根癌农杆菌介导转化番茄的影响因素

综述影响根癌农杆菌介导番茄转化效率的因素,包括根癌农杆菌菌株类型、Vir基因的活化、选择标记基因、植物基因型、外植体类型、培养基中是否附加植物激素和抑菌抗生素、菌液浓度、侵染时间长短,是否预培养和共培养天数等;同时不同的培养方式也是影响番茄转化效率的主要因素,包括液体培养法、农杆菌介导的floral-dip转化法、超声波辅助农杆菌介导法、农杆菌介导与基因枪轰击结合法等.     

农杆菌介导的木本植物遗传转化

木本植物由于本身的特殊性使其遗传转化较为困难,但根据植物类型及其生长特点,选取不同的受体系统,采用农杆菌介导进行转化,已在多种木本植物上获得成功。本文综述了农杆菌介导的木本植物在转化方法、受体系统和转化机理方面的研究,对Ｖｉｒ 基因活化、外植体预培养、共培养方法等影响转化的一些因素及存在的问题进行了探讨。     

观赏兰科植物组培快繁及遗传转化的研究进展

兰花作为一种高档花卉,近年来其组培快繁和基因工程研究取得了比较大的进展。综述的观赏兰科植物组织培养内容包括外植体、培养基及培养方式等,遗传转化内容包括靶材料、选择标记基因、报告基因、启动子和转化方法等并总结了兰科植物基因工程研究的成果、最新进展及存在的问题 。     

带内含子卡那霉素抗性基因双元载体构建及烟草转化

农杆菌介导法是植物基因转化的常用方法,然而由于筛选培养基中常用的抗生素头孢霉素和羧苄青霉素具有类植物激素活性,影响外植体的再生和转化频率。将一个植物的内含子插入卡那霉素抗性基因编码区的N端,合成了一个带内含子的卡那霉素抗性基因。构建带该基因的植物双元表达栽体pYP1202并转化烟草,受侵外植体在含卡那霉素50～200mg/L的选择培养基中抗性芽分化频率不受卡那霉素浓度影响,然而具有GUS活性的转化子占分化芽的比例却随着卡那霉素浓度的增加而升高。当培养基中加入500mg/L羧苄青霉素后受侵外植体产生的抗性芽频率比单一的卡那霉素筛选提高近1倍,高达91.4％,然而具GUS活性的转化子占抗性芽的比例仅有26.7％,在200m/L的卡那霉素筛选下,比例升至93.3％。用带内含子卡那霉素抗性基因构建的植物表达载体转化植物可以减少假抗性芽的产生。     

带内含子卡那霉素抗性基因双元载体构建及烟草转化

农杆菌介导法是植物基因转化的常用方法,然而由于筛选培养基中常用的抗生素头孢霉素和羧苄青霉素具有类植物激素活性,影响外植体的再生和转化频率,将一个植物的内含子插入卡那霉素抗性基因编码区的N端。合成了一个带内含子的卡那霉素抗性基因。构建带该基因的植物双元表达载体pYP1202并转化烟草,受外植体在含卡那霉素50－200mg/L的选择培养基中抗性芽分化频率不受卡那霉素浓度影响,然而具有GUS活性的转化子占分化芽的比例却随着卡那霉素浓度的增加而升高。当培养基中加入500mg/L羧苄青霉素后受侵外植体产生的抗性芽频率比单一的卡那霉素筛选提高近1倍,高达91.4％,然而具GUS活性的转化子占抗性芽的比较仅有26.7％,在200mg/L的卡那霉素筛选下,比例升至93.3％。用带内含子卡那霉素抗性基因构建的植物表达载体转化植物可以减少假抗性芽的产生。     

农杆菌介导的芸苔属植物遗传转化技术的研究进展

本文从芸苔属植物遗传转化技术的几大要素：植物基因型,农杆菌株系,外植体生理状态及培养条件;筛选标记及抗生生素类型,ＡｇＮＯ３的作用;褐化及玻璃苗的防治等方面对农杆菌介导的转化技术的研究进展进行了综述。     

