草菇(Volvariella volvacea)、银丝草菇(V.bombycina)原生质体分离研究

本文对草菇、银丝草菇菌丝原生质体制备的最佳条件作了探讨。结果表明,培养两天的草菇菌丝,以0.5MKCI或0.5MMgSO_(4.7)H_2O作渗透压稳定剂,1.5％Lywallzyme(v/m),35℃下酶解1.5—2小时,原生质体产量可达1.5×10~6个/ml以上,培养3天的银丝草菇菌丝,以0.5MKCl作渗透压稳定剂,1.5％Lywallzyme,28℃酶解2—3小时,原生质体产量可达2.8×10~6个/ml以上。此研究对以后的原生质体融合育种打下了基础。     

皱叶甘蓝的原生质体培养与植株再生

皱叶甘蓝(Brassica oleracea L. var. subauda)“SA61”(SV)的叶及下胚轴分离的原生质体在 MS_1(修改的MS)培养基上细胞壁再生和分裂启动较快。叶原生质体在 DPD_1(修改的 DPD)培养基上获得了最高的分裂率和植板率;下胚轴原生质体在MS_1上获得最佳的培养效果。叶原生质体培养3—4天后见到一次分裂;下胚轴原生质体在48小时左右即可发生一次分裂。原生质体培养 20—30天后形成肉眼可见的微愈伤颗粒,40天左右即可达1mm大小。在7种不同培养基上增殖微愈伤组织,MB_2、MB_3表现了优良的效果。在MS_2培养基上的芽分化效果最为理想。在不加任何激素的MS培养基上诱导生根,2周后得到再生植株。     

酿酒酵母原生质体融合及其融合子的鉴定

应用原生质体融合技术,得到乙醇产量较高的两株多倍体酵母菌融合子F_(28)和F_(38),融合频率为2×10~(-5)。测定融合子F_(28)、F_(38)每个细胞内DNA含量分别为2.14×10~(-8)、2.34×10~(-8)μg,而亲株DNA含量分别为0.78×10~(-8)和1.24×10~(-8)μg。并进行了融合子细胞增殖率、乙醇发酵能力及同功酶分析等试验。用固定化细胞发酵乙醇试验结果表明,在pH4.0,17％糖蜜为基质情况下,发酵3小时,融合子F_(28)、F_(38)的乙醇产量分别为77.21和77.09mg/ml;亲株乙醇产量仅为65.00mg/ml和68.40mg/ml。为固定化细胞发酵乙醇提供优良菌株。     

赤霉素产生菌——藤仓赤霉菌融合重组的研究

以一对营养互补的缺陷型突变株作为亲本,酶法去除细胞壁制成原生质体,以等量相混,用30%PEG 4000诱导融合,在最低营养再生培养基上直接选择原养型融合重组子,重组频率约为10~-,同时产生一定数量的不稳定异核体,频率约为10~(-5)—10~(-6).融合重组导致色素产生和菌丝形态及赤霉素产生能力的多种变异.融合重组子中赤霉素产量的正变率为15.3%.其中RN2和RG14菌株的赤霉素产量比原养型出发菌株207提高25%以上.     

金钗石斛原生质体的制备与融合

以金钗石斛(Dendrobium nobile Lindl.)再生苗的幼嫩叶片为实验材料,以原生质体活力率为评价指标,用正交试验设计对原生质体制备过程中的纤维素酶浓度、果胶酶浓度、离析酶浓度和酶解时间四个关键因素进行最佳组合筛选,发现当纤维素酶为9.5 mg/m L、果胶酶为5.0 mg/m L、离析酶为6.0 mg/m L、酶解时间为3.0 h的组合条件下,制备的原生质体活力率最高,为94.42%。在此基础上,采用高钙高pH值结合PEG4000诱导法进行原生质体融合研究,以高活力的赤水和瑞丽金钗石斛原生质体为材料,以异核体融合率为评价指标,用响应面法优化设计对原生质体融合过程中的PEG浓度、CaCl_2浓度、pH和融合时间四个因素进行最佳组合筛选,发现当PEG4000浓度为38.41%、CaCl_2浓度为0.44 mol/L、pH为9.26、融合时间为28.87 min的组合条件下,异核体融合率达到最高,为0.924%。同时,响应面法分析还发现,PEG浓度和CaCl_2浓度之间、PEG浓度和pH之间、CaCl_2浓度和融合时间两个因素共同对异核体融合率有显著影响。此外,响应面分析还发现,当pH为9.26时,PEG浓度、CaCl_2浓度和融合时间的值在一定范围内变化并不会显著影响异核体融合率的值,数值均维持在0.94%左右。该研究完善了金钗石斛原生质体制备和融合的技术体系,为金钗石斛种质资源的保护、拓展和新品种选育提供了实验材料,为其他近缘物种的相关研究提供了实验参考。     

