Protoplast Culture and Plant Regeneration of Winter Wheat (Triticum aestivum L.)

Suspension cultures were established from embryogenic calli derived from cultured anthers of cv. Jinghua No.1 and mature embryos of cv. Youmangbai No. 7, respectively. After being isolated and cultured in WPMI, protoplasts began to form cell walls within 1 day post-isolation, followed by cell division observed between 2–3 days. A division frequency of 22.0% was estimated on the 7th day of culture, and 43.7% on the 14th day. During 10–15 days after the initiation of culture, a large number of cell aggregates emerged, with 0.5–0.8% of plating efficiency. Protoplast-derived calli grew up to lmm or more in diameter when cultured for 4 weeks, and eventually gave rise to green plants through embryogenesis and organogenesis after being transferred to differentiation media. Plant regeneration from protoplasts was already obtained from Jinghua No.l, and protoplast-derived calli from Youmangbai No.7; an experiment on organ differentiation for the latter is under way. A few factors affecting the protoplast cultures were also studied.     

新疆甜瓜子叶原生质体的培养和植株再生

从新疆甜瓜(Cucumts melo L.)的无菌苗子叶游离原生质体,用改良的 Miller 培养基(Ma)培养,得到了再生细胞的高频率分裂。比较了液体浅层培养、双层培养与琼脂糖珠看护培养等方法,发现由烟草瘤细胞 B_6S_3看护的琼脂培珠培养,最宜于新疆甜瓜子叶的原生质体。再生的愈伤组织经液体与固体两步培养程序分化出完整的小植株。     

Protoplast Culture and Plant Regeneration of Ramie

Calli were induced and suspension cell lines were established from cotyledones of ramie (Boehmeria nivea). Protoplasts (2 × 10 6/g fr. wt) were isolated from suspension cell cultures in enzyme mixture solution containing 4. 5 % cellulase Onozuka R-10 and 0. 8 % Macerozyme R-10, 0.8 % hemicellulase. When cultivated on KM8p medium containing 2, 4-D 0.5 mg/L, KT 0.5 mg/L with alginate embedding method, they grew vigorously and produced microcalli within fifty days. After subcultured, the protoplast-derived ~alli produced shoots and roots on different differentiation media, then complete plants were formed. Protoplasts from cotyledones divided only several times.     

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。     

Protoplast Culture and Plant Regeneration of Angelica dahurica

White and soft calli were induced from the stemnodes of Angelica dahurica on MS medium containing lmg/L 2,4-D, and subcultured on the same medium with decreased concentration of the hormone for about half a year, until quite a number of embryogenic cell clusters were produced in calli. Protoplasts prepared only from this kind of callus were regenerable. The protoplasts-derived colonies were able to develop into embryos directly or to grow continously into calli as affected by the hormone and, in particular, by osmotic pressure in the culture medium. The embryos either formed directly or via callus stage were all capable of regenerating complete plants under proper culture conditions.     

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.     

Plant Regeneration from Protoplast of Peucedanum praeruptorum Dunn

Calll were initiated from the seedling segment of Peucedanum praeruptorum Dunn and subcultured on the MS agar medium with 0.5 mg/L 2,4-D. Cell suspension culture with a lot of embryogenic cell clumps was obtained in liquid medium. Protoplasts were isolated from the cell clumps in enzyme mixture solution containing 1.5% Onozuka R-10, 0.3% Macerozyme R-10, 0.5% helicase, 5 mmol/L CaCl2 and 0.6 mol/L mannital, at pH 5.6 and shaking for 5- hours at 25℃. Helicase is necessary for isolation. After purified by washing, the protoplasts were cultured in liquid medium containing 1 mg/L 2,4-D +0.5 mg/L zeatin. First cell division was observed after four days. Large cell clumps were formed after thirty days. Microcalli of 1 mm in size was formed after about fifty days, and continued to grow on the MS solid medium containing 0.5 mg/L 2,4-D and 200 mg/L casein hydrolysate, and later differentiated into embryoids when transferred to MS agar medium with 0.1 mg/L zeatin. Eventually, embryoids developed into whole plantlets on the MS solid medium without phytohormones.     

Embryogenic Suspension Culture and Plant Regeneration from Suspension-Derived Proto-plasts of Cucumber (Cucumis sativus L.)

Fast growing embryogenic cell suspension culture was established when embryogenic callus derived from cotyledon protoplasts of cucumber was transferred into a liquid culture. So far the cell line has been subcultured for two years and retained the ability of embryogenesis and plant regeneration. Experimental data showed that the concentration of ABA or sucrose had a dramatic effect on embryogenesis and synchronization of embryoid development. Low level of sucrose concentration (1%) facilitated the precocious germination of the embryoids while 1 mg/l of ABA or 7–9% of sucrose was found to be effective for reducing callusing of the cultures and synchronisticly controlling the embryoids at globular or late globular stage. Embryogenic cells taken from 3–5 days after subculture were enzymatically digested. A large amount of viable protoplasts was isolated. Protoplasts were cultured in a DPDK1 medium either by means of drop or thin layer liquid culture or by means of sodium alginate encapsulation culture. Actively dividing cells formed cell colonies and globular embryoids which were transferred onto a solidified agar medium or directly into a liquid medium to form a shaken culture. The embryoids would proliferated continuously. Embryoids eventually developed into plantlets when they were transferred onto a 1/2 MSO medium devoid of phytohormones.     

