Development of Alfalfa （Medicago sativa L.） Regeneration System and Agrobacterium-Mediated Genetic Transformation

The regeneration ability of four alfalfa （Medicago sativa L.） cultivars, Xinjiang Daye, Longdong, Gannong 1 and Gannong 3, was studied, and the effects of various cultivars, explant sources and medium recipes on regeneration were compared. The better callus forming frequency obtained from hypocotyls of Xinjiang Daye is 88.5% and regeneration frequency is 9.8% in our initial experiments. To further optimize regeneration system for genetic transformation, we therefore changed concentrations of plant growth regulators and supplemented with glutamine into callus-induction and shoot-regeneration media. Callus forming frequency and shoot differentiation frequency were increased to 100%. The time taken to generate transgenic plants （16 weeks） was shorter than that for previouse procedure （25 weeks） and regeneration frequency was promoted to 15.1%. The results show that addition of glutamine is particularly important for shortening period of regeneration and promoting regeneration frequency. For study of genetic transformation of alfalfa, Agrobacterium tumefaciens-mediated transformation of Xinjiang Daye was developed based on this optimized regeneration system. The plant expression vector carrying two glutamine synthetases （GS 1 and GS2） and △1-pyrroline-5-carboxylate synthetase （P5CS） gene was used for alfalfa in vitro transformation. Six transgenic alfalfa plantlets with resistance to PPT were obtained. The introduction of foreign genes into plants was assessed in the transformants by PCR analysis and Southern hybridizations.     

Optimization of Agrobacterium tumefaciens-Mediated Immature Embryo Transformation System and Transformation of Glyphosate-Resistant Gene 2mG2-EPSPS in Maize（Zea mays L.）

Since maize is one of the most important cereal crops in the world,establishment of an efficient genetic transformation system is critical for its improvement.In the current study,several elite corn lines were tested for suitability of Agrobacterium tumefaciens-mediated transformation by using immature embryos as explants.Infection ability and efficiency of transformation of A.tumefaciens sp.strains EHA105 and LBA4404,different heat treatment times of immature embryos before infection,influence of L-cysteine addition in co-cultivation medium after transformation,and how different ways of selection and cultivation influence the efficiency of transformation were compared.Glyphosate-resistant gene 2mG2-EPSPS was transformed into several typical maize genotypes including 78599,Zong 31 and BA,under the optimum conditions.Results showed that the hypervirulent Agrobacterium tumefaciens sp.strain EHA105 was more infectious than LBA4404.Inclusion of L-cysteine（100 mg L-1） in co-cultivation medium,and heating of the immature embryos for 3 min prior to infection led to a significant increase in the transformation efficiency.Growth in resting medium for 4-10 d and delaying selection was beneficial to the survival of resistant calli.During induction of germination,adding a high concentration of 6-BA（5 mg L-1） and a low concentration of 2,4-D（0.2 mg L-1） to regeneration medium significantly enhanced germination percentage.Using the optimized transformation procedure,more than 800 transgenic plants were obtained from 78599,Zong 31 and BA.By spraying herbicide glyphosate on leaves of transgenic lines,we identified 66 primary glyphosate-resistant plants.The transformation efficiency was 8.2%.PCR and Southern-blot analyses confirmed the integration of the transgenes in the maize genome.     

Effects of Environmental Temperature on the Regeneration Frequency of the Immature Embryos of Wheat （Trificum aestivum L.）

