趋势,说明抑制GbHCT13使得棉花生长代谢受阻,影响纤维发育起始。研究表明,GbHCT13基因能影响棉花植株中木质素合成从而调控纤维的生长发育,其功能与GbHCT13基因在模式植物拟南芥中的基本一致。     

Transgenic Plants of Colonial Bentgrass from Embryogenic Callus via Agrobacterium-mediated Transformation

Colonial bentgrass (Agrostis tenuis Sibth. Fl. Oxen.) is a cool-season turfgrass used on fairways in golf courses. The object of this study was to develop a more efficient, reliable and repeatable approach in transforming the grass using Agrobacterium (strain LBA4404), in which -glucuronidase (gus) gene was used as a reporter and hygromycin phosphotransferase (hpt) gene as a selectable marker. This vector was effective in transforming 7-week-old calluses derived from mature seeds cultured on MS medium supplemented with 2,4-D. A two-step solid medium selection with increasing hygromycin concentration (from 50 to 70 mg l^–1) was used to obtain resistant calluses. Hundreds of transgenic plants have been produced from several independent transformed calluses. The presence of functional -glucuronidase (GUS) was detected in hygromycin-resistant calluses, young leaves and roots of transgenic plants. The transgenic plants collected from greenhouse showed strong resistance to 50 mg l^–1 hygromycin solution. Four putative transgenic plants and one control plant were randomly chosen and analyzed by Southern blot analysis. Bands corresponding to the hpt gene were clearly shown in transgenic plants.     

Transformation of the wild tomatoLycopersicon chilense Dun. byAgrobacterium tumefaciens

Summary Leaf disc transformation-regeneration technique was applied to the drought tolerant wild relative of cultivated tomato,Lycopersicon chilense, using a plasmid construct which contained the coding sequences of neomycin phosphotransferase (NPTII) and chloramphenicol acetyltransferase (CAT) genes. The two genotypes used, LA2747 and LA1930, showed a distinct difference in their aptitude to transformation; a higher success rate was obtained for the first genotype in every stage of the process. Shoots were formed on the regeneration medium containing 100 g/ml kanamycin through direct or indirect organogenesis. Root formation became only possible when the concentration of kanamycin was reduced to 50 g/ml. Expression of chloramphenicol acetyltransferase gene was observed in all of the kanamycin-screened plants after they matured; the activity of the gene was absent or low in some of the young plants. The presence of the CAT gene in transgenic plants was further confirmed by Southern blot analysis. Although transgenic plants grew to maturity, they did not produce fruit, owing to the self incompatibility ofL. chilense. Abbreviations BAP 6-benzylaminopurine - CAT chloramphenicol acetyltransferase - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - LB Luria Broth - EDTA ethylenediamine-tetraacetic acid     

Silicon Carbide Whisker-Mediated Embryogenic Callus Transformation of Cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.) and Regeneration of Salt Tolerant Plants

A silicon carbide whisker-mediated gene transfer system with recovery of fertile and stable transformants was developed for cotton (Gossypium hirsutum L.) cv. Coker-312. Two-month-old hypocotyl-derived embryogenic/non-embryogenic calli at different days after subculture were treated with silicon carbide whiskers for 2 min in order to deliver pGreen0029 encoding GUS gene and pRG229 AVP1 gene, encoding Arabidopsis vacuolar pyrophosphatase, having neomycin phosphotransferaseII (nptII) genes as plant-selectable markers. Three crucial transformation parameters, i.e., callus type, days after subculture and selection marker concentration for transformation of cotton calli were evaluated for optimum efficiency of cotton embryogenic callus transformation giving upto 94% transformation efficiency. Within six weeks, emergence of kanamycin-resistant (km^r) callus colonies was noted on selection medium. GUS and Southern blot analysis showed expression of intact and multiple transgene copies in the transformed tissues. Kanamycin wiping of leaves from T₁, T₂, and T₃ progeny plants revealed that transgenes were inherited in a Mendelian fashion. Salt treatment of T₁ AVP1 transgenic cotton plants showed significant enhancement in salt tolerance as compared to control plants. Thus far, this is first viable physical procedure after particle bombardment available for cotton that successfully can be used to generate fertile cotton transformants.     