Culture of Cotyledon and Hypocotyl Protoplasts from True Potato Seedlings in Solanum tuberosum L.

Culture of protoplast using cotyledon and hypocotyl as the donor tissue from true potato seedlings (TPSs) of 3 breeding lines (DTO-33, ND 860-2 and BN 9815-3) of Solanum tuberosum L. was studied. The cotyledons and hypocotyls of TPSs just extended were excised and digested in an enzyme solution containing 1 % cellulase and 0. 5 % macerozyme for 17—20 h after vacuum infiltration of the tissue in the solution. The protoplasts were cultured in an improved liquid medium and transferred onto solid media for callus culture and shoot regeneration. Some factors affecting the efficiency of cotyledon and hypocotyl protoplast culture were studied. The results showed that using the cotyledons and hypocotyls as donor tissues for protoplast isolation and culture in potato, the division frequency of protoplast derived cells was significantly higher than that using the leaves and shoot-tips of the test-tube plantlets: the yield and quality of the protoplast from TPSs cultured under continuous high light intensity (3000 Ix) were much higher than the TPSs cultured under low light intensity (1000 Ix), and no intact protoplast was ever obtained from the TPSs cultured in continuous dark condition. Vacuum infiltration of the cotyledon and hypocotyl segments in enzyme solution before digestion increased protoplast yield. The yield of protoplasts from hypocotyl tissue was significantly higher than from the cotyledon, but there was no significant difference in quality between the protoplast derived from the two tissues. The significance, advantages and shortcomings of using the cotyledons and hypocotyls as the donor tissues for isolation and culture of potato protoplasts are dicussed.     

Plant Regeneration from Protoplasts of Corn(Zea mays L.)

Maize embryogenic calli induced from pollen were subcultured for one and one half years on N, basic medium supplemented with 2 mg/1 kinetin, 1 mg/l 6-benzyl-aminopurine, 0.3 mg/l 2,4-D, 500 mg/l casein hydrolysate and 250 mg/l glutamine. These embryogenic calli were used for protoplast isolation. Protoplasts were cultured on Z2 medium (Table 1) which is composed of rice protoplast culture basic medium 1 supplemented with 0.2 mg/l kinetin, 0.1 mg/l 6-benzyl-aminopurine, 0.5 mg/l 2,4-D, 200 mg/l casein hydrolysate, 100 mg/l glutamine and 2% coconut milk. The first division of regenerated cell occurred after 4-6 days in culture. After 3 weeks later, small calli could be seen with naked eyes. At this moment, addition of the same Z2 medium with decreased osmoticum twice for the protoplast culture is necessary. Regenerated calli, 2–4 mm in diameter, were transferred in succession on differentiation medium Z3 and Z4 for organogenesis. Embryogenesis and plant regeneration could occur simultaneously on Z4 differentiation medium. It seems that except the cultural conditions genotype and using of embryogenic materials are the two key factors for plant regeneration of maize protoplast and the former may be the critical one.     

茄子子叶原生质体再生可育植株

将茄子子叶原生质体放在0.75％纤维素酶R-10、0.2％半纤维素酶Rhozyme和0.2％果胶酶溶液中分离。原生质体在培养基中诱导出小愈伤组织。愈伤组织在Ms+2mg/l KT+0.005mg/l NAA+2％蔗糖的固体培养基中,一个月后分化出芽。芽生长至3—4厘米高,转接在Ms+0.1mg/l 1AA+1％活性炭+2％蔗糖的培养基上,一个星期后可长出根,继而形成完整植株。随后移栽至灭菌的混合土壤中长到开花结果。     

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.     

PEG－mediated Gene Transfer into Orychophragmus Violaceus Cotyledon Protoplast and Regeneration of Transgenic Plants

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Mesophyll Protoplast Culture and Plant Regeneration of Oriental Planetree (Platanus orientalis)

Large populations of mesophyll protoplasts were released from the leaves of 1.5–2 month old sterile seedlings, with a high protoplast yield (3.7× 10 6g-1FW) after protoplast purification. The purified protoplasts were cultured in a modified K8p liquid medium supplemented with 0.5 mg/L 2,4-D, 1 mg/L NAA and 0.5 mg/L BA. Higher density (1× 106/ml) in the initial culture of protoplasts is favourable to the division of cultured mesophyll protoplasts of this woody species among the densities tested. The protoplasts started to divide after 6 days of culture, and achieved 26.8% division frequency by 14 days. Sustained divisions resulted in mass production of cell colonies and small calli in 8 weeks. The calli further grew to 2–3mm on the gelrite-solidified K8 medium supplemented with 0.2 mg/L NAA aud 0.5 mg/L BA. Then, they were transferred onto the MSB proliferation medium with 0.1 mg/L NAA and 0.25 mg/L BA, where compact and cream-coloured calli were formed. Shoot formation was initiated on MSB differentiation medium coraming 0.5 mg/L IAA, 1 mg/L each of BA and ZT. It was observed that the frequency of shoot formation was about 28.7%. Whole plantlets were regenerated upon transferring 3 cm shoots to 1/2MS medium with 0.5mg/L IBA and 0.1mg/L BA, from which they were already transplanted into pots and grew well in the phytotron of Shanghai Institute of Plant Physiology.     

