花烟草叶肉原生质体再生植株

用自制的纤维素酶(EA3-867)从花烟草(Nicotiana alata)叶肉细胞制备大量有活力的原生质体。在NT培养基(内含2,4-D2,KT0.25毫克/升)上观察到原生质体长大,分裂,形成愈伤组织。愈伤组织悬浮在含有2,4-D2mg/升的MS培养基上诱导出球形胚,移入MS(BA2,IAA 0.2 mg/l)培养基上出苗。小苗移植土壤中正常生长、开花、结实。     

北美鹅掌楸原生质体的分离与培养

以鹅掌楸属植物北美鹅掌楸的悬浮细胞和组培苗叶片为材料,对北美鹅掌楸原生质体分离、纯化与培养条件进行研究.结果表明:叶片和悬浮细胞用含有0.1％2-吗啉乙磺酸(MES)和0.6 mol/L甘露醇的Cell ProtoplastWash(60M-CPW)溶液25℃预处理lh效果最好;悬浮细胞最佳酶解液为60M-CPW+ 1％纤维素酶+1％半纤维素酶+0.2％果胶酶Y-23+0.1％ MES,每克材料25℃酶解6h有效原生质体产量可以达到3×106个;叶片最佳酶解液为60M-CPW+2％纤维素酶+1％半纤维素酶+0.2％果胶酶Y-23+0.1％ MES,每克材料25℃酶解10 h有效原生质体产量可以达到11×106个;悬浮细胞原生质体易于培养,在KM8p+1.0 mg/L 2,4-D+0.5 mg/L 6-BA培养基中培养25 d可形成肉眼可见的愈伤组织.     

沙打旺原生质体培养再生植株

用1%半纤维素酶,0.4%纤维素酶,0.1%果胶离析酶,CPW9M酶液分离沙打旺无菌苗下胚轴和子叶原生质体。K8P原生质体培养基悬滴培养。下胚轴原生质体形成小细胞团后用琼脂糖包埋培养,形成小块愈伤组织后转入增殖培养基M1、M2(改良MS培养基)上形成大块愈伤组织。经过两次诱导分化,在分化培养基M3(MS 0.7mg/L BA 0.2mg/L NAA),M4(MS 0.5mg/L BA 0.5mg/L KT 0.5mg/L ZT 0.2mg/L NAA)和M6(MS 3mg/L ZT 0.2mg/L IAA)上分化出苗,再生植株。由子叶分离的原生质体未能形成愈伤组织。     

玉米原生质体的植株再生

以玉米花粉诱导产生的胚性愈伤组织,在 N6基本培养基附加激动素2 mg/l,6-苄基氨基嘌呤1mg/l,2,4-D 0.3 mg/l,水解酪蛋白500 mg*l 及谷酰胺250 mg/l 的培养基上进行转代培养。用转代培养一年半后的胚性愈伤组织分离原生质体,原生质体培养在附加激动素0.2 mg/l,6-苄基氨基嘌呤0.1 mg/l,2,4-D 0.5 mg/1,水解酪蛋白200 mg/l,谷酰胺100 mg/l及椰乳296的原生质体培养基 Z_2中。培养4—6天后,原生质体的再生细胞进行第一次分裂;培养3星期后发育成肉眼可见的小愈伤组织。此后,需添加降低糖浓度的同样原生质体培养基 Z_2共两次。待再生愈伤组织长到直径2—4 mm 大小时,把它们先后转经第一及第二(即Z_3及 Z_4)分化培养基上诱导器官分化。最后在 Z_4分化培养基上同时有胚状体的发生及植株的分化。     