苏云金芽孢杆菌Bt-3701与巨大芽孢杆菌Bm-107种间原生质体融合

具有杀虫活性的苏云金芽孢杆菌(B. thuringensis subsp kurstaki)Bt-3701与具有解磷活性的巨大芽孢杆菌(B. megaterium var. phosphaticum)Bm-107原生质体进行了融合。Bt-3701原生质体形成率为99.4％,再生率为21.4％;Bm-107原生质体形成率为97.5％,再生率为23.8％。以PEG为助融剂进行原生质体融合,得到22个融合子,融合率达6.03×10~(-3)。融合子在双抗培养基(DR-CNB)上连续传代12次,得到4个稳定的融合于生物测定表明,融合子既具有一定的杀虫活性又具有一定的解磷活性。     

埃塞俄比亚芥的原生质体培养及植株再生

以埃塞俄比亚芥“84A165”为材料,从3—5日龄无菌苗的子叶、下胚轴或温室生长的三叶期苗的第一真叶游离原生质体,悬浮于 P-B 液体培养基中,用0.15—0.3%低融点琼脂糖固化,薄层漂浮,暗培养。原生质体密度为5×10~3—1×10~4/ml。下胚轴原生质体在培养后48小时出现一次分裂,3天后分裂频率达21%,6天后达34%。子叶和真叶原生质体起始分裂稍晚(第5天),分裂频率也较低。培养1周后,加入稀释培养基 DPDK_3,并转至光下,以后每隔一周加液一次。一个月后获得肉眼可见的小愈伤组织。植板率为2—3%。小愈伤组织在固体增殖培养基上继代长大。子叶和真叶原生质体来源的愈伤组织在转至补加 NAA0.1、BA3mg/1的分化培养基上后获得芽分化,把这些芽切离,转至 IAA0.2mg/l 的生根培养基上,再生出完整植株。     

用由植物与哺乳动物细胞融合物衍生的“植物”生产哺乳动物免疫球蛋白

虽然有从动物细胞和植物原生质体融合物形成活的、可分裂的界间异核体的报道,但这些融合产物中没有一个被报道产生了动物蛋白。而现在,泰国Chiang Mai大学的S.Makonkaw keyooa、P. Smitam-     

苗龄与红光对向日葵原生质体分离和培养的影响

用1.0—1.5%(W/V)纤维素酶(Onozuka R-10)和0.3—0.5%(W/V)果胶酶[Pectinasc (Serva)]配合分离到大量有活力的向日葵下胚轴原生质体,经液体浅层培养或琼脂糖小块培养7—10天后,均能持续分裂到细胞团或体细胞胚,至14—21天形成大量肉眼可见的小愈伤组织(直径0.5—2.0mm)。比较试验表明:(1)影响向日葵下胚轴原生质体分裂生长的首要因素是起始材料无菌苗的生理状态。用红光照射无菌苗,能明显地促使下胚轴原生质体在较低密度(1×10~4/ml)培养时,也能持续分裂,再生小愈伤组织;(2)在MS培养基上添加5mmol/L谷氨酰胺或以7.5mmol/L谷氨酰胺代替原培养基中的无机氮,能促使原生质体高频率(44.4%左右)分裂,再生愈伤组织。     