The immature embryos （IEs） of wheat are the most widely used tissues for in vitro culture and genetic transformation due to its high regeneration competency. However, this explant can only be maintained in 4℃ daily cooler for a short period time for its use in plant tissue culture or transformation experiments. This study aimed to investigate the effects of environmental temperature, cryopreservation storage temperature, and heat shock culture （HSC） temperature on the regeneration frequency of wheat IEs. Results indicated that environmental temperature significantly affected the induction of embryonic calli. The optimum total accumulated temperature （TAT） during the time of anthesis and sampling for regeneration of these tissues was around 280℃ for spring wheat type cv. CB037 and approximately 300℃ for winter wheat type cv. Kenong 199. Regeneration ability obviously declined when the highest environmental temperature was over 35℃ for 1 d or a high temperature between 30 and 33℃ lasted for 5 d during anthesis and sampling. This finding was verified by culturing the freshly isolated IEs under different temperatures from 29 to 37℃ in different controlled growth incubators for 5 d; the IEs almost completely lost regeneration ability when the temperature rose to 37℃. Cryopreservation of-20℃ caused the wheat samples lost ability of producing callus or embryonic callus in a few days, and cryopreservation of-10℃ more than 10 d made the regeneration potential of the tissues dramatically declined. Comparatively, the temperature that best maintained high regeneration ability was -5℃, at which the materials can be maintained for around 1 mon. In addition, the preservation of the immature samples at -5 or -10℃ inhibited the direct germination of the IEs, avoiding the embryo axis removing process. Our results are useful for ensuring that field collection and cryopreservation of the wheat IEs are done correctly to enable tissue culture and genetic transformation.     

A Review on Regeneration in Cowpea （Vigna unguiculata L. Walp）

Vigna unguiculata L. Walp is a recalcitrant plant in terms of in vitro cell, tissue and organ differentiation, which makes it difficult to apply tissue-culture dependant approaches for obtaining stable genetic transformation in cowpea. Despite this, sporadic efforts have been made to develop regeneration systems in cowpea during the past 40 years. This review presents the considerable progress on cowpea regeneration （organogenesis and embryogenesis） and especially focuses on the regeneration mode of organogenesis, including highlights of the effect of genotypes, explants, medium and plant hormones used in tissue culture. The existing problems and the future research directions were also discussed.     

QTLs for Sugar Content of Stalk in Sweet Sorghum （Sorghum bicolor L.Moench）

High sugar content of sorghum stalk is an important factor in the sorghum silage production. To identify the genomic regions controlling sugar content and to develop molecular markers linked to sugar content in sweet sorghum, we used an F2：3 segregating population consisting of 207 individuals derived from a cross between a high sugar content inbred line, Early Folger, and a normal inbred line, N32B, for genetic linkage mapping and quantitative trait locus （QTL） analysis. We constructed a genetic linkage map spanning 983.5 cM based on a total of 327 markers comprising 31 restriction fragment length polymorphism （RFLP） markers, 254 amplified fragment length polymorphism （AFLP） markers, and 42 simple sequence repeat （SSR） markers. In the 20 linkage groups detected, 98.2% of markers aligned to the 10 linkage groups of sorghum. Variations in sugar content at different growth stages and among internodes suggested that the sugar content of middle internodes is stable and suitable for measuring at early dough stage. The broad sense heritability （hB0 of sugar content was 0.64 and 0.62 estimated from the data of F3 families and each parent in 2003 and 2004. We identified one and two QTLs accounting for 22.2 to 25.0% of phenotypic variance using simple interval mapping method in 2003 and 2004, respectively. These two QTLs showed a negative additive effect, and over-dominance effect. A QTL on LG-D was detected in both two years. Above results will be help us to understand the genetic mechanism of sugar content in sorghum and the QTL detected in this study might be useful in the improvement of sugar content by marker-assisted selection.     

In vitro antioxidant activity of callus culture of carrot （Daucus carom）

Carrot （Daucus carom） is a valuable source of health promoting ingredients such as anthocyanin, carotenes, phenolic compounds etc. These substances are important to man as a source of pharmaceuticals, fragrance, agrochemicals as well as food additives and used for prevention of many chronic diseases. Since these activities may be correlated with the presence of antioxidant compounds, extract of carrot and carrot callus were evaluated for their anthocyanin, flavonoids and total phenolic content as well as total antioxidant activity. Anthocyanin content was measured by spectrophotometric method. Total phenols and flavonoids were analyzed according to the Folin-Ciocalteu method and total antioxidant activity was assessed by ferric reducing/antioxidant power （FRAP） assay. Anthocyanin, flavonoids and total phenolic content were estimated to be 9.36 mg%, 46.96 mg% and 57.01 mg% for callus and 6.82 mg%, 32.96 mg% and 42.69 mg% for carrot, respectively, on fresh weight basis. The total antioxidant activity for the callus and carrot was found to be 51.13 mg, 118.77 mg, 91.08 mg and 140.08 mg equivalent and 79.40 mg, 184.44 mg, 141.43 mg and 217.52 mg equivalent to gallic acid, vitamin C, butylated hydroxyanisole （BHA） and trolox, respectively, when expressed per 100 g on fresh weight basis. The antioxidant activity of fresh carrot was found to be higher compared to its callus.     