Recovery of phenotypically normal transgenic plants of Brassica oleracea upon Agrobacterium rhizogenes-mediated co-transformation and selection of transformed hairy roots by GUS assay

In this paper we describe the production of transgenic broccoli and cauliflower with normal phenotype using an Agrobacterium rhizogenes-mediated transformation system with efficient selection for transgenic hairy-roots. Hypocotyls were inoculated with Agrobacterium strain A4T harbouring the bacterial plasmid pRiA4 and a binary vector pMaspro::GUS whose T-DNA region carried the gus reporter gene. pRiA4 transfers T_L sequences carrying the rol genes that induce hairy root formation. Transgenic hairy-root production was increased in a difficult-to-transform cultivar by inclusion of 2,4-D in the medium used to resuspend the Agrobacterium prior to inoculation. Transgenic hairy roots could be selected from inoculated explants by screening root sections for GUS activity; this method eliminated the use of antibiotic resistance marker genes for selection. Transgenic hairy roots were produced from two cauliflower and four broccoli culivars. Shoots were regenerated from transgenic hairy root cultures of all four cultivars tested and successfully acclimatized to glasshouse conditions, although some plants had higher than diploid ploidy levels. Southern analysis confirmed the transgenic nature of these plants. T₀ plants from seven transgenic lines were crossed or selfed to produce viable seed. Genetic analysis of T₁ progeny confirmed the transmission of traits and revealed both independent and co-segregation of Ri T_L-DNA and vector T-DNA. GUS-positive phenotypically normal progeny free of T_L-DNA were identified in three transgenic lines out of the six tested representing all the cultivars regenerated including both cauliflower and broccoli.     

转OvDREB2B基因陆地棉鉴定及其对水分渗透胁迫的分析

通过花粉管通道技术,以该实验室自育陆地棉品系TH₁和TH₂为材料,将诸葛菜(Orychophragmus vidaceus)抗逆转录因子OvDREB2B基因构建到植物表达载体后,导入棉花基因组,经卡那霉素筛选和分子鉴定表明目的基因已整合到棉花基因组中并表达。将T₁代转基因植株和受体对照在温室中栽培,待植株生长至四叶一心时,用不同渗透势的PEG-6000水溶液进行渗透胁迫处理,分析探讨转基因植株的抗旱效果及其抗旱机理。结果显示:当渗透势为0和0.5 MPa处理时,转基因植株和对照无明显差异;当渗透势为0.8 MPa和1.1 MPa处理时转基因植株较对照抗旱性明显提高。当渗透势为1.1 MPa处理96 h时,对照植株F_v/F_m降至0.2左右,而转基因植株仍正常生长,F_v/F_m值约为0.51,而且初始荧光（F₀）值、净光合速率（P_n）、胞间CO₂浓度（C_i）、蒸腾速率（T_r）等一系列参数转基因植株都明显优于对照,表明DREB2B基因能够提高棉花对水分胁迫的耐受性。     

Improvement of cotton fiber quality by transforming the<Emphasis Type="Italic"> acsA</Emphasis> and<Emphasis Type="Italic"> acsB</Emphasis> genes into<Emphasis Type="Italic"> Gossypium hirsutum</Emphasis> L. by means of vacuum infiltration

A novel method for the genetic transformation of cotton pollen by means of vacuum infiltration and Agrobacterium-mediated transformation is reported. The acsA and acsB genes, which are involved in cellulose synthesis in Acetobacter xylinum, were transferred into pollen grains of brown cotton with the aim of improving its fiber quality by incorporating useful prokaryotic features into the colored cotton plants. Transformation was carried out in cotton pollen-germinating medium, and transformation was mediated by vector pCAMBIA1301, which contains a reporter gene -glucuronidase (GUS), a selectable marker gene, hpt, for hygromycin resistance and the genes of interest, acsA and acsB. The integration and expression of acsA, acsB and GUS in the genome of transgenic plants were analyzed with Southern blot hybridization, PCR, histochemical GUS assay and Northern blot hybridization. We found that following pollination on the cotton stigma transformed pollen retained its capability of double-fertilization and that normal cotton seeds were produced in the cotton ovary. Of 1,039 seeds from 312 bolls pollinated with transformed pollen grains, 17 were able to germinate and grow into seedlings for more than 3 weeks in a nutrient medium containing 50 mg/l hygromycin; eight of these were transgenic plants integrated with acsA and acsB, yielding a 0.77% transformation rate. Fiber strength and length from the most positive transformants was 15% greater than those of the control (non-transformed), a significant difference, as was cellulose content between the transformed and control plants. Our study suggests that transformation through vacuum infiltration and Agrobacterium mediated transformation can be an efficient way to introduce foreign genes into the cotton pollen grain and that cotton fiber quality can be improved with the incorporation of the prokaryotic genes acsA and acsB.Communicated by D. Bartels