甘蓝型油菜与蔊菜的原生质体融合与植株再生

以甘蓝型油菜下胚轴和蔊菜叶片为外植体提取原生质体, 采用PEG-高pH、高Ca²⁺附加DMSO的原生质体融合方法, 用液体浅层静置培养融合体, 获得了10株融合杂种, 观察了杂种形态学和细胞学。结果表明：1%纤维素酶+0.2%离析酶+3 mmol/L MES 酶解14 h 可获得较高产率的油菜原生质体, 0.25% 纤维素酶+0.5%离析酶+5 mmol/L MES酶解12 h可获得较高产率的蔊菜原生质体; 30% PEG + 0.3 mol/L葡萄糖+50 mmol/L CaCl₂•2H₂O +15%DMSO的融合条件下, 获得了10.4%的融合率; 实验所获的原生质体融合材料可作为新种质。     

利用荧光染色和流式细胞技术辅助卵孢小奥德蘑原生质体制备与再生研究

本研究以卵孢小奥德蘑液体培养菌丝作为实验材料,利用单因子变量法探索研究了菌丝培养时间、酶浓度、酶解时间、酶解温度、稳渗剂类型对卵孢小奥德蘑原生质体制备的影响,并对原生质体再生培养基进行选择和优化。通过荧光染色,利用激光共聚焦显微镜和流式细胞仪对原生质体的制备过程、得率和活力进行研究。结果表明,将卵孢小奥德蘑菌丝在液体培养基中培养5d收集菌丝体,以甘露醇作为渗透压稳定剂,在溶壁酶浓度2%、30℃条件下酶解5h,获得的原生质体得率最高,达2.0×10 ⁷个/mL;通过流式细胞仪分析,约57.69%的原生质体细胞为活细胞;在RM培养基中再生效果最好,再生率为(0.103±0.025)%。研究结果可以为卵孢小奥德蘑育种与食用菌原生质体制备再生提供研究基础。     

蓝色犁头霉原生质体的制备与再生

研究了氢化可的松生产菌蓝色犁头霉原生质体的形成与再生。通过对溶解酶系统的选择,影响原生质体形成的因素如渗透压稳定剂、酶浓度、菌龄、菌丝培养基和培养方式等因素进行考察,发现以0．4mol/L NH4Cl做为稳定剂、2．5mg/mL溶壁酶和5mg/mL纤维素酶组成的混合酶液溶解菌丝,4h后原生质体量可达10^6cell/mL。通过显微镜观察原生质体的形成过程以及在高渗培养基上的再生情况,再生率为15．6％。     

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2.4×106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。     

The isolation, culture and division of protoplasts from citrus cotyledons

High yields (2.3 × 10⁵ to 1.3 × 10⁶ protoplasts/g.f.wt.) of isolated protoplasts were obtained from cotyledons of Cirus sinensis (L.) Osb. 'Valencia'. Osmotic potential of the medium and enzyme concentrations were important in obtaining high viability of preparations as indicated by FDA fluorescence. Adding malt extract to a Murashige-Tucker basal medium increased plating efficiencies somewhat, but not the rate or duration of cell division. However, modifying the NAA and kinetin concentration optimized plating efficiencies (up to 20%) of protoplasts and also the rate or duration of cell division. The highest plating efficiency and number of cells per colony were obtained on a defined medium containing NAA (15 μ M ). and kinetin (4.6 μ M ). Coincidence of percentage protoplast viability after 13 days (assessed by FDA fluorescence) with plating efficiency after 21 days indicates that FDA fluorescence is an accurate indicator of citrus protoplast viability.     

金龟子绿僵菌原生质体的制备和再生及其羟化酶活性研究

对金龟子绿僵菌(Metarhizium anisopliae)原生质体的制备和再生的影响因素进行实验,并在此基础上考察了甾体底物对原生质体羟化酶的诱导作用。结果表明,原生质体制备的合适条件是:42 h的菌丝体用纤维素酶(10 mg/mL)和蜗牛酶(5 mg/mL)的混合酶在含有0.8 mol/L甘露醇的pH 5.8磷酸缓冲液中,28℃震荡(80 r/min)酶解3 h,原生质体产量可达到6.12×10~7/mL,在含有0.6 mol/L KCl的双层马铃薯培养基上再生率达到7.79%。经过6 h底物诱导的菌丝体制备的原生质体细胞色素P450的表达量比没经过诱导的菌丝体制备的原生质体高约40%,证明该菌羟化酶系统的可诱导性。由于没有细胞壁的阻碍经过底物诱导的原生质体能够高效的将底物转化为产物,且副产物相对较少。     

Regeneration of Hybrid Plantlets Via Pollen-Hypocotyl Protoplast Fusion in Brassica spp.

