甘薯叶柄原生质体有效植株再生

将甘薯(Ipomoea batatas (L．)Lam．)‘元气’和‘白星’(‘White Star’)的叶柄原生质体培养在含有0.05 mg·L-1 2,4-D和0.5 mg·L-1 KT的改良MS液体培养基中,3～4 d后细胞开始分裂。培养8～9周后,将直径达1～2 mm的愈伤组织转移到添加0.05～0.2 mg· L-1 2,4-D和0～0.5 mg·L-1 KT或添加0.5～2.0 mg ·L-1 NAA和1.0～3.0 mg·L-1 BAP 的MS固体增殖培养基上使其增殖。转移3～5周后,将愈伤组织再转移到MS基本培养基或转移到添加2.0～3.0 mg·L-1 BAP的MS培养基上。当进一步转移到MS基本培养基上后,从愈伤组织或从愈伤组织形成的不定根上再生出植株。‘元气’植株再生率高达60.0 ％,White Star高达43.4％。     

诸葛菜叶柄原生质体培养再生植株

本文首次报道用诸葛菜(Orychophragmus violaceus)试管苗叶牺为材料分离原生质体,经培养再生了植株。用于原生质体培养的基本培养基为Nitsch培养基,附加1OOmg／L丝氨酸,800mg／L谷氨酰胺和13％的蔗糖,激素成分为0.5mg／L BA,0．5mg／L NAA和lmg／L2,4一D(或0．5mg／L BA和2mg／L 2,4-D)。原生质体的培养密度为2×10⁵／ml。培葬7天的原生质体分裂频率约为40％。在附加O．05mg／L NAA和3mg／L BA的MS分化培养基上,愈伤组织可分化出大量的芽和苗,分化频率为100％。     

甘蓝型油菜叶柄原生质体培养再生植株

Two cultivars of Brassica napus,Altex and Canadian twins,were used as materials.Protoplasts isolated from petioles of plants grown in vitro were cultured in Nitsch mediumsupplemented with 0.5mg/L BA,0.5mg/L NAA,1mg/L 2,4-D,100mg/L serine,800mg/Lglutamine,4% sucrose and 0.4mol/L mannitol.After 2 days of culture,the first division wasobserved.The division frequency estimated after 10 days of culture was 30—60%.One weekafter transferring onto MS medium containing 6mg/L GA_3 and 3mg/L BA,protoplast-derivedcalli regenerated into shoots.The regeneration frequency of the two cultivars was 24% and31% respectively.It was found that the protoplasts isolated from petioles could float on thesurface of the 3% sucrose contained solution which was very favourable both to purificationand culture of the protoplasts.     

党参原生质体再生植株

党参下胚轴愈伤组织原生质体在附加１．２ｍｇ／Ｌ２,４－Ｄ,０．２ｍｇ／Ｌ ＮＡＡ,０．２ｍｇ／Ｌ ＢＡＰ和０．１ｍｇ／Ｌ ＺＴ的ＭＳ,Ｃ８１Ｖ,ＤＰＤ及ＫＭ８ｐ培养基中进行液体体层培养。在ＫＭ８ｐ中获得了最高的分裂频率。葡萄糖作渗透剂优于甘露醇,两结合使用效果更好。在合适的条件下,原生质体培养３天出现第１次分裂,４周内形成大细胞团,培养６周后形成０．５－１．０ｍｍ大小的小愈伤组织。在附加２％蔗糖     

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。     

番茄子叶原生质体再生植株

从番茄2—3周苗龄的子叶游离原生质体,在 MS 液体培养基中(附加2,4-D 1,6-BA 0.1mg/l)培养;在培养过程中经常不断添加新鲜培养液。6周后将细胞团移到半固体 MS 培养基上(附加成份同上,琼脂0.3%)。然后将肉眼可见的愈伤组织再移入 MS 固体培养基上,愈伤组织长到直径为5 mm 大小时,转到 MS 分化培养基上(附加6-BA 2 mg/1,[AA 0.2 mg/l)诱导分化,得到了再生植株。比较了固体培养、悬浮培养和双层培养三种方法,观察原生质体生长情况,以双层培养为好。     

