大白菜生物技术研究进展

综述了国内外在大白菜(Brassica rapa ssp. pekinensis)单倍体培养、基因工程和分子标记方面的最新研究成果。重点讨论了大白菜小孢子培养的影响因素、取材要点、培养条件、植株再生和倍性鉴定以及大白菜的转基因载体选择、受体系统建立、遗传转化与转基因植株鉴定等。对小孢子培养及转基因研究的原理、方法和关键技术进行了总结。展望了大白菜生物技术研究的应用前景。     

水稻游离小孢子培养最新研究进展

水稻小孢子培养技术不但可用来生产单倍体植株和加倍单倍体植株,而且可望成为转基因的理想受体系统。目前转基因技术、分子杂交技术与小孢子培育技术的有机结合已成为加快水稻育种速度的有效途径。本文从水稻小孢子分离纯化、影响愈伤组织或胚状体发生的因素以及植株再生三个方面综述了水稻游离小孢子培养的最新研究进展。最后就水稻小孢子培育技术急需解决的问题进行了讨论,并对小孢子培育技术发展前景进行了展望。     

大白菜与结球甘蓝异源三倍体小孢子植株的获得与鉴定

以6个不同基因型的大白菜四倍体(AAAA,2n=4x=40)品系9401、9402、9403、9404、9405、9406为母本,结球甘蓝二倍体(CC,2n=2x=18)自交系9501为父本配制杂交组合得到的6个杂种一代为试材,进行了游离小孢子培养研究,成功诱导出胚状体,获得了再生植株,并对部分再生株进行了染色体数鉴定和性状调查。结果表明:不同组合小孢子胚胎发生能力不同,各组合产胚率均较低;小孢子再生植株中,染色体数为18的个体所占比例最大,达46.7%;小孢子植株减数分裂行为复杂,终变期除二价体和单价体外,还有三价体等联会形式;小孢子植株性状表现各异。     

抗菌肽基因转化大白菜获得抗病转基因植株及稳定遗传

软腐病是大白菜( Brassica pekinensis Rupr.)的三大病害之一.抗菌肽对软腐病菌有很强的杀伤作用.建立了根癌土壤杆菌( Agrobacterium tumefaciens ) EHA105(pMOG410)工程菌的高频转化载体系统,将抗菌肽基因导入目前推广种植的大白菜AB-81自交系,获得了转基因植株.PCR及 Southern blotting分子杂交鉴定表明抗菌肽基因已整合到白菜基因组.转基因植株提取液的体外抑菌实验、试管苗及盆栽转基因植株的病原菌接种抗病测试结果表明转基因植株具有明显的抗病特性,并且能稳定遗传,转基因植株R1自交分离比为3∶1,R5的转基因植株保持抗Km和抗病特性,可望以其为亲本选育出大白菜抗软腐病的新品种.     

麦类作物小孢子培养与遗传转化研究进展

小孢子作为单个单倍体细胞,通过诱导培养能再生成纯合的二倍体植株,不仅为分子生物学、遗传学、形态发生学研究提供稳定的材料,也对小麦等作物的转基因研究提供新方向和新思路。本文主要介绍麦类作物小孢子培养的基本步骤及关键因素,简要介绍了以小孢子为材料进行小麦基因遗传转化的方法。     

不结球白菜小孢子胚植株再生及倍性研究

以不结球白菜子叶型小孢子胚为外植体,研究冷处理、活性炭及AgNO3对小孢子植株再生的影响,并对再生植株染色体倍性进行鉴定.结果表明:对小孢子胚进行5 d的4℃冷处理培养能提高其胚芽诱导率和胚芽数;培养基中添加1.0 g/L的活性炭对提高小孢子胚芽诱导率没有明显效果,但能有效减轻胚芽的玻璃化;添加5.0或7.0 mg/L的AgNO3对小孢子胚芽诱导有显著效果.染色体倍性鉴定结果表明:不结球白菜小孢子植株的染色体自然加倍率较高,在50%～100%之间;不同基因型不结球白菜小孢子植株的倍性变异具有多样性;在部分基因型中嵌合体占较高比例,最高达到42.86%.     