It has been reported that "gameto-somatic hybridization" was induced by fusion of microspore tetrad protoplasts with somatic protoplasts in Nicotiana and Petunia. However, since the success of isolation of pollen protoplasts in recent years, the use of protoplasts at pollen stage as one of the fusion partners in such hybridization is a novel experimentation. Young pollen protoplasts were isolated from the pollen grains of Brassica chinensis at mid-late unicellular to early bicellular stage the pollens for 1.5--2.5 h at 25℃ in a CPW solution containing 0.8 % of eellulase, 0.5 % pectinase, 0.1% pectolyase, 1 3 % mannitol, 1 0 % glucose, 0. 3% potassium dextran sulphate and 3 mmol/L MES. The purified pollen protoplasts were then fused with the hypocotyl protoplasts of B. napus by PEG method. Heterokaryons were identified by means of visualization of the fluorescence from FITC-prela-beled pollen protoplasts. In order to increase heterokaryons and reduce hypocotyls homokaryons, the denstity of hypocotyl protoplasts were lowered and the ratio of the number of hypocotyl vs. pollen protoplasts were adjusted from 1 : 3 to 1 : 6. The fusion products were cultured in a liquid KM8p medium supplemented with 0.4 mol/L glucose, 0.8 mg/L 2, 4-D, 0.25 mg/L NAA. 0. 5 mg/L BA, 500 mg/L glutamine and 3 mmol/L MES where cell division and callus formation took place. The calli, after being transferred to a MS medium supplemented with 2.0 mg/L BA, 3 % sucrose and 0.4 % agarose, differentiated into a few shoots. The shoots were transferred onto a half-strength MS medium supplemented with 2% sucrose, 0.1--0. 2 mg/L NAA, 0.5 mg/L IBA and 20% potato juice for root formation. Finally, three plantlets were regenerated. Chromosome counts by roottip squash method revealed that one plantlet was 2n= 48, corresponding to an allotriploid resulted from a fusion between one pollen protoplast of B. chinensis (2n = 20) and one hypocotyl protoplast of B. napus (2n = 38), and the other two plantlets were 2n = 58, which might be an allotetraploid originated from a fusion between two pollen protoplasts and one hypocotyl protoplast. The isozyme patterns of leaf esterases showed that all the three plantlets had bands characteristic of both parents. This is the first case of success in "gameto-somatic hybridization" by using pollen protoplasts rather than tetrad protoplasts as the haploid partner.     

Enhanced protoplast division by media ultrafiltration

Leaf protoplasts of lucerne (alfalfa-Medicago sativa L.) and tobacco (Nicotiana tabacum L.) were cultured in standard liquid culture medium and in medium that had been passed through a 10-kDa cut-off ultrafilter. The proportion of lucerne cells that divided was increased by 50–400% in ultrafiltered medium over that in standard medium. The effect was seen in six independent experiments performed over a period of 9 months. The inhibitory effect was detected in each of four separate batches of glucose that were examined from the same manufacturer. Ultrafiltration of medium used to culture tobacco protoplasts gave a 10% increase in the proportion of cells that divided. High molecular weight inhibitors of protoplast division were detected as contaminants in a number of components of the lucerne protoplast culture medium, including glucose, minor sugars and sugar alocohols, coconut water and casein hydrosylate. Gel filtration showed that the major inhibitory contaminant in glucose had a molecular weight greater than 200 000.     