Dicamba and Sugar Effects on Callus Induction and Plant Regeneration from Mature Embryo Culture of Wheat

To establish a highly efficient plant regeneration system for wheat genetic transformation, the effects of three different concentrations of dicamba and two different sugar types on callus induction and plant regeneration from mature embryo cultures were evaluated. Callus induction and plant regeneration were obtained from mature embryos of two commercial cultivars Zhoumai 18 and Yumai 34 （Triticum aestivum L.） cultured on L3 basal medium. The results showed that the efficiency of mature embryo culture was significantly influenced by the genotypes, sugar types and dicamba concentrations. 4 mg L^-1 dicamba proved the best effective for inducing embryogenic callus and also gave the highest proportion of plants regenerated across the two cultivars. Substitution of maltose by sucrose significantly improved the plant regeneration efficiency in both cultivars. There was a significant interaction between genotype-by-sugar types, and sugar types-bydicamba concentrations. Overall, Zhoumai 18 gave the highest frequency of plant regeneration （82.65%） when dicamba concentration was 4.0 mg L^-1 and with sucrose in initial callus induction. These results will facilitate genetic transformation work with elite wheat.     

Development of Highly Efficient Transformation System of Yeast-Like Conidia of Tremella fuciformis

Tremellafuciformis is one of higher basidiomycetes. Its basidiospore can reproduce yeast-like conidia, which is also called the blastospore by budding. The yeast-like conidia of T. fuciformis is monokaryotic and easy to culture by submerged fermentation similar to yeast. Thus, it is a good recipient cell for exogenous gene expression. In this study, the expression plasmid pAN7-1 （containing promoter gpd-An derived from Aspergillus nidulans and selectable marker gene hph conferring resistance to hygromycin B） and plasmid pLg-hph （containing promoter gpd-Le derived from Lentinula edodes and selectable marker gene hph） were transformed into the yeast-like conidia of T. fuciformis by PEG-mediated protoplast transformation, respectively. The primary putative transformants were selected by the sandwich screening method with the selective medium containing 50 μg mL^-1 hygromycin. The putative transformants were obtained from the primary putative transformants transferred on PDSA plates containing 100 μg mL^-1 hygromycin for second round selection. Experimental results showed that the optimal concentration of PEG 4000 for mediating protoplast transformation was 25%. PCR and Southern blotting confirmed that the selectable marker gene hph was integrated effectively into the genome of the yeast-like conidia of T. fuciformis with plasmid pLg-hph transformation. Its transformation efficiency was 110 transformants per μg DNA, and the hph gene was integrated into the genome of some yeast-like conidia with plasmid pAN7-1 transformation. However, its transformation efficiency was only 9 transformants per μg DNA. The presence of hph gene in the genome of transformants after 5 generations of sub- culturing on PDSB medium was confirmed by PCR, suggesting that the foreign gene hph was stable during subculture.     

Establishment and Optimization of the Regeneration System of Mature Embryos of Maize （Zea mays L.）

A reliable system was developed for regeneration from mature embryos derived from callus of four maize inbred lines （Liao 7980, Dan 9818, Dan 340, and Dan 5026）. The protocol was mainly based on a series of experiments involving the composition of culture medium. We found that 9 pM 2,4-dichlorophenoxyacetic acid in MS medium was optimum for the induction of callus. The induction frequency of primary calli was over 85% for four inbred lines tested. The addition of L- proline （12 mM） in subculture medium significantly promoted the formation of embryogenic callus but it did not significantly enhance growth rate of callus. Efficient shoot regeneration was obtained on regeneration medium containing 2.22 μM 6- benzylaminopurine in combinations with 4.64 μM Kinetin. Regenerated shoots were rooted on half-strength MS medium containing 2.85 μM indole-3-butyric acid. This plant regeneration system provides a foundation for genetic transformation of maize.     

