海岛棉子叶原生质体游离和再生愈伤的获得

对１０个海岛棉品种的子叶进行了原生质体的游离和培养。培养３～５天的无菌苗子叶经１２～１６小时的酶解，获得了一定数量的原生质体。不同的酶液浓度组成对原生质体的产量有较大的影响。纯化的原生质体在修改的Ｋ３培养基上进行培养，３～５天后再生细胞开始分裂，２个星期后形成小细胞团，ｌ个月后获得了小愈伤组织。     

新牧4号紫花苜蓿原生质体游离和培养条件的研究

主要探讨影响苜蓿原生质体游离和培养的条件,为原生质体的培养体系的建立提供了依据。以‘新牧4号紫花苜蓿’的子叶、下胚轴和愈伤组织为材料,研究了酶液的pH值,酶解时间,酶液组合和不同培养方法对原生质体游离和培养效果的影响。结果表明：当酶液pH值为5.8时,有活力的原生质体的产量最高,同时细胞碎片少;子叶、下胚轴和愈伤组织适宜的酶解时间均为10 h;酶液组合为2%纤维素酶＋0.5%果胶酶＋0.3%或0.5%离析酶,游离出的有活力的原生质体产量较高;采用液体浅层培养和固液双层培养,均观察到原生质体发生分裂,但固液双层培养法更有利于‘新牧4号紫花苜蓿’原生质体的分裂和培养。     

‘新牧4号’紫花苜蓿原生质体游离和培养因素的研究

主要探讨影响苜蓿原生质体游离和培养的因素,为原生质体培养体系的建立提供依据。以‘新牧4号’紫花苜蓿的子叶、下胚轴和愈伤组织为材料,研究了酶液的p H值,酶解时间,酶液组合和不同培养方法对原生质体游离和培养效果的影响。结果表明:当酶液p H 5.8时,有活力的原生质体的产量最高,同时细胞碎片少;子叶、下胚轴和愈伤组织适宜的酶解时间均为10 h;酶液组合为2%纤维素酶+0.5%果胶酶+0.3%或0.5%离析酶,游离出的有活力的原生质体产量较高;采用液体浅层培养和固液双层培养,均观察到原生质体发生分裂,但固液双层培养法更有利于‘新牧4号’紫花苜蓿原生质体的分裂和培养。     

洗液对拟南芥叶原生质体分离的影响

原生质体分离过程中洗液对原生质体的纯化和保存起着重要的作用。本试验通过比较30 h内WI、W5、MMg三种洗液中原生质体数量与活力的变化以及分析不同浓度CaCl2处理WI、W5、MMg后对原生质体的影响,选择出获得最多活性原生质体数量的洗液。结果表明,WI在维持质膜稳定性上较有优势,而W5则更有利于保持原生质体活力且30 h后得到的活性原生质体最多,较适宜在短期内保存原生质体;CaCl2处理洗液对原生质体活力无显著影响,且其对原生质体数量的影响取决于CaCl2处理浓度及洗液种类,MMg洗液在补充0~50 mM CaCl2后原生质体数量呈上升趋势。     

决明原生质体的分离与培养研究

用决明（ＣａｓｉａｏｂｔｕｓｉｆｏｌｉａＬ．）子叶和下胚轴为材料,游离和培养原生质体。结果表明,１５日龄无菌苗的子叶和下胚轴比较适于游离原生质体,每１ｇ材料的原生质体产量高达１．６×１０４个;ＰｅｃｔｏｌｙａｓｅＹ－２３是分离原生质体所必需的;用含２,４－Ｄ０．４ｍｇ／Ｌ（以下单位同）,ＮＡＡ１．０与ＫＴ０．１的ＫＭ８Ｐ漂浮培养利于原生质体的分裂。培养４ｄ后,原生质体开始分裂,１５ｄ后其植板率约为１９．２％,３０ｄ形成了细胞团或小愈伤组织。增殖愈伤组织在分化培养基上培养,可分化出芽。芽在生根培养基上培养１４ｄ生根,从而再生决明小植株。     

小麦愈伤组织诱导及原生质体的分离与纯化

摘 要：以普通小麦的幼穗和幼胚为外植体进行愈伤组织诱导,研究了外植体及低温预处理等因素对小麦愈伤组织诱导的影响;并由小麦幼胚形成的愈伤组织进行了原生质体分离和纯化实验,研究了酶浓度及配比、酶解时间、蔗糖浓度等因素对原生质体分离和纯化效果的影响。结果表明：小麦幼穗离体培养时,以1.0-1.5cm长度的幼穗有利于愈伤组织的诱导;小麦幼穗和幼胚外植体采后低温储藏时不易超过3d,时间太长会影响愈伤组织诱导;2.0%纤维素酶＋0.5％果胶酶+0.6M甘露醇分离原生质体较好,最佳酶解时间为4-6h,原生质体产量和活力较高。     