Isolation,Culture and Plant Regeneration of Protoplasts from an Embryogenic Callus Tissue of Gossypium hirsutum

Protoplasts were isolated and cultured from hypocotyl embryogenic callus tissue of Gossypium hirsutum L. cv. "Lumian 6". The highest yields of viable protoplasts were obtained from a vigorous embryogenic callus 7 to 9 d old subcultured on MS medium supplemented with 2 mg/L IAA and 1 mg/L KT using a solution of 1% cellulase Onozuka R-10, 1% pectinase, 0.7 mmol/L KH2PO4, 2.5 mmol/L Ca2+ , and 0.5 mol/L osmoticum (mannitol), at pH 5.8 and at a temperature of 30 ℃. After separation and purification (in 21% sucrose floatation medium), the protoplasts were laid up in a quiet liquid protoplast culture medium containing K3 salts, NT vitamins with 0.1 mg/L 2,4-D, 0.2 mg/L KT and 0.45 mol/L glucose for 10 to 15 min. The protoplasts were fractioned into an upper and a lower layer in the centrifugal tube. Most of the protoplasts in the lower layer were smaller, round and rich in cytoplasts in which contain many granular substances. When this kind of protoplasts were cultured in the thin liquid protoplast culture medium with a density of 1 x l0s to 5 x los protoplasts/mL, the division and the callus formation of the regenerated cells were easily observed. The first divisions occurred in 3 days and small cell clusters could be seen after 2 to 3 weeks in the culture. At this moment, the addition of the protoplast culture medium with decreased osmoticum once or twice is needed for the continuous protoplasts division to form calli. Regenerated calli, 3 to 5 mm in diameter, were transferred in succession on MS medium with 2 mg/L IAA and 1 mg/L KT for the initiation of embryogenesis. The embryoids germinated on the hormonefree MS medium and a number of plantlets were obtained. It seems that using vigorous embryogenic callus and decreasing osmoticum are the two critical factors for plant regeneration of cotton protoplasts.     

疏绵状嗜热丝孢菌原生质体的制备与再生

以疏绵状嗜热丝孢菌(Thermomyces lanuginosus)为供试菌株,研究了菌龄、酶的种类及浓度、酶解时间、酶解温度和稳渗剂对原生质体制备的影响及稳渗剂对原生质体再生的影响。结果表明,制备嗜热丝孢菌原生质体比较适宜的条件为:PDB液体培养基培养28 h,以0.7 mol/L NaCl为稳渗剂,0.15 mol/L的溶壁酶,30℃酶解4 h。原生质体再生以0.7 mol/L蔗糖作稳渗剂为最佳。     

草木樨状黄芪甲硫氨酸抗性系的原生质体培养及植株再生

建立了草木樨状黄芪(Astragalus melilotoides Pall．)甲硫氨酸抗性系原生质体再生植株的实验体系。以茎切段诱导的松软愈伤组织为材料,通过酶法分离出大量有活力的原生质体。原生质体经培养持续分裂形成了愈伤组织,并高频率地分化出再生苗。比较了不同培养基、培养方法和培养密度对原生质体分裂和再生的影响。结果表明,原生质体以3×10⁵/mL的植板密度,采用琼脂糖岛法培养在附加1．0mg／L 2,4-二氯苯氧乙酸(2,4-D)、0．5mg／L 6-苄氨基嘌呤(6BA)、500mg／L水解酪蛋白、3％蔗糖、0．3mol／L甘露醇的KM_8p培养基中,可获得最佳效果,其细胞分裂频率达38％左右。原生质体培养后仍然保持对甲硫氨酸的抗性,同时对乙硫氨酸表现交叉抗性。     

玉米、小麦、水稻原生质体制备条件优化

玉米Zea mays L.、小麦Triticum aestivum L.、水稻Oryza sativaL.是三大重要粮食作物,对其原生质体制备条件的优化具有重要意义.以玉米(综3)、小麦(中国春)、水稻(日本晴)10日龄幼苗为材料,研究了叶肉细胞原生质体分离过程中的酶浓度、酶解时间和离心力大小等因素对产量和活力的影响.结果表明:酶浓度和酶解时间对原生质体产量影响显著,随着酶解液浓度和酶解时间的提高,原生质体产量增加,但细胞碎片同时增多.水稻经真空处理后,原生质体产量大幅度提高.通过正交实验设计得出如下结果:玉米叶肉细胞原生质体分离的最佳条件为:纤维素酶1.5％,离析酶0.5％,50 r/min酶解7h,100×g离心2 min收集,原生质体产量为7×106/g FW;小麦叶肉细胞原生质体分离的最佳条件为:纤维素酶1.5％,离析酶0.5％,50 r/min酶解5h,100×g离心2 min收集,原生质体产量为6×106/g FW;水稻叶肉细胞原生质体分离的最佳条件为:纤维素酶2.0％,离析酶0.7％,50 r/min酶解7h,1 000×g离心2 min收集,得到的原生质体产量为6×106/g FW.通过二乙酸荧光素染色发现原生质体活力均在90％以上.用PEG-Ca2+介导法将含有绿色荧光蛋白的质粒转化入原生质体,转化率可达50％ ～80％.     

