抗氧化剂对苜蓿原生质体早期培养的影响

研究发现,分离原生质体的酶解脱壁处理可以诱导苜蓿细胞产生活氧。培养基中添加抗氧化剂,有助于提高培养原生质体的分裂频率,缓解褐化现象的出现。经紫外照射处理的培养基不利于苜蓿原生质体的生长和分裂,添加抗氧化剂后,紫外辐射所引起的不良效应则被抵消。因而,通过抗氧化剂对活性氧的清除,有助于早期原生质体的培养。     

谷子胚性愈伤组织粘液提取物对原生质体培养的影响

将谷子胚性愈伤组织粘液提取物添加到谷子原生质体培养基中,其对原生质体培养的影响表明该提取物有助于原生质体形成细胞壁;并且该类有粘液分泌的念伤组织的原生质体游离所需的酶液浓度低、处理时间短。由原生质体形成完整细胞的数量在一定范围内与谷子原生质体培养的植板率相对应;通过增加形成完整细胞的数量可较大幅度地提高原生质体培养的植板率。     

谷子原生质体的分离和培养(简报)

取继代培养后4—5天的谷子(Setariaitalica,品种豫谷一号)悬浮培养细胞,用含2%纤维素酶(Onozuka Rs)和0.1%果胶酶(Pectolyase Y 23)的酶溶液酶解分离原生质体。原生质体纯化后的产量在2—6×10~6原生质体/克鲜重。原生质体在培养2天后形成细胞壁并开始进行分裂。在培养6天时的分裂频率达5—12%。此后随添加新鲜培养基并降低培养基的渗透压,形成细胞团。培养一个月后,可将可见的细胞团转移到附加2,4-D和激动素的MS琼脂培养基上,即形成旺盛生长的愈伤组织。     

培养基对烟草叶肉原生质体再生细胞的分裂及植株再生的影响

以普通烟草的一种香料烟品种为材料,用国产酶一步法游离叶肉原生质体。以液体浅层静置培养的方法培养原生质体,14天左右添加一次新鲜的不含甘露醇的培养液,使渗透压减半。以后转入完全没有甘露醇的固体培养基里。将形成的直径为1—2毫米的小愈伤组织转至分化培养基,再生了完整植株。而后将苗转至花盆里能正常生长。 比较了原生质体培养基的不同成分,发现将大量元素中两个成分的量略作改变,并增加水解乳蛋白及一些有机成分能加速分裂速度,分裂频率由对照NT培养基的50％左右提高到80％以上。讨论了出芽现象及培养条件对培养的影响。     

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。     

抗坏血酸对沙打旺原生质体分离和培养中膜损伤及相关酶活性的影响

抗坏血酸（ＡＳＡ） 能减轻沙打旺原生质体的褐化,改善原生质体的培养状况。ＡＳＡ的作用可能与它增强原生质体抗过氧化能力有关。酶解处理诱导原生质体超氧化物歧化酶（ＳＯＤ） 和抗坏血酸过氧化物酶（ＡＰＸ）活性升高,但培养过程使ＡＰＸ 活性明显下降,原生质体清除过氧化物能力减弱,膜脂过氧化产物丙二醛（ ＭＤＡ） 积累增加,膜发生损伤。向酶溶液和培养基中添加ＡＳＡ 可显著提高ＳＯＤ 尤其是ＡＰＸ 活性,减轻膜脂过氧化,增强原生质体的存活力,促进原生质体的分裂和细胞克隆的形成。所有处理中过氧化氢酶（ＣＡＴ） 活性变化不大,表明它在原生质体清除过氧化物过程中不具主要作用。     