马铃薯遗传转化体系的优化建立及其主要影响因素

植物基因转化的成功依赖于一个良好的转化系统,能有效地将外源基因导入受体细胞,并得以表达。通过农杆菌介导的马铃薯遗传转化体系主要受基因型、预培养、菌液浓度及侵染时间、共培养等因素的影响,由于转化受体的异质性,有必要根据实际情况进行验证和改进,以获得最适转化条件。本研究以马铃薯无菌苗的叶片、茎段为外植体,通过农杆菌介导法,将抗马铃薯X病毒和Y病毒的RNA干扰型基因结构转入马铃薯。通过研究外植体预培养、菌液浓度及侵染时间、共培养等不同转化条件及影响因素对马铃薯遗传转化的影响,建立一种高效的马铃薯遗传转化体系,试验得出最佳转化条件为外植体预培养2 d,然后用OD600=0.5的农杆菌液侵染10 min,共培养2 d。本研究为下一步的对PVX和PVY双抗的马铃薯无标记转基因研究提供技术参考。     

小麦遗传转化研究进展

小麦作为最重要的3大禾谷作物之一,其离体培养具有很强的惰性,再生频率与水稻、玉米相比要低一些,目前大多通过对基因型和外植体的选择来达到植株的高频再生分化,因此其遗传转化就远远滞后于水稻和玉米,更不用说与其它双子叶植物相比了.重点综述了小麦转基因技术和外源基因在小麦中的遗传转化研究现状,其内容包括几种主要的小麦转基因方法和以基因枪法为主的各种转化技术对品质基因、抗除草剂基因和抗病等基因在小麦中的遗传转化研究进展,并对存在的一些问题进行了简要的论述.     

植物胚性愈伤诱导的机理及提高途径

对植物胚性愈伤诱导的研究结果进行综合分析,寻找植物胚性愈伤诱导参与的基因、诱导的机理、甲基化与提高植物胚性愈伤诱导途径之间的联系,探索提高植物胚性愈伤诱导几率的可能途径,为转化困难的植物的基因工程操作奠定基础。LBD (laterial organ boundaries domain)家族基因、伤口诱导脱分化蛋白WIND1 (wound induced dedifferentiation 1)、AP2/ERF转录因子BABYBOOM等参与了植物胚性愈伤诱导的过程;DNA甲基化在这个过程中也发挥了重要作用,植物胚性细胞通过PCG和PKL通路两种表观遗传途径促使胚性细胞保持胚性。这些研究结果表明,通过筛选距离合子胚最近的体细胞作为外植体,利用分子标记进行筛选具有胚性的细胞或组织作为外植体进行培养,在培养基中添加去甲基化的试剂等方法可能能够提高植物胚性愈伤诱导的比例,转化困难的农作物的基因工程育种有望得到显著改进。     

纳米载体介导的植物遗传转化研究现状和前景

高效遗传转化技术是植物重要性状功能基因鉴定的前提和转基因育种的基础。随着纳米生物技术的发展,以纳米载体介导的植物转基因技术已显示出巨大的应用潜力。综述了国内外应用于植物纳米载体的类型、与外源基因的结合方式以及传输细胞的原理,重点阐述了影响纳米基因载体性能与转化效率的重要因素,以及纳米载体介导外源基因转化植物细胞的方法,分析了纳米载体介导法与其他转基因方法的特点和优势,并提出纳米载体介导的转化技术应加强稳定遗传转化、基因编辑与植物原位转化等方面探索研究,旨为植物遗传转化技术和方法提供新的思路。     

An examination of factors affecting the efficiency ofAgrobacterium-mediated transformation of tomato

An improved protocol forAgrobacterium-mediated transformation of the tomato cultivar Moneymaker was developed by examining the effects of six different factors on the efficiency of transformation. Explant size, explant orientation, gelling agent and plate sealant were found to affect transformation efficiency. Two other factors, type of explant (hypocotyl or cotyledon) and frequency of transfer to fresh selective regeneration medium, did not have any effect on transformation efficiency. By combining the best treatments for each factor, an average transformation efficiency of 10.6% was obtained for Moneymaker.     

Improved genetic transformation protocol for cork oak (<Emphasis Type="Italic">Quercus suber</Emphasis> L.)

An optimized protocol for Agrobacterium tumefaciens-mediated transformation of mature Quercus suber L. embryogenic masses is reported. In this work several variables were tested. Plant genotype, explant type and time elapsed between the last subculture and inoculation, i.e. the explant preculture period, were found to be very important. Interaction between inoculum density and cocultivation period influenced the transformation efficiency as well. A transformation efficiency (i.e. percentage of the inoculated explants that yielded independent transgenic embryogenic lines) of up to 43% was obtained, greatly improving the previously described method for plant transformation of adult-selected cork oak. It was also shown that this protocol could be applied to various genotypes. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Production of transgenic diploid <Emphasis Type="Italic">Cucumis melo</Emphasis> plants