Isolation of Pollen Protoplasts in Nicotiana tabacum

A new method for isolation of quantities of mature pollen protoplasts in Nicotiana tabacum has been established. The first step was to germinate mature pollen in Brewbaker and Kwack medium containing 20% sucrose. When most of the pollen grains had just germinated short pollen tubes, they were transferred to an enzymatic solution for the second step. The enzymatic solution contained 1% pectinase, 1% cellulase, 0.5% potassium dextran sulfate, 1 mol/L mannitol, 0.4 mol/L sorbitol in Dx medium with or without 15% Ficoll. The enzymes firstly degraded the pollen tube wall and then the intine. As a result, intact pollen protoplasts were released with the isolation rate up to 50%-70%. Factors affecting pollen protoplast isolation during the germination and maceration of pollen grains were studied. The suceees depended on two key points:pollen germination duration and osmotieum concentration. The optimal germination duration was 30 rain at 30℃. When it was too long, long pollen tubes formed and subsequently, large number of subprotoplasts instead of whole protoplasts were yielded, as the case reported by previous investigators. The optimal concentration of mannitol and sorbitol in enzyme solution was as high as 1.4 mol/L in total. Lowering of the osmoticum concentration resulted in decrease of percentage of pollen protoplasts.     

菌龄与培养基组分对曲霉原生质体释放的影响

培养基组分显著影响曲霉菌丝原生质体的释放,降低培养基中碳素或氮素养分供给量能显著促进原生质体的释放,且降低碳素养分的促效更大。菌龄与原生质体释放量之间的关系因培养基种类而异,在完全培养基上营养体生物量累积曲线具典型正S型曲线特征,原生质体释放量与菌龄呈明显反比例关系,在本研究条件下12 h龄固培菌丝体最宜制备原生质体。在酪素培养基上,原生质体释放量与菌龄无关,12~26 h菌龄期间的固培菌丝体均可释放出大量原生质体。     

毛栓菌原生质体制备和再生及单核菌株产漆酶特性

毛栓菌Trametes hirsuta能有效地降解木质素,在生物燃料、制浆和饲料工业等方面具有很高应用价值。为了获得遗传性能稳定的T. hirsuta单核菌株,研究了其菌丝生长培养基的类型、菌丝生长时间（菌龄）、酶解时间、原生质体纯化离心速度和原生质体再生培养基类型对T. hirsuta YJ-9-1原生质体制备与再生的影响;采用DAPI染色和锁状联合缺失的观察,从再生株中筛选单核菌株并考察其产酶特性。结果表明：采用YGM菌丝生长培养基、88h菌龄、1h酶解时间、4,000r/min原生质体纯化离心速度以及YGMS再生培养基,最终可获得密度大约为5.0×106个/mL的原生质体悬浮液和9.1%的再生率;从200株再生菌株中筛选出了3株单核菌株,其中一株单核菌株D-2-1的漆酶产量比原菌T. hirsuta YJ-9-1明显提高,在第12天其漆酶酶活为771.67U/L,是原菌的1.51倍。     

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。     

Cytoplasmic-nuclear interaction and medium effect for protoplast culture in sunflower

