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.     

Plant Regeneration from Cotyledon Protoplasts of Tomato (Lycopersicon esculentum L.)

otyledon protoplasts of tomato (Lycopersicon esculentum L.) isolated from 2–3 week grown seedlings were cultured in MS liquid medium (2,4-D 1, 6-BA 0.1 mg/l) and fresh medium added subsequently. After 6 weeks culture, the cell clusters were transferred to semisolid medium (the additive same as in liquid medium, agar 0.3%). When the calli grew to 0.5 cm in diameter, transfer them to MS medium (6-BA 2, IAA 0.2 mg/l) for differentiation. The regenerated plants were obtained. After comparing different culture methods, tomato protoplasts grew better in double layers than in agar plate and hanging drops.     

Plantlet Regeneration from Leaf Protoplasts in Seedling of Actinidia eriantha Benth.

Newly extended leaves of in vitro seedling of Actinidia eriantha Benth. were used for protoplast isolation. Protoplasts were cultured in liquid MS medium (devoid of NH4NO3) supplemented with 1.0 mg/L 2, 4-D and 0.4 mol/L glucose. The plating efficiency after 3 weeks of culture was about 19.4 %. Protoplasts-derived cells divided sustainably and developed into calli of 2 mm in size in the original protoplast-culture-medium without adding fresh medium so to decrease the osmotic pressure. These calli regenerated shoots when being transfered to MS medium with 0. 5 mg/L zeatin and 0. 1 mg/L IAA. Regenerated shoots were rooted by immersion in 20 ppm IBA solution before culturing on half-strength MS medium devoid of growth regulators.     

沙葱叶基愈伤组织原生质体再生体系的建立

沙葱是一种具有抗旱抗寒、抗病性和适应性强等生理特性的荒漠植物.为开发利用其固有的遗传资源,本研究利用细胞工程技术建立了沙葱(Allium mongolicum Regel)叶基愈伤组织原生质体的分离、培养和植株再生实验体系.研究结果表明,酶法分离原生质体的产率和分裂频率明显取决于用于制各原生质体的愈伤组织的状态.转代培养7～10d的松软愈伤组织可分离出大量有活性的.在附加2.0mg/L2,4-D、0.2mg/L激动素、500 mg/L水解乳蛋白、0.4 mol/L甘露醇和2%蔗糖的MS培养基中进行液体浅层培养,4～5 d后出现第一次原生质体分裂;7～10d出现第二次分裂.结果显示原生质体的分裂频率大约为5%;4周后,可见到小愈伤组织.当将原生质体分裂形成的愈伤组织转移到附加2.0mg/L6-苄氨基嘌呤(或激动素)和0.4mg/L萘乙酸(NAA)的MS固体培养基上,并在低光照条件下培养后,从愈伤组织上分化出了不定芽,进而发展成小植株,并移栽成活.本研究对沙葱抗逆遗传品质用于经济植物遗传改良的研究奠定了可行的实验基础.     

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 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 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 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.     

甘薯叶柄原生质体有效植株再生

将甘薯（Ｉｐｏｍｏｅａｂａｔａｔａｓ（Ｌ．）Ｌａｍ．）‘元气’和‘白星’（‘ＷｈｉｔｅＳｔａｒ’）的叶柄原生质体培养在含有０．０５ｍｇ·Ｌ－１２,４Ｄ和０．５ｍｇ·Ｌ－１ＫＴ的改良ＭＳ液体培养基中,３～４ｄ后细胞开始分裂。培养８～９周后,将直径达１～２ｍｍ的愈伤组织转移到添加０．０５～０．２ｍｇ·Ｌ－１２,４Ｄ和０～０．５ｍｇ·Ｌ－１ＫＴ或添加０．５～２．０ｍｇ·Ｌ－１ＮＡＡ和１．０～３．０ｍｇ·Ｌ－１ＢＡＰ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２．０～３．０ｍｇ·Ｌ－１ＢＡＰ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％,ＷｈｉｔｅＳｔａｒ高达４３．４％。     

有机及生态绿色栽培对猕猴桃果实品质的影响

以中华猕猴桃（Actinidia chinensis Planch.）品种‘H-1’为材料,研究有机栽培和生态绿色栽培模式对其可溶性固形物、总糖、总酸、Vc、氨基酸、矿物元素和香气等果实品质性状的影响。结果显示：生态绿色栽培的‘H-1’中,果实可溶性固形物、总糖和Vc含量比有机栽培的果实分别高出15.90%、26.30%和29.25%,其中Vc的差异最为明显;总酸含量与有机栽培种相差不大;有机栽培的‘H-1’中必需氨基酸的含量是生态绿色栽培种的1.69倍,且锌和钙的含量也高于生态绿色栽培,但钾元素含量相差不明显;生态绿色栽培的‘H-1’果实中可检出的果香成分比有机栽培多5种。研究结果表明栽培方式不同,猕猴桃果实品质表现出一定差异,可根据果实鲜食及加工用途的不同选择合适的栽培方式。     

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 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.     