马氏钳蝎毒对大鼠神经和骨胳肌的作用

本工作在大鼠膈神经膈肌标本上观察马氏钳蝎(Buthus marensi Karsch)毒对神经、肌肉和神经肌肉接头传递的作用,结果如下:1.在蝎毒(3×10~(-5)g/ml)作用下,由吸附电极引导的膈神经单相动作电位的下降相逐渐延长,形成平台,3小时后电位时程可达100ms 以上。2.蝎毒(5×10~(-7)—6×10~(-5)g/ml)显著改变肌纤维动作电位的波形,降低肌细胞的静息膜电位。如用6×10~(-5)g/ml 浓度蝎毒处理膈肌,半小时内即可使肌纤维动作电位下降相大大延长,形成平台。一小时后膜电位由对照的78±4mV 降低至59±13mV,2小时后至55±8mV(平均值±S.D.)。3.河豚毒(3μM)可使被蝎毒降低了的肌细胞膜电位迅速复原,但随着时间的延续还可以再出现缓慢的轻度下降。4.在河豚毒(3μM)使肌细胞动作电位消失后,向溶液中加入蝎毒,半小时后将二者一并洗去,重新出现的动作电位亦带有明显的平台。5.在接头传递已被高 Mg~(++)或筒箭毒碱阻遏的标本上加蝎毒后,单个间接刺激可诱发出一串终板电位,甚至引起肌肉收缩。6.蝎毒(1×10~(-5)g/ml)明显增加小终板电位的发放频率,作用1小时后其频率可高达每秒100次以上。7.肌肉对间接刺激的收缩反应在加入蝎毒后首先增大,然后逐渐下降,在1×10~(-5)g/ml 浓度蝎毒作用下1.5—2小时传递阻遏,此时肌肉对直接刺激     

Culture of plant somatic hybrids following electrical fusion

Summary Electrically-induced protoplast fusion has been used to produce somatic hybrids between Nicotiana plumbaginifolia and Nicotiana tabacum. Following fusion of suspension culture protoplasts (N. plumbaginifolia) with mesophyll protoplasts (N. tabacum) heterokaryons were identified visually and their development was followed in culture. Because electrical fusion is a microtechnique, procedures were developed for culturing the heterokaryons in small numbers and at low density. The fusion and culture procedures described are rapid, uncomplicated and repeatable. Good cell viabilities indicate that the fusion procedure is not cytotoxic. Fused material was cultured 1–2 days at high density in modified K3 medium (Nagy and Maliga 1976). The heterokaryons were isolated manually and grown, at low density in conditioned media. Calli have been regenerated. Esterase isozyme patterns confirm the hybrid character of calli and clonally-derived plantlets recovered from these fusions.     

Optimization of Electrofusion Parameters and Interspecific Somatic Hybrid Regeneration in Citrus

This study was primarily attempted to optimize the electrofusion parameters using protoplasts isolated from cell suspension cultures of "Page" tangelo ( Citrus reticulata Blanco x C. paradisi Macf) and mesophyll protoplasts of rough lemon ( Citrus jambhiri Lush) as fusion partners. It was shown that the binuclear heterokaryons frequency reached 15% with the following parameters: alternate current (AC) 125 V/cm, AC time 60 s, direct current (DC) 1 250 V/cm, DC pulse width 50 μs, DC pulse interval O. 5 s, No. of DC pulse 3. Considering the fact that different types of protoplasts have different specific weights, higher frequency of the binuclear heterokaryons was obtained by controlling the centrifugation time after fusion. The fusion products regenerated into plantlets after 3 to 4 months of culture. Chromosome counting of the root tips and morphological observation of the regenerants verified that 78% were tetraploids and the rest were diploids with the leaf morphology of mesophyll parent. Peroxidase (POX) isozyme and RAPD analysis indicated that interspecific somatic hybrids were obtained and an autotetraploid plant of mesophyll parent type was also verified.     

