大豆和小麦原生质体的分离、融合及融合体的筛选技术

本文提供了大豆(Glycine max)根尖分生组织细胞原生质体和小麦叶栅栏细胞原生质体的分离方法。建立了大豆和小麦原生质体融合的实验程序和融合体筛选技术。     

芸薹属作物原生质体融合及基因转移

用于芸薹属作物原生质体融合的主要方法有PEG诱导融合法和电场诱导融合法，融合方式有对称融合与非对称融合。已在芸薹属种内、种间、属间及族间通过体细胞杂交实现了胞质基因组的转移，改良或创造了细胞质雄性不育系。从远缘物种中转移了一些有用的外源基因到芸薹属作物中，如抗性基因、品质性状基因及控制其它一些重要农艺性状的基因。讨论了体细胞杂种鉴定技术及原生质体融合技术应用于芸薹属作物改良的前景和局限性。     

大豆与豌豆原生质体融合的初步研究

我们采用PEG—高pH—高钙的方法对大豆和豌豆原生质体进行融合,获得了10—36％的融合率。大豆原生质体游离的混合酶液为2％ Cellulase ONOZUKA R-10,2％ Hemicellulase-Rhozyme, 1％ Pectinase Sigma;豌豆原生质体游离的混合酶液为2％Cellulase ONOZUKA R-10,2％ Hemicellulase sigma,1％ Pectinase Sigma。原生质体的密度对融合的影响很大,以10~4—10~(?)/毫升的密度适合原生质体融合及有利细胞分裂。品种间融合率有差异。融合体用高国楠的KM8P培养基培养,经过多次分裂,获得了愈伤组织。     

原生质体融合技术在烟草品种改良中的研究与应用前景

主要论述了原生质体融合技术在烟草品种改良中取得的成就,以及应用原生质体融合技术常采用的方法和存在的问题,讨论了原生质体融合技术在烟草抗生性育种上应用的前景。     

甘蔗原生质体RNA提取条件和方法的优化

为了获得高产量和高纯度的甘蔗原生质体RNA,本研究以新台糖22号（ROC22）和桂糖28号（GT28）甘蔗原生质体为材料,探究了渗透压和原生质体活力对甘蔗原生质体RNA提取效果的影响,并比较了Trizol法、改良Trizol法、改良CTAB法、试剂盒法4种RNA提取方法。结果表明：原生质体渗透压与RNase活性呈显著正相关,原生质体活力与RNase活性呈极显著负相关;RNase活性越高,RNA提取效果越差;使用0.5 mol/L的甘露醇作为渗透压调节剂可获取活力最高的甘蔗原生质体;原生质体活力为70%和90%时所提的RNA完整性好且纯度高,符合后续分子实验的需求,原生质体活力为90%时,RNA产量最高。因此,需要提取甘蔗原生质RNA时,至少需要保证原生质体的活力在70%以上,且原生质体活力越高,RNA的产量越高;采用改良Trizol法,可以大量制备能满足后续分子实验的完整性好且纯度高的RNA,利用该条件和方法,可以确保获得大量高质量的甘蔗原生质体RNA。本研究结果为甘蔗体细胞融合育种过程中的分子生物学研究提供了方法和技术支撑。     

预处理方式对大豆原生质体产率及其脂肪酸类型的影响

本文研究了不同预处理条件对大豆原生质体的产率和脂肪酸类型的影响。研究结果指出:新生叶片及子叶的原生质体产率较高。缺水处理使子叶细胞、原生质体中的脂肪酸脱饱和程度减弱,不饱和脂肪酸向饱和脂肪酸转化,饱和脂肪酸含量增加。而叶肉细胞原生质体中则不存在这种变化趋势。     

大豆原生质体培养经胚胎发生高频率再生植株

大豆原生质体培养诱导再生植株一直为国内外学者所关注。如Kao(1970)和Miller等(1971)从悬浮培养的大豆细胞分离出原生质体,经培养获得了愈伤组织。但在以后的多年中进展不够大。据报道,已从幼荚子叶、幼苗根、叶肉组织和悬浮培养细胞等外植体游离出原生质体,经培养获得愈伤组织,并进行了大量的分化研究,但最终都未能得到再生植株。卫志明和许智宏于1988年首次由大豆幼荚子叶分离出     

植物原生质体融合研究进展及其在育种中的应用

植物原生质体融合即体细胞杂交是有性杂交的重要有益补充.本文主要介绍植物原生质体融合技术(包括融合方法和融合方式)的研究进展及其在植物育种中的主要应用,并分析和探讨体细胞杂交育种在实践中存在的一些问题,同时对其应用前景作出展望.     

