应用ImageMaster^TM图象分析系统计算原生质体活力

植物原生质体的活力高低是代表原生质体样品质量的重要指标之一,它也反应了用于游离原生质体的原始植物材料本身的质量高低。原生质体活力高证明所用的原始材料质量高,原生质体活力低说明这种材料质量低。因此估算样品中原生质体的活力,是原生质体培养、原生质体融合、基因转化等涉及原生质体的各项生物工程中的常规工作。可是原生质体的计数却是十分费力。为了克服这一困难,我们试图借用Pharmacia公司的ImageMaster~(TM)图象分析系统来完成这一过程。     

花菸草(Nicotiana alata)叶肉原生质体生成植株

普通菸草(Nicotiana tabacum L.)的原生质体培养成植株的研究已有不少报道。另外一种花菸草(Nicotiana alata Link et Otto)染色体数目较少(2n=18),植株形态与其他种有显著的差异。如果它的原生质体培养具有分裂快、分化好、能生成植株的特点,那将是研究体细胞杂交的一个好材料。目前,对它的原生质体培养的研究仅在个别报道中提到过。本文应用我们已建立的酶分离及通底离心管漂洗纯化原生质体的方法,制备了原生质体,并进行了培养试验,获得了较为满意的结果。     

人参原生质体培养再生愈伤组织

人参原生质体培养未见报道成功。Harn(1974)曾用人参幼叶,幼根和上胚轴进行原生质体分离,但得到的数量很少,无法进行培养。本实验以人参培养细胞为材料,通过培养再生了愈伤组织。     

一种溶高等担子菌细胞壁酶的制备

我们试图以高等担子菌(主要是食用菌)为材料进行原生质体融合,以期获得新型杂种菌株。我们先后采用过制备植物原生质体的纤维素酶(上海东风生化试剂厂生产和从日本进口)、制备酵母菌原生质体的蜗牛酶(来源于中国科学院生物物理研究所)、和制备细菌原生质体的溶菌酶(上海禽蛋二厂生产)单独或混合酶解担子菌菌丝,均难以获得担子菌原生质体。其     

CAT和GUS基因在水稻玉米等作物原生质体中的瞬间表达

使用电击法和 PEG 法将 CAT 和 GUS 基因分别与 CaMV 35S 启动子构成的两种嵌合基因,转移到水稻、玉米、烟草、胡萝卜和香石竹(Dianthus caryophyllus)等品种的原生质体中,均得到了瞬间表达。在电击法中,大多数转化后玉米、烟草和胡萝卜原生质体表现出明显的基因活性。转化效率与所用仪器的电击参数有关。PEG 转化中 PEG 处理时间较国外报道稍有缩短,但转化后的水稻、烟草、胡萝卜和香石竹样品中均得到了阳性结果。     

转正义和反义abp基因烟草原生质体质膜超极化特性及其培养

以转生长素结合蛋白 (ABP)基因烟草为材料 ,通过原生质体培养和测定原生质体的跨膜电位 ,研究了abp基因转移与生长素诱导的烟草原生质体质膜超极化特性、原生质体培养过程中对生长素的敏感性三者之间的关系 .结果表明 :转正义abp基因烟草达到最大超极化的NAA浓度小于对照 ,原生质体培养中对生长素的敏感性增加 ;而转反义abp基因烟草达到最大超极化的NAA浓度则大于对照 ,原生质体培养中对生长素的敏感性降低 .这为ABP在内质网上合成后需运输到细胞膜上发挥作用提供了证据     

野生种菸草无菌株的原生质体游离培养技术的改进(英文)

