Effect of ethylene on Agrobacterium tumefaciens-mediated gene transfer to melon

The effect of ethylene on gene transfer mediated by an Agrobacterium tumefaciens harbouring a binary vector with the β‐glucuronidase (uid A) gene was investigated in melon, Cucumis melo L. Explants excised from melon cotyledons produced ethylene, the production of which was increased by the addition of 1‐aminocyclopropane‐1‐carboxylic acid (ACC, 20 or 200 μM), and inhibited by the addition of aminoethoxy‐vinylglycin (AVG, 10 μM). Agrobacterium inoculation of explants increased ethylene production, while application of AVG during inoculation reduced it. After 4 days of co‐cultivation with Agrobacterium, gene transfer in the explants was assayed by transient uid A expression. Application of ACC to the co‐cultivation medium reduced Agrobacterium‐mediated gene transfer to explants and that of AVG increased it. These results suggest that ethylene affects the A. tumefaciens‐mediated gene transfer to the explants excised from melon cotyledons, and the efficiency of Agrobacterium‐mediated gene transfer can be improved by inhibiting ethylene production from the explants.     

Genetic variability in melon based on microsatellite variation

A set of 18 simple‐sequence repeat (SSR or microsatellite) markers was used to study genetic diversity in a collection of 27 melon (Cucumis melo L.) accessions, representing a broad range of wild and cultivated melons. The materials studied were highly polymorphic for SSRs and a total of 114 alleles were detected (average of 6.3 alleles per locus). Cluster analysis suggests the division of these accessions into two major groups, largely corresponding to the division of C. melo in the two subspecies agrestis and melo. The assignment of the accession to the subspecies was generally in agreement with published reports, except for those corresponding to the ‘dudaim’ and ‘chito’ cultivar groups, which, according to the observed SSR variability, should be included in subspecies agrestis. Based on cluster analysis, five groups of accessions were defined. The two most divergent groups include mainly accessions from the Mediterranean which form one group, and accessions from China, Japan, Korea and India forming the other. Both groups shared a low level of intra‐accession variation compared with the other groups, which suggests an erosion of their genetic variability because of drift and/or inbreeding. The remaining accessions, mainly from Central Africa and India, were more variable and may be an important source of genetic variation for melon breeding.     

转Cry1A基因海岛棉的获得和鉴定

进行农杆菌介导转抗虫基因试验,以期选育抗虫性优良的海岛棉新品系(品种);本实验在海岛棉体细胞胚再生体系的基础上,将携带Cry1A基因的表达载体pBI121通过农杆菌介导法转化到‘新海33号’的胚性愈伤组织中。经培养基筛选、PCR检测、RT-PCR检测、抗虫性鉴定等筛选抗虫后代。经培养基筛选共获得12棵抗性植株。PCR检测结果显示,这12棵抗性植株均能扩增出了大小相符的片段,检测结果呈阳性;RT-PCR结果显示,12棵转基因植株目的基因已经整合入基因组,且能反转录出mRNA;Bt-Cry1Ab/Ac转基因试纸条检测结果表明这12棵转基因植株含有目的蛋白;抗虫性鉴定结果证实了这些转基因植株对棉铃虫幼虫具有一定的抗性。本研究成功将抗虫基因导入至海岛棉的体内,获得了具有抗虫性的植株,其结果对转基因抗虫海岛棉新品种的培育具有一定的参考价值。     

农杆菌介导大豆遗传转化研究进展及其应用

农杆菌介导是大豆遗传转化的主要手段。近几年来大豆遗传转化方法得到了快速发展,但是仍然存在受体基因型匮乏、转化效率低,再生系统不完善等问题,为了进一步探讨大豆遗传转化的主要问题和策略,本研究归纳了近年来农杆菌介导大豆遗传转化在受体基因型及外植体选择,菌株筛选,标记基因选用和培养基组成等方面所做的改进,以得出有效的解决方法。并且介绍了农杆菌介导大豆遗传转化在作物改良和功能基因组学上的应用。     