不同激素条件下大豆原生质体培养和植株再生

以栽培大豆(Glycine max(L.) Merr.)南农86-21、南农86-4、南农73-935为材料，用K8/K8p基本培养基比较了不同原生质体密度和不同激素种类、水平对未成熟子叶原生质体培养的影响，发现它们的差异表现在植板率、产生愈伤组织的速度和频率、愈伤组织的颜色和结构等几方面。对低激素来源南农73-935的愈伤组织的速度和频率、愈伤组     

茶树叶片和胚根原生质体的分离及PEG诱导融合

植物原生质体是细胞培养和体细胞融合等细胞水平研究及植物遗传育种的重要材料。本研究用福鼎大白茶茶树的幼嫩叶片及胚根, 分析了原生质体分离过程中的材料、酶解液组成及酶解时间、纯化方法等影响因子, 建立了最佳原生质体分离体系, 为茶树体细胞杂交等细胞水平的研究提供了高效获取大量高活力原生质体的方法。结果表明, 23°C恒温黑暗或遮光培养的茶树实生苗的5周叶龄以内的幼嫩叶片是茶树原生质体分离的最佳材料, 其次是茶树种子萌发后的幼嫩胚根; 而以茶园健康生长的5周叶龄以内的幼嫩叶片为材料时, 只能获得混有大量细胞碎片的少量具有活力的原生质体。以茶树幼嫩叶片为分离材料的酶解液组成为1.5%纤维素酶+0.1%离析酶+0.5%果胶酶+0.4 mol L^-1甘露醇+20 mmol L^-1 MES; 以茶树幼嫩胚根为分离材料的酶解液组成为1.5%纤维素酶+0.3%离析酶+0.5%果胶酶+0.4 mol L^-1甘露醇+20 mmol L^-1 MES。分离茶树幼嫩叶和幼嫩胚根原生质体时, 宜采用低速(分别为55 r min^-1和50 r min^-1)恒温(23°C)摇床振荡酶解培养, 时间分别为7 h和8 h; 最适宜采用15×g的转速, 离心4 min可纯化获得高产量和活力的原生质体。用40% PEG-6000诱导20 min后可使茶树原生质体融合, 融合率达10%。     

洋葱愈伤原生质体分离和纯化研究

建立高效原生质体分离和植株再生体系是进行体细胞杂交的基础性工作。以洋葱多次继代的愈伤组织为研究试材,对原生质体分离纯化技术进行研究。结果表明：2.0%纤维素酶Onozuka R-10+0.1%果胶酶Y-23+0.5%离析酶 R-10+0.60 mol/L甘露醇+CPW+0.1% MES的酶液,酶解6 h,洋葱原生质体分离效果最好。纯化时,适当降低离心力的短时间离心有利于原生质体纯化过程中产量和活力的保持,以600 r/min离心5 min效果为好。     

玉米胚乳细胞原生质体的分离与流式纯化

玉米胚乳是籽粒淀粉形成的主要场所,胚乳细胞的发育对籽粒的器官建成具有重要意义。分离胚乳细胞原生质体,能为胚乳培养、转录组分析等后续研究提供均一实验材料。本文在借鉴前人研究的基础上,通过调整酶的搭配种类、浓度以及质膜稳定剂、渗透压稳定剂的使用,优化了一套有效的玉米籽粒胚乳原生质体分离方法,并进一步运用流式分选技术纯化原生质体。结果表明,以1%纤维素酶、0.5％离析酶、0.5%半纤维素酶,在渗透压调节剂0.7~0.8 mol L^-1和质膜稳定剂0.8mol L^-1的MS培养液内,30℃消解4 h,能够获得大量完整的粗制原生质体,二乙酸荧光素(FDA)染色表明纯化的原生质体仍能保持90%以上的生活力。流式分选术可将有活力原生质体从粗制原生质体悬浮液中富集、纯化出来。对一些原生质体分离和流式分选时的技术参数和影响因素进行了讨论。     

A comparison of somaclonal variation in rice plants derived and not derived from protoplasts

To clarify the effect of the type of in vitro culture on the generation of somaclonal variation, protoplast-derived rice plants were compared with rice plants derived from suspension culture or primary calli (not derived from protoplasts). Regenerated plants showing polyploid-like phenotypes appeared at a higher frequency (33–70%) in plants derived from protoplasts than in those not derived from protoplasts (3–6%). In the first progeny (R₁) generation of all regenerated plants, 120 of 368 lines (33%) segregated plants with mutated characters such as albino, dwarf and sterile. In quantitative traits, 62 (21%) and 144 (50%) of 290 Rj lines showed shorter culm and lower seed-fertility, respectively, compared with the control line derived from the selfed seeds of the original cultivar. The frequency of the mutant-possessing R₁ lines among lines derived from protoplasts was not significantly different from those not derived from protoplasts. These results indicate that isolation and culture of protoplasts does not enhance genetic changes other than cytogenetical changes.     