以野生种菸草Nicotiana sylvestris(2n=24)和N. plumbaginifolia(2n=20)的无菌小植株为实验材料,比较了影响原生质体植板率的各种因素,改进了叶肉原生质体的游离和培养,使这两种野生菸草的原生质体植板率分别为40％和70％。使这两种菸草的原生质体培养系统可以用于细胞放射生物学的领域,例如研究突变效应。 对游离原生质体的方法作了改进,其主要优点是不需要撕表皮和离心。先在叶片的上表面轻轻地铺上金刚砂粉末,用刷子刷10—20秒,然后洗去粉末,在质壁分离剂中处理几小时,再在酶溶液里保温培养过夜(16小时),小心地吸去酶液后加入由0.5克分子浓度蔗糖和T_2无机盐组成的溶液,得到原生质体的悬浮液。再将悬浮液移入试管,在上面轻轻地加入培养基,沉淀1—2小时后从溶液界面或上层培养液中分离出原生质体,离心提纯并进行培养。 比较了供试材料的各种因素对原生质体植板率的影响。发现同种供试材料的基因型不同,植板率也有明显差异,因此实验前要筛选好的基因型;籽苗的嫩叶最适合原生质体的游离和培养,挑选2—3厘米长的幼叶为宜。因为这时细胞核有95％左右处于G_1期,并且不存在内多倍性;无菌小植株的培养器皿不要密封;供试材料的培养基中蔗糖浓度尽可能降低,5克/升可能是较好的;供试材料的培养以3,000 lux的低光     

影响决明无菌苗子叶原生质体分离和培养因素的研究

以决明(Cassia obtusi folia)无菌苗子叶为材料,对酶组合、无菌苗日龄,植物激素组合和培养方法对其原生质体的分离和培养的影响进行了研究。结果表明:用3％的纤维素酶和0.2％Pectinase Y-23的酶组合处理决明无菌苗子叶块8小时可以高效分离出有活力的原生质体;约14日龄的决明无菌苗子叶比较适合于原生质体的分离;适当浓度的2,4-D 有利于原生质体的分离。促进原生质体分裂的理想的植物激素组合为0.4 mg/L 2,4-D,1.0 mg/L NAA and 0.1 mg/L KT;漂浮培养法最有利于原生质体的分裂和发育。找出了适合于决明无菌苗子叶原生质体的分离和培养的酶组合、植物激索组合、有效培养方法和决明无菌苗子叶日龄。这为有效地从决明无菌苗子叶原生质体再生植株奠定了基础。     

二倍体马铃薯叶肉细胞原生质体培养的研究

以马铃薯抗青枯病二倍体材料ED13和CE171、炸片颜色好的二倍体材料HS66以及优良性状双单体材料DH401和DH405为供体材料,对马铃薯叶肉原生质体培养进行研究。叶片悬浮黑暗预处理和试管苗黑暗预处理两种预处理方式对原生质体活力无显著影响。以0.5 mol/L甘露醇为渗透调节剂,25℃酶解12 h条件下,适宜CE171和DH401纤维酶浓度略高于ED13、HS66和DH405,分别为0.3%和0.4%。ED13和DH401原生质体在VKM液体培养基中培养3～4周,经愈伤组织生长培养基培养2周,转至芽诱导培养基培养,2～3个月后形成具根茎叶的完整植株。HS66和CE171原生质体培养6～8周也能形3～4 mm愈伤组织,但没有分化出芽;DH405的原生质体不分裂。     

石牌广藿香原生质体的分离及培养研究

以石牌广藿香悬浮细胞为材料,对影响其原生质体分离和培养的酶浓度、作用时间、溶液渗透压和材料的生理状态等因素进行了研究。结果表明:以0.5%的果胶酶、0.2%离析酶和0.8%的纤维素酶组合处理继代培养3～11d的悬浮细胞8h,渗透压调节剂为9%甘露醇,原生质体产量达1.65×106 protoplasts·mL-1 PCV,活力超过86%。在原生质体的液体浅层培养中,细胞分裂频率为13.5%。     

Plant Regeneration from Protoplasts of Corn(Zea mays L.)

Maize embryogenic calli induced from pollen were subcultured for one and one half years on N, basic medium supplemented with 2 mg/1 kinetin, 1 mg/l 6-benzyl-aminopurine, 0.3 mg/l 2,4-D, 500 mg/l casein hydrolysate and 250 mg/l glutamine. These embryogenic calli were used for protoplast isolation. Protoplasts were cultured on Z2 medium (Table 1) which is composed of rice protoplast culture basic medium 1 supplemented with 0.2 mg/l kinetin, 0.1 mg/l 6-benzyl-aminopurine, 0.5 mg/l 2,4-D, 200 mg/l casein hydrolysate, 100 mg/l glutamine and 2% coconut milk. The first division of regenerated cell occurred after 4-6 days in culture. After 3 weeks later, small calli could be seen with naked eyes. At this moment, addition of the same Z2 medium with decreased osmoticum twice for the protoplast culture is necessary. Regenerated calli, 2–4 mm in diameter, were transferred in succession on differentiation medium Z3 and Z4 for organogenesis. Embryogenesis and plant regeneration could occur simultaneously on Z4 differentiation medium. It seems that except the cultural conditions genotype and using of embryogenic materials are the two key factors for plant regeneration of maize protoplast and the former may be the critical one.     