根癌农杆菌介导ACO基因对亚洲百合的转化

以亚洲百合‘普利安娜’的鳞块为基因转化的受体材料,利用根癌农杆菌介导法将ACO基因导入百合,研究了影响其转化的因素。结果表明,以鳞块为受体材料,外植体预培养0天的污染率最低,有利于农杆菌的侵染;菌液浓度OD600=0.8左右时侵染30 min效果较好;重悬后菌液活化1~2 h有利于抗性芽的分化;MS重悬液和共培养基中均添加20 mg/L乙酰丁香酮（AS）可提高抗性芽分化率;去除大量元素中的NH4NO3可提高转化率;抗性植株经PCR检测部分呈现阳性,初步证明外源基因已整合到百合基因组中。     

Effect of genotype and light intensity on somatic embryogenesis and plant regeneration in melon (Cucumis melo L.)

The efficiency of 14 commercial cultivars of melon (Cucumis melo L.) for callus induction, plant regeneration and somatic embryogenesis under different photosynthetic photon flux densities (PPFDs) (150 or 50μmol m^?2 s^?1) was investigated. Cotyledonal explants were cultured on a Murashige and Skoog (MS) medium supplemented either with 9.0 μM 2,4-dichlorophenoxyacetic acid and 23.2μM kinetin or with 0.05 μM 2,4-dichlorophenoxyacetic acid and 0.26 μM 6-benzyladenine for the induction of somatic embryogenesis and shoots, respectively. For embryo maturation and root induction, growing callus tissues were transferred on growth regulator-free MS medium. Both genotype and the intensity of light significantly affected the rate of somatic embryo-genesis, embryo maturation and plant regeneration. On average, 12–47 primary globular-stage embryos were produced per mm² of explant surface. Fully developed, cotyledonary-stage somatic embryos were obtained from only three cultivars. Relatively high root induction rates were observed both on the shoot induction medium (11 cultivars) and on growth regulator-free medium (seven cultivars). In contrast, only six cultivars responded positively to the shoot induction treatment. Callus growth and somatic embryogenesis were significantly improved if cultures were incubated under higher PPFD values, although plant regeneration from all cultivars was significantly reduced under the same conditions.     

Genetic transformation of pigeonpea with rice chitinase gene

With a long‐term plan to develop transgenic pigeonpea with resistance to fungal disease, the transfer of a rice chitinase gene to pigeonpea [Cajanus cajan (L.) Millsp.] is reported here. The rice chitinase gene harboured in the plasmid pCAMBIA 1302:RChit was delivered via the Agrobacterium‐mediated method to the cotyledonary node explants followed by subsequent regeneration of complete plants on selection media containing hygromycin. Putative transformed pigeonpea plants were recovered with stringent selection pressure and confirmed using molecular techniques. Stable integration and expression of the chitinase gene has been confirmed in the T0 and T1 transgenics through molecular analysis.     

根癌农杆菌介导的苹果遗传转化研究进展

农杆菌介导的遗传转化方法是苹果进行遗传改良的主要方法,本文综述了影响农杆菌转化苹果的各个因素,指出今后的研究重点应放在新的转化方法的提出及进一步提高转化效率的研究上。     

小对叶植株再生及遗传转化体系构建

为了利用生物技术方法开发及改良水生植物小对叶,以小对叶（Bacopa monnieri）叶片为外植体,建立其组织培养再生体系及根癌农杆菌介导的遗传转化体系。结果表明：小对叶叶盘最佳愈伤组织诱导培养基是MS+6-BA 0.5 mg/L+NAA 0.02 mg/L;最佳分化培养基是MS+6-BA 0.5 mg/L+NAA 0.05 mg/L;最佳生根培养基是1/2MS+IBA 0.1 mg/L。最佳的根癌农杆菌介导的小对叶遗传转化条件是：外植体预培养2天,侵染菌液OD600为0.5,侵染时间7 min、共培养4天和延迟筛选4天,可获得最高转化率21.4%。     