小偃麦原生质体培养及植株再生

硬粒小麦(Triticum durum Desf.AABB)和中间偃麦革[Elytrigia intermedium(Host)Nevski BBEEFF]的杂种 F_1——小偃麦的幼穗诱导的胚性愈伤组织继代培养近两年后,转入修改的 MS 液体培养基建成胚性细胞悬浮系。从此悬浮系分离的原生质体在修改的 KM_(8p)培养基中培养48小时后出现第一次分裂。15天后,在液体浅层培养条件下的细胞分裂频率为2%;而用1.2%琼脂糖固化进行固体平板培养时,细胞的分裂频率则为12.14%。20—30天后,添加渗透压降低的原生质体培养液。当从原生质体再生的愈伤组织长至2—4mm 大小时,逐步转至生长及分化培养基上再生出完整植株。     

霞草原生质体培养和植株再生

用霞草胚性悬浮细胞分离原生质体,以含0.2%琼脂糖的KM 8p培养基薄层漂浮培养,原生质体培养密度6×10~3-1×10~4/ml。培养3天再生细胞开始分裂,7天统计分裂频率最高达25.4%,10天形成小细胞团,并加降低渗透压的稀释培养基,每周一次。20—25天形成肉眼可见的小愈伤组织,植板率达3.5%。原生质体衍生的愈伤组织在增殖培养时加入0.3%-0.4%活性炭有利于生长及分化。在含6-BA 3.5 mg/L,IBA 0.8 mg/L的培养基上,再生芽的分化频率可达85%。再生芽在添加NAA 0.5 mg/L,6-BA 0.05 mg/L的1/2 MS生根培养基中2周内形成具根的再生小植株。     

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     

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。     

结球白菜下胚轴原生质体培养及其体细胞胚植株再生

结球白菜（Brassica campestris ssp．pekinensis）的原生质体培养由于基因型依赖性强,细胞易褐化,愈伤组织的芽诱导率低等而难于再生植株。本实验以结球白菜的下胚轴原生质体为试材,研究了影响其细胞分裂及愈伤组织形成的因素,探索了经过体细胞胚发生途径获得再生植株的技术。结果表明,试材的基因型及培养基组成影响细胞分裂及褐化;KM8P是结球白菜原生质体培养更适宜的培养基,能显著减轻细胞的褐化;液体培养基中一定浓度的活性炭能在一定程度上减轻细胞褐化进程,并有利于星状细胞团的形成;基因型Asko中,愈伤组织形成体细胞胚的结构,其发生的频率约为5％,该类体细胞胚能全部顺利地发育成完整植株。本技术具有再生植株形成容易、频率较高且通过体细胞胚发生途径等优点。     

结球白菜下胚轴原生质体培养及其体细胞胚植株再生

结球白菜(Brassica campestris ssp. pekinensis)的原生质体培养由于基因型依赖性强, 细胞易褐化,愈伤组织的芽诱导率低等而难于再生植株。本实验以结球白菜的下胚轴原生质体为试材, 研究了影响其细胞分裂及愈伤组织形成的因素, 探索了经过体细胞胚发生途径获得再生植株的技术。结果表明, 试材的基因型及培养基组成影响细胞分裂及褐化; KM8P是结球白菜原生质体培养更适宜的培养基, 能显著减轻细胞的褐化; 液体培养基中一定浓度的活性炭能在一定程度上减轻细胞褐化进程, 并有利于星状细胞团的形成; 基因型Asko中, 愈伤组织形成体细胞胚的结构, 其发生的频率约为5%, 该类体细胞胚能全部顺利地发育成完整植株。本技术具有再生植株形成容易、频率较高且通过体细胞胚发生途径等优点。     

Removal of browning and growth enhancement by polyvinylpolypyrrolidone in protoplast cultures ofCyamopsis tetragonoloba L.

The occurrence of browning in protoplast cultures ofCyamopsis tetragonoloba completely inhibited the growth of protoplast derived colonies. Of the various additives employed to counteract the problem of browning and subsequent necrosis, polyvinylpolypyrrolidone (PVPP) was found most effective. Simultaneous addition of polyvinylpyrrolidone (PVP) to the protoplast culture medium accentuated the effect of PVPP and also improved the frequency of protoplast division.     