Protoplasts of 6 alloplasmic and 2 euplasmic sunflower inbred lines were isolated from dark grown seedling hypocotyls with a density of 2×10⁴ protoplasts/ml. The protoplast suspension was mixed with a solution of 0.5% agarose (sigma – type 1), then pipetted in droplets of about 1000 protoplasts. Droplets were surrounded by two different liquid media. After 30 days droplets from both media were transferred to solid differentiation medium. Protoplast division, microcolony frequency and the number of calluses produced were strongly dependent on medium composition and genotype. The number of calluses per 1000 protoplasts plated range from 0.3 to 5.0 according to the genotype and the method used. The alloplasmic line RHA274-PEF1, was the best responding genotype for calluses produced in both media used. In all cases, the percentage of calluses for alloplasmic lines were significantly higher when compared with the nucleus donor genotype. H. petiolaris fallax cytoplasm increased both the number of calluses produced and the percentage of microcolonies. The complex interaction among genotypes tested indicates that protoplast culture responses are affected independently by nuclear-cytoplasm interactions. Some nucleus-cytoplasm combinations can improve the protoplast culture responses in sunflower. This revised version was published online in June 2006 with corrections to the Cover Date.     

红曲霉原生质体的制备、再生及其遗传转化系统

原生质体是研究和建立真菌遗传转化系统的重要工具。为了建立原生质体介导的红曲霉遗传转化系统,考察了各种细胞壁裂解酶和渗透压稳定剂等对红曲霉原生质体形成和再生的影响。将红曲霉分生孢子在铺有玻璃纸的平板上30℃培养30~40 h收获的菌丝体最有利于原生质体的形成和释放。红曲霉菌丝体形成和释放原生质体最适裂解酶和酶解时间分别为：0.3 % lysing enzyme、0.1 % cellulase和1 % snailase的酶组合,30℃作用2.5 h;最适渗透压稳定剂是：1mol /L MgSO4。最适合原生质体再生的培养基为含0.6 mol/L蔗糖的CM培养基。原生质体液涂布单层再生培养基的方法,再生率最高,菌株M34和N18分别为8.5 %和36.4 %。在PEG和CaCl2存在下,以潮霉素B为抗生素选择标记,用质粒pBC-Hygro和pNL1共转化菌株M34原生质体,每微克DNA克获得100个稳定转化子。     

Somatic embryogenesis and plant regeneration from isolated protoplasts of Lavatera thuringiaca

Embryogenic cell suspensions of Lavatera thuringiaca L. were established from leaf petiole and shoot regeneration was achieved when cells were plated on medium without growth regulators. We tested three methods for protoplast culture, isolated from a one-year old embryogenic cell suspension, to determine the best conditions for L. thuringiaca protoplast culture and shoot regeneration. The highest protoplast plating efficiency was obtained with the agaroseembedded method, reaching 30%, while the nursing culture method gave 5% when the protoplasts were plated over Whatman paper No. 2. However, the same nursing culture failed to produce protoplast-derived microcalluses when the protoplasts were plated on a nitrocellulose filter. The liquid thin layer method gave the lowest plating efficiency with only 0.5%. Shoot regeneration from protoplast-derived microcalluses was achieved in two steps; first, globular embryo development was favored in medium low in auxin (2,4-d and BA at 0.01 and 0.05 mg 1^-1, respectively), second, the globular embryos further differentiate into shoots in medium without growth regulators or in medium containing GA₃ (0.5 to 1.0 mg 1^-1).Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - BA benzyladenine - GA₃ gibberellic acid - NAA -naphthaleneacetic acid - IBA indole-3-butyric acid     

The effect of a calcium-channel antagonist, nifedipine and agonist, Bay K-8644, on the phytochrome-controlled swelling of etiolated wheat protoplasts

The effect of external Ca²⁺ and Ca²⁺-channel modulators on the phytochrome-controlled swelling of etiolated wheat ( Triticum aestivum L. cv. Arminda) mesophyll protoplasts has been studied. The red light (R)-stimulated swelling of the protoplasts requires Ca²⁺ in the surrounding medium and maximum response was observed in a medium containing I m M CaCI₂. Far-red light (FR) irradiation of protoplasts in the presence or absence of Ca²⁺ does not influence the protoplast volume. The Ca²⁺-channel antagonist nifedipine prevents R-induced protoplast swelling at very low concentrations (0.1 μ M ). The Ca²⁺ -channel agonist Bay K-8644 stimulates the swelling of protoplasts incubated in darkness or irradiated with FR. Action of nifedipine depends on whether it is applied before or after the R pulse. The results are compatible with the hypothesis that phytochrome controls the activity of dihydropyridine-sensitive L-type Ca²⁺ channels.