Isolation,Culture and Callus Formation of lpomoea batatas Protoplasts

Numerous viable protoplasts from stem callus cells of Ipomoea batatas tissue culture have been isolated by enzyme treatment involving cellulase EA3 867 (2.0%), CaC12·2H2O (20 mM) and mineral constituent of medium A at pH5.4 in 0.8 M mannitol in 5 hours at 25±1℃. The protoplasts were cultured at a density of 1-2 × 105/ml in solid agar medium E supplemented with 2, 4-D (0.1mg/l) and kinetin (0.1 mg/l), or NAA (0.3 mg/l) and kinetin (0.1 mg/l) in petri dishes, and placed in a controlled growth cabinet maintained at 27 ℃, and illuminated with floureseent light. They regenerated new cell wails after 7 days of culture. The first cell division was observed after 10 days. Ceil division continued thereafter, and after 40 days of culture small white calli (size about 0.2–0.3 mm) were visible in the petri dishes small calli were inoculated in the same nutrients as the protoplasts culture media, but without mannitol. They developed into large calli.     

骆驼刺发根农杆菌转化系的原生质体培养和植株再生

从发根农杆菌A4转化的荒漠植物—璐驼刺毛状根愈伤组织中分离的原生质体培养的结果表明,酶解新转代7～10d的淡黄色松软愈伤组织,可获得大量有活力的原生质体。原生质体在附加有1．5mg．L-1 2,4．D、0．2mg．L-1 6．BA、0．3m01．L-1甘露醇、2％（W/V）蔗糖和500mg·L-1水解酪蛋白的DPD培养基中进行液体浅层培养可持续分裂。培养基的最适渗透压为（450±3）mOsm·kg-1,原生质体的最适植板密度为4×10^5个．mL-1。制备原生质体的愈伤组织以低温（4℃）预处理后,原生质体的产率和分裂频率均提高,分裂频率最高可达50％。原生质体分裂形成的愈伤组织转移在附加1-2mg．L-1 6-BA（或KT）和0．2mg·L-1NAA的MS培养基上培养后,可以分化并获得再生植株。纸电泳检测表明,原生质体再生的愈伤组织和分化植株仍然含有毛状根转化系的特异产物——冠瘿碱。     

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.     

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.     

Culture of the Cotyledon Protoplast of Xinjiang Muskmelon(Cucumis melo L.) and Plant Regeneration

The cotyledon protoplasts were isolated from sterile seedling of Xinjiang muskmelon (Cucumis melo L.) and cultured in modified Miller medium. The high frequency division of the regenerated cells was observed It was indicated that the agarose bead culture with B6S3 nurse cells is the most suitable for the cotyledon protoplast of Xinjiang muskmelon when compared with thin liquid culture and double layer culture. The intact plants were differentiated from the regenerated calli by two steps of culture with liquid first and then solid medium.     

Callus Formation from Protoplasts and Plant Regeneration from Tissue Culture of Silybum marianum Gaertn

Leaf calli of Silybum marianum Gaertn. subcultured for one year were used for protoplast isolation and culture. First division was observed three days after culture on medium M12, and the highest division frequency was 35.4%. One to three months later, small ralli were seen with naked eyes, and grew up gradually. Upon transferring them onto D6 differentiation medium, the green bud apices were observed two months later. However, no shoot differentiation was obtained. Hypocotyl calli were induced on MS+NAA 0.8mg/1, 6-BA 0.5mg/1. Two months after transferring calli onto D6 medium, shoots were regenerated from the surface of the calli. The freqency of shoot differentiation was 75%. On a MS rooting medium containing NAA 0.5 mg/1, IBA 0.1 mg/1, whole plants with healthy roots were obtained.     

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。     

水稻原生质体培养再生植株程序化的初步研究

从12个品种水稻成熟种子诱发愈伤组织并继代培养,通过MS培养基中2,4-D浓度的变换,研究了2,4-D对水稻愈伤组织生长的影响。用AA培养基建立适合原生质体培养的胚性细胞悬浮系仅需3个月。由悬浮细胞系游离的原生质体在改良的KPR培养基中进行液体浅层培养,有10个品种获得高植板率的细胞团。变换使用不同的分化培养基,从7个品种得到再生植株。实验重复性达到80%,初步实现了水稻原生质体培养的程序化。     

Plantlet regeneration from protoplast-derived haploid embryogenic calli of wheat

To search for an alternative method for protoplast culture, regenerable embryogenic calli were obtained from anther culture of three wheat cultivars, Karl 92, Jinghua #1, and Pavon 76. Protoplasts were isolated directly from the haploid embryogenic calli and cultured in modified PMI and LM8P media without going through cell suspension culture. After 8–11 days of subculture, the embryogenic calli produced the maximum yield of protoplasts and cell division was at the highest frequency when plated at a density of 3–4 × 10⁵ protoplasts ml⁻¹. Frequency of colony formation varied from 0.2% to 0.5% for Jinghua #1 and from 0.1% to 2% for Pavon 76, while Karl 92 failed to produce colonies, even though its embryogenic calli were friable and fast-growing on the maintenance medium. Green haploid plantlets of Jinghua #1 and Pavon 76 have been regenerated from protoplasts, which were cultured on a differentiation medium first and then on a rooting medium. This revised version was published online in June 2006 with corrections to the Cover Date.     