甘蓝型油菜与芝麻菜体的细胞杂交

为拓宽油菜育种的基因资源库, 改良油菜品种, 以甘蓝型油菜(Brassica napus)花油3号下胚轴和芝麻菜(Eruca sativa)下胚轴为材料分离制备原生质体; 然后采用PEG-高Ca2+-高pH法进行原生质体融合, 当PEG浓度为35%, 原生质体融合密度为5×105个/mL时, 融合25 min时, 融合率可达18.2%。融合后在培养密度为1×105个/mL时, 以附加1.0 mg/L 2,4-D +0.5 mg/L 6-BA+0.5 mg/L NAA+ 200 mg/L肌醇+300 mg/L水解酪蛋白的改良的KM8p为融合体培养基, 以0.1 mol/L 蔗糖+0.2 mol/L葡萄糖+0.2 mol/L甘露醇作渗透稳定剂进行液体浅层培养, 效果较好, 愈伤组织再生率最高为6.8%。将融合体再生的小愈伤组织转移至培养基(B5无机盐+0.087 mol/L蔗糖+0.2 mg/L 2, 4-D+0.5 mg/L NAA+0.2 mg/L 6-BA+ 0.5% Agar, pH 5.8)上增殖培养, 待愈伤组织长至直径为3~5 mm时, 及时将其转至分化培养基(MS无机盐+0.087 mol/L 蔗糖+0.1 mg/L IAA+0.8 mg/L 6-BA+0.8% Agar, pH 5.8)中诱导不定芽再生, 芽分化率为35.7%。当不定芽长为2~3 cm时, 将其切下转入附加0.5 mg/L IBA+0.2 mg/L 6-BA的1/2MS生根培养基中诱导生根, 14 d左右即可形成再生植株, 生根率可达88%。同时, 以紫外线(60 μW/cm2)照射芝麻菜原生质体, 进行不对称融合, 照射2 min的获得了愈伤组织和再生植株, 照射4 min的只获得愈伤组织, 而照射5 min以上的没有获得愈伤组织, 但其愈伤组织再生、增殖及植株再生均不如对称融合。从细胞学鉴定的21块杂种愈伤组织上再生出16株杂种植株。     

甘蓝型油菜与芝麻菜体的细胞杂交

为拓宽油菜育种的基因资源库, 改良油菜品种, 以甘蓝型油菜(Brassica napus)花油3号下胚轴和芝麻菜(Eruca sativa)下胚轴为材料分离制备原生质体; 然后采用PEG-高Ca2+-高pH法进行原生质体融合, 当PEG浓度为35%, 原生质体融合密度为5×105个/mL时, 融合25 min时, 融合率可达18.2%。融合后在培养密度为1×105个/mL时, 以附加1.0 mg/L 2,4-D +0.5 mg/L 6-BA+0.5 mg/L NAA+ 200 mg/L肌醇+300 mg/L水解酪蛋白的改良的KM8p为融合体培养基, 以0.1 mol/L 蔗糖+0.2 mol/L葡萄糖+0.2 mol/L甘露醇作渗透稳定剂进行液体浅层培养, 效果较好, 愈伤组织再生率最高为6.8%。将融合体再生的小愈伤组织转移至培养基(B5无机盐+0.087 mol/L蔗糖+0.2 mg/L 2, 4-D+0.5 mg/L NAA+0.2 mg/L 6-BA+ 0.5% Agar, pH 5.8)上增殖培养, 待愈伤组织长至直径为3~5 mm时, 及时将其转至分化培养基(MS无机盐+0.087 mol/L 蔗糖+0.1 mg/L IAA+0.8 mg/L 6-BA+0.8% Agar, pH 5.8)中诱导不定芽再生, 芽分化率为35.7%。当不定芽长为2~3 cm时, 将其切下转入附加0.5 mg/L IBA+0.2 mg/L 6-BA的1/2MS生根培养基中诱导生根, 14 d左右即可形成再生植株, 生根率可达88%。同时, 以紫外线(60 μW/cm2)照射芝麻菜原生质体, 进行不对称融合, 照射2 min的获得了愈伤组织和再生植株, 照射4 min的只获得愈伤组织, 而照射5 min以上的没有获得愈伤组织, 但其愈伤组织再生、增殖及植株再生均不如对称融合。从细胞学鉴定的21块杂种愈伤组织上再生出16株杂种植株。     

茎用芥菜细胞质雄性不育系子叶原生质体培养和高效植株再生

利用茎用芥菜细胞质雄性不育系原生质体培养获得了再生植株,并研究了影响原生质体培养的因素.结果表明,子叶是茎用芥菜原生质体培养最佳的外植体,10 d苗龄的子叶原生质体在改良MS培养基上培养3 d后发生第1次细胞分裂,6 d后发生第2次分裂,3周后形成细胞团,5周后形成肉眼可见的小愈伤.培养基中缺少NAA或2,4-D都会降低愈伤组织的再生能力.在含一定浓度的NAA(0.25 mg/L)和2,4-D(0.25 mg/L)培养基上诱导的愈伤组织质地致密且有光泽,芽的分化能力高;在MS+BA l mg/L+NAA 0.2 mg/L的培养基上芽的分化频率高达近29%,再生芽在1/2MS+NAA0.1 mg/L培养基上生根,形成完整植株.     

