月季的植株再生及遗传转化研究进展

本文对近20年月季植株再生和转基因研究进展进行了较为系统的回顾和总结。月季通过器官和体细胞胚发生途径都能再生植株,但遗传转化主要是利用体细胞胚发生途径。通过农杆菌介导法和基因枪法,外源基因如报告基因、抗病基因和改变花色的基因等已转化成功。文章还对今后月季转基因研究的方向进行了讨论。     

月季组织培养和遗传转化体系的研究进展

月季通过器官和体细胞胚发生途径都可以获得再生植株,在遗传转化中主要是利用体细胞胚作为转化受体。目前,利用农杆菌介导法和基因枪法已成功将外源基因如报告基因、抗病基因和改变花色的基因等导入月季基因组中。本文对近年来月季组织培养和转基因研究进展进行了综述,为建立月季高效遗传转化体系奠定了理论基础。     

人参的遗传改良*

遗传改良是人参育种的重要手段之一,而遗传转化和再生体系的建立是开展人参遗传改良工作的前提和基础。人参植株再生可以通过器官发生和体细胞胚发生,间接体细胞胚发生是人参植株再生的主要途径,从不同外植体,不同碳源,体细胞胚优化和无激素再生等方面进行了综述。在人参遗传转化方面,发根农杆菌和根癌农杆菌对人参的遗传转化均已成功,人参皂苷合成途径中的关键酶基因和抗除草剂基因也已陆续导入人参,得到了遗传改良的转化人参。发根培养系统可用于大量生产人参皂苷,讨论了rolC基因对人参发根诱导的作用,发根植株再生能力及生物反应器培养,最后指出了人参基因工程研究中存在的问题。     

月季遗传转化研究进展

月季再生方式主要有直接再生、芽体再生、体细胞胚再生。主要从品种、外植体、基本培养基成分、激素浓度及配比、碳水化合物、培养条件等影响因素对其进行了论述 ;同时从外源基因、转化方法、转基因植物再生方式和检测等方面对月季遗传转化进行综述。最后展望了基因工程在月季育种中的应用。     

苏云金芽孢杆菌杀虫晶体蛋白基因导入大豆的研究

用苏云金芽孢杆菌(BacillusthuringiensisBerliner)杀虫晶体蛋白(Bt)基因和葡糖苷酸酶(GUS)基因通过基因枪轰击和根癌土壤杆菌(Agrobacteriumtumefaciens(SmithetTownsend)Conn)介导转入大豆(Glycinemax(L.)Merr.),诱导大豆转基因植株再生。大豆主栽品种“中黄4号”和品系8502未成熟子叶有较强体细胞胚分化能力。体细胞胚的脱水处理显著促进“中黄4号”体细胞胚的萌发。未成熟子叶的预培养有利于根癌土壤杆菌感染子叶外植体体细胞胚的分化。基因型和受体的选择,转基因体系的改进,体细胞胚的脱水处理等是提高大豆转基因效率的重要因素。     

抗冻蛋白(afp)基因表达载体的构建及对香蕉胚性细胞悬浮系的转化

通过PCR从‘京都七寸人参'胡萝卜基因组DNA中扩增抗冻蛋白基因,测序结果表明该基因的核苷酸序列与从宁夏‘吴忠'胡萝卜中克隆的完全一致。先后将获得的胡萝卜afp基因克隆和亚克隆至pMD18-T和pBI121,构建植物表达载体pBI121-afp。通过冻融法将pBI121-afp导入根癌农杆菌EHA105中。以香蕉栽培品种‘北大矮蕉'的胚性细胞悬浮系为受体,采用农杆菌介导法将胡萝卜afp基因导入其中,然后在Kanamycin的选择压力下通过体细胞胚发生途径进行植株再生。共获得抗性再生植株9株,其中两株经PCR检测呈阳性,可初步确定目的基因已经整合到这两株转基因香蕉植株的基因组中。     

植物体细胞胚发生的分子基础

植物通过体细胞胚胎发生途径形成再生植株已是及其普遍的现象,这一发育途径为研究植物细胞的分化、发育、全能性表达和作物品种改良、突变体筛选等提供了良好的实验体系,在理论上和应用中都具重大意义.体细胞分化为胚性细胞是受细胞内外多种因子所调控[1, 2],其中最重要的是受特定基因的调控,在胚性细胞的分化过程中伴随着特定的遗传信息表达,其分化过程的实质是基因按顺序表达调控,是相应基因产物作为胚性细胞形成的分子基础.本文结合我们的工作来探讨体细胞胚发生分子基础研究现状.     

