马铃薯无菌苗叶肉原生质体再生植株

商用马铃薯叶肉原生质体,在不同的液体培养基中浅层培养,耳生细胞分裂,并获得愈伤组织。将愈伤组织转到固体培养基上诱导分化,已从克新四号和68—62两个马铃薯品种的原生质体得到再生的完整植株。再生细胞的分裂频率受原生质体培养密度的影响。细胞团的生氏对液体培养基中的蔗糖浓度敏感。不同的原生质体培养基对愈伤组织的分化频率的影响非常显著。在分比培养基中加入3％的甘露醇,能提高愈伤组织的分化频率,并缩短分化期。     

基因组对芸苔属作物原生质体培养及植株再生的影响

本文以包心菜、芜菁油菜、浙油６０１的无菌苗叶肉原生质体为材料,经不同液体培养基浅层培养,细胞分裂并形成愈伤组织。愈伤组织经增殖后,转到分化培养基上诱导分化,均获得了再生植株。本文着重研究了植物基因组对原生质体分裂频率及植株再生的影响。研究结果表明：（１）植物基因组对原生质体分裂频率的影响随原生质体培养基的不同而异;（２）植物基因组对原生质体再生植株影响显著,芜菁油菜的Ａ基因组不利于原生质体再生植株     

黄花烟草(Nicotiana rustica L.)叶肉原生质体培养花芽形成的观察

黄花烟草(Nicotiana rustica L.)叶肉原生质体于 NT培养基上形成愈伤组织。愈伤组织于MSBI分化培养基上形成绿色突起。将绿色突起转移到MS培养基上于10小时光照下形成花芽。一部分在试管中从愈伤组织直接形成不带叶片的花芽并开花结籽,其他都形成有叶片的开花植株。对此种现象进行讨论。     

分离植物原生质体的纤维素酶的制备及性质

EA_3-867纤维素酶是由绿色木霉变异株EA_3-867,用稻草粉发酵提取,经过饱和硫酸铵沉淀,分子筛脱盐,冰冻干燥制成的。酶制剂在水中溶解度高,酶活力较稳定,它含有纤维素酶(C_1,C_x)、果胶酶、半纤维素酶等组分,是一种比较理想的分离植物原生质体的复合酶。我们用EA_3-867酶制剂已从20多种植物材料中分离出大量完整的原生质体,并把烟草的叶肉组织或愈伤组织的原生质体培养分化成植株。EA_3-867纤维素酶制剂的成功制备为我国进一步开展植物原生质体和体细胞杂交等研究提供了有利条件。     

LpDH活性作为一种遗传标记在体细胞融合中的转移

用具有LpDH活性,但不能分化植株的烟草冠瘿瘤B 6 S 3为亲本的原生质体,和与之有相反特点的正常烟草xanthi品科叶肉原生质体间融合,由融合处理的原生质体形成了愈伤组织并再生了植株。对56株叶片的LpDH活性电脉分析表明,有75％植株含有不同程度的LpDH活性,即能合成章鱼碱。随植株发育成长,一些植株的LpDH活性有减弱或丢失现象。但叶片形态具有双亲部分特征,表明烟草冠瘿瘤的LpDHT活性标记可通过原生质体融合转移 到烟草xanthi细胞中。     

兰属、兜兰属、石斛属植物叶片的扫描电镜观察

对兰科植物的兰属、兜兰属及石斛属１６个种折叶片及其横断面进行了扫描电镜的观察。兰属各种叶片上表皮细胞均为矩形,上表皮细胞表面具小乳突或不明显突起。石斛属及兜兰属的各个种上下表皮细胞均为多边形,但石斛属表皮细胞表面无坦无纹饰,而兜兰属花叶类上表皮细胞表面明显呈乳突状,绿叶类呈龟背状隆起。兰属及石斛属叶片叶肉组织没有栅栏组织及海绵组织的分化,而兜兰属的绿叶类叶肉不分化;花叶类叶肉有分化。     

大麦叶肉原生质体培养中核酸酶活性的研究

在利用原生质体技术来改良谷类作物的工作中,掌握其分离的原生质体再生完整植株的技术是非常重要的一环。由一些禾谷类作物愈伤组织来源的原生质体,已能连续产生细胞分裂,并进而形成愈伤组织或小植株;然而由叶肉来源的原生质体,在培     