茎用芥菜细胞质雄性不育系子叶原生质体培养和高效植株再生（英文）

利用茎用芥菜细胞质雄性不育系原生质体培养获得了再生植株,并研究了影响原生质体培养的因素.结果表明,子叶是茎用芥菜原生质体培养最佳的外植体,10 d苗龄的子叶原生质体在改良MS培养基上培养3 d后发生第1次细胞分裂,6 d后发生第2次分裂,3周后形成细胞团,5周后形成肉眼可见的小愈伤.培养基中缺少NAA或2,4-D都会降低愈伤组织的再生能力.在含一定浓度的NAA(0.25 mg/L)和2,4-D(0.25 mg/L)培养基上诱导的愈伤组织质地致密且有光泽,芽的分化能力高;在MS+BA l mg/L+NAA 0.2 mg/L的培养基上芽的分化频率高达近29%,再生芽在1/2MS+NAA0.1 mg/L培养基上生根,形成完整植株.     

马铃薯叶肉原生质体再生植株的研究

马铃薯两个品系小叶子x多子白和乌盟601的叶肉原生质体在原生质体培养基中诱导出愈伤组织,叶肉原生质体来源的愈伤组织转移到MS＋2mg/1ZT+0.1mg/1 IAA培养基中,培养至70天以后,开始发生芽的分化,待芽生长到2－3cm高度时,转入MS＋0．05mg/1 NAA培养基中,很快出根长成完整植株,带1－2片叶的茎段移栽入灭菌的混合土壤中生长并结出薯块。     

鹰嘴紫云英甲硫氨酸抗性系原生质体培养及植株再生

本研究建立了鹰嘴紫云英(AstragaluscicerL.)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,通过酶法游离出大量有活力的原生质体。原生质体经培养持续细胞分裂形成了愈伤组织,并分化出再生苗。比较了不同培养基、培养密度对原生质体形成细胞分裂和再生的影响。结果表明,原生质体以2×105个/ml的植板密度,在附加2.0mg/L2,4-二氯苯氧乙酸(2,4-D)、0.2mg/L6-苄氨基嘌呤(6-BA)、200mg/L水解酪蛋白、2%蔗糖和0.3mol/L甘露醇DPD培养基中培养后,其分裂频率达38.3%。原生质体培养形成的愈伤组织仍具有对甲硫氨酸的抗性。转移到附加10mg/LKT、0.5mg/LNAA的MS分化培养基上,获得大量的再生苗。     

Somatic embryogenesis and plant regeneration from protoplasts of asparagus (Asparagus officinalis L.)

Protoplasts were isolated from embryogenic calli of Asparagus officinalis L. cv. Mary Washington and cultured in 1/2 MS medium with 1 mg/l NAA, 0.5 mg/l zeatin, 1 g/l L-glutamine, 0.6 M glucose and 0.1% Gellan Gum. Protoplasts started to divide after 3–4 d of culture and formed visible colonies after 30 d of culture. The percentage of colony formation (plating efficiency) was 7.2%. The colonies were then transferred onto Gellan Gum-solidified MS medium containing 1 mg/l 2,4-D and 3% sucrose for further growth. Somatic embryos were induced from all colonies of 0.5–1.0 mm size after transferring to 1/2 MS medium lacking growth regulators. After treating these somatic embryos (1–3 mm) in distilled water for a week, 30–40% of them germinated normally and grew into plantlets 20–30 d after transplanting on 1/2 MS medium containing 1 mg/l IBA, 1 mg/l GA₃ and 1% sucrose. These protoplast-derived plants were diploid with 20 chromosomes.Abbreviations BA 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - GA₃ gibberellic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962)     

Plant Regeneration from Protoplasts of Suspension Cells of Saposhnilkovia divaricata (Turcz.) Schischk

Embryogenic calli were produced from the segments of the young roots, hypocotyls or petioles of test-tube seedlings on MS agar medium containing 1 mg/1 2,4-D. When shaken in the MS liquid medium, the calli formed cell suspension with many embryogenic cell clumps.Using the enzyme mixture: Onozuka R-10 1.5%+MacerozymeR-10 0.3%+Snailase 0.5%+CaCl2 5 mmol/l + Mannitol 0.6 mol/1 (pH=5.8), protoplasts were obtained from the cell clumps which had been subcultured for three to' seven days. When cultivated, the protoplasts grew and began to divide after four days, and formed cell clumps about l—2 mm within fifty days. Protoplast-derived calli were formed from the cell clumps on the MS agar medium with 0.5 mg/l 2,4-D. When transferred onto the MS agar medium containing 0.1 mg/1 6-BA or 0.1 mg/1 2,4-D and 0.5 mg/1 6-BA, the calli differentiated into embryoids. On the MS agar medium without phytohormone, the embryoids grew into plantlets.     