甲基氨草磷对橡胶树悬浮细胞同步化及微核诱导的影响

植物微原生质体融合(microprotoplast fusion)是将部分基因组转移而又避免染色体损伤的不对称融合技术,微原生质体成功诱导是微原生质体融合的先决条件。本研究在摸索甲基氨草磷(amiprophos-methyl,APM)对橡胶树悬浮细胞有丝分裂中期指数和染色体形态影响基础上,研究了酶解时间及酶解过程中添加APM对微原生质体产量的影响。结果发现,悬浮细胞随APM处理浓度和时间的增加,有丝分裂中期指数先升高再下降,处理12 h时达到最大值;染色体聚集程度也先增加再降低,处理16 h时染色体开始解聚。在酶解过程中添加APM和延长酶解时间会大大降低微原生质体的产量。悬浮细胞经过1 μmol/L APM处理10 h后,酶解10 h,中期细胞可达到79.4%,微核化细胞指数达到7.3%,从而建立了APM诱导橡胶树悬浮细胞同步化及高效获得橡胶树微化细胞的方法。     

播娘蒿与甘蓝型油菜的原生质体融合与植株再生

以播娘蒿叶片和甘蓝型油菜子叶为材料提取原生质体.采用PEG-高pH、高钙附加DMSO融合方法,液体浅层静置培养,研究影响播娘蒿与甘蓝型油菜原生质体融合的因素,获得了再生植株.结果表明:4%纤维素酶 0.5%离析酶 5mmol/L MES酶解10b可获得高产率播娘蒿原生质体,1%纤维素酶 0.2%离析酶 3mmol/LMES酶解14h可获得高产率油菜原生质体;30%PEG 0.3mol/L葡萄糖 50mmoL/L CaCl2·2H2O 15%DMSO可获得10%的融合率;改良B5培养基的原生质体培养效果最好;最佳分化培养基为MS 4.0mg/L 6-BA 0.3mg/LNAA 5.0mg/L AgNO3;最佳生根培养基为MS 0.1mg/LIBA 0.1mg/L NAA.     

Aspects of fusion combining ability of dihaploidSolanum tuberosum L.: influence of the cytoplasm

Summary Creation from 4x hybrid clones from protoplast fusion of 2x clones of potato was evaluated. Besides combined nuclear genomes, composition of the cytoplasm significantly influenced the phenotypic traits of hybrid clones. To ascertain the influence of parental cytoplasm on the success of protoplast fusion and regeneration of hybrid plants, data from 74 fusion combinations of 50 dihaploid clones were analyzed. The majority of dihaploid breeding clones belonged to the cytoplasm types Wα, Tβ and Wγ. When the closely related mt types α, β and γ were used, fusion combinations had a better combining ability compared with more distantly related cytoplasms δ and ⃛. Fusions containing the same mitochondrial type (homofusions) were not superior to closely related mitochondrial types. However, homofusions of cytoplasm type Wα yielded significantly more hybrids than homofusions of type Tβ. In general, parental cytoplasm types had little impact on the fusion combining behaviour. Thus the cytoplasm type of the fusion parents is not a suitable marker for predicting the combining ability in protoplast fusion experiments.     

荔枝种内原生质体电融合参数研究

选择“桂味”和“妃子笑”两个荔枝(Litchi chinensis Sonn.)品种的花药胚性细胞悬浮系来源的原生质体作融合亲本,在优化电融合参数(排列电压5V,脉冲电压80V,脉冲持续时间45～65 μs,原生质体密度5.0×105～7.5×105个/mL,融合液Ca2+浓度0.2 mmol/L,甘露醇浓度10％～11％)条件下,可获得20％以上双元异核融合率.在亲本原生质体混合前,以0.02％中性红10 min染色“桂味”原生质体,有助于双元异核融合体的有效确认.     

