转基因技术在大豆育种上的应用与研究

近年来,转基因技术在大豆上的研究重点主要集中在建立高效再生体系和稳定地遗传转化体系方面,随着遗传转化技术的发展,我国已获得了抗病、抗虫转基因的大豆植株并取得突破性进展。笔者就大豆遗传转化在受体系统(器官发生受体系统、体细胞胚胎发生受体系统、原生质体受体系统)以及转化方法(农杆菌介导法、基因枪法)等方面的研究进展情况进行了综述,并对今后大豆转基因研究方向进行了探讨。     

大豆体细胞胚胎组织培养及遗传转化研究进展

大豆体细胞胚胎遗传转化体系是提高外源基因遗传稳定性,克服嵌合体的有效途径,因此建立高效稳定的大豆体细胞胚胎组织培养体系并广泛应用于大豆的遗传转化是完善大豆遗传转化研究的重要研究内容。本文从大豆体细胞胚胎再生体系研究和转基因研究方法两个方面阐述了大豆体细胞胚胎再生体系的研究进展,并对影响大豆体细胞胚胎遗传转化的影响因素进行了探讨。     

大豆组织和细胞培养研究进展

从大豆组织培养的培养基选择、基因型和外植体类型培养差异、器官和胚胎发生再生植株、花药和花粉及 原生的南体培养再生植株、体细胞无性系变异体选择和基因转化等方面综述了大豆作物组织和细胞培养所取得的进展，并对今后的研究提出了建议。     

茶树体细胞胚发生的研究进展

体细胞胚胎发生(somatic embryogenesis, SE)是植物特异性的发育过程和植株再生的关键因子,其对于茶树的育种及繁殖具有重要意义。为了系统认识茶树体细胞胚胎发生的适宜培养条件,本研究总结了茶树体细胞胚发生的研究进展;综述了影响茶树体细胞胚胎发生的主要因素;剖析了茶树体细胞胚胎诱导过程中存在的问题;并对如何解决这些问题提出了研究展望。以期为茶树遗传转化体系的建立和新品种培育提供重要的理论和技术支撑。     

红掌(Anthurium andraeanum)细胞工程研究进展及其在育种上的应用

红掌是一种重要的观花和观叶兼具的草本植物。植物离体培养技术已经成为红掌繁殖主要采用的方法,绝大部分品种可以通过组织培养实现再生,其推动了红掌细胞工程育种的发展。本综述总结了红掌细胞组织培养繁殖途径,包括直接获得无菌苗途径、愈伤组织诱导和不定芽再生途径、体细胞胚胎发生途径、原球茎和类原球茎途径、原生质体途径等方面的研究进展,及其花器官培养,多倍体诱导,物理化学诱变,遗传转化,体细胞无性系变异及筛选的研究在育种中的应用。并提出了红掌细胞工程研究当前存在的问题和以后的研究方向,以期为将来红掌的研究提供借鉴。     

杉木愈伤组织的高效诱导和瞬时转化体系的建立

本研究参考已有的杉木(Cunninghamia lanceolata)体细胞胚胎发生系统,建立了以杉木针叶为外植体的愈伤组织高效诱导体系和原生质体瞬时转化体系,并初步探讨了细胞壁再生和以茎段为受体材料的稳定遗传转化技术。以杉木针叶为外植体,在添加有1.0 mg/L 2,4-二氯苯氧乙酸(2,4-D)、2.0 mg/L 6-苄氨基嘌呤(6-BA)、1.0 mg/L 6-糠基氨基嘌呤(KT)以及0.5 mg/L萘乙酸(NAA)的MS培养基诱导愈伤组织,愈伤组织形成率可以达到87.5%;对分离得到的杉木次生木质部原生质体采用40%PEG-4000介导法进行原生质体瞬时转化,外源基因的转化率达到30%,并实现了原生质体多次分裂形成细胞团;以上结果表明,以杉木针叶作为外植体可高效诱导形成愈伤组织,且在增殖培养基上生长状态良好。本研究建立了杉木原生质体瞬时转化体系并实现了原生质体的细胞壁再生和分裂。     