Current status of genetic transformation technology developed in cucumber (Cucumis sativus L.)

Genetic transformation is an important technique for functional genomics study and genetic improvement of plants. Until now, Agrobacterium-mediated transformation methods using cotyledon as explants has been the major approach for cucumber, and its frequency has been up to 23%. For example, significantly enhancement of the transformation efficiency of this plant species was achieved from the cotyledon explants of the cultivar Poinsett 76 infected by Agrobacterium strains EHA105 with efficient positive selection system in lots of experiments. This review is to summarize some key factors influencing cucumber regeneration and genetic transformation, including target genes, selection systems and the ways of transgene introduction, and then to put forward some strategies for the increasing of cucumber transformation efficiency. In the future, it is high possible for cucumber to be potential bioreactor to produce vaccine and biomaterials for human beings.     

Early Identification of Stable Transformation Events by Combined Use of Antibiotic Selection and Vital Detection of Green Fluorescent Protein (GFP) in Carrot (Daucus carota L.) Callus

Genetic transformation is a useful technique to complement conventional breeding in crop improvement. Although carrot has been a model organism for in vitro embryogenesis study, genetic transformation of carrot is still lengthy and labor intensive. An efficient transformation and detection system is desirable. Direct infection of Agrobacterium to carrot calli has provided an easy way for carrot genetic transformation. To improve the efficiency of antibiotic selection in this method, we report the combined use of an improved green-fluorescent protein, referred to as smGFP, to establish a versatile selection method for carrot callus transformation system. By combining antibiotic selection with the bright fluorescence observed in the callus tissue, we were able to easily identify stable transformants in early stage of the transformation process. In addition to the GFP expression of the callus cells, the transgenic nature of callus cells was confirmed with Southern and Western analysis. We found we can link the simplicity of carrot-callus-cell transformation, early detection of stable transformants with antibiotic selection, visualization of GFP fluorescence, and molecular analysis (Southern and Western) of callus tissue (non-photosynthetic tissue) to provide a more efficient way in identifying stable transformants at early stage of carrot transformation.     

链格孢菌苹果致病型的ATMT转化体系的建立

【目的】链格孢菌（Alternaria alternate）苹果致病型是苹果上的重要致病菌,可以侵染苹果叶片和果实。论文旨在建立农杆菌介导的链格孢菌苹果致病型高效稳定的分子转化体系,为在分子水平上研究链格孢苹果致病型病菌的致病机制提供技术支持。【方法】质粒pKO1-HPH是以pCAMBIA1300骨架为基础构建的穿梭质粒,含有潮霉素抗性基因,并在其多克隆位点上插入了绿色荧光蛋白基因,这个质粒在大肠杆菌和农杆菌细胞中都能够稳定地复制繁殖。将质粒pKO1-HPH采用冻融法转化到农杆菌菌株AGL1中,然后与链格孢苹果致病型菌株XP-1的分生孢子在诱导培养基上共培养进行基因转化,转化子在含有潮霉素的马铃薯-葡萄糖-琼脂培养基上进行筛选;在含有50 μg·mL^-1潮霉素的马铃薯-葡萄糖-琼脂培养基上检测转化子细胞中抗性基因在转化子中的表达情况;用荧光显微镜检测绿色荧光蛋白基因在转化子细胞中的表达情况。在初步确认可以将绿色荧光蛋白和潮霉素B磷酸转移酶基因转入链格孢苹果致病型菌株中后,对菌落培养时间、转化前分生孢子的预处理方法对转化效率的影响进一步优化。转化子的稳定性采用分生孢子5次继代培养后,能否在含有潮霉素的马铃薯-葡萄糖-琼脂培养基上稳定生长来检测;采用PCR方法检测绿色荧光蛋白外源基因和潮霉素B磷酸转移酶基因是否存在于转化子基因组中;用Southern杂交检测外源基因在转化子基因组中插入的拷贝数;在苹果果实和叶片上接种检测转化子致病性的变化。【结果】将100 μL孢子悬浮液（含10⁵孢子）涂布于马铃薯-胡萝卜-琼脂平板上菌落培养36-48 h后,链格孢苹果致病型菌株XP-1可以产生足量的孢子用于农杆菌介导的分子转化。将分生孢子用无菌水洗下,在4℃处理6 h,再与含质粒pKO1-HPH的农杆菌进行诱导共培养,转化子在含有50 μg·mL^-1潮霉素的马铃薯-葡萄糖-琼脂培养基上进行筛选,转化效率较好,10⁷个分生孢子转化可获得约200个转化子。转化子经过5次继代培养,对潮霉素B的抗性没有改变。特异性引物PCR检测表明外源基因能够成功地整合到供试菌株的基因组中;Southern杂交表明外源基因多以单拷贝形式随机插入到链格孢苹果致病型菌株XP-1细胞的基因组中;转化子的菌丝和分生孢子在荧光显微镜下可以观察到明显的绿色荧光,说明荧光蛋白基因在菌丝及孢子中均能够成功表达。通过部分转化子的致病性试验,筛选获得数个在苹果果实和叶片上致病性都显著降低的菌株。【结论】建立了农杆菌介导的链格孢菌苹果致病型稳定高效的分子转化体系,为进一步开展此病菌与其寄主的分子互作研究打下基础。     