小麦叶肉原生质体在马铃薯简化培养基上的分裂

近年来,在小麦、水稻花药培养的研究中, 使用了马铃薯简化培养基,已取得很大成 效^[1-3]。我们在大麦、小麦叶肉原生质体培养的 研究中,为了寻找能促使其再生细胞进行连续 的细胞分裂,除了采用各种合成培养基外,也使 用了马铃薯培养基。本文简要报道小麦叶肉原 生质体在马铃薯简化培养基中能有规则地进行 有限的几次细胞分裂的结果     

烟草叶肉细胞原生质体培养成植株的研究

应用自制的纤维素酶从三个烟草品种的叶肉组织获得大量完整的有活力的原生质体,阐述了影响原生质体活力的某些因素。详细叙述了烟草原生质体的培养过程和方法,由于培养技术的改进,试验所用三个烟草品种的叶肉原生质体已经生长分裂形成愈伤组织,并分化成再生植株。此外,观察到“金星”的双倍体与单倍体叶肉原生质体在同一培养基上生长的差异。并对自制纤维素酶与日本纤维素酶(Onozuka)进行了比较。讨论了烟草原生质体从体积增大到细胞分裂形成愈伤组织过程中必须移贴的原因。     

植物原生质体质膜表面电荷性质的研究——Ⅰ.pH及某些酶制剂对元麦叶肉原生质体电泳率的影响

用细胞电泳方法研究了pH 及胰蛋白酶,脂—蛋白脂酶、碱性磷酸酶等酶制剂对元麦叶肉原生质体电泳率的影响。结果表明,新鲜制备的元麦叶肉原生质体表面具负电性,但在低pH(<2.8)条件下其表面具正电荷;当pH>2.8时,其表面电性才由具正电性转变为具负电性。上述三种酶制剂处理原生质体后,均能改变原生质体的电泳率,随酶浓度增高均使原生质体的电泳速度减慢。文章并对所获得的结果在阐明质膜表面电荷的化学特性上的意义进行了讨论。     

Culture of Cotyledon and Hypocotyl Protoplasts from True Potato Seedlings in Solanum tuberosum L.

Culture of protoplast using cotyledon and hypocotyl as the donor tissue from true potato seedlings (TPSs) of 3 breeding lines (DTO-33, ND 860-2 and BN 9815-3) of Solanum tuberosum L. was studied. The cotyledons and hypocotyls of TPSs just extended were excised and digested in an enzyme solution containing 1 % cellulase and 0. 5 % macerozyme for 17—20 h after vacuum infiltration of the tissue in the solution. The protoplasts were cultured in an improved liquid medium and transferred onto solid media for callus culture and shoot regeneration. Some factors affecting the efficiency of cotyledon and hypocotyl protoplast culture were studied. The results showed that using the cotyledons and hypocotyls as donor tissues for protoplast isolation and culture in potato, the division frequency of protoplast derived cells was significantly higher than that using the leaves and shoot-tips of the test-tube plantlets: the yield and quality of the protoplast from TPSs cultured under continuous high light intensity (3000 Ix) were much higher than the TPSs cultured under low light intensity (1000 Ix), and no intact protoplast was ever obtained from the TPSs cultured in continuous dark condition. Vacuum infiltration of the cotyledon and hypocotyl segments in enzyme solution before digestion increased protoplast yield. The yield of protoplasts from hypocotyl tissue was significantly higher than from the cotyledon, but there was no significant difference in quality between the protoplast derived from the two tissues. The significance, advantages and shortcomings of using the cotyledons and hypocotyls as the donor tissues for isolation and culture of potato protoplasts are dicussed.     

Iron deficiency decreases the Fe(III)-chelate reducing activity of leaf protoplasts

The ferric-chelate reductase (FC-R) activity of mesophyll protoplasts isolated from Fe-sufficient (control) and Fe-deficient sugar beet (Beta vulgaris L.) leaves has been characterized. Measurements were made in an ionic environment similar to that in the apoplastic space of the sugar beet mesophyll cells. The FC-R activity of Fe-sufficient and Fe-deficient protoplasts was dependent on light. Fe deficiency decreased markedly the FC-R activity per protoplast surface unit. The optimal pH for the activity of the FC-R in mesophyll protoplasts was in the range 5.5 to 6.0, typical of the apoplastic space. Beyond pH 6.0, the activity of the FC-R in mesophyll protoplasts decreased markedly in both Fe-sufficient and Fe-deficient protoplasts. These data suggest that both the intrinsic decrease in FC-R activity per protoplast surface and a possible shift in the pH of the apoplastic space could lead to the accumulation of physiologically inactive Fe pools in chlorotic leaves.     