Plant Regeneration from Petiole Protoplasts of Brassica napus

Two cultivars of Brassica napus, Altex and Canadian twins, were used as materials. Protoplasts isolated from petioles of plants grown in vitro were cultured in Nitsch medium supplemented with 0.5mg/L BA, 0.5mg/L NAA, lmg/L 2,4-D, 100mg/L serine, 800mg/L glutamine, 4% sucrose and 0.4mol/L mannitol. After 2 days of culture, the first division was observed. The division frequency estimated after 10 days of culture was 30-60%. One week after transferring onto MS medium containing 6mg/L GA3. and 3mg/L BA, protoplast-derived calli regenerated into shoots. The regeneration frequency of the two cultivars was 24% and 31% respectively. It was found that the protoplasts isolated from petioles could float on the surface of the 3% sucrose contained solution which was very favourable both to purification, and culture of the protoplasts.     

Plant Regeneration from Protoplasts of Coriandrum sativum

Calli produced from stem segments of seedling of Coriandrum satwum which were cultured on MS agar medium containing NAA 1.0mg/L. The embryogenic cell colony suspension was estabilished on MS liquid medium containing NAA 1.0mg/L%2,4-D 0.2mg/L+BA 0.5 mg/L. The cell suspension culture was used for protoplast preparation. Protoplasts were obtained in the enzyme mixture containing 2.0% Onozuka R-10, 1.0% pectinase, 0.5% snailase, 0.5% dextran sulfate potassium Salt, 0.6mol/L mannital CPW solution at pH 5.8 and 25℃. Cultured in a KM8P liquid medium containing NAA 1.0mg/L+2,4-D 0.2mg/L+6-BA 0.5 mg/L, glucose 0.4mol/L and CM 20mi/L; the protoplasts entered the stage of derision after three days, cell clusters formed in 10 days and calli formed after about 50 days. When the calli were transferred to MS agar medium containing many growth substances, they differentiated into embryoids, and then developed into plantlet with many green leaves and roots on the 1/2 MS agar medium.     

Plant Regeneration from Protoplasts of Brassica Juncea L.

Protoplasts isolated enzymatically from epicotyl and growing tip of Bressica juncea divide to form callus on Kp8 medium. Plant regeneration is obtained from protoplast- derived callus of on MSD3 medium. High concentration of inositol in differentiation medium stimulates plant or shoot regeneration from the epicoty protoplast origin.     

Plant Regeneration from Callus Protoplasts of Codonopsis pilosula

Embryogenic cell line was established from hypocotyl segments of Codonopsis pilosula (Franch.)Nannf. 4--8 day old embryogenic callus was used to isolate protoplasts in an enzyme solution containing 1.5 % cellulase Onozuka R-10 and 3 % pectinase. Protoplasts were cultured in MS,C81V,DPD and KMSp basal medium supplemented with 1.2 mg/L 2,4-D, 0.2 mg/L NAA, 0. 2 mg/L BAP, 0. 1 mg/L ZT,and different combinations of glucose and mannitol . Protoplast-derived cells underwent sustained divisions in KM8p medium. As an osmoticum, glucose was more beneficial to protoplast division. A combination of 0. 30 mol/L glucose with 0.10 mol/L mannitol gave the best result. Under proper conditions , protoplasts underwent the first division on the 3rd day of culture,formed colonies within 30 days , and developed into microcalli in 6 weeks. Plantlets were regenerated from protoplast-derived calli through somatic embryogenesis. 0.2 % activated charcoal promoted embryoid formation and root development.     