怀黄菊间接体胚受体再生体系的建立及CmTGA1的遗传转化

植物受体再生及遗传转化体系的建立是对植物进行转基因操作时至关重要的一个技术环节。以菊花优良种质怀黄菊(Chrysanthemum morifolium cv. ‘Huaihuang’)为试材, 探讨胚状体诱导所需的最佳培养基及芽分化和根生长的最适抗生素选择压。在此基础上, 将抗病基因CmTGA1通过同源转化的方法转入怀黄菊, 获得再生植株。结果表明, 在附加1.5 mg·L⁻¹IAA、0.5 mg·L⁻¹ 6-BA和1.0 mg·L⁻¹ 2,4-D的MS培养基上诱导培养15天后, 再去除2,4-D进行分生培养, 并进一步诱导芽再生, 最终86%的供试外植体通过胚状体途径获得再生芽; 在芽分化时所需潮霉素选择压为5.0 mg·L⁻¹, 生根时潮霉素选择压为4.5 mg·L⁻¹。对转化植株进行半定量PCR (semi-quantitative PCR)和实时荧光定量PCR (real-time quantitative PCR)检测, 结果表明, CmTGA1基因成功整合到转化植株基因组中, 从而建立了怀黄菊间接体细胞胚途径转基因受体再生体系。该技术的建立为通过转基因手段解决生产中存在的怀黄菊病害感染严重和种质退化等问题奠定了基础。     

紫花苜蓿外源基因共转化植株的再生

高含硫氨基酸蛋白(HNP)基因和发根(rol)基因由发根农杆菌介导转入紫花苜蓿, 成功地从子叶毛根组织诱导转基因植株再生. 子叶是较理想的转化受体, 毛根年龄与紫花苜蓿体细胞胚分化频率呈负相关. 共转化植株高产、优质性状的产生对紫花苜蓿新品种的培育有重要意义.     

糜子离体体细胞胚胎发生的组织学研究

利用组织学连续石腊切片的研究方法,观察了糜子组织培养中植株再生的过程,从而证明了植株再生是通过体细胞胚胎发生途径的。结果表明:(1) 糜子成熟胚培养首先从下胚轴及胚根区愈伤组织化;(2) 体细胞胚起源于下胚轴及胚根维管束周围愈伤组织中单个离散的胚性细胞;(3) 糜子离体体细胞胚有与典型禾谷类作物合子胚大致相似的发育过程。     

松杉类植物体细胞胚发育机理的研究进展

植物体细胞胚胎发生不仅可作为其繁育的重要手段,而且也是研究胚胎发育过程的一种重要模式系统.体细胞胚在形态和生理上的成熟,直接影响到植株的萌发和再生频率.本文综述了近年来国内外有关裸子植物中几种松杉类植物体细胞胚发育过程的研究报道,其中主要涉及培养基成分和脱落酸(ABA)对体细胞胚发育的影响,以及体细胞胚发育在细胞学、细胞程序性死亡、相关基因和蛋白质组学等方面的研究进展,并进一步讨论了松杉类植物体细胞胚的发育机理,以及体细胞胚在遗传转化系统中的作用.     

棉花体细胞胚发生机理的研究进展

棉花是一种较难通过体细胞胚发生完成植株再生的作物,而体细胞胚发生是限制棉花基因工程和细胞工程得以广泛应用的主要因素.研究者从不同角度探讨了棉花体细胞胚发生机理并取得了很大的进展,为棉花的遗传转化提供了重要的技术支持,同时成功建立了一些棉花植株再生体系.本文分别从基因型限制、胚胎学、生理生化变化、分子机理等方面阐述了近年来棉花体细胞发生机制的研究进展,并讨论了本研究领域目前存在的主要问题及相应对策.     

Inheritance of somatic embryogenesis and plantlet regeneration from primary (type 1) callus in maize

Summary Genetic factors controlling the differential expression of somatic embryogenesis and plant regeneration of maize from tissue culture were studied in two crosses. Inbred, hybrid, F2 and backcross generations developed from crossing maize inbred A188 with two commercially important inbred maize lines (B73 and Mo17) demonstrated genetic and environmental effects on somatic embryogenesis and plant regeneration when immature zygotic embryos were cultured on MS medium. Additive gene effects were more important in both crosses than dominant gene effects for precent somatic embryogenesis and percent or number of plants regenerated per embryo when generation means were analyzed. In backcross generations of each cross, cytoplasmic, maternal and/or paternal effects were significant for frequency of somatic embryos three weeks after culture as well as frequency, or number of plants regenerated per embryo, nine weeks after culture. Analysis of genetic variances suggests at least one gene (or block of genes) controls the expression of the frequency of somatic embryogenesis in these crosses. Differences in somatic embryogenesis and plant regeneration between B73 and Mo17 are discussed. This is Journal Paper No. 11,435 of the Purdue University Agricultural Experiment Station.     

