Somatic cell cryopreservation and protoplast regeneration of important disease-resistant wild rice Oryza meyeriana Baill

Oryza meyeriana Baill is one of the three wild rice species found in Chiia.O. mcyeriana possesses valuable characteristics but is reluctant in cell culturein vitro. In a series of experiments, callus with no regeneration ability was induced from young panicle ofO. meyeriana. The callus was subcultured and propagated. Embryogenic cell clones were obtained after cryopreswation. Suspension cultures were established and protoplasts were isolated and regenerated into plants. Results of artificial inoculation ofXanthomonas campestris pv.Oryzae showed that the strong resistance did not change in the regenerated plants. The development of protoplast-to-plant system is an important progress towards utilization ofO. meyeriana via cellular engineering. The experiments demonstrated that cryopreservation of plant calli was a new way to obtain embryogenic cell line. Project partially supported by the National Natural Science Foundation of China (Grant No. 392704361, Foundation for Outstanding Young Teachers of China, State Key Program of China and Natural Science Foundation of Hubei Province, China.     

Callus formation from protoplasys of Sorghum cell suspension cultures

Cell suspension cultures have been obtained from three cultivars of Sorghum bicolor L. Moench. Protoplasts readily obtained from these cultures underwent sustained cell division and callus formation.     

拟青霉属虫生真菌核型的脉冲电泳分析

利用钳位均匀电场脉冲电泳系统,从电泳的时间、角度、电压、转换时间间隔等方面优化电泳参数,确定了适宜拟青霉属内4种虫生真菌染色体DNA电泳的最佳条件。根据电泳及软件分析结果,估算出斜链拟青霉和环链拟青霉的染色体数目和染色体组型大小,即核型(karyotype)特征。通过进行拟青霉属内4种7株虫生真菌染色体DNA核型相关的比较和分析,表明拟青霉菌种间染色体数目差异较小,核型差异较大;相同菌种不同菌株间的染色体数、核型大小并不完全相同,存在不同程度的差异;且菌株间核型大小差异程度明显小于菌种间的差异程度。     

A novel method for the isolation and purification of protoplasts from friable,embryogenic corn (Zea Mays L.) callus

A method is described for the isolation of protoplasts from rapidly-growing, friable embryogenic and organogenic cell cultures of corn. A Sepharose 6MB cyanogen-bromide-activated macrobead column coupled with Cellulase RS was used to separate contaminating cells from protoplasts. The column consists of layering 1.5 cm of the coupled-macrobeads into a 2.2-cm diameter column. Contamination of protoplasts by cells possessing partial or complete walls was reduced from 25% to near zero after a single passage through the column. The column was capable of retaining in excess of 30 million cells and recovering 99% cell-free preparations from culture material consisting of less than 1% protoplasts. Coupled-macrobeads were easily recovered, washed free of cells and stored for repeated use. Corn protoplasts appeared undamaged by the column and rapeseed (Brassica napus) protoplasts which were passed through the column have divided and formed colonies in culture. Uncoupled macrobeads were not as efficient as coupled macrobeads in reducing cellular contamination.     

以潮霉素B抗性为选择标记的深黄被孢霉原生质体转化

利用亚硝基胍(MNNG)诱变方法筛选了一株深黄被孢霉潮霉素B敏感型菌株M6-22-4。采用PEG介导的方法,将含有E.coli潮霉素B抗性标记的PD4质粒转入敏感株M6-22-4原生质体,并在潮霉素B浓度为400μg/mL的选择培养基上筛选转化子,获得了1.6~2.8个转化子/μg质粒DNA的转化频率。稳定性实验表明,质粒线性化后所获得的转化子在PDA培养基上传代10代以后,转接到选择平板上有31.6%仍具有HmB抗性;随机挑选了3个转化子,通过PCR方法检测到潮霉素抗性基因的存在,Southern杂交发现,潮霉素抗性基因已经以1~2拷贝数整合到深黄被孢霉M6-22-4染色体上,这是深黄被孢霉转化系统的首次报道。     

原生质体培养在芸薹属蔬菜作物中的研究进展

原生质体是遗传转化的优良受体。在原生质体培养过程中会产生很多无性系变异,还可以产生体细胞杂种,这些都为蔬菜育种提供了新的途径。介绍了原生质体培养及原生质体融合方法的研究进展,综述了原生质体培养在芸薹属蔬菜育种中所取得的研究成果,并对今后研究的方向进行了展望。     