探析植物体胚发生的关键基因

体细胞胚胎是植物离体再生的高效方式及遗传转化的理想受体,也是进行科学研究的理想体系;但是从体细胞转变为胚性细胞的过程是复杂的基因调控网络调控的,在这其中,BABY BOOM/WUSCHEL/LEAFY COTYLEDON/MYB115等转录因子处于体胚调控网络的上游,能够带动下游相关基因的响应从而启动了体细胞胚胎的发生,起着"画龙点睛"的关键作用,是启动体胚发生"按钮"和调控体胚发育的"开关"。本综述分析了上述关键基因在启动体细胞胚胎发生及体胚发育和器官再生等过程中的作用,及探讨了从分子水平调控体胚发生的研究体系。认为,在化学诱导表达体系下操纵BABY BOOM和WUSCHEL基因的表达是启动体胚发生及提高植物再生能力的最有效的调控系统,无论同源和异源表达均表现出明显的功能和诱导体胚发生形成的作用,对于科研和生产均具有重要的价值和意义。     

棉花组织培养体细胞胚胎发生的扫描电镜观察

近年来棉花组织培养发展很快, 先后从陆地棉、 海岛棉、 克劳茨基棉、 戴维逊氏棉、 拟似棉等不同棉种的胚轴、 子叶、 茎段、 花药、 原生质体等诱导获得了体细胞胚胎发生和植株再生。 但是对棉花体细胞胚胎发生的机制还需要探索, 本文采用扫描电镜技术对棉花体细胞胚胎发生的形成过程和形态特征进行了研究, 旨在进一步了     

高渗处理对不同基因型大豆未成熟子叶基因枪法转化效果的影响

为了进一步优化以未成熟子叶为外植体的大豆遗传转化技术体系,研究了高渗剂种类、高渗处理时间,以及轰击后高渗培养时间对不同基因型大豆未成熟子叶基因枪转化效果的影响。结果表明,高渗剂种类极显著地影响体细胞胚数,基因型间体细胞胚胎发生率存在显著差异,而大豆基因型与高渗剂种类间在体细胞胚胎发生率和胚数上都存在极显著的互作。对大豆品种合丰25未成熟子叶进行高渗处理24h时,体细胞胚胎发生率极显著地高于7h和0h处理;胚数在3种高渗时间处理间没有显著差异。基因枪轰击后高渗培养5d和7d时大豆5个基因型间在体细胞胚胎发生率方面存在极显著差异,均以吉育71最高,其次是合丰25和黑农51,绥农28和吉育75较低。吉育71的体细胞胚胎发生率在轰击后高渗培养5d处理中最高,而其它基因型的体细胞胚胎发生率不受轰击后高渗时间的显著影响。     

葡萄体细胞胚发生的研究进展

主要综述了近年来葡萄胚性愈伤组织的诱导、体细胞胚胎发生的诱导、体细胞胚长期保存和体细胞胚的萌发与成苗的研究进展,并对研究中的主要影响因素进行叙述。此外,还介绍了体细胞胚在转基因上的应用,指出葡萄体细胞胚胎发生过程还存在胚状体诱导难,再生率低等问题。对葡萄胚性悬浮培养液的建立及体细胞胚发生在转基因、原生质体培养等上的应用进行了展望。     

棉花原生质体培养与体细胞杂交研究进展

棉花原生质体培养已在多个棉种上取得成功,体细胞杂交也取得了较大的进展,创造出多个种间体细胞杂种,为棉花遗传改良提供了丰富的种质资源.结合本实验室的研究进展,综述了棉花原生质体培养和体细胞杂交的基本技术、研究现状、存在问题和应用前景,为棉花基因工程和细胞工程研究提供有益的借鉴.     

植物组织培养再生相关基因鉴定、克隆和应用研究进展

离体植物组织体细胞胚胎发生是一个复杂的无性繁殖过程，依次经历外源植物激素信号应答、已分化细胞的脱分化、静止细胞的再分裂以及特定组织、器官原基或分生组织的形成等，是多个基因在外界因素刺激下协调、有序表达和互作的结果，不但受培养基中植物激素和营养成分的影响，也与外植体的生理状态关系密切。本文综述了外源激素和内源激素在植物组织培养中的作用，以及外源激素对内源激素的调节功能；重点介绍了5类与植物体细胞胚胎发生有关的候选基因，包括体细胞胚胎发生相关类受体蛋白激酶、阿拉伯葡聚糖酶、亚硝酸还原酶、生长素结合蛋白和抗氧化酶。再生相关基因的利用不但有助于提高植物组织培养植株再生率和遗传转化率，而且有助于获得安全型转基因植物，在基因工程育种中具有潜在应用前景。不同植物和同种植物不同外植体组织培养中调控体细胞胚胎发生的主效基因可能不同，关键再生相关基因的克隆和功能鉴定是今后需要加强的方向。     