胡萝卜愈伤组织上长期培养和红掌上复壮繁殖对相似穿孔线虫耐低温能力的影响

利用从观赏植物上截获的相似穿孔线虫Radopholus similis 6个种群,研究在胡萝卜愈伤组织上长期培养繁殖和在红掌Anthurium andraeanum上接种繁殖复壮后,相似穿孔线虫耐低温能力的变化.将经2种方式培养繁殖的相似穿孔线虫接种在胡萝卜愈伤组织上,测定其耐低温能力.结果表明,在胡萝卜愈伤组织上长期(6代)连续培养后,相似穿孔线虫的耐低温能力降低,在低温下的生存能力和繁殖能力明显降低;通过在自然寄主植物红掌根系上接种繁殖后,相似穿孔线虫在低温下的生存和繁殖能力又可得到不同程度的恢复,即耐低温能力得到复壮.胡萝卜愈伤组织上长期培养繁殖对相似穿孔线虫不同群体耐低温能力的影响不同,相似穿孔线虫不同种群在红掌上繁殖后耐低温能力的恢复程度也有所不同.     

水稻基因遗传转化方法研究进展

介绍水稻遗传转化方法的发展历程和科研成果，为水稻遗传转化体系的研究和应用提供借鉴。从生物介导转化法和非生物介导转化法2类方法出发，介绍各种转化方法在水稻上的首次报道和重要进展并进行了展望。生物介导转化法中，农杆菌Agrobacterium介导转化法通过侵染种胚、稻穗、愈伤组织和茎尖进行转化，种胚及其诱导的愈伤组织作为材料的转化体系较为成熟，稻穗和茎尖转化法则操作简便、转化再生周期短；此外，有研究尝试用根瘤菌Sinorhizobium和Rhizobium以及附着剑菌Ensifer adhaerens转化水稻。非生物介导转化法中，物理方法转化法(基因枪法、电击法、花粉管通道法和显微注射法)是较为传统的转化方法，基因枪法应用较为成熟，花粉管通道法则取得较多育种成果；介质介导转化法中，纳米材料的应用正逐步成为研究热点。水稻遗传转化体系的发展可从转化材料的筛选和优化介导转化的载体入手，同时将转化体系和DNA-free、单倍体诱导等技术结合起来，以提高转化效率和安全性，缩短转化再生周期。     

以诺丽茎段为外植体的无籽基因转化研究

以诺丽无菌苗不带腋芽的茎段为转化材料,采用根癌农杆菌介导法,将含有无籽基因的质粒pCAMBIA1305.1导入到诺丽细胞中,通过Kan快速筛选、PCR检测等方法对转化子进行鉴定.实验结果表明:抗性芽的平均分化频率为42%,经过PCR检测,以茎为外植体进行遗传转化的转化率为5.4%.在转化过程中,有些植株可能会发生饰变.该实验初步证实已把无籽基因转入诺丽,后续需Southern杂交进一步验证.     

