百脉根原生质体制备酶解液优化及再生壁纤维素含量分析

为了解百脉根细胞壁形成中纤维素含量的变化,以其成熟叶片为材料,筛选优化原生质体分离制备的酶解液组成和制备培养原生质体,并测定原生质体再生壁形成过程中的纤维素含量。结果表明,优化后的酶解液配比为:1.00%纤维素酶+1.00%果胶酶+0.50%半纤维素酶,该酶液制备的原生质体产量和活力分别为1.12×106个/g·FW和68.7%。纤维素含量随着细胞壁的形成而逐渐增加,原生质体在培养1 d、3 d、5 d和7 d时,其纤维素含量分别占再生壁干重的1.13%、4.17%、8.08%和12.79%。     

原生质体和细胞融合新技术在作物育种中的应用

在植物细胞的外部,有一层由纤维素组成的细胞壁。用酶把细胞壁溶化掉以后,这种裸露着的细胞就叫原生质体。原生质体从植物的根、茎、花瓣等器官上都可以取得,但一般是在展开叶上制取。这样得到的原生质体在适宜生育的培养基和环境条件下培养以后,细胞壁能够再生,继而开始发育,细胞分裂,最后分化成从前那样的植物体细胞。这种现象,已经被烟草,胡萝卜等若干植物的试验所证实。     

植物原生质体分离及培养研究进展

原生质体分离及再生体系现已被广泛用于生理、生化、遗传、分子生物学、基因组学、蛋白质组学、代谢组学的研究中,并能够为植物遗传转化提供良好的受体系统,从而达到优化遗传性状,丰富种质资源的目的等。园艺植物由于其观赏性强、倍性高的特点,具有很高的研究价值,但目前具有稳定的原生质体分离体系的园艺植物数量很少。本研究总结了园艺植物原生质体分离及培养时的各种影响因素：原生质体分离的植物材料;原生质体的酶解条件和纯化;原生质体的培养方法;原生质体的培养基成分,以及原生质体在各领域的应用,为建立更多植物原生质体分离及再生体系提供参考,为将原生质体系统更广泛的应用于多种植物中提供帮助。     

草菇原生质体制备、再生条件的响应面法优化及诱变效应

为了提高草菇原生质体在筛选耐低温突变筛选的诱变效率,建立基于草菇原生质体制备产量及再生率的响应面模型,优化草菇原生质体制备体系。本研究以草菇V23为材料,利用Design-Expert 8.0.6软件进行二次回归分析对参数菌龄、酶的浓度、酶解时间、酶解温度等进行了系统的优化研究。结果表明,溶壁酶的浓度为1.71%,酶解温度为31.4℃,菌丝体菌龄为3.42 d,酶解时间为3.21 h,草菇原生质体产量最高,达到2.57×107个/m L;用1.84%的溶壁酶在31.2℃条件下对菌龄为3.68 d的菌丝体酶解3.38 h,原生质体再生率最高,为14.1%。并利用紫外诱变和化学诱变技术对草菇原生质体进行了耐低温诱变初探,筛选获得5株抗冻能力提高的突变菌株。草菇V23原生质体产量和再生率的优化条件的建立,为提高草菇在遗传转化、基因组重排、基因编辑技术等后续分子遗传学操作实验打下了良好的基础。     

植物原生质体分离方法及其应用研究进展

植物原生质体是进行细胞工程遗传操作和植物育种的的良好材料。由于部分植物的不亲和障碍和特殊细胞信号传导途径研究等方面的需要,使得植物原生质体操作的相关研究变得独居优势。我们对近年来植物原生质体分离方法及应用等方面的研究进展进行了综述。并对植物原生质体研究未来的发展趋势进行了展望。指出在植物遗传操作及育种等方面,植物原生质体研究将变得不可或缺。研究影响植物原生质体再生的因素可能是未来植物原生质体研究的重要方向。     

原生质体技术在园艺植物育种中的应用

原生质体技术包括原生质体植株再生、原生质体融合技术等,在园艺植物育种应用工作中取得了一定的成果,原生质体再生植株的变异为园艺植物育种工作提供了大量材料;原生质体融合技术则是育种工作的新途径。二者都对育种工作的发展起到了非常重要的作用。     

果树原生质体培养研究进展

原生质体培养是进行植物育种和细胞各种生理生化特性研究的平台.本文对近些年在果树原生质体分离培养条件、方法和植株再生技术方面的研究进展进行了综述,分析了影响原生质体的分离、纯化、培养的有关因素,并根据有关文献讨论了今后果树原生质体研究的重点方向.     

烟草原生质体再生植株的影响因子

原生质体培养和植株再生技术是植物生物工程的基础，植物原生质体的来源、酶解的条件、培养基的组成、培养方法等对原生质体的培养有很大的影响。本文着重对影响烟草原生质体培养再生植株的相关因素进行了综合论述，并讨论了原生质体培养技术在植物育种上的应用潜力。     

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。     

谷子原生质体植株再生研究

原生质体作为植物遗传操作的理想受体,在创造新种质,培育新品种的研究中,具有特殊的应用价值.近10年来,禾谷类作物原生质体再生植株的研究已取得明显进展,但重复性、实用性尚不理想.我们通过愈伤组织改造程序获得了高频率原生质体分裂和植株再生的结果.113个谷子代表品种的幼穗与成熟种子作外植体被接种在含2,4-D 2,0mg/l、KT0.2     