Studies on Plant Regeneration from Mesophyll Protoplasts of Solanum tuberosum L.

Protoplasts from potato mesophyll of two strains (Solannum tuberosum L. cv. Xiao Yie Zi x Duo Zi Bia and Solanum tuberosum L. cv. Wu Meng 601) were induced to callus in culture medium of protoplasts. The callus derived from mesophyll protoplasts were transferred to MS medium with 2 mg/l ZT+0.1 mg/L IAA. Shoots regenerated from the callus were detected after 70 days of culture.The shoots which had grown to a height of 2–3 cm were transferred to MS medium with 0.05 mg/L NAA. Roots were coming out in a few days.Complete plantlets were achieved. Stern segments with 1–2 leaves were then transferred to a mixture of sterilized soil and grown, and produced tuber.     

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 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.     

蚕豆未成熟子叶原生质体再生植株

近年来,豆科植物原生质体诱导再生植株的研究越来越受到国内外学者关注。但在籽粒型食用豆科植物中,迄今成功的种类仍然不多,在文献记载中仅见有大豆、赤豆、豇豆、豌豆和刀豆,说明籽粒型食用豆科植物原生质体再生植株仍有较大困难,要取得禾谷类作物那样的重大进展,尚需作出巨大努力。蚕豆原生质体培养仅见有从预培养的叶肉原生质体再生细胞团、从茎尖和叶肉原生质体再生愈伤组织和从     

Triterpenoids from the Roots of Actinidia chinensis

Two new triterpenoids, 1 and 2 , were isolated from the hepatoprotective AcOEt fraction of the roots of Actinidia chinensis, together with eight known 12‐en‐28‐oic acids of oleanane or ursane type, 3 – 10 . The two new compounds were elucidated as 2α,3β‐dihydroxyurs‐12‐en‐28,30‐olide ( 1 ) and 2α,3β,24‐trihydroxyurs‐12‐en‐28,30‐olide ( 2 ), on the basis of spectroscopic (IR, NMR, and MS) analyses. The chemotaxonomic significances of some triterpenoids were also discussed.     

Protoplast Culture and Plant Regeneration of Malus pumila

Calli were induced and suspension cell lines were set up from ovule of Malus pumila Mill. Protoplasts (5.40 × 106/g fr. wt) were isolated from suspension cell lines in enzyme mixture solution containing 2.0% Onozuka R-10, 0.5% pectinase, 0. 65 mol/L mannital, 0.01 mol/L CaC12, 0.7 mmol/L KH2 PO4, 0.3% dextran sulfate potassium salt, at pH 5.8 for 6 h at 26℃. The cell clumps were formed from protoplasts cultured in modified MS, K8p, D2 media. Calli were formed on MS solid medium containing 2.0 mg/L IAA, 2.0 mg/L NAA, 0.1 mg/L BA. Shoots were differentiated on differentiated medium after several changes of the medium. Eventually, shoots rooted and developed into whole plantlets on a rooting medium.     

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 Cell Supension Derived Protoplasts of Heracleum moellendorffii

Heracleum moellendorffiz Hance is a herb belonging to Umbelliferae used in traditional medicine in China. The young stem-nodes were induced for callus formation on MS medium containing 1 mg/L 2,4-D. After subcultured for about five months, the embryogenic calli were used for cell suspension culture. The protoplasts were prepared from this suspension by digestion with enzyme mixture containing 1. 5% cellulase Onozuka R-10 +0. 3% macerozyme R-10 + 0. 5% snailase + 5 mmol CaCl2 + 0. 6 mol/L mannitol, at pH 5.8, and cultured in modified MS and modified N6 media with 0.3 % agarose. They divided after 3 days and developed into small cell colonies after about 2 weeks. From this time on, the glucose concentration in the culture media was decreased to 0. 2 mol/L,which led to futher growth of the colonies to small calf . After a period of proliferation on solid medium with 0. 5 mg/L 2,4-D, the calli were transferred to a medium with 0. 1 mg/L zeatin on which somatic embryos differentiated and developed to plantlets