Somatic Embryogenesis and Plant Regeneration from Protoplasts of Cowpea (Vigna sinensis)

Immature cotyledons of cowpea (Vigna sinensis Endlo) were used for protoplast isolation. Enzyme solution for protoplast isolation contained 40% cellulase Onozuka R-10,0.30% Macerozyme R-10 and 2% hemicellulase. The purified protoplasts were cultured in Bs,MS or KM8p liquid medium in dark (25℃) at a density of 1 × 105–5 × 105/ml. The protoplasts started cell division in 3–5 days . Sustained cell divisions resulted ill formation of cell clusters and small calli,with cell division frequency reaching 23%–28% in MS medium . Calli of 2 mm in size were transferred onto MSB (MS salts+B5 vitamins) medium with 2 mg/L 2,4-D, 0. 5mg /L BA forfurther growth. Embryogenic calli appeared on this medium. After passage to fresh medium with the same composition, the embryogenic calli were transferred into MSB liquid medium to establish suspension culture. When the suspended calli were transferred back onto MSB agar medium with 0. 1 mg /L IAA, 0.5mg/L KT, 5% mannitol (cultured in light,2000 lx,12h/d), a lot of adventitious roots formed in 7–10 days, and then somatic embryos formed from the protoplast derived calli. But only a few embryoids developed further into the cotyledonary stage ,and the others died at globular, heart-shaped, or torpeto stage . Finally, some cotyledonary embryoids germinated and developed into plantlets or shoots with leaves.     

Efficient callus formation and plant regeneration of goosegrass [<Emphasis Type="Italic">Eleusine indica </Emphasis>(L.) Gaertn.]

Efficient methods in totipotent callus formation, cell suspension culture establishment and whole-plant regeneration have been developed for the goosegrass [ Eleusine indica (L.) Gaertn.] and its dinitroaniline-resistant biotypes. The optimum medium for inducing morphogenic calli consisted of N6 basal salts and B5 vitamins supplemented with 1-2 mg l(-1) 2,4-dichlorophenoxyacetic acid (2,4-D), 2 mg l(-1) glycine, 100 mg l(-1) asparagine, 100 mg l(-1) casein hydrolysate, 30 g l(-1) sucrose and 0.6% agar, pH 5.7. The presence of organogenic and embryogenic structures in these calli was histologically documented. Cell suspension cultures derived from young calli were established in a liquid medium with the same composition. Morphogenic structures of direct shoots and somatic embryos were grown into rooted plantlets on medium containing MS basal salts, B5 vitamins, 1 mg l(-1) kinetin (Kn) and 0.1 mg l(-1) indole-3-acetic acid (IAA), 3% sucrose, 0.6% agar, pH 5.7. Calli derived from the R-biotype of E. indica possessed a high resistance to trifluralin (dinitroaniline herbicide) and cross-resistance to a structurally non-related herbicide, amiprophosmethyl (phosphorothioamidate herbicide), as did the original resistant plants. Embryogenic cell suspension culture was a better source of E. indica protoplasts than callus or mesophyll tissue. The enzyme solution containing 1.5% cellulase Onozuka R-10, 0.5% driselase, 1% pectolyase Y-23, 0.5% hemicellulase and N(6) mineral salts with an additional 0.2 M KCl and 0.1 M CaCl(2) (pH 5.4-5.5) was used for protoplast isolation. The purified protoplasts were cultivated in KM8p liquid medium supplemented with 2 mg l(-1) 2,4-D and 0.2 mg l(-1) Kn.     

Plant regeneration from protoplasts of peppermint (Mentha piperita L.)