Plant regeneration from protoplasts of immature Vigna sinensis cotyledons via somatic embryogenesis

Summary Protoplasts were isolated from immature cotyledons of Vigna sinensis and cultured in a modified MS Liquid medium containing 0. 2 mg/l 2, 4-dichlorophenoxyacetic acid (2, 4-D), 1 mg/l naphthaleneacetic acid (NAA) and 0. 5 mg/l 6-benzylaminopurine (BAP) in the dark at a density of 1 × 10⁵/ml. The protoplasts began to divide in 3–5 days. Sustained cell division resulted in formation of cell clusters and small calli, with the cell division frequency and plating efficiency of cell colonies reaching 27. 7% and 1. 7% respectively. When calli of 2 mm in size were transferred onto MSB medium (MS salts and B5 vitamins) containing 500mg/l NaCl, 500 mg/ 1 casein hydrolysate (CH), 2 mg/l 2,4-D and 0. 5 mg/l BAP for further growth, approximately 5% of the calli developed embryogenically. The embryogenic calli were selected and subcultured on the same composition of MSB medium and were able to maintain somatic embryogenesis capacity in subculture for a long time. When the calli were moved to MSB medium with 0. 1 mg/l indole-3-acetic acid (IAA), 0. 5mg/l kinetin(KT), 3–5% mannitol and 2% sucrose in the light, many somatic embryos formed from the calli. Only part of the embryoids developed further to the cotyledonary stage, and the others died at the globular, heart-shaped or torpedo stages. Finally, some cotyledonary embryoids germinated and developed into plants or shoots. The shoots were readily rooted on 1/2 strength MS medium with 0. 1–0.3 mg/l indole-3-butyric acid (IBA). The plants grew well in soil and were fertile.Abbreviations 2, 4-D dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - BAP 6-benzylaminopurine - IAA indole-3-acetic acid - KT kinetin - IBA indole-3-butyric acid - CH casein hydrolysate - CM coconut milk - ZT zeatin     

白Pian体细胞胚悬浮培养的动力学研究

白(ＰｉｃｅａｍｅｙｅｒｉＲｅｈｄ.ｅｔＷｉｌｓ.)是我国特有的云杉属树种,在林业生产和环境绿化中均具有重要地位。其体细胞胚胎发生的研究,一方面可用于优良种质的大规模快速繁殖,为植树造林和园林绿化提供优质苗木;另一方面可作为遗传转化的再生系统,进行树种遗传...     

江西铅山红芽芋胚性愈伤组织的包埋玻璃化超低温保存

为长期安全保存江西铅山红芽芋种质资源,本文以江西铅山红芽芋的胚性愈伤组织为对象,研究了包埋玻璃化冻存过程中各因素对细胞活力和愈伤组织成活率的影响,优化建立了江西铅山红芽芋胚性愈伤组织包埋玻璃化超低温保存体系。将约0.2 g胚性愈伤组织块包埋成海藻酸钙凝胶珠后,在25℃下转入MS+2 mg/L TDZ+1 mg/L NAA+0.75 mol/L蔗糖的培养基中于14 h/d光周期下预培养1 d;预培养后的胚性愈伤组织块用2 mol/L甘油和0.4 mol/L蔗糖的混合物在25℃下装载40 min;采用PVS2在25℃下脱水30 min,更换PVS2后直接投入液氮保存1 d;再将胚性愈伤组织块置于37℃恒温水浴中化冻3 min,然后用MS+2 mg/L TDZ+1 mg/L NAA+1.2 mol/L蔗糖的液体培养基洗涤3次,每次10 min;洗涤后的胚性愈伤组块转入MS+2 mg/L TDZ+1mg/L NAA固体培养基上先暗培养7 d再转到14 h/d光周期中培养。7 d后胚性愈伤组织块开始恢复生长,并且在30 d内分化出胚状体;将胚状体再次转入MS+2 mg/L TDZ+1 mg/L NAA固体培养基上,60 d后形成完整的植株。红芽芋胚性愈伤组织包埋玻璃化超低温保存后的平均成活率约为60%,并且红芽芋胚性愈伤组织冻后再生苗没有发生形态性状和染色体数目的变异,此结果为长期安全保存江西铅山红芽芋种质资源奠定了良好的基础。     

Somatic embryogenesis and plant regeneration from immature seed-derived calli of rugosa rose (Rosa rugosa Thunb.)