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.     

籼稻原生质体培养和植株再生

用籼稻IR52、IR8和IR45的幼花序和幼胚愈伤组织在LS培养基建立了稳定的悬浮培养物。悬浮系的建立经历三个阶段:褐变期,长根期,成熟期。建立了适合籼稻原生质体生长的Y8培养基,其植板率显著高于KPR和PCM培养基。悬浮细胞系间差异明显,只有部份系可以提供有分裂能力的原生质体或具看护活性。以上三个品种的原生质体均分裂良好,但只有IR52和IR8分化出苗,其中IR52分化率1.25%,得再生植株50余株,移至田间生长结实正常。     

Protoplast culture and plant regeneration ofPinellia ternata

A study was undertaken to develop a protoplast regeneration system for pinellia. A yield of 19 29 x 10⁵ protoplasts/g F. W. could be obtained from cell suspension cultures incubated in a digestion enzyme solution with 2% cellulase Onzuka R-10, 10% pectinase (Sigma), 0.01% pectolyase Y23. K8P and modified MS media were used to culture protoplasts in: a) liquid, b) liquid-solid double layer, or c) agarose embedded protoplast culture. The former two were conducive to colony formation from protoplast-derived cells. The frequency of cell division was about 8% after 3 days in culture. Gradually adding fresh medium of lower osmotic pressure into the medium for protoplast culture favored cell division. Calli (1–2 mm in diameter) formed after 30–40 days in culture. The calli transferred onto medium supplemented with KT (0.5 mg 1^–1) and NAA (0.2 mg 1)^–1) could regenerate plants after 40–50 days. Of 47 plantlets transplanted into plots, 29 flowered and were fertile.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - KT kinetin - CH casein hydrolysate     

Fertile wheat (Triticum aestivum L.) regenerants from protoplasts of embryogenic suspension culture

We report regeneration of fertile, green plants from wheat (Triticum aestivum L. cv. Aura) protoplasts isolated from an embryogenic suspension initiated from somatic early-embryogenic callus. The present approach combines the optimization of protoplast culture conditions with screening for responsive genotypes. In addition to the dominant effect of the culture media, the increase in fresh mass and the embryogenic potential of somatic callus cultures varied considerably between the various genotypes tested. Establishment of suspension cultures with the required characters for protoplast isolation was improved by reduction of the ratio between cells and medium and by less frequent (monthly) transfer into fresh medium. A new washing solution was introduced to avoid the aggregation of protoplasts. However, the influence of the culture medium on cell division was variable in the different genotypes. We could identify cultures from cultivar Aura that showed approximately a 9% cell division frequency and morphogenic response. The protoplast-derived microcolonies formed both early and late-embryogenic callus on regeneration medium and green fertile plants were obtained through somatic embryogenesis. The reproducibility of plant regeneration from protoplast culture based on the cultivar Aura was demonstrated by several independent experiments. The maintenance of regeneration potential in Aura suspension cultures required establishment of new cultures within a 9-month period.     

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.     

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。     

Differentiation of potato (Solanum tuberosum L.) plants from cultured leaf protoplasts

Through induction of totipotent stage in cultured cells plants were regenerated from protoplast derived callus colonies of potato. Growing of the plants in vitro with optimal aeration and pretreatment of the leaves in dark and cold prior to protoplast isolation has improved the protoplast yield and frequency of cell division. Protoplasts of four potato genotypes have started to divide within 2-5 days after plating them into culture medium containing 2,4-D (0.2 mg/l); ZEA (0.5 mg/l); and NAA (1 mg/l) as growth regulators. Embedding of the cells into agarose proved to be favourable to avoid cell browning and to increase colony formation. The series of hormone treatments based on complex action of NAA and BAP promoting colony growth and greening, ZEA and IAA inducing shoot redifferentiation, and GA3 plus NAA supporting shoot elongation and rooting, finally resulted in high frequency of plant regeneration from microcolonies.