Plant regeneration from cell suspension-derived protoplasts of Nicotiana africana

Plants have been regenerated from Nicotiana africana Merxm. protoplasts isolated from cell suspensions. Two different sequences of media were assayed, one usually used to regenerate tobacco mesophyll protoplasts (K3,RMO) the other previously recommended for potato mesophyll protoplast regeneration (W-S-S, ST-1, ST-2, S-3). Only the media for potato protoplasts were efficient for African tobacco plant regeneration. The regeneration efficiency was 6.3 plants per 1000 plated protoplasts. This revised version was published online in June 2006 with corrections to the Cover Date.     

植物单盐毒害和离子拮抗机理研究Ⅰ.单盐和混合盐对植物原生质体膜某些特性、超氧物歧化酶和过氧化氢酶活性的影响

单盐(KCl, CaCl_2或MgCl_2)和混合盐(KC_1+CaCl_2或KCl+MgCl_2)对植物原生质体完整率、存活率和膜透性等均有明显影响。K~+、Ca~(2+)或Mg~(2+)等单种阳离子明显降低原生质体膜完整率和存活率而增加其物质渗漏量,其中以单价阳离子K~+的影响为甚。上述单种阳离子还明显降低小麦幼叶超氧物歧化酶(SOD)和过氧化氢酶活性。只有由单价和二价阳离子组成的平衡混合盐才能使原生质体维持较高的完整率、存活率和较正常的膜透性.并能使细胞维持较高的SOD和过氧化氢酶活性。 认为单盐毒害机理可能是首先引起细胞膜发生不正常的膜相变或细胞累积较多的有害氧自由基,引起膜脂发生过氧化或脱酯化而破坏膜结构。在离子平衡混合盐作用下,膜系才能维持正常液晶相,具有较高活性的SOD和过氧化氢酶等生物保护性酶系是离子拮抗作用之原因。     

Ribosomal RNA metabolism in cucumber leaf mesophyll protoplasts.

Aspects of the metabolism of RNA have been studied in enzymatically isolated protoplasts from cotyledon and first leaf mesophyll tissue of two cultivars of cucumber. The first leaf mesophyll protoplasts incorporated (3H)-uridine into ribosomal RNA at a constant rate for up to 25 hr in a simple salts medium and for up to 45 hr in a growth medium. Pulse-chase labelling experiments on such preparations showed a rapid dilution of the intracellular (3H)-uridine pool(s) and a high metabolic rate in the cells in one cultivar but not in another. Gel electrophoretic analysis of the RNA from both cotyledon and first leaf protoplasts showed that both protoplast types incorporated either (14C)- or (3H)-uridine into ribosomal RNA species. Incorporation of (3H)-uridine into chloroplasts RNA was minimal in cotyledon protoplasts, but significant in leaf protoplasts. Greater incorporation into the chloroplast RNA species could be achieved by longer pulses. Synthesis of all of the ribosomal RNA species was sensitive to actinomycin D at 10 and 25 mug/ml concentrations in all protoplasts tested.     

几种植物组织及其原生质体过氧化物酶同工酶的比较研究

对马尾松幼苗子叶和胚轴、甘蔗、小麦、烟草、黄花菜等幼叶及其原生质体的过氧化物酶同工酶(POI)分别作比较研究。观察到凡经纤维素酶处理的各植物组织POI酶带数均多于未经纤维素酶处理的组织的酶带数;除个别例外,后者一般又比无壁原生质体的酶带数多。此种差异随植株生长年龄而增大,表明植物组织内大部分POI主要存于质外体中。     

Regeneration of protoplasts of dihaploid potato plants bleached by a herbicide (SAN 6706)

Summary Bleaching of dihaploid potato shoots by application of the herbicide SAN 6706 did not affect the ability for cell division of subsequently isolated mesophyll protoplasts. The development of green calluses from bleached protoplasts shows that bleaching is reversable. The positive effects of SAN 6706 on cell division activity in protoplasts are discussed and the application of the bleaching method in experiments to select somatic hybrids after protoplast fusion.     