Summary Enzymatically isolated leaf-derived protoplasts of peppermint (Mentha piperita L.) were cultured in modified B5 medium containing 1 mg/l NAA, 0.4 mg/l BA, 0.5% sucrose, 0.5 M mannitol and 0.1% Gelrite (first medium). After 30 d culture at 25°C in the dark, protoplasts formed colonies consisting of about 100 cells. Gelrite medium blocks were transferred into liquid medium to promote further growth. Colonies of 0.5 mm transferred to 0.2% Gelrite solidified medium (same components as first medium) formed green calli (1–2 mm) under incubation in the light. Green calli transferred to differentiation medium (B5, 0.1 mg/l NAA, 5 mg/l BA, 2% sucrose, 0.2 M mannitol, 0.2% Gelrite) developed shoot buds after 3–4 weeks. Whole plants were recovered following rooting of shoots in B5 medium without hormones.Abbreviations BA 6-benzylaminopurine - NAA -naphthaleneacetic acid - KIN kinetin - ZEA zeatin - CPW cell and protoplast wash solution - B5 Gamborg et al. (1968) mineral elements - MS Murashige and Skoog (1962) mineral elements     

Plants Regenerated from Mesophyll Protoplasts of Cottonwood New Clone

Poplar NL-80106 (Populus deltoides×P, simonii) mesophyll protoplasts were isolated from leaves of 30 days-old sterile shoot, with 4 × 107/g fr. wt of protoplast yield after purification. The protoplasts were cultured in KM8p and MS liquid media containing 2 mg/L 2, 4-D, 0. 5 mg/L NAA and 0.5 mg/L KT. Higher plating density and lower osmatic pressure (0.45 mol/L) were proved to be favourable to division of protoplast-derived cells. The first division initiated 5 days after culture, and the division frequency reached 4.5 % on the 10th day. A number'of cell colonies and microcalli was formed in 12 weeks. Using organic nitrates and glucose in protoplast culture medium was beneficial to increase division frequency and plating efficiency. The calli were allowed to grow to 4--6 mm in height with red colour and compact structure on the gelrite-sohdified NLZ1 proliferation medium in 3 weeks and were transferred onto NLF differentiation medium where the frequency of shoot formation could reach 100%. The 3 cm high shoots were then cut off from the callus and rooted on 1/2 MS medium.     

Effect of sugar type on the efficiency of plant regeneration from protoplasts isolated from shoot tip-derived meristematic nodular cell clumps of Lilium x formolongi hort.

Summary Suspension cultures composed of meristematic nodular cell clumps of Lilium x formolongi hort were established from shoot tips placed on MS medium supplemented with 1 mg/l picloram and 30 g/l sucrose, glucose, fructose or sorbitol. Protoplasts isolated from these cultures were embedded in 1 g/l gellan gumsolidified 1/2MS medium with 1 mg/l picloram and the different kinds of sugars at 0.5 M, and cultured at 25 °C in the dark. The highest plating efficiency (13.7%) was obtained when the protoplasts were isolated from the cell clumps which had been subcultured in MS medium containing glucose and were likewise cultured in MS medium supplemented with 0.5 M glucose. Plants were regenerated from the protoplast-derived calli on 1/2MS medium containing 2.5–10 g/l sucrose or 5–10 g/l glucose. These results suggest that the kinds of sugar and concentration are important parameters affecting protoplast isolation, proliferation and plant regeneration in L. x formolomgi hort.Abbreviations FW fresh weight - MS Murashige and Skoog (1962) - 1/2MS medium MS medium containing half strength mineral salts - 1/2MS-0 1/2MS medium containing no growth regulators - NAA 1-naphthaleneacetic acid - p-calli protoplast-derived calli - PE plating efficiency - picloram 4-amino-3,5,6-trichloro-picolinic acid     

霸王的原生质体培养的研究

目的：为利用原生质体融合技术转移霸王抗旱基因。方法：采用酶解法分离霸王原生质体,比较了霸王子叶和愈伤组织游离原生质体的产量和活力,不同渗透压和起始密度对原生质体分裂频率的影响。结果：愈伤组织游离的原生质体产量和活力均高于子叶,原生质体产率可达2.4&#215;106个/g.FW,活力达89%。采用液体浅层培养,在附加2,4-D（2mg/L）、6-BA（1.0mg/L）、2%蔗糖和甘露醇（0.4mol/L）的DPD培养基中,原生质体分裂频率最高,达68.6%。转移到附加2-iP（3mg/L）、KT（1.0mg/L）、6-BA（1.0mg/L）的分化培养基上,获得2个再生苗。结论：采用酶解法游离霸王愈伤组织,可获得高活力和高分裂频率的霸王原生质体。     