霸王的原生质体培养的研究

目的：为利用原生质体融合技术转移霸王抗旱基因。方法：采用酶解法分离霸王原生质体,比较了霸王子叶和愈伤组织游离原生质体的产量和活力,不同渗透压和起始密度对原生质体分裂频率的影响。结果：愈伤组织游离的原生质体产量和活力均高于子叶,原生质体产率可达2.4×106个/g.FW,活力达89%。采用液体浅层培养,在附加2,4-D（2mg/L）、6-BA（1.0mg/L）、2%蔗糖和甘露醇（0.4mol/L）的DPD培养基中,原生质体分裂频率最高,达68.6%。转移到附加2-iP（3mg/L）、KT（1.0mg/L）、6-BA（1.0mg/L）的分化培养基上,获得2个再生苗。结论：采用酶解法游离霸王愈伤组织,可获得高活力和高分裂频率的霸王原生质体。     

黑果枸杞原生质体培养及植株再生

该研究以黑果枸杞(Lycium ruthenicum)无菌苗为材料,建立了愈伤组织来源的原生质体再生体系,采用ISSR和FCM技术对再生植株进行了遗传稳定性分析。结果表明：(1)黑果枸杞叶片愈伤组织是产生原生质体的最好材料,在含0.5 mg·mL^-1甘露醇的酶液中,继代1次的叶片愈伤组织中原生质体产量为7.77×10⁶个·g^-1,活力为92%。(2)改良MS培养基 固体液体双层培养(MS₂ 固液双层)是培养原生质体的最好方式,培养10 d的原生质体分裂频率为45.9%,培养20 d的细胞团形成频率为22.9%。(3)在1.5 mg·mL^-1 6 BA+0.1 mg·mL^-1 IBA+MS培养基中,叶片愈伤组织产生的原生质体可分化获得再生植株。(4)ISSR分析显示,再生植株的平均遗传相似系数为0.88;FCM显示再生植株为二倍体,与亲本植株一致。该研究结果为进一步研究枸杞体细胞杂交技术转移野生植物抗逆遗传性状提供科学依据,为枸杞优良品种的选育奠定了基础。     

草木樨状黄芪单细胞培养再生植株及其影响因素的研究

从草木樨状黄芪的子叶和胚轴诱导的愈伤组织中选取结构松脆的颗粒状愈伤组织,用液体培养基Ｓ１２进行悬浮培养建立了悬浮细胞。取换液３天左右生长旺盛的悬浮系材料分离单细胞,用基本培养基Ｓ１２与不同浓度的植物血球凝集素组成的系列培养基ＭＣ１－ＭＣ６进行液体浅层静置暗培养;形成小愈伤组织后,经Ｍ３（ＭＳ＋２,４－Ｄ ２ｍｇ／Ｌ,ＮＡＡ ０．２ｍｇ／Ｌ,ＫＴ １ｍｇ／Ｌ）培养基增殖,ＭＲ２培养基诱导分化出苗,再     

Cauliflower inflorescence protoplast culture and plant regeneration

A yield of 2–4×10⁶ protoplasts/g F.W. could be obtained when fresh cauliflower inflorescence segments were digested with 2% cellulase Onozuka R-10, 1% cellulase RS and 0.4% Macerozyme R-10 in CPW18S for 7 to 10 h. Purified protoplasts were cultured in K8p liquid and agarose medium. Although protoplasts in liquid medium divided earlier than in agarose, protoplast-derived cells in liquid culture could not avoid browning. With agarose culture, sustained division and callus formation could be achieved. After 20 days, calli were transferred onto B5 agar medium with ZT 1.5 mg l^-1, BA 0.5 mg l^-1 and IAA 0.1 mg l^-1 for shoot formation. The frequency of bud formation varied from 56.7% for calli of 1mm in size to 5.6% for 5mm calli. The shoots formed were rooted in B5 medium containing 0.5 mg l^-1 IBA, and the regenerated plants were transplanted to pots and grew normally. It took about two months from protoplasts to the regenerated plants.Abbreviations Ade adenine - BA 6-benzyl aminopurine - CH casein hydrolysate - CM coconut milk - 2,4-D 2,4,-dichlorophenoxyacetic acid - GA₃ gibberellic acid - Gln glutamine - NAA -naphthylacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - ZT zeatin     

Isolation,Culture and Plant Regeneration of Protoplasts from an Embryogenic Callus Tissue of Gossypium hirsutum

Protoplasts were isolated and cultured from hypocotyl embryogenic callus tissue of Gossypium hirsutum L. cv. "Lumian 6". The highest yields of viable protoplasts were obtained from a vigorous embryogenic callus 7 to 9 d old subcultured on MS medium supplemented with 2 mg/L IAA and 1 mg/L KT using a solution of 1% cellulase Onozuka R-10, 1% pectinase, 0.7 mmol/L KH2PO4, 2.5 mmol/L Ca2+ , and 0.5 mol/L osmoticum (mannitol), at pH 5.8 and at a temperature of 30 ℃. After separation and purification (in 21% sucrose floatation medium), the protoplasts were laid up in a quiet liquid protoplast culture medium containing K3 salts, NT vitamins with 0.1 mg/L 2,4-D, 0.2 mg/L KT and 0.45 mol/L glucose for 10 to 15 min. The protoplasts were fractioned into an upper and a lower layer in the centrifugal tube. Most of the protoplasts in the lower layer were smaller, round and rich in cytoplasts in which contain many granular substances. When this kind of protoplasts were cultured in the thin liquid protoplast culture medium with a density of 1 x l0s to 5 x los protoplasts/mL, the division and the callus formation of the regenerated cells were easily observed. The first divisions occurred in 3 days and small cell clusters could be seen after 2 to 3 weeks in the culture. At this moment, the addition of the protoplast culture medium with decreased osmoticum once or twice is needed for the continuous protoplasts division to form calli. Regenerated calli, 3 to 5 mm in diameter, were transferred in succession on MS medium with 2 mg/L IAA and 1 mg/L KT for the initiation of embryogenesis. The embryoids germinated on the hormonefree MS medium and a number of plantlets were obtained. It seems that using vigorous embryogenic callus and decreasing osmoticum are the two critical factors for plant regeneration