Effect of cellulase fractionation on the viability of cultured barley (Hordeum vulgare) protoplasts

The age of the stock plants was important for the barley ( Hordeum vulgare L. cv. Perth) protoplast viability. Light conditions under which the stock plants were grown also affected the viability of the protoplasts. Greenhouse-grown plants yielded much higher number of protoplasts than dark-grown plants, but protoplast viability was better when protoplasts were isolated from etiolated plants. Light supplied during protoplast culture affected protoplast viability within the first 24 h of culture. Cellulase R-10 (Onozuka) was better than Cellulysin (Calbiochem) and Cellulase ⁺ Macerozyme R-10 (Onozuka) for barley mesophyll protoplast isolation. Cellulase R-10 (Onozuka) was fractionated on a G-75 Sephadex column. The eluted fractions were tested for their ability to release barley mesophyll protoplasts and for their toxicity towards the protoplasts. Only a small part of the Cellulase R-10 was necessary for protoplast isolation from barley leaves. When the fractionated cellulase was analysed by isoelectric focusing, this part of the cellolase appeared as a single band.     

紫云英叶片及叶肉原生质体的培养

本工作研究了豆科植物紫云英的叶片及叶肉原生质体的培养。叶片培养实验表明,诱导愈伤组织的最适培养基为MS加1.0-2.0毫克/升2,4-D和0.25毫克/升KT;诱导根分化需加1.0—5.0毫克/升NAA和0.5毫克/升BA;而苗分化则以0—0.5毫克/升IAA和0.5毫克/升BA为好。高浓度的NAA有利于根分化而抑制茎芽形成;高浓度的IAA对根和芽分化都有抑制作用。叶肉原生质体分离和培养试验表明,紫云英叶肉原生质体的释放及其培养活力受叶龄、植株生理状态和酶浓度的影响。叶肉原生质体在改良的KM8P培养基中能分裂。用改良KM8细胞培养基定期稀释,可使分裂持续进行而得到细胞团。BA和2,4-D为诱导紫云英叶肉原生质体分裂所必需。其最佳组合激素为BA 0.21毫克/升和2,4-D 1.13毫克/升。葡萄糖作为渗透压稳定剂时,其浓度明显影响原生质体的存活率。弱光条件下培养比黑暗培养有利于叶肉原生质体分裂。由叶肉原生质体形成的愈伤组织能形成瘤状结构和根。     

Culture and fusion of pollen protoplasts of Brassica oleracea L. var. italica with haploid mesophyll protoplasts of B. rapa L. ssp. pekinensis

Hybrid callus was formed from the successful protoplast fusion between pollen protoplasts of Brassica oleracea var. italica and haploid mesophyll protoplasts of Brassica rapa. The pollen protoplast isolation frequency in broccoli was highly related to the ratio of trinucleate pollens in the male gametophyte population. Large quantities of pollen protoplasts with high vigor could be isolated, and the isolation frequency reached up to 90% in 6.0-7.0 mm long flower buds with about 94.7% trinucleate-stage pollens. Pollen protoplasts could be collected and purified by discontinuous gradient centrifugation. In 1% Na-alginate embedding culture, cell divisions were observed but no further development was found. The haploid mesophyll protoplasts were isolated from in vitro haploid plants of B. rapa. Results strongly showed the variability in culturability of mesophyll protoplasts from different haploid lines. Both pollen protoplasts and haploid mesophyll protoplasts retained a stable round shape in the designed prefusion solution with an osmotic pressure of 0.74 osmol/kg. Polyethylene glycol was used for the protoplast fusion, and 40% polyethylene glycol 4000 enabled the highest fusion frequency of about 20%. Some postfusion protoplasts showed cell divisions up to callus proliferation. Calli were screened by random amplified polymorphic DNA analysis for their hybrid character. Results revealed the existence of the hybrid calli. Some of the hybrid calli grew well with green color and shoot primordia. According to our knowledge, this is the first report about a hybrid formation between two haploid protoplasts. Potential comprehensive applications, as well as problems of this technique, are discussed.