Melon (Cucumis melo L.) is considered to be a recalcitrant species for genetic transformation. Additionally, many studies have observed that regenerated transgenic plants are frequently polyploids. Here we have studied several aspects of melon transformation with the aim of improving transformation efficiency and producing diploid transformed plants. The protocol was based on using cotyledon explants from quiescent seeds that retain meristematic cells, which facilitated the regeneration of transformed diploid melon plants. In this study we evaluated the effect of using two different explant types from the proximal portion of melon seeds on the ploidy status (evaluated by flow cytometry) of regenerated plants. We also determined the transformation efficiencies obtained with these types of explants from four different genotypes. Regeneration was obtained from all explant types. Using quiescent seeds the percentage of diploid plants produced ranged from 85.2 to 94.1%, depending on the type of explant. On the other hand, only half of the plants regenerated from older-seed cotyledons (2- or 3-day-old) were diploids. Transgenic plants were produced with variable transformation efficiencies depending on the explant and which of the four melon genotypes was used. The explants with the best behavior produced transgenic plants with the highest efficiencies ever published both, in terms of plants expressing the visual marker transgenes (ranging from 4.5 to 15.4%) and the number of rooted plants in selective medium (ranging from 1.3 to 3.8%). Although the transformation efficiencies were still relatively low, they were consistent for the four very different melon genotypes tested. Furthermore, at least 85% of plants produced were diploid.     

农杆菌介导的小麦遗传转化几个影响因素的研究

采用携带gus和（或）bar基因双元表达载体（p3301,pBTAaB)的3个根癌农杆菌（Agrobacterium tumefaciens)菌株（AGL-1,EHA105和LBA4404）对普通小麦（Triticum aestivumL.)冬性栽培品种农大170和农大146的幼胚及幼胚愈伤组织进行了遗传转化,结果表明,菌液浓度OD6001.0和侵染时间1h对外植体的生存和转化最为有利;侵染前对外植体进行高渗处理较明显地提高了抗性愈伤获得率;乙酰丁香酮（AS）对小麦转化的作用随菌株和外植体的不同而异;菌株/质粒组合,受体基因型及外植体的类型,年龄和生理状态对转化效率有很大的影响,条件优化后,得到大量具有PPT抗性的愈伤和一些抗性植株,抗性愈伤的GUS染色阳性率在50%-60%之间,所检测的抗性苗呈GUS阳性,对6株抗性苗的PCR和Southern检测初步证明,外源基因已经整合到其中3株的基因组中。     

Transgenic caraway, Carum carvi L.: a model species for metabolic engineering

Regeneration in caraway was obtained via two different routes. Hypocotyls showed delayed shoot formation after a callus phase and at relatively low frequencies. In contrast, high-frequency, direct regeneration occurred when cotyledonary node explants were used. Transient expression of β-glucuronidase was monitored after inoculation of both explant types with Agrobacterium tumefaciens AGL0(pMOG410). Gene transfer was more efficient when using cotyledonary node explants. This explant type also proved to be the best for stable transformation resulting in transgenic plants. Several parameters determining regeneration and transformation efficiency were tested. The percentage of explants giving one to numerous transgenic plants could be as high as 13%. This system for the rapid production of many transgenic caraway plants opens up possibilities for studying metabolic engineering with this crop. Received: 8 October 1996 / Revision received: 2 January 1997 / Accepted: 2 February 1997     

Transformation of potato (Solanum tuberosum) using particle bombardment

Internodes, leaves and tuber slices from potato (Solanum tuberosum), genotype 1024-2, were subjected to particle bombardment. Transient expression was optimized using the uidA and the luc reporter genes that encode #-glucuronidase (GUS) and luciferase, respectively. Stable transformation was achieved using the neomycin phosphotransferase (nptII) gene, which confers resistance to the antibiotic kanamycin. The influence of biological parameters (tissue type, growth period before bombardment, pre- and post-bombardment osmoticum treatment) and physical parameters (helium pressure, tissue distance) that are known to possibly affect stable transformation were investigated. Putative transgenic plants, which rooted in media containing kanamycin, were obtained from all of the tissues tested although there were large differences in the efficiency: internodes (0.77 plants per bombarded explant), microtuber slices (0.10 plants per bombarded explant) and leaves (0.02 plants per bombarded explant). Southern blot analysis of putative transgenic plants confirmed the integration of the transgenes into plant DNA. The results indicate that an efficient particle bombardment protocol is now available for both transient and stable transformation of potato internodal segments, thus contributing to an enhanced flexibility in the delivery of transgenes to this important food crop.     