Plantlet regeneration from protoplast-derived embryogenic calli of Crocus cancellatus

Plant regeneration from protoplast culture of Crocus cancellatus was investigated using regenerable embryogenic calli obtained from shoot meristem culture on LS (Linsmaier and Skoog, 1965) medium containing 4 mg l⁻¹ kinetin and 1 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D). Protoplasts were isolated directly from embryogenic calli. The best protoplast growth was found on those embedded in Ca-alginate beads and cultured with nurse cells in MS (Murashige and Skoog, 1962) medium supplemented with 2 mg l⁻¹ kinetin, 1 mg l⁻¹ 2,4-D and 100 mg l⁻¹ ascorbic acid at 25 °C in darkness. After 4–5 weeks of culture, microcalli appeared on the surface of the Ca-alginate beads, but the protoplasts without immobilization in Ca-alginate beads showed very low cell division. Growth of the microcalli in the medium with nurse cells was much better than in the medium without nurse cells. Transferring beads onto half strength MS medium supplemented with 0.2 mg l⁻¹ kinetin and 0.1 mg l⁻¹ 2,4-D, increased the growth of embryogenic calli. Somatic embryo development was observed either on half strength MS medium growth regulator free or with 1 mg l⁻¹ abscisic acid. Matured embryos germinated on half strength MS medium containing 25 mg l⁻¹ of gibberelic acid. Plantlet formation was obtained on half strength MS medium containing 1 mg l⁻¹ 6-benzyladenine and 1 mg l⁻¹ α-naphthaleneacetic acid at 20 °C in a 16/8 h light/dark cycle. This revised version was published online in June 2006 with corrections to the Cover Date.     

骆驼刺发根农杆菌转化系的原生质体培养和植株再生

从发根农杆菌A4转化的荒漠植物—璐驼刺毛状根愈伤组织中分离的原生质体培养的结果表明,酶解新转代7～10d的淡黄色松软愈伤组织,可获得大量有活力的原生质体。原生质体在附加有1．5mg．L-1 2,4．D、0．2mg．L-1 6．BA、0．3m01．L-1甘露醇、2％（W/V）蔗糖和500mg·L-1水解酪蛋白的DPD培养基中进行液体浅层培养可持续分裂。培养基的最适渗透压为（450±3）mOsm·kg-1,原生质体的最适植板密度为4×10^5个．mL-1。制备原生质体的愈伤组织以低温（4℃）预处理后,原生质体的产率和分裂频率均提高,分裂频率最高可达50％。原生质体分裂形成的愈伤组织转移在附加1-2mg．L-1 6-BA（或KT）和0．2mg·L-1NAA的MS培养基上培养后,可以分化并获得再生植株。纸电泳检测表明,原生质体再生的愈伤组织和分化植株仍然含有毛状根转化系的特异产物——冠瘿碱。     

毛花猕猴桃原生质体再生植株

从毛花猕猴桃（Ａｃｔｉｎｉｄｉａ ｅｒｉａｎｔｈａ Ｂｅｎｔｈ．）试管培养的实生苗新展开叶片分离的原生质体,培养在液体ＭＳ（除去ＮＨ４ＮＯ３）附加２,４－Ｄ １．０ ｍ ｇ／Ｌ和葡萄糖０．４ ｍ ｏｌ／Ｌ的培养基上。培养３周后植板率达到１９．４％ 。在未添加新鲜培养基的情况下,原生质体再生的细胞可持续分裂,并于３个月时长成２ ｍ ｍ 大小的愈伤组织。将该愈伤组织转移到附加玉米素０．５ ｍ ｇ／Ｌ和ＩＡＡ ０．１ ｍ ｇ／Ｌ的固体ＭＳ培养基上,分化出苗。试管苗经诱导生根,长成完整小植株