抗菌肽基因转化大白菜获得抗病转基因植株及稳定遗传

软腐病是大白菜 (BrassicapekinensisRupr.)的三大病害之一。抗菌肽对软腐病菌有很强的杀伤作用。建立了根癌土壤杆菌 (Agrobacteriumtumefaciens)EHA10 5 (pMOG4 10 )工程菌的高频转化载体系统 ,将抗菌肽基因导入目前推广种植的大白菜AB_81自交系 ,获得了转基因植株。PCR及Southernblotting分子杂交鉴定表明抗菌肽基因已整合到白菜基因组。转基因植株提取液的体外抑菌实验、试管苗及盆栽转基因植株的病原菌接种抗病测试结果表明转基因植株具有明显的抗病特性 ,并且能稳定遗传 ,转基因植株R1自交分离比为 3∶1,R5的转基因植株保持抗Km和抗病特性 ,可望以其为亲本选育出大白菜抗软腐病的新品种。     

用激光微束穿刺法转化甘蓝型油菜小孢子的研究

建立了油菜小孢子再生胚状体的实验体系,掌握了受体最佳发育时期,使再生频率高达２１．２个胚状体／花蕾。以油菜游离小孢子为受体,用微束激光穿刺法导入芜菁花叶病毒外壳蛋白基因（ＴｕＭＶ）,成功地获得了转基因植株。研究结果表明,用预培养３天的小孢子进行转化得到的１５个胚状体,再生出一株绿苗,２株白苗。而未经预培养的小孢子未能得到转基因植株。用植物总ＤＮＡ以ＰＣＲ法扩增ＴｕＭＶ基因产物片断,然后进行电泳检测,证明该绿苗带有芜菁花叶病毒外壳蛋白基因。     

结球甘蓝和青花菜小孢子胚植株再生

结球甘蓝（Brassica oleracea var.capitata）和青花菜（Brassica oleracea var.italica）小孢子胚再生植株频率低是目前影响游离小孢子培养技术有效应用的关键问题之一,研究其小孢子胚植株再生频率的影响因素,提高胚再生植株频率,对促进游离小孢子培养技术在甘蓝类蔬菜育种中更好地应用具有重要意义。该文以结球甘蓝中甘11和青花菜TI-111等基因型为试材,对影响游离小孢子胚再生成植株的固体培养基类型、琼脂浓度、胚的类型及胚在液体培养基中的滞留时间等因素进行了研究。结果表明：游离小孢子培养25天的子叶胚在琼脂浓度为1%–1.25%的B5培养基上植株再生频率最高。进一步通过8个不同基因型对上述实验结果进行了验证,结果显示,游离小孢子培养25天的子叶胚在1%琼脂浓度的B5培养基上植株再生频率达77.8%–97.2%。     

结球甘蓝和青花菜小孢子胚植株再生

结球甘蓝(Brassica oleracea var. capitata)和青花菜(Brassica oleracea var. italica)小孢子胚再生植株频率低是目前影响游离小孢子培养技术有效应用的关键问题之一, 研究其小孢子胚植株再生频率的影响因素, 提高胚再生植株频率, 对促进游离小孢子培养技术在甘蓝类蔬菜育种中更好地应用具有重要意义。该文以结球甘蓝中甘11和青花菜TI-111等基因型为试材, 对影响游离小孢子胚再生成植株的固体培养基类型、琼脂浓度、胚的类型及胚在液体培养基中的滞留时间等因素进行了研究。结果表明: 游离小孢子培养25天的子叶胚在琼脂浓度为1%–1.25%的B5培养基上植株再生频率最高。进一步通过8个不同基因型对上述实验结果进行了验证, 结果显示, 游离小孢子培养25天的子叶胚在1%琼脂浓度的B5培养基上植株再生频率达77.8%–97.2%。     

Factors affecting the regeneration capacity of isolated barley microspores (Hordeum vulgare L.)

The culture of isolated microspores of barley (Hordeum vulgare L. cv. Kymppi, an elite malting barley cultivar) was studied. A careful choice of culture steps resulted in an average regeneration frequency of 300 green plants per starting material spike. Strong seasonal variation in regeneration capacity was observed. The choice of a cold pretreatment method affected the viability of microspores. A cold pretreatment of the collected starting material at +4°C for 4 weeks was needed for the efficient regeneration of green plants from isolated microspore cultures. Glutamine omission from and copper additions to microspore culture were studied. The omission of glutamine did not affect the number of regenerated green plants but did result in an increase in the number of regenerated albino plants. The addition of copper did not improve the regeneration capacity of isolated barley microspores. Transformation by particle bombardment of isolated microspores did not result in the production of transgenic plants.     

Regeneration of androgenic embryos in apple (<Emphasis Type="Italic">Malus x domestica</Emphasis> Brokh.) <Emphasis Type="Italic">via</Emphasis> anther and microspore culture

Based on optimized protocols for anther and microspore culture in apple (Malus x domestica Borkh.), the regeneration phase and the efficiency of the processes in general were compared by using the same androgenic material of two experimental years. Microspore culture resulted in an increase in embryo induction depending on the genotype (Höfer 2004), however anther culture was superior to microspore culture in the total number of regenerated plants. The regeneration process in anther and microspore culture is similar. Two developmental pathways were observed: 1) secondary embryogenesis followed by adventitious shoot formation and 2) direct adventitious shoot formation from primary embryos. Induction and regeneration processes are delayed in microspore culture as compared with anther culture. The reasons for the reduced regeneration efficiency in microspore culture are discussed.     