旱稻原生质体培养和植株再生的研究

旱稻仿野生87-707品系具有多年生趋势，是我院研究多年生旱稻的一个材料。我们用它进行原生质体培养，想为研究多年生旱稻基因改良打下基础。现用该品系的成熟胚诱导的愈伤组织，在含有氨基酸丰富的AA培养基中进行细胞悬浮培养。取该细胞悬浮培养物游离的原生质体，用琼脂糖包埋于改良的KM8P培养基中，发生了持续分裂。经一系     

Plant regeneration from protoplasts isolated from primary calli using mature embryos of two Brazilian rice cultivars

A plant regeneration system from rice protoplasts using calli derived from mature embryos was established for the two Brazilian modern rice cultivars IAC-201 and IAC-165. After 30 to 40 days of in vitro culture it was possible to obtain on average 6 million protoplasts per gram of callus. Microscopic selection of embryogenic calli was a key step for protoplast isolation. The production of embryogenic calli increased when L-proline and casein hydrolysate were used in the callus induction medium. The Oc or IR52 nurse cell lines were essential for protoplast division. Different regeneration media were studied and 139 plants were regenerated which set seed. Some of the regenerated plants showed morphological variation such as the presence of awns in spite of the short time of the in vitro culture. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

棉属G染色体组野生棉种的体细胞胚胎发生与植株再生研究

通过培养基激素配比、碳源种类等培养条件的筛选，对2个G染色体组棉种（纳尔逊氏棉，Gossypium nelsonii Fryx.；澳洲棉，Gossypium australe F. Muell.）进行体细胞培养并获得了体细胞胚，其中纳尔逊氏棉获得了再生植株，澳洲棉获得了大量的胚状体。与D染色体组棉种克劳茨基棉（Gossypium klotzschianum Anderss）相比，G染色体组棉种再生时间长，且体细胞胚畸形严重、萌发困难，但通过培养条件的调控可以得到大量胚状体和少量再生植株。激素组合0.1 mg L^-1 KT+0.1 mg L^-1 2, 4-D诱导的愈伤组织较松软，分化潜力高；胚性愈伤组织的增殖使用0.2 mg L^-1 KT+0.5 mg L^-1 IBA的激素组合；组合0.25 mg L^-1 IBA＋0.3 mg L^-1 KT有利于体细胞胚胎发生，而含激素组合MSB5+0.15 mg L^-1 KT+0.5 mg L^-1 NAA的培养基适合体细胞胚胎的萌发和植株再生。此外，愈伤组织诱导宜用葡萄糖作为碳源，而在胚性愈伤组织的增殖及保存和胚状体的萌发过程中用麦芽糖作碳源的效果更佳。     

Research in Tissue Culture of Diploid Cotton Species

Diploid cotton species provide parental (ancestral) species for tetraploid cultivars and are the key germplasm resources for cotton genetic improvement. In addition, diploid cotton species supply excellent materials for mutant library construction and functional genomics. Diploid cotton species originated from Asia- Africa, America, Oceania regions with rich genetic diversity. The tissue culture research mainly involves regeneration of plant organs and cell culture, protoplast culture, somatic culture and fusion. The regenerated plants have been obtained in Gossypium klotzschianum, G. gossypioides, G. arboreum, G. davidsonii, G. raimondii, G. stocksii, G. nelsonii, G. bickii, etc. Among them, regenerated plants in shoot tip culture were only obtained in G. arboreum, G. bickii. Regenerated plantlets in protoplast culture were obtained in G. klotzschianum, G. davidsonii. Interspecific hybrids between G. hirsutum and diploid cotton of G. klotzschianum, G. stocksii and G. bickii were obtained, respectively. The tissue culture systems of diploid cotton were different from each other, and the strategy is similar to that in woody plants. This paper summarized the progresses in related researches and discussed the future research.     

玫瑰黄链霉菌Men-myco-93-63原生质体形成 和再生条件的初探

玫瑰黄链霉菌Men-myco-93-63在温室及大田试验中都对棉花黄萎病菌表现出很强的拮抗作用，是一株很有应用潜力的生防菌株。不论是菌丝体本身还是其代谢产物都对多种植物病原菌有很强的抑制作用。对该生防菌株进行深入研究，尤其是分子水平的研究尤为重要。笔者通过研究影响链霉菌原生质体形成的多种条件和因素，包括培养基的选择、培养时间、甘氨酸浓度、溶菌酶浓度、酶解时间等，最终成功的制备出了生防菌Men-myco-93-63的原生质体，初步确定了其形成条件：TSB培养基一级培养24h，用SGGP作为二级培养基培养菌丝体40h（补加甘氨酸2%），将菌丝体加入2mg/ml溶菌酶37℃水浴60min后即可得到原生质体，涂在再生培养基R5上再生培养，再生数可达4.8×106个/ml。为该生防菌原生质体的转化、基因表达等研究奠定了重要的基础。     

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.     

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.     

禾谷类作物原生质体培养研究进展

植物原生质体在品种改良、基因遗传转化、远缘杂交的应用研究以及生物学的基础理论研究中有着重要的研究价值。为了给禾谷类作物原生质体培养体系的建立提供理论依据,推动禾谷类作物品种遗传改良工作的开展,从原生质体制备、培养、再生、应用四个方面对禾谷类作物原生质体的培养研究进行了详细的阐述,分析了禾谷类作物原生质体制备和培养的主要影响因素,归纳了禾谷类作物原生质体再生的3种途径,对其在遗传改良和理论研究方面的应用进行了总结,并提出了禾谷类作物原生质体培养、遗传转化、细胞融合三个方面存在的问题和今后努力的方向。