Distribution of two groups of melon landraces and inter-group hybridization enhanced genetic diversity in Vietnam

To understand the genetic diversity and differentiation of Vietnamese melon (Cucumis melo L.), we collected 64 landraces from the central and southern parts of the country and assessed molecular polymorphism using simple sequence repeat and random amplified polymorphic DNA markers. The Vietnamese melon was divided into seven cultivar groups, namely “Dua le”, “Dua vang”, “Dua bo”, “Dua gang-andromonoecious”, “Dua gang-monoecious”, “Dua thom”, “Montok”, and the weedy-type melon “Dua dai”. Among these, Dua le, Dua vang, Dua bo, and Dua gang-andromonoecious are cultivated on plains and they formed cluster II along with the reference accessions of Conomon and Makuwa. Based on genetic distance, Dua le and Dua vang were regarded as Makuwa and Dua bo and Dua gang-andromonoecious as Conomon. In contrast, Dua thom and Montok are cultivated in highlands, and they formed cluster III along with landraces from the southern and eastern foot of the Himalayas. Dua gang-monoecious which is commonly cultivated in the southern parts of Vietnam, exhibited the greatest genetic diversity, as explained by its possible origin through the hybridization between Dua gang-andromonoecious and Montok. Genetic differences in melon landraces between plains and highlands and hybridization between these two geographical groups have contributed to the enhancement of genetic diversity in Vietnamese melon.     

农杆菌介导外源基因在棉花中的表达

用 5～ 6d的棉花无菌苗下胚轴切段与农杆菌共培养 ,菌株质粒中 Gus基因为标记基因 ,NPT 基因为选择基因。在含 0 .1 mg· L- 12 ,4- D和 0 .1 mg· L- 1KT的 MS培养基上共培 48h,转移到添加头孢霉素 50 0 mg· L- 1、卡那霉素 50 mg· L- 1MS培养基中诱导和筛选抗性愈伤组织 ,70～ 80 d时 ,进行 Gus检测 ,选择 Gus阳性愈伤组织 ,经体细胞分化生成转基因再生植株     

Production of fertile transgenic Brassica napus by Agrobacterium-mediated transformation of protoplasts

A protocol for Agrobacterium tumefaciens‐mediated transformation of Brassica napus mesophyll protoplasts is described. A strain with a neomycin phosphotransferase (nptII) gene and a KCS gene under control of a napin promoter was used at co‐cultivation. Transformed protoplasts were regenerated to fertile and morphologically normal transgenic plants. Transformants were confirmed by PCR of the nptII gene and NAP/KCS expression cassette, and Southern blot analysis. Seeds of the transformants showed a changed fatty acid profile: two transformants had a higher erucic acid level and differed significantly from that of B. napus. Genetic analysis of the progeny revealed that the kanamycin resistance introduced was inherited in a Mendelian fashion.     

Phenotypic expression of quantitative and qualitative components of partial resistance to powdery mildew (Sphaerotheca fuliginea race 1) in melon (Cucumis melo) germplasm

A study was conducted to investigate the expression of four components of partial resistance to Sphaerotheca fuliginea race 1 in selected melon (Cucumis melo L.) lines viz. infection frequency, latent period, spore production, and disease-severity score. Those components were evaluated at two developmental stages of the host: the cotyledon stage and the stage of the first two true leaves. Detached plant parts (disks of cotyledons and true leaves) were inoculated using a vacuum-operated settling tower. All four components showed significant variation among genotypes, and correlations between components at both developmental stages were large and significant. The line ‘CNPH 83–095’ (without any major resistance gene to powdery mildew) presented the highest level of partial resistance in both vegetative stages for almost all components evaluated. The lines ‘W-6’ (Pm1Pm1, Pm2Pm2), ‘Cinco’ (Pm1Pm1, Pm2Pm2), and CNPH ‘84–147’ (Pm1Pm1), even though carrying the major gene Pm1 for complete resistance to race 1 of the fungus, showed slight but significant differences for quantitative components of partial resistance at the cotyledonal stage. Different levels of partial resistance may be expressed, even in lines with a major race-specific resistance gene to powdery mildew, in specific developmental stages of the melon plants.     