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.     

Protoplast Culture and Plantlet Regeneration from Hypocotyls of Brassica campestris var. parachinesis

Protoplasts isolated from 3--4 day-old (ca 4 cm in length) etiolated hypocotyls of Brassica carnpestris var. parachinesis (Bally) Tsen et Lee and purified with 20% sucrose were cultured on K8p medium suplemented with 0. 5 mg/L ZT, 0.5 mg/L 2, 4-D, 1.0 mg/L NAA and 0. 4 mol/L glucose. When initially cultured for 14-18 hours the protoplasts formed new walls and by first division after 36 hours. The divided protoplasts reached 35 % after being cultured for three days. When cultured under optimum conditions for 8-9 days, the proto plasts formed 8-16 cell colonies with a plate effeciency as high as 15%-18%. Rapidly growing and dividing calli of 2 mm in diameter were transferred onto semisold gelrite media with 0.3 mg/L 2, 4-D enabling them to proliferate further towards the size of 4-5 mm in diameter. Shoot differentiation was carried out in MS medium with 3.2 (or 1.6) mg/L BA, 1.6 (or 0.8) mg/L ZT, 0.01 mg/L NAA, 0. 1 mg/L GA3 and 0.2 % sucrose. Shoots were cut down and rooted on medium with 0.2 mg/L IAA and 2 % sucrose where whole plants were evatually developed.     

Plant regeneration in pigeonpea [Cajanus cajan (L.) Millsp.] by organogenesis

A method for regenerating pigeonpea [Cajanus cajan (L.) Millsp.] plants has been developed using distal cotyledonary segments of mature seeds as explants. A large number of shoot buds were induced directly from explants of genotypes T-15-15 and GAUT-82-90 when cultured on six different basal media fortified with 22.2 μm N⁶-benzylaminopurine, 2.3 μm kinetin, and 271 μm adenine sulfate. The shoot buds developed into shoots when they were subcultured on the same medium but with one-tenth concentrations of cytokinins and adenine sulfate. The shoots elongated by subculturing first two to three times on Murashige and Skoog (MS) basal medium supplemented with 2.22 μm N⁶-benzylaminopurine and 0.54 μm α-naphthaleneacetic acid or on half-strength MS medium containing 2.89 μm gibberellic acid, and then once on the same medium without growth regulators. Elongated shoots were rooted with 80–85% efficiency on MS medium with 4.92 μm indole-3-butyric acid and the plantlets were transferred for hardening. Plants survival in pots was 70–75%. This method may be useful for improving the crop through genetic manipulations. Received: 11 August 1997 / Revision received: 12 January 1998 / Accepted: 30 January 1998     

水稻原生质体培养及植株再生的研究

由粳稻77-170品系及籼稻品种IR-50的细胞悬浮培养物游离的原生质体,用琼脂糖包埋于RY-2培养基中,发生了持续分裂。前者植板率达2.5％以上,二者最后都再生出植株。对游离和培养方法做了如下改进:1)采用两步法,即先用果胶酶,再用果胶酶和纤维素酶的混合酶进行游离,可避免原生质体发生融合并获得高质量的原生质体;2)悬浮细胞培养基中加入ABA有利于原生质体的存活和分裂;3)琼脂糖包埋培养可大大提高植板率;4)用较高渗透压的培养基培养原生质体再生的细胞团及愈伤组织,可提高植株再生频率。由于这两个品种(系)的培养物都已继代一年半之久,再生植株均为白化苗。这是迄今第一个由籼稻原生质体再生植株的报道。     

Plant Regeneration from the Protoplast Culture of Multitiller and Multispike Forage Maize and Seeding of the Regenerated Plant

This paper deals with systematic studies on the protoplast culture of multitiller and multispike forage maize, including selection of genotypes, induction and subculture of embryogenic calli, cell suspansion culture and protoplast culture. Methods such as liquid culture, nurse culture and agarose bead culture were used for protoplast culture. Four cell clones were obtained. Of which calli and embryoids derived from protoplasts were induced from 79, 55 and 79-8 three genotypes, and regenerated plants were acquired from 79 and 79-8. One of the regenerated plants, after being transplanted into soil, developed normally with flowering and earing. After self and cross pollinations three ears with normally developed seeds were obtained. These seeds germinated well in germination test.     

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