Plant Eegeneration from Protoplasts of Brassica carinata Braun

Protoplasts of Brassica carinata Braun. (accession No. 84A165) were enzymatically isolated from hypocotyls and cotyledons of 3–5 day-old test-tube seedlings or first true leaves taken from greenhouse grown plants at a three-leaf stage. The protoplasts were suspended in a P-B liquid medium solidified with 0.15%–0.3% low melting agarose which formed a thin layer floating on the surface of the liquid medium. The optimum protoplast density was ranging from 5× l03 to 1 × 104/ml. As for the hypocotyl protoplasts, the first division was observed after 48 h in the culture. The division frequency reached 21% and 34% at day 3 and 6 respectively. The initiation of cell division in the case of cotyledon and mesophyll protoplast culture was late, usually at day 5, and the division frequency was also somewhat lower. One week after culture, the cultures were transferred to fluorescent light condition with an intensity of about 1000 lx. A dilution medium DPDK3 was then added and the dilution procedure was repeated at one week interval thereafter. One month after culture, microcalli with 300–500 μm in size were formed. It was also found that in some cases globular embryoid structure protruded on the callus surface. Totally, a 2%–3% plating efficiency was achieved. Shoot regeneration occurred when cotyledon and mesophyll protoplast-derived calli were transferred onto a modified MS medium supplemented with NAA 0.1, BA 3 mg/l. Individual shoots were rooted on a rooting medium supplemented with 0.2 mg/l of IAA. Intact plants with normal morphology were eventually produced.     

An efficient and rapid procedure for plantlet regeneration from chicory mesophyll protoplasts

An efficient procedure for plantlet regeneration from chicory mesophyll protoplasts has been developed in order to perform protoplast fusion experiments. Protoplasts were isolated from a genotype of Italian red chicory (CH 363) and purified by centrifugation in a solution containing 13% (w/v) sucrose to collect uniform protoplasts in size. After 2 days culture at a density of 2×10⁴ protoplasts ml⁻¹ of liquid medium, protoplasts were cultured following three different procedures: in liquid medium, stratified in semi-solid medium, and embedded in Ca-alginate droplets. Four different media were used and culture procedures were evaluated recording the protoplast viability, protoplast division frequency and plating efficiency for each experiment. The embedding of protoplasts in Ca-alginate droplets enhanced both division frequency and plating efficiency for chicory mesophyll cells. Furthermore, this procedure shortened the cycle of plant regeneration from protoplasts, which could be completed in eight weeks. This revised version was published online in June 2006 with corrections to the Cover Date.     

Winged-bean protoplasts: Isolation and culture to callus

A system was developed for protoplast isolation and culture from suspension cultured cells of winged bean,Psophocarpus tetragonolobus. Cells from a three-day-old suspension were incubated in an enzyme mixture containing 6% cullulysin, 1% Macerase, 1% desalted Rhozyme, 0.4M sorbitol, and 0.1M CaCl₂ at pH 5.5. Average yields of protoplasts were 6.5 × 10⁶ per gram fresh weight of cells. Protoplasts were cultured in modified B5 medium containing 68.4 g/l glucose, 250 mg/l xylose, 0.1 mg/l 2,4-D, 0.5 mg/l BAP, 250 mg/l N-Z amine type AS, and 20 ml/l coconut water. After 24 h of culture, the protoplasts had synthesized a new wall, and in three days had begun division. The optimum plating density was 1–2 × 10³ protoplasts/ml. The division frequency ranged between 40%–60% for most experiments with a high of 72% in one experiment. After three weeks, cell colonies could be transferred to solid MS medium containing N-Z amine and coconut water where callus developed. This protoplast system is technically comparable to soybean for experiments concerned with genetic manipulation involving legumes.     

Isolation,culture and plantlet regeneration from cotyledon and mesophyll protoplasts of two pickling cucumber (Cucumis sativus L.) genotypes

Optimal protoplast yields from cotyledons (2.0×10⁶ protoplasts/ 0.5 g tissue) and from true leaves (5.0×10⁶ protoplasts/g tissue) of two Cucumis sativus genotypes were obtained following a 16 h digestion with, respectively, 1.25% pectinase+0.5% Cellulysin and 0.5 % pectinase+ 1.0% Cellulysin. Enzyme solutions were prepared in modified MS medium containing half-strength major salts, full complement of minor salts and vitamins, 2% sucrose and 0.25 M mannitol. A plating density of 3.5–4.0× 10⁴ protoplasts/ml or higher was required for sustained division, with first division occurring in 6–7 days, second-third division in 8–9 days, and minicalli formation by day 13. Embedding in 0.4% agarose provided the highest plating efficiency (proportion that formed minicalli) of mesophyll protoplasts, which was 28.3% for genotype 3672 and 15% for genotype 3676. By comparison, liquid culture and droplet culture gave lower plating efficiencies (10–19%). Cotyledon and mesophyll protoplasts of one genotype formed minicalli on MS medium containing 2,4-D/BA at 1.0/2.5 M and 5.0/5.0 M, respectively, within 21 days, while mesophyll protoplasts of the second genotype formed minicalli on MS medium containing NAA/BA at 5.0/5.0 M within 12 days. Shoot buds or somatic embryos were obtained upon subculture of calli to MS medium containing lower concentrations (0.05–0.01 M) of 2,4-D/BA or NAA/BA and a few plantlets, ca.18, were recovered on hormone-free medium.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog medium - NAA naphthaleneacetic acid     