Inheritance of resistance to races 1, 2 and 5 of powdery mildew in the melon TGR-1551

The inheritance of resistance to races 1, 2 and 5 of Podosphaera xanthii in the melon TGR-1551 was studied in a cross with the susceptible cultivar 'Bola de oro'. Under artificial inoculation conditions, four phenotypic classes were defined according to the level of fungal sporulation. The segregation ratios for resistance/susceptibility observed in the different populations and the results of the progeny test indicated that resistance to races 1, 2 and 5 of P. xanthii in TGR-1551 was governed by two independent genes, one dominant and one recessive, which means that the genetic control is a dominant–recessive epistasis. This is the first time that a recessive gene that confers resistance to more than one race of powdery mildew has been identified in melon. TGR-1551 could be considered a good genetic source for the development of powdery mildew resistant breeding lines.     

Generation means analysis of plant architectural traits and fruit yield in melon

Unique architectural phenotypes have the potential for increasing yield in commercial melon (Cucumis melo L.). Therefore, a generation means analysis was conducted to investigate the inheritance of architectural traits (days to anthesis, primary branch number, fruit number and weight, and average weight per fruit). Progeny (F₁, F₂, BC₁P₁ and BC₁P₂) from a cross between US Department of Agriculture (USDA) line, USDA 846‐1 (P₁) and ‘TopMark’ (P₂) were evaluated at Arlington (AR) and Hancock (HCK), Wisconsin in 2001. Significant (P ≤ 0.05) environment effects and genotype × environment interactions (G × E) analyses necessitated analysis by location. Significant differences (P ≤ 0.05) among parents and generations were observed for all traits, and the two parental lines differed significantly for primary branch number, fruit number and average weight per fruit. Additive gene effects were most important in governing primary branch number and fruit number per plant, while dominance and epistatic genetic effects mainly controlled days to anthesis, fruit weight per plant and average weight per fruit. Narrow‐sense heritabilities were 0.62 (AR) for days to anthesis, 0.71 (AR) and 0.76 (HCK) for primary branch number, 0.68 (AR) and 0.70 (HCK) for fruit weight per plant, 0.33 (AR) and 0.45 (HCK) for fruit weight per plant, and 0.06 (AR) and 0.79 (HCK) for average weight per fruit. Estimations of the least number of effective factors for primary branch number were relatively consistent at both AR (approx. 4) and HCK (approx. 2). Results suggest that introgression of yield‐related genes from highly branched melon types (e.g. USDA 846‐1) into US Western Shipping germplasm may aid in the development of high‐yielding cultivars with concentrated fruit set suitable for machine and/or hand‐harvesting operations.     

根癌农杆菌介导马齿苋遗传转化体系的研究

利用马齿苋的叶片诱导出愈伤组织,再以愈伤组织为受体建立了根癌农杆菌介导的遗传转化体系。利用该转化体系得到抗性愈伤组织后,对其进行GUS组织化学染色和PCR扩增鉴定,证实为转化子,表明根癌农杆菌介导外源基因转化马齿苋的愈伤组织是完全可行的,为以后通过基因工程手段进行马齿苋的改良奠定了基础。     

Agrobacterium-mediated transformation of Japanese lawngrass (Zoysia japonica Steud.) containing a synthetic cryIA(b) gene from Bacillus thuringiensis