青花菜与白菜间体细胞杂种获得与遗传特性鉴定

为获得芸薹属白菜Brassica campestris与青花菜Brassica oleracea var. botrytis的种间体细胞杂交体,以青花菜和白菜的子叶与下胚轴为材料,分离制备原生质体,用40％聚乙二醇 (Polyethylene glycol,PEG) 进行原生质体融合。融合细胞在以0.3 mol/L 蔗糖、0.3 mol/L葡萄糖为渗透稳定剂,附加0.2 mg/L 2,4-D+0.5 mg/L 6-苄氨基嘌呤 (6-BA) +0.1 mg/L 1-萘乙酸 (NAA) +0.1 mg/L激动素 (Kinetin,Kin) 的改良K8p培养基中液体浅层培养。将包埋于0.1％琼脂糖的8~10个细胞期的细胞在添加0.3 mol/L蔗糖和2 mg/L 6-BA+2 mg/L玉米素 (Zeatin,ZEA) +1 mg/L NAA+0.5 mg/L Kin的Kao培养基中诱导愈伤组织。愈伤组织转到MS+5 mg/L ZEA+2 mg/L IAA诱导不定芽。将长1～2 cm的不定芽转到1/2 MS+0.2 mg/L NAA诱导生根。将生根的植株转移到花盆,并对其杂种性质进行形态学、细胞学和分子生物学鉴定。结果表明,融合细胞培养2～7 d后发生第1次分裂,培养35 d后植板率为0.66％,不定芽再生率达3.7％。形态学观察显示,绝大多数再生植株的叶面积较大,株型和叶型为两种杂交亲本的中间型。染色体计数结果显示,再生植株染色体数目为2n=38。流式细胞仪测定DNA含量显示,再生植株DNA含量是亲本之和。随机扩增多态性DNA (Random amplified polymorphic DNA,RAPD) 和基因组原位杂交 (Genomic in situ hybridization,GISH) 分析结果证明再生植株具有双亲基因组。体细胞杂种花粉育性比较低,杂交、回交后其育性逐渐获得恢复。     

黑曲霉原生质体诱变选育果胶酶高产菌株

通过UV和NTG诱变筛选获得了2株高产果胶酶突变株。以果胶酶产生菌黑曲霉EIM6为诱变材料,采用1．5％的溶壁酶和1．5％的纤维素酶处理其对教生长期菌丝体2h获得高质量的原生质体。采用UV25S或50μg／mL NTG诱变30min,构建原生质体突变库,经刚果红果胶平板筛选获得果胶酶突变株,通过液体深层培养复筛获得高产突变株EIM6-U11、EIM6-N5,酶活力分别从46598．08、46598．08U／mL提高至68596．57、68879．56U／mL,分别提高了47．21％、47．82％。连续8次传代经发酵测酶活力表明高产突变株EIM6-U11、EIM6-N5具有较高的遗传稳定性。     

Somatic hybrids between potato <Emphasis Type="Italic">Solanum tuberosum</Emphasis> and wild species <Emphasis Type="Italic">Solanum verneï</Emphasis> exhibit a recombination in the plastome

In the present survey, interspecific somatic protoplast fusion between a dihaploid cultivated potato Solanum L. tuberosum (BF15) and a wild species Solanum verneï (V3) has been performed using the PEG method. Five putative hybrids were first selected. Among them, only two were retained. DNA analysis, using flow cytometry, showed that the first hybrid (VB2) was dihaploid (2n=2x=24 chromosomes), whereas the second (VB1) was hexaploid (2n=6x=72 chromosomes). In the greenhouse, these putative hybrids showed that they were able of raising and producing large tubers but with a modified shape compared to parental ones. Furthermore, they harbored an intermediate leaf morphology compared to their parents. The hybrid nature of these plants was first confirmed according to their esterase and peroxidase isoenzyme patterns. The RAPD analysis of genomic DNA and microsatellite based amplification (I-SSR) showed that both clones VB1 and VB2 are asymmetric somatic hybrids. They seem to have eliminated the major part of V3 parental nucleus but not the totality. The analysis of chloroplast DNA suggests that both hybrid plastomes were the result of a recombination between parental ones.     

2004 SIVB Congress Symposium Proceedings “Thinking Outside the Cell”: Applications of Somatic Hybridization and Cybridization in Crop Improvement,with Citrus as a Model

Summary Although somatic hybridization techniques are being ignored by variety improvement programs for most commodities, their contribution to citrus variety improvement continnes to expland and with increasing complexity. Citrus is, one of the few commodities where somatic hybridization is reaching its predicted potential, as somatic hybrids are now possible from most desirable parental combinations. Somatic hybrid citrus plants have been produced from more than 250 parental combinations, including more than 130 at the CREC. The CREC hybrids include 34 from sexually compatible intergeneric combinations, 16 from sexually incompatible combinations, and 81 interspecific combinations. The objective of this report is to demonstrate the impact of somatic hybridization on citrus improvement programs, and to discuss its potential with other commodities. For citrus scion improvement, several applications are aimed at the development of improved seedless fresh fruit varieties, and these include symmetric somatic hybridization, haploid+diploid fusion, targeted cybridization to transfer cytoplasmic male sterility (mtCMS) from Satsuma mandarin, and triploidy via interploid crosses using somatic hybrid allotetrapoid breeding parents. For rootstock improvement symmetric somatic hybridization provides an opportunity to hybridize complementary rootstocks without breaking up successful gene combinations. Rootstock somatic hybridization is providing opportunities for improving disease and inseet resistance, soil adaptation, and tree size control. Wide somatic hybridization provides an opportunity for gene transfer from related species, including some that are sexually incompatible. Extensive field research on citrus somatic hybrid rootstocks combined with emerging molecular analyses of citrus has allowed for the development of additional strategies for rootstock improvement. These include rootstock breeding and selection, at the tetraploid level using somatic hybrid parents, and the resynthesis of important rootstocks at the tetraploid level via fusion of selected superior parents. Ongoing examples of each strategy will be provided, along with ideas for extending the technology to other commodities.