Plant regeneration from protoplasts of mango (Mangifera indica L.) through somatic embryogenesis

 This report describes a protocol for the regeneration of plants from protoplasts isolated from proembryogenic masses (PEMs) in a suspension culture derived from the nucellar callus of mango (Mangifera indica L. cv 'Amrapali'). The maximum yield (24.6±1.1×10⁶), with 81.04±4.1% viable protoplasts per gram PEMs, was obtained with an enzyme mixture containing 1.2% cellulase, 1.0% hemicellulase and 0.6% pectinase. An optimum density of 5×10⁴ cultured protoplasts per milliliter culture medium was required for the highest frequency (88.89±5.40%) of division. Dividing protoplasts developed into microcalli that proliferated on medium supplemented with growth regulators (auxins or kinetin alone, or auxins with kinetin) and produced somatic embryos after transfer to a growth regulator-free medium. The protocallus on 2,4-D-containing medium produced the maximum number (102.50±6.93) of somatic embryos. Maturation of somatic embryos depended upon the presence, and the nature and combination of growth regulators in the medium during proliferation of the callus. The mature somatic embryos germinated and developed into plants that were transferred to soil. Received: 1 April 1999 / Revision received: 27 July 1999 / Accepted: 23 August 1999     

Differentiation in calli originated from isolated protoplasts of rice (Oryza sativa L.) through plating technique

Summary Protoplasts from mesophyll cells and callus cells of rice (Oryza sativa L.) have been isolated by enzyme treatment involving 2% pectinase followed by 3% cellulase at pH 5.4 in 0.45 M mannitol (viable protoplasts from mesophyll cells in 50–60% yield, 60–70% yield of viable protoplasts from callus cells through treatment with the mixture of the above mentioned enzymes at the same concentration). Our completely defined medium is the combination of three established media (Table 1). Culture conditions are: soft agar in petri dishes at 26° C, where they regenerated cell walls after 24 h. The first cell division was observed after 4 days in culture for callus protoplasts and after 5 days in culture for mesophyll protoplasts. Cell division continues thereafter, and after 4 weeks of culture small white calli were visible in the petri dishes. The type of plant material (whitish leaf sheaths) and cell density are important factors for the efficiency of colony formation (30% plating efficiency). Healthy root formation through transfer to suitable medium is up to now the morphogenetic reaction of the calli.Work carried out at Molecular Cytogenetics Research Unit, Deptt, of Genetics and Plant Breeding, Banaras Hindu University, Varanasi, India     

Electrical fusion for optimal formation of protoplast heterokaryons in Nicotiana

The electrical fusion of protoplasts has been studied in order to maximize the formation of heterokaryons for culture. Heterokaryons of Nicotiana tabacum L. mesophyll protoplasts and N. plumbaginifolia Viviani supension-cell protoplasts were identified in fixed and stained as well as living material; a quantitative fusion index was thereby developed. With this index the efficiencies of various electric fields and fusion-chamber designs have been determined. Optimal fusion was obtained with an alternating-current (AC) field of 150 V/cm and direct-current (DC) square-wave pulses of 1000 V/cm. A new, simple-to-use, largescale fusion chamber is described in which batches of up to 5·10⁵ protoplasts (0.5 ml of cells at 10⁶/ml) can be fused in 5–7 min with efficiencies approaching 40%. Half of the fusion products are heterokaryons, thus fusion is random. Of the fusion products, 60% are bi- or trinucleate. Using fusion procedures similar to those described here Bates and C. Hasenkampf (1985, Theor. Appl. Genet., in press) have recovered viable somatic hybrids which have been regenerated.Abbreviations AC alternating current - DC direct current - PEG polyethylene glycol