草坪草生物技术研究进展

概述了草坪草植株再生体系和遗传转化体系建立的方法和进展.通过愈伤组织培养、悬浮细胞培养和原生质体培养方法对草坪草的一些种已建立较为完善的植株再生体系.在建立再生体系的基础上,利用原生质体融合、农杆菌介导、基因枪和碳化硅纤维介导等转基因方法在一些草坪草种上建立了遗传转化体系并获得了有一定价值的转基因植株.最后,对草坪草转基因存在的问题和前景作了讨论.     

Microspore embryogenesis and fertile plantlet regeneration in a salt susceptible × salt tolerant rice hybrid

Shed microspore embryogenesis and fertile plantlet regeneration were observed in a salt susceptible × salt tolerant indica rice F₁ hybrid involving IR 24 and CRM 30. The in vitro culture response and regeneration of green plantlets in the hybrid were superior to those of the parents. Direct embryogenesis and plantlet regeneration with multiple tillers were observed in shed microspore embryos. In intact anther culture, plantlet development from microspore involved a callus phase. The number of multiple tillers developed through secondary embryogenesis was almost equal in both the cases. However, the results indicate that regeneration of green plantlets was higher in case of shed microspore culture in liquid medium containing the synthetic polymer Ficoll 400 than from intact anthers cultured on a semi-solid system. Shed microspore culture produced a number of double haploids, which may result in far reaching consequences in genetic improvement of rice. This revised version was published online in June 2006 with corrections to the Cover Date.     

Inducing homozygosity in transgenic barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) by microspore culture

Homozygosity was induced in transgenic barley by microspore culture. Spikes of transgenic barley plants carrying microspores in the late uni-nucleate stage were cold pretreated. Teflon rod maceration and a density of 100 000 viable micropores per plate were used. The developed calli were regenerated and plantlets were treated with colchicine. The microspore culture of 16 mother plants (three transgenic lines) resulted in 927 green regenerants. Of these plants, 476 were transferred to soil, 380 were transgenic, 358 reached maturity and 350 were fertile with a normal seed-set carrying a yield of 6.9 kg. A production efficiency of 0.8 fertile transgenic doubled haploid barley plants per spike used for microspore isolation was recorded. The produced transgenic seeds were used in malting experiments.     

Isolated microspore culture and plant regeneration in rye (Secale cereale L.)

 An isolated microspore culture and green plant regeneration method for rye (Secale cereale L.) was established. Rye isolated microspore androgenesis was genotype-dependent. PG-96M medium supplemented with 6% maltose gave the highest microspore survival rate after 48 h of culture and the highest embryo/callus yield (930 embryos/calli per 100 anthers from cv. Florida 401). Osmotic pressure in the induction medium played an important role. Pretreatment of the anthers with mannitol was beneficial for the microspore culture. Embryos/calli of a relatively younger age and smaller size had a higher regeneration ability, with the best green plant regeneration rate being 6%. Over 150 microspore-derived green plants have been obtained so far. About 90% of the regenerated plants were spontaneous doubled haploids. This is the first report of isolated microspore culture in true rye resulting in androgenic embryogenesis and plant regeneration. Received: 26 April 1999 / Accepted: 23 November 1999     

Microspore culture of small grain cereals

Androgenesis of wheat, rice and triticale was studied in isolated microspore culture. It is the first publication which studies microspore culture reaction of Hungarian rice varieties. The effect of different basic media, lack and absence of growth regulators in culture media were tested on important parameters of microspore culture. Direct embryogenesis was observed in microspore culture of wheat and triticale genotypes. In the case of rice, calli were induced in isolated rice microspore culture and haploid rice plantlets were regenerated via organogenesis.In wheat, the effect of basic media (W₁₄, A2, CHB₃, P4-m) was compared and among them the W₁₄, and A2 had a superior effect on embryo production and albino and green plantlet regeneration. In rice the C, CHB₃ and MS_m media were tested in microspore culture and the significantly highest numbers of calli were achieved by using C and CHB₃ media depending on the genotypes. The lack of exogenous growth regulators was observed in isolated microspore culture of triticale and rice. Growth regulator-free medium had a positive effect on embryo production and plant regeneration of triticale genotypes, whereas in rice microspore culture multicellular structures did not continue their division without growth regulators from the third week of microspore culture. Developing of microspore-origin calli was maintained by supplement of 2,4-D and Kinetin combination in the microspore culture medium.