农杆菌介导法在橡胶树遗传转化中的应用与展望

综述农杆菌介导法在巴西橡胶树遗传转化中的应用进展,分析影响农杆菌转化的关键因素,如植物基因型与外植体、菌株与载体类型、菌液浓度与侵染时间、vir诱导物、筛选剂与抑菌剂、培养基的组成和附加成分等,并对提高巴西橡胶树转化效率的策略进行探讨。     

Genetic transformation of Robinia pseudoacacia by Agrobacterium tumefaciens

Transgenic Robinia pseudoacacia plants were obtained by Agrobacterium tumefaciens mediated gene transfer. Agrobacterium strain LBA4404 harbouring a binary vector that contained the chimeric neomycin phosphotransferase II (NPTII) and beta-glucuronidase (GUS) genes was co-cultivated with hypocotyl segments of in vitro raised seedlings of Robinia. Parameters important for high efficiency regeneration and transformation rates included type of explant, pre-conditioning of explants and appropriate length of co-cultivation period with Agrobacterium. A transformation frequency 16.67% was obtained by 48 hr of pre-conditioning followed by 48 hr of co-cultivation. Transformed tissue was selected by the ability to grow on kanamycin containing medium. Successful regeneration was followed after histochemical GUS assay for the detection of transgenic tissue. This transformation procedure has the potential to expand the range of genetic variation in Robinia.     

Factors influencing the regeneration capacity of oilseed rape and cauliflower in transformation experiments

The efficiency ofAgrobacterium-based transformation technique in oilseed rape and cauliflower was influenced by cultivar specificity, donor plant age and explant type. Marked differences in demands for plant hormone contents in the regeneration medium were recorded already among different types of nontransformed explants. The highest regeneration capacity was recorded with stem and leaf segments isolated from one-month-old aseptically grown plants. The regeneration was markedly species-dependent. Regeneration of transformed plants from stem segments and thin layers isolated from field-grown oilseed rape plants (at the most 2% of regenerating explants) and from oilseed rape hypocotyls (0.8% of regenerating explants) and cauliflower (1.2% of explant regenerated transformed shoots) was achieved after disarmedAgrobacterium treatment. Hypersensitive reaction of explants could be prevented by using prolongedin vitro precultivation and delayed application of the selective agent.     

Highly efficient Agrobacterium-mediated transformation and plant regeneration system for genome engineering in tomato

Tomato (Solanum lycopersicum L.) is an important vegetable and nutritious crop plant worldwide. They are rich sources of several indispensable compounds such as lycopene, minerals, vitamins, carotenoids, essential amino acids, and bioactive polyphenols. Plant regeneration and Agrobacterium-mediated genetic transformation system from different explants in various genotypes of tomato are necessary for genetic improvement. Among diverse plant growth regulator (PGR) combinations and concentrations tested, Zeatin (ZEA) at 2.0 mg l^?1 in combination with 0.1 mg l^?1 indole-3-acetic acid (IAA) generated the most shoots/explant from the cotyledon of Arka Vikas (36.48 shoots/explant) and PED (24.68 shoots/explant), respectively. The hypocotyl explant produced 28.76 shoots/explant in Arka Vikas and 19.44 shoots/explant in PED. In contrast, leaf explant induced 23.54 shoots/explant in Arka Vikas and 17.64 shoots/explant in PED. The obtained multiple shoot buds from three explant types were elongated on a medium fortified with Gibberellic acid (GA₃) (1.0 mg l^?1), IAA (0.5 mg l^?1), and ZEA (0.5 mg l^?1) in both the cultivars. The rooting was observed on a medium amended with 0.5 mg l^?1 indole 3-butyric acid (IBA). The transformation efficiency was significantly improved by optimizing the pre-culture of explants, co-cultivation duration, bacterial density and infection time, and acetosyringone concentration. The presence of transgenes in the plant genome was validated using different methods like histochemical GUS assay, Polymerase Chain Reaction (PCR), and Southern blotting. The transformation efficiency was 42.8% in PED and 64.6% in Arka Vikas. A highly repeatable plant regeneration protocol was established by manipulating various plant growth regulators (PGRs) in two tomato cultivars (Arka Vikas and PED). The Agrobacterium-mediated transformation method was optimized using different explants like cotyledon, hypocotyl, and leaf of two tomato genotypes. The present study could be favourable to transferring desirable traits and precise genome editing techniques to develop superior tomato genotypes.