Plant regeneration via organogenesis and somatic embryogenesis in callus cultures derived from mature zygotic embryos of leek (Allium ampeloprasum L.)

Summary A high frequency plant regeneration system via organogenesis and somatic embryogenesis was established with callus cultures derived from mature zygotic embryos of different leek genotypes (Allium ampeloprasum L.). Four different callus types with varying morphogenetic potential were obtained. Relatively high concentrations of the auxin 2,4-dichlorophenoxy-acetic acid reduced callus weight and subsequent shoot regeneration and primordia formation of the callus. Shoot regeneration and primordia formation of the callus decreased after prolonged subculture on media containing 2,4-dichlorophenoxy acetic acid. A callus growth period of six weeks on Murashige and Skoog medium with 0.25–0.5 mg l^-1 2,4-dichlorophenoxy acetic acid showed the highest rate of shoot regeneration after transfer of callus to regeneration medium with 1 mg l^-1 kinetin.Differences between leek genotypes in callus type, callus weight, shoot regeneration and primordia formation were observed. Histological observations showed that plant regeneration took place, both via the pathway of somatic embryogenesis and organogenesis.Abbreviation 2,4-D 2,4-dichlorophenoxy acetic acid - MS Murashige and Skoog (1962) medium     

Regeneration and transformation of cassava

A prerequisite for the development of a successful transformation system is the availability of efficient regeneration systems. Up to 1995 the only available regeneration system in cassava was an organized type of somatic embryogenesis. Transformation of these organized somatic embryogenic cultures with particle bombardment or Agrobacterium tumefaciens resulted in chimeric transformed embryos. However, the transformed sector was lost after repeated cycles of secondary somatic embryogenesis. After 1995 a less organized system of somatic embryogenesis was developed, so called friable embryogenic callus (FEC) and a system of adventitious shoot regeneration. The FEC regeneration system was combined successfully with particle bombardment. Selection of transgenic plants was based on either luciferase activity, or resistance to the aminoglycoside paromomycin or the herbicide phosphinothricin. Furthermore, protoplasts of FEC are able to regenerate into plants and can be transformed by electroporation. The adventitious shoot regeneration system was combined successfully with Agrobacterium tumefaciens. For this mature somatic embryos were cocultivated with Agrobacterium and cultured for adventitious shoot development. After selection based on the aminoglycoside geneticin or on hygromycin transgenic plants were formed. This revised version was published online in July 2006 with corrections to the Cover Date.     

菜豆再生体系及遗传转化体系研究进展

菜豆分子育种及功能基因组学分析需要高效稳定的再生体系和遗传转化体系。归纳了近年来国内外菜豆再生体系及遗传转化体系的研究进展。首先阐述了菜豆再生方法及研究现状,其中包括基于器官发生和体细胞胚发生不同途径菜豆再生体系的建立和相关的影响因素。其次概括了农杆菌介导法在菜豆遗传转化方面的研究进展,并分析了影响农杆菌介导遗传转化效率的主要因素,包括受体外植体类型和外植体损伤、农杆菌菌株及菌液浓度、共培养条件(时间、温度、光照时间)、乙酰丁香醇浓度等。然后叙述了有关基因枪介导菜豆遗传转化的研究进展。最后对目前菜豆再生体系和遗传转化体系存在的难题进行了总结,以期为建立菜豆的高效离体再生体系和遗传转化体系提供重要参考。     