Transgenic Japanese lawngrass plants containing a synthetic cryIA(b) gene from Bacillus thuringiensis under the control of a maize ubiquitin promoter were developed by Agrobacterium -mediated transformation. A total of 1540 embryogenic calluses derived from dormancy-removal mature seeds were co-cultured with the disarmed strain EHA105 harbouring the binary vector pKUB. Three days after co-culture with EHA105 in the dark at 21°C, transient β -glucuronidase (GUS) expression frequency was 74.2%. After selection with 100 mg/l hygromycin B, a total of over 50 independent resistant cell clones and 25 regenerated plants were obtained. The integration and expression of the cryIA(b) gene into the genome was confirmed in 22 regenerated plants by the GUS histochemical assay, PCR amplification, Southern blotting and Western blotting analysis, with a transformation efficiency of 1.4%. The entire process from callus induction of mature seeds to production of transgenic plantlets was 80–100 days. T₁ progeny segregation analysis of these transgenic lines demonstrated that 59.1% of the transgenic events were inherited in a typical Mendelian fashion.     

A temporal genetic analysis of disease resistance genes: resistance to melon vine decline derived from Cucumis melo var. agrestis

Melon vine decline causes severe economic losses to melon crops all over the world. Two fungi. Acremonium cucurbitacearum and Monosporascus cannonballus, have been reported as the main causal agents of root damage, responsible for the decrease in root water and nutrient uptake capacity. However, melon vine decline aetiology is not completely understood, so screening assays must be conducted under natural infestation conditions. In field assays, the erratic effect of uncontrolled environmental factors makes the study of the trait's genetics difficult. The percentage of asymptomatic plants scored at individual moments during the infection process in the field is an imprecise indicator of the resistance level of each genotype. The analysis of disease progress curves does allow for the minimization of the stochastic fluctuations caused by environmental factors at various moments in time. Using this analysis the mode of inheritance of the partial resistance to melon vine decline derived from the accession Pat 81 of Cucumis melo var. agrestis was further studied. Data from the six generations assayed (susceptible and resistant parents. F₁, F₂, and backcrosses BC₁ and BC₂ were fitted to an additive/dominance model without epistatic effects using a scaling test. Monogenic control was proposed as the simplest model to explain the data. Assuming this simple model, the method of analysis also allowed for the characterization of the incomplete penetrance of resistance, which varies as a log probit function of the time. Simple genetic control would simplify the use of Pat 81 in breeding programmes aimed at developing commercial melon varieties with resistance to vine decline.     

High-efficiency transformation of Gossypium hirsutum embryogenic calli mediated by Agrobacterium tumefaciens and regeneration of insect-resistant plants

A simple protocol of transformation of cotton (Gossypium hirsutum L.) at a high frequency has been developed via Agrobacterium mediation, coupled with the use of embryogenic calli as explants. Embryogenic calli derived from only one to two somatic embryogenic calli lines of two Chinese cotton cultivars, the cvs. Ekang 9 and Jihe 321 which have low embryogenic potency were first inoculated with the A. tumefaciens strain LBA4404 harbouring binary vector pBin438 carrying a synthetic Bacillus thuringiensis‐active Cry1Ac and API‐B chimeric gene. Infected embryogenic calli were co‐cultivated for 48 h and were then moved on to the selection medium with kanamycin (100 mg/l) for 7‐8 weeks. Then, the kanamycin‐resistant calli (Km¹) subcultured in proliferation medium would re‐differentiate to form somatic embryos in 30 days. Cotyledon embryos were transferred to 100‐ml Erlenmeyer flasks for germination and regeneration. Putative transformants were confirmed by polymerase chain reaction and Southern blot analysis. Forty‐five regenerated plants were successfully transferred to soil, of which 12 proved to have the active Cry1Ac and API‐B chimeric gene. Insect resistance was tested by bioassay. The transgenic plants were highly resistant to cotton bollworm (Heliothis armigera) larvae, with mortality (insect resistance) ranging from 95.8 to 100%. In comparison with the methods used in Agrobacterium‐mediated transformation of cotton hypocotyls or cotyledons, about 6 months are saved by using the method presented in this paper to obtain a large number of transgenic plants.