Biotechnological advances in mango (Mangifera indica L.) and their future implication in crop improvement: a review

Biotechnology can complement conventional breeding and expedite the mango improvement programmes. Studies involving in vitro culture and selection, micropropagation, embryo rescue, genetic transformation, marker-assisted characterization and DNA fingerprinting, etc. are underway at different centers worldwide. In vitro culture and somatic embryogenesis of several different genotypes have been achieved. The nucellus excised from immature fruitlets is the appropriate explant for induction of embryogenic cultures. High frequency somatic embryogenesis has been achieved in some genotypes; however, some abnormalities can occur during somatic embryo germination. Embryo rescue from young and dropped fruitlets can improve the hybridization success in a limited flowering season. Protocols for protoplast culture and regeneration have also been developed. In vitro selections for antibiotic tolerance and fungal toxin resistance have been very promising for germplasm screening. Genetic transformation using Agrobacterium tumefaciens has been reported. Genes that are involved with fruit ripening have been cloned and there have been attempts to deliver these genes into plants. DNA fingerprinting and studies on genetic diversity of mango cultivars and Mangifera species are also being conducted at several research stations. The purpose of this review is to focus upon contemporary information on biotechnological advances made in mango. It also describes some ways of overcoming the problems encountered during in vitro propagation of mango.     

Efficient somatic embryogenesis in sugar beet (<Emphasis Type="Italic">Beta vulgaris</Emphasis> L.) breeding lines

Efficient regeneration via somatic embryogenesis (SE) would be a valuable system for the micropropagation and genetic transformation of sugar beet. This study evaluated the effects of basic culture media (MS and PGo), plant growth regulators, sugars and the starting plant material on somatic embryogenesis in nine sugar beet breeding lines. Somatic embryos were induced from seedlings of several genotypes via an intervening callus phase on PGo medium containing N⁶-benzylaminopurine (BAP). Calli were mainly induced from cotyledons. Maltose was more effective for the induction of somatic embryogenesis than was sucrose. There were significant differences between genotypes. HB 526 and SDM 3, which produced embryogenic calli at frequencies of 25–50%, performed better than SDM 2, 8, 9 and 11. The embryogenic calli and embryos produced by this method were multiplied by repeated subculture. Histological analysis of embryogenic callus cultures indicated that somatic embryos were derived from single- or a small number of cells. 2,4-dichlorophenoxyacetic acid (2,4-D) was ineffective for the induction of somatic embryogenesis from seedlings but induced direct somatic embryogenesis from immature zygotic embryos (IEs). Somatic embryos were mainly initiated from hypocotyls derived from the cultured IEs in line HB 526. Rapid and efficient regeneration of plants via somatic embryogenesis may provide a system for studying the molecular mechanism of SE and a route for the genetic transformation of sugar beet.     

New insights into plant somatic embryogenesis: an epigenetic view

Somatic embryogenesis plays a significant role in plant regeneration and requires complex cellular, molecular, and biochemical processes for embryo initiation and development associated with plant epigenetics. Epigenetic regulation encompasses many sensitive events and plays a vital role in gene expression through DNA methylation, chromatin remodelling, and small RNAs. Recently, regulation of epigenetic mechanisms has been recognized as the most promising occurrences during somatic embryogenesis in plants. A few reports demonstrated that the level of DNA methylation can alter in embryogenic cells under in vitro environments. Changes or modification in DNA methylation patterns is linked with regulatory mechanisms of various candidate marker genes, involved in the initiation and development of somatic embryogenesis in plants. This review summarizes the current scenario of the role of epigenetic mechanisms as candidate markers during somatic embryogenesis. It also delivers a comprehensive and systematic analysis of more recent discoveries on expression of embryogenic-regulating genes during somatic embryogenesis, epigenetic variation. Biotechnological applications of epigenetics as well as new opportunities or future perspectives in the development of somatic embryogenesis studies are covered. Further research on such strategies may serve as exciting interaction models of epigenetic regulation in plant embryogenesis and designing novel approaches for plant productivity and crop improvement at molecular levels.     

Chromosomal location of genes controlling short-term and long-term somatic embryogenesis in wheat revealed by immature embryo culture of aneuploid lines

The expression of essential genes during somatic embryogenesis can be analysed by inducing aneuploid cells to undergo embryogenesis during immature embryo culture and then determining whether defects occur. Triticum aestivum disomic and aneuploid stocks, including 36 ditelosomics and 7 nullitetrasomic Chinese Spring wheats, were compared for their ability to undergo somatic embryogenesis after 2 months of in vitro immature embryo culture. Their regeneration capacity was observed after 4 and 14 months of in vitro culture to determine which chromosome arms influence the process. The large range of variation found among the tested aneuploids suggested that genetic control of the somatic tissue culture ability is polygenic. Our results indicate that genes affecting somatic embryo-genesis and regeneration are located in all of the homoeologous chromosome groups. The lack of chromosome arms 1AL (DT 1AS) and 3DL (DT 3DS) practically suppresses somatic embryogenesis, demonstrating that major genes on wheat chromosome arms 1AL and 3DL control regeneration capacity. Results suggest that plants were mainly produced from somatic embryo development. Although the control of somatic embryogenesis and regeneration is polygenic, the genes located on the long arms of homoeologous group 3 chromosomes have a major effect. We also have evidence of chromosome arms that determine the time required for regeneration.