Plant tissue culture of Secale: A review

Summary Plant tissue culture of rye employs different parts of plant bodies originating from various stages of rye ontogenesis. For the culture initiation mainly diploid and after that tetraploid forms were used, however, haploids and triploids were subject of investigation. The development of plant regeneration via somatic embryogenesis or organogenesis required the using of numerous basal media with various plant growth hormones. Haploidization appeared to be the most difficult problem and only interspecies hybridization helped to overcome this problem. Wide sexual intergenera and interspecies hybridization in cereals require the development of proembryo and immature embryo culture system. Using the nurse culture basing on immature endosperm resulted in getting fully formed rye plants. There is no progress in somatic cell genetic manipulation of rye because of lack of plant regeneration system in mesophyll green leaf and suspension protoplast cultures.Abbreviations 2, 4, 5, Cl₃POP... 2, 4, 5-Cl₃-phenoxypropionic acid, dicamba...-3, 6-dichloroasinic acid - GA₃... gibberellic acid - BA... benzyladenine - IBA... 3-indolebutric acid - IAA... indoleacetic acid - NAA... 1-naphtylacetic acid - 2, 4-D... 2, 4-dichlorophenoxyacetic acid - 2, 4, 5-T... 2, 4, 5-trichloroacetic acid - p-CPA... parachlorophenoxyacetic acid - CM... coconut milk - CW... deproteined coconut milk - Kin Kinetin     

Plant regeneration from protoplasts of Iris germanica L.

Summary Protoplasts were isolated enzymatically from suspension cultures derived from embryogenic calli induced by leaf base culture of Iris germanica. In protoplast culture, the effects of glucose concentration, different sugars and combinations of 2,4-D and KIN on protoplast division and colony formation were examined. N6 medium supplemented with 0.1–1 mg/l 2,4-D, 1 mg/l KIN, 200mg/l casein hydrolysate, 250 mg/l proline, 0.2 M glucose and 20 g/l agarose was suitable for protoplast division and colony formation. When colonies formed were transferred onto hormone-free MS medium, many plantlets were regenerated through somatic embryogenesis. Thus, we could establish a plant regeneration system from protoplasts of I. germanica.Contribution from the Laboratory of Plant Breeding, Faculty of Agriculture, Miyazaki University, Japan, No. 95.     

Plant regeneration in sweet potato (Ipomoea batatas L., Convolvulaceae)

The application of new techniques for improvement of sweet potato crops, particularly including the exploitation of somaclonal variation, gene transfer by genetic transformation and somatic hybridization, requires the control of plant regeneration from tissue cultures. Shoots can easily be regenerated from explants of stems, petioles, leaves and roots, while callus cultures do not produce any shoots. The potential of somatic embryogenesis and plant regeneration via embryogenesis was evaluated for 10 cultivars of sweet potato. Protocols for plant regeneration from cultured protoplasts have also been developed. Since mesophyll was resistant to enzyme digestion, fragments of stems and petioles, callus and cell suspensions were used as source of protoplasts of sweet potato. Series of transfers of protoplast-derived calluses, particularly those which had been obtained from in vitro plants, to media containing a high level of zeatin resulted in successful formation of shoots in only two sweet potato cultivars. In addition, the embryogenic potential was irreversibly lost through protoplast culture, since protoplasts isolated from embryogenic cell suspensions developed into non-embryogenic callus. Consequently, an alternative protocol is being successfully developed to improve plant regeneration from cultured protoplasts of sweet potato, involving first root formation from which shoots can then be regenerated. Preliminary evaluation in field conditions in Gabon revealed that plants regenerated from cultured protoplasts exhibited a great genetic variability in their growth and tuber formation in particular. This revised version was published online in July 2006 with corrections to the Cover Date.     

RAPD-based detection of genomic instability in soybean plants derived from somatic embryogenesis

Random amplified polymorphic DNA (RAPD) markers were used to evaluate genetic stability of regenerants of soybean plants obtained through somatic embryogenesis using 180 μM 2,4‐dichloro‐phenoxy acetic acid. Twenty primers were used to screen 44 regenerants from the cultivar ‘Spring’ and 28 from the cultivar ‘CAC‐1’. Three primers were polymorphic for two of the ‘Spring’‐derived regenerants, with a somaclonal frequency of 4.5%. Four primers were polymorphic for the ‘CAC’‐l‐derived regenerant, with a somaclonal frequency of 3.57%. The results indicate the usefulness of RAPD markers to detect genetic instability in soybean primary regenerant plants derived from somatic embryogenesis, and as a certification tool for monitoring genetic stability during the regeneration process.