大豆组织培养的研究进展

概述了大豆原生质体和单细胞的组织培养，以及外植体经器官发生和体细胞胚胎发生途径再生植株的研究进展，对不同再生途径进行了比较，并指出了大豆组织培养存在的主要问题和今后研究方向。     

Interspecific somatic hybridization betweenSolanum tuberosum L. andS. bulbocastanum dun. as a means of transferring nematode resistance

Interspecific somatic hybrids were produced between tetraploidSolanum tuberosum and a nematode-resistant accession of the diploid speciesSolanum bulbocastanum by protoplast fusion. Hybrid cells were selected using dual fluorescent labeling of protoplast preparations prior to fusion. Hybridity of regenerated plants was confirmed with a combination of morphological assessment, chromosome counting and isozyme analysis. Somatic hybrids had the same level of resistance to infection by race 1 of the nematodeMeloidogyne chitwoodi as theS. bulbocastanum parent used in the fusion. Some of the somatic hybrids were fertile as females when crossed with tetraploidS. tuberosum breeding lines. Thus, these hybrids can be used in a potato improvement program to incorporate a valuable pest resistance     

Differences between hybrids ofSolanum tuberosum L. andSolanum circaeifolium Bitt. obtained from symmetric and asymmetric fusion experiments

Summary Potato plants obtained by symmetric and asymmetric protoplast fusion experiments withSolanum tuberosum andSolanum circaeifolium were compared and their potential use for plant breeding was investigated. For asymmetric hybridisation wild species tissue was irradiated with X-rays. Irradiation enhanced regeneration frequency. RFLP analysis showed that both techniques gave approx. 95% hybrids among the regenerates. Asymmetric protoplast fusion gave 22% regenerates with a ploidy level between 2x and 4x or 4x and 6x. None of the regenerates obtained from symmetric fusion had these ploidy levels. The tetraploid hybrids showed good male and female fertility. Male fertility was highest for the eutetraploid regenerates. With increasing asymmetry and with increasing ploidy level fertility decreased. Berry induction and seed production were observed for the eutetraploid and, to a lesser degree, for the hypotetraploid and hypohexaploid hybrids.     

基于多元素和脂肪酸指纹特征的中国北方大豆产地鉴别研究

为探讨数据融合技术用于邻近区域大豆产地鉴别的可行性,在黑龙江省农垦九三管理局和绥化地区采集216份大豆样品,测定镁（Mg）、铝（Al）、磷（P）等13种矿物质元素含量和棕榈酸、硬脂酸、油酸等5种脂肪酸含量。分别利用矿物质元素、脂肪酸、数据级融合和特征级融合数据建立4种核函数的支持向量机（Support Vector Machine,SVM）模型。通过网格搜索算法结合五折交叉验证进行参数优化后,模型识别准确率分别提升至90.77%、92.31%、89.23%、95.38%。结果表明,特征级数据融合技术对邻近区域大豆产地鉴别效果显著,优于其他三种数据识别技术。采用特征级数据融合技术建立支持向量机产地鉴别模型,能够对邻近区域大豆产地进行准确、有效的区分,为地理标志产品保护技术提供了新的研究方向。     

马铃薯抗青枯病和低温糖化体细胞杂种的鉴定

ACC-1-1和ACC-1-2再生株系由来自马铃薯二倍体栽培种(Solanum tuberosum)品系AC142-01(2n=2x=24)和二倍体野生种S.chacoense的一个无性系C9701(2n=2x=24)经原生质体融合获得,经鉴定为体细胞杂种。流式细胞仪分析和染色体计数显示,ACC-1-1为混倍体,ACC-1-2为八倍体。两个株系均能正常开花但花粉活力较低。花粉母细胞减数分裂观察显示,两个系在减数分裂各个时期均出现广泛的异常染色体行为,可能是花粉活力低的原因。青枯病接种鉴定表明,ACC-1-2对青枯病表现为高抗,而ACC-1-1表现为中感,同时这两个株系均具有"低温糖化"抗性,证明体细胞融合加上适当选择可以有效聚合有性杂交不亲和双亲的优良性状。     

Ultrastructural Analysis of Electrofused Protoplasts from Pansy and Wild Viola by Scanning Electron Microscopy

Summary

Ultrastructural changes during the fusion of the protoplasts isolated from wild viola callus (without chloroplasts) and pansy mesophyll cells (with chloroplasts) were examined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The presence of pansy protoplasts in the fusion products was proved by the presence of chloroplasts lying beneath the plasma membrane identified by SEM. Involvement of pansy chloroplasts in the fusion products was confirmed by TEM. Thus it was possible to distinguish between viola protoplasts and pansy protoplasts by SEM. During the process of protoplast fusion, adhesion of two protoplasts, the rupture and recovery of the membrane between them, and a single-paired spherical fusion body were clearly observed by SEM using our preparation method. Fused protoplasts divided and a microcolony was produced.