Plant Regeneration from Protoplasts of Medicago lupulina

Protoplasts isolated from suspension cell lumps of Medicago lupulina L. started to divide after 2 clays in K8p culture medium containing 0. 1～2.0 mg/L of 2, 4-D, with a maximum division frequency of 38. 35%. After S weeks of culture, the protoplast-derived cell lumps were transferred to liquid/solid double-layer media for microcallus regeneration, with a maximum frequency of 0.58%. The whole plants were regenerated from protoplastderived calli via somatic embryogenesis and organogenesis. In somatic embryogenesis, the embryoids were induced on MS and W14 media with rather wide range (1. 0420.0 mg/L) of 2, 4-D concentration. The highest induction frequency of embryoids was 71.0%. In organogenesis, the differentiation media containing lower concentration of 6-BA (0. 5～0. 7 mg/L) were suitable for adventitious bud formation. The highest frequency of adventitious bud formation from calli was 27. 8%. The mature protoplast-regenerated plants were obtained 3 months after transplanting the plantlets into soil.     

Plant Regeneration of Protoplasts of Trifolium lupinaster L. from Cell Suspension Cultures

paper deals with regeneration of protoplasts in cell suspension cultures of hypocothl from Trifolium lupinaster L. on the SL2 basal medium with BA 0.1 mg/L and picloram 0.06 mg/L for 3--4 month,s. The protopiasts were isolated from suspensions cells subcultured for 3 days and were recuhured in modified liguid medium 8p. The first division of the regenerated cell occurred 3 days after being cultured in medium Bp. Small calli could be seen with naked eyes by one month. The calli when grew up to 2 mm long, were transferred in succession differentiation medium A and B for organ differentiation. The differentiated shoots formed their roots on 1/2 MS supplamented with NAA 1.0mg/L and then grew into plantlets.     

Plant Regeneration from Protoplasts Derived from Suspension Cultures in Leymus racemosus

Calli initiated from mature embryos of Leymus racemosus (Lam. Tzvel. =L. giganteus were transferred onto the AA and DM media to produce friable embryogenic callus,from which embryogenic suspension cultures were established. Protoplasts were isolated from the embryogenic suspension cultures and were cultured either in thin-layer liquid medium or in double-layer (agar/liquid) medium. When visible calli were formed they were transferred onto the NBI agar medium or into the MBL liquid medium for further proliferation. These calli were transferred onto differentiation media of NBII and NR, where green spots were developed. Plants with both shoots and roots can be recovered from these green spots on MS Ⅱ medium containing 0.5 mg/L NAA. The results showed that the Km8p basal medium was favourable to the culture of L. racemosus protoplasts during the early stages of culture. In addition, the composition of the media added to the cultures had a marked influence on the growth of protoplasts, indicating that the nutritional requirements in this plant were different at various stages of protoplast growth and differentiation.     

Plant Regeneration from Protoplasts of Talinum paniculatum

The protoplasts of Talinum paniculaturn (Jaeq.) Gaertn. were isolated from leaves and calli. The mesophyll protoplasts did not undergo normal division and lived one week at the longest in culture. However, the callus protoplasts, cultured in P4 medium (K8p+2, 4-D 0.2 mg/L, NAA 1.0 mg/L, ZT 0.5 mg/L, coconut milk 50 mL/L, glucose 0.5 mol/L), underwent first division after 3 d of culture. The division frequency was 36.7 % after 7 d of culture. The regeneration frequencies of callus were 0.31% in liquid culture and 0.34% in double-layer culture. Shoots differentiated on regeneration media and rooted on R3 and R7 media. Mature plants were obtained 2～3 months after transplanting the protoplast-derived plantlets into flower pot or successive subculturing in test tubes. The results also indicated that: (1) Too long a period of callus culture in liquid medium or in solid proliferation medium was unfavorable to differentiation. (2) Low concentration of 6-BA in medium was suitable for callus differentiation. (3) GA3 promoted development of young adventitious bud. (4) Multi-effect triazole significantly strengthened sprout and root development in test tube cultures.