A procedure for plant regeneration from immature seed-derived calli of rugosa rose (Rosa rugosa Thunb.) via somatic embryogenesis is described. Embryogenic calli were initiated from immature seeds 2–3 weeks after anthesis on Murashige and Skoog (MS) medium without growth regulators. Induced calli had a white, friable and nodular appearance with several proembryos. These calli were subcultured at 20-day intervals on MS medium containing 0.1–0.2 M galactose on which they grew rapidly; but somatic embryogenesis was inhibited. Somatic embryos were again induced from the subcultured calli after transferring to MS medium containing 0.1 M M fructose or sucrose but lacking growth regulators. After transferring these embryos (1–2 mm) to MS medium containing 0.1 M sorbitol, 3% of them germinated and grew into plantlets which showed sustained growth on the MS medium containing only 0.1 M sorbitol as the sole carbon source.     

Plant Regeneration from Protoplasts of Ligusticurn wallichii Franch

The hypocotyls of the embryoid derived plantlets of Ligusticum wallichii Franch were used for protoplast preparation. Protoplasts were obtained with the enzyme mixture containing 1.5% Onozuka R-10, 0.3% Macerozyme R-10, 0.5% Snailase, 5 mmol/l CaCl2, 1 mmol/l KH2PO4, 0.6 mol/l manitol, at pH 5.6–5.8 and 27℃. Protoplasts were cultured in a modified MS liquid medium containing 1 mg/l 2,4-D + 0.5 mg/l 6- BA. The first divisions were found after twelve days, and the dividing cells formed cell colonies of 0.5–1 mm after about fourty days. When they were transferred to MS agar medium (with half quantity of macronutrients) supplemented with 2,4-D (0.5mg/l) and 6-BA(0.5mg/l), they grew into calli. At last, on the medium without any phytohormones, the growing calli differentiated embryoids which developed into plantlets with many green leaves and roots.     

Protoplasts Culture and Plant Regeneration of Soybean (Glycine max L. and G. soja)

Protoplasts were isolated enzymatically from immature cotyledons of soybean. The protoplasts divided to form calli in the K8P liquid medium. The calli further grew to 2–3 mm on the solid K8 medium and were transferred onto the MSB medium (MS minerals+B5 organic components+0.5–1.0 mg/l 2,4-D+0.2–0.5 mg/l BA) to obtain compact and nodular calli. Shoot formation was initiated on M1 medium (MSB medium with 0.15 mg/1 NAA, and BA, KT and ZT, 0.5 mg/l of each, 500 mg/1 CH). Differentiation frequency was 13.6–24.2%. Plants have been regenerated from protoplasts of immature cotyledons in 2 cultivars, and normal pods were obtained from them.     

Plant Regeneration from Protoplasts of Soybean (Glycine max L.)

Protoplasts were isolated enzymatically from immature cotyledons of soybean. The protoplasts divided to form calli in the K8P liquid medium. The calli further grew to 2–3 mm on the solid K8 medium and were transferred onto the MSB medium (MS minerals+B5 organic components+0.5–1.0 mg/l 2,4-D+0.2–0.5 mg/l BA) to obtain compact and nodular calli. Shoot formation was initiated on M1 medium (MSB medium with 0.15 mg/1 NAA, and BA, KT and ZT, 0.5 mg/l of each, 500 mg/1 CH). Differentiation frequency was 13.6–24.2%. Plants have been regenerated from protoplasts of immature cotyledons in 2 cultivars, and normal pods were obtained from them.     

An efficient and reproducible method for regeneration of whole plants from mature seeds of a high yielding Indica rice (Oryza sativa L.) variety PAU 201

Tissue culture is one of the tools necessary for genetic engineering and many other breeding programs. Moreover, selection of high regenerating rice varieties is a pre-requisite for success in rice biotechnology. In this report we established a reproducible plant regeneration system through somatic embryogenesis. The explants used for regeneration were embryogenic calli derived from mature seeds cultured on callus induction media. For callus induction mature seeds were cultured on MS medium containing 30 g/l sucrose combined with 560 mg/l proline and 1.5-3.5 mg/l 2,4-D and 0.5-1.5 mg/l Kin. For plant regeneration, embryogenic calli were transferred to MS medium containing 30 g/l sucrose, supplemented with 1.0-3.0 mg/l BAP, 0.5-1.5 mg/l Kin and 0.5-1.5 mg/l NAA. The highest frequency of callus induction (44.4%) was observed on the MS medium supplemented with 2.5 mg/l 2,4-D, 0.5 mg/l Kin, 560 mg/l proline and 30 g/l sucrose. The highest frequency of shoot regeneration (42.5%) was observed on the MS medium supplemented with 2.0 mg/l BAP, 0.5 mg/l NAA and 0.5 mg/l Kin. The plantlets were hardened and transferred to soil in earthen pots. The developed method was highly reproducible. The in vitro developed plants showed normal growth and flowering under glasshouse conditions.