橡胶树原生质体的分离及红色荧光蛋白的瞬时表达研究

目的：分离巴西橡胶树松散愈伤组织原生质体,建立目标基因在橡胶原生质体的瞬时表达系统。方法：以巴西橡胶树松散愈伤组织为材料,酶解分离原生质体,通过电击介导的转化方法,将编码红色荧光蛋白（RFP）的植物表达载体转入原生质体中,用激光扫描共聚焦显微镜检测原生质体中RFP的表达情况。结果：分离出大量高纯度的橡胶原生质体,成功获得转基因原生质体细胞,RFP在原生质体中得到成功表达。初步建立了橡胶原生质体瞬时表达系统。     

酸枣幼根原生质体的制备

以生长6～9 d的酸枣种子幼根为试验材料,0.7 mol·L-1甘露醇作为渗透压稳定剂、pH值5.5～6.0、28℃恒温水浴条件下,以光学显微镜观察的水解总时间、初始产生原生质体的时间、原生质体产量及细胞碎片量为指标,研究通过酶水解来制备酸枣幼根原生质体的方法.结果表明,在含1％果胶酶+4％纤维素酶R-10+3％纤维素...     

Protoplast Fusion in the Genus Medicago and Isoenzyme Analysis of Parental and Somatic Hybrid Cell Lines

Leaf mesophyll protoplasts of Medicago arborea (2n = 32) were electrofused with cell suspension and/or callus protoplasts of Medicago sativa. Heterokaryons were identified in agarose beds by their dual fluorescein isothiocianate—chlorophyll fluorescence and their coordinates were recorded. Hybrid minicalli were manually picked up and grown first in nurse culture and then in callus induction medium. Hybrid nature of the selected calli was confirmed by isoenzyme analyses. In order to verify whether morphogenesis of somatic hybrid calli was affected by cell incompatibility, mesophyll and cell suspension protoplasts, derived from the same plant of M. sativa with high embryogenic capacity, were fused. Only callus tissues derived from mesophyll protoplasts retained the highly embryogenic character of the M. sativa genotype, while hybrid cell lines were non-morphogenic and showed isoenzyme patterns similar to tissues derived from cell suspension protoplasts. The achievement of somatic hybrid plants in the genus Medicago is discussed.     

Isolation and culture of grape vine cv. Chardonnay leaf protoplasts

Summary Experimental conditions were established that resulted in high yields and good viability of the protoplasts obtained from leaves of Vitis vinifera, cv. Chardonnay regenerated in vitro by somatic embryogenesis. The effect of factors of the culture medium and various environmental conditions upon the frequency of cell division has been examined, and a method of culture is described by which protoplasts were induced to begin division. Most protoplasts obtained in this way regenerated cell walls within the first few days and cell division occurred after 10 days of culture in a liquid medium. Some cells have divided two or even three times. Nevertheless, the cells did not continue dividing beyond this stage.     

Production of somatic hybrid plants between Iris ensata Thunb. and I. germanica

Wide hybridization that cannot be attained through conventional sexual crosses, can now be approached by somatic hybridization. Protoplasts of I. ensata and I. germanica were fused by electrofusion. For the selection of somatic hybrids, protoplasts of I. ensata which did not form colonies in protoplast culture and protoplasts of I. germanica which had regeneration ability for only albino shoot were used in symmetric fusion. On the other hand, the protoplasts of I. ensata and I. germanica protoplasts which were inactivated by iodoacetamide (IOA) treatment were used in asymmetric fusion. Five-six months after cell fusion, green plants were obtained in the symmetric and asymmetric fusion. In the random amplified polymorphic DNA (RAPD) analysis, the green plants had bands specific to both parental species. Therefore, these plants were somatic hybrids between I. ensata and I. germanica. This revised version was published online in July 2006 with corrections to the Cover Date.     

小青杨叶肉原生质体分离条件的研究

为获得大量、有活力的原生质体,建立起高效的小青杨（Populus pseudo-simonii Kitag.）原生体分离体系,本研究以小青杨无菌苗叶片为材料进行原生质体分离条件的研究。结果表明：酶的种类和浓度、渗透压稳定剂浓度、酶解时间、叶龄是影响原生质体分离的重要因素;将叶龄35天的无菌苗第2~3片叶片置于酶液组成为CPW+3.0% 纤维素酶R-10+0.5% 离析酶 R-10+0.1% 果胶酶Y-23+0.6 mol/L甘露醇+0.6 g/L MES+1 g/L BSA中酶解8 h,原生质体产量和活力分别为2.44×107个/g和78.7%。通过该分离体系稳定的获得了高产量、高活力的原生质体,可以满足进一步的原生质体培养等技术的要求。     

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.     

叶片的生理状态及添加物对大麦叶肉原生质体培养的影响

作为原生质体源的叶片的生理状态及培养基中的添加物对大麦叶肉原生质体培养均有明显的影响。试验中观察到叶片中、上段的原生质体比基部的具较强的感应力。叶片经黑暗预处理后，其原生质体培养时呈较好的稳定状态，并观察到添加１．０，２．０ｍｍｏｌ／Ｌ的抗坏血酸能提高原生质体的活力。在添加０．５ｍｇ／Ｌ２，４－Ｄ、１．０ｍｇ／ＬＮＡＡ及０．５ｍｇ／ＬＺＴ的ＲＭＳ培养基中，经微弱光照（１５０ｘ）诱导了细胞持续分裂并