双价抗寒基因CBF3/COR15A转化大白菜的初步研究

为探索拟南芥的冷诱导转录因子CBF3和抗寒基因COR15A在"黔白"系列大白菜的转化体系,以"黔白"系列大白菜3个优良自交系作为研究对象,分别用大白菜4d苗龄的带柄子叶、半子叶、下胚轴作为外植体,利用农杆菌介导的方法将同时含有CBF3和COR15A双价抗寒基因的表达载体转入大白菜,就影响大白菜再生和遗传转化的因素进行研究。通过抗生素筛选得到大白菜T0代转基因抗性植株36株,经PCR检测后获得转基因阳性植株6株。经抗寒性初步鉴定,转基因大白菜植株抗寒性超过对照。     

根癌农杆菌介导 hGLP1基因转化摩尔多瓦葡萄的研究

以摩尔多瓦葡萄早期体细胞胚为受体,通过农杆菌介导辅以超声波处理转化 hGLP1基因。结果表明,摩尔多瓦葡萄体细胞胚的卡那霉素临界致死质量浓度为10 mg/L,农杆菌浸染时辅以超声波处理可显著提高GUS瞬时表达效率（高达69.4%）。采用此体系对摩尔多瓦葡萄进行转化,获得高瞬时表达和稳定表达的转化子,进一步筛选培养获得7株抗性再生植株。利用PCR扩增的方法进行检测,其中2株PCR检测呈阳性,初步证实 hGLP1基因已整合到摩尔多瓦葡萄基因组。     

茶树离体再生和遗传转化的研究进展

茶树是我国重要的经济作物之一,通过组织培养技术,利用茶树茎段、腋芽、叶、花粉、种子等器官作为外植体建立茶树遗传转化的再生系统,对获得优质茶树种质资源至关重要.目前关于茶树遗传转化方法的研究,主要集中在农杆菌介导法和基因枪法.综述当前茶树遗传转化再生系统的研究进展,并对今后茶树遗传转化提出展望.     

甘蓝抗枯萎病基因FOC1植物表达载体构建及遗传转化

为研究甘蓝枯萎病抗性基因FOC1的抗性功能,利用前期克隆的FOC1基因,以pBI121质粒为植物表达载体,采用同源重组法构建FOC1基因的过表达载体;将构建好的重组质粒采用冻融法转入根癌农杆菌LBA4404菌株中,并通过农杆菌介导的甘蓝外植体转化法对感病甘蓝进行遗传转化,利用载体特异引物对获得的转基因植株进行PCR鉴定。结果表明,最终成功构建FOC1基因的过表达载体pBI121-35S-FOC1,并已成功整合到受体甘蓝基因组中。     

生防改良菌株F1-35的GFP标记及其对西瓜幼苗生理及防御系统的影响

为探究生防改良菌株F1-35的绿色荧光蛋白标记及其在标记后相关性状的改变情况,采用PEGCaCl2法介导原生质体转化,成功地将GFP基因转入到生防改良菌株F1-35中,转化率为每微克质粒DNA6个转化子,转化子经继代培养5代后仍能在含潮霉素B的培养基上正常生长,且其荧光稳定,强度良好;其分生孢子也能表达强烈的荧光。灌根处理西瓜幼苗发现,与原始生防改良菌株F1-35比较,经GFP标记后的菌株F1-35-GFP对西瓜的促生、抗逆作用以及其在西瓜幼苗根部的定殖情况和对西瓜枯萎病的防治效果未发生改变,防治效果仍能达60%以上;通过分析F1-35对西瓜枯萎病的防治效果及其对西瓜幼苗防御酶的影响关系发现,F1-35能提升PPO等防御酶的活性,从而抑制西瓜枯萎病的发生。可见,GFP能稳定存在于生防改良菌株F1-35中,且不对其促生、抗逆作用及防效产生影响,F1-35可通过提升西瓜幼苗防御酶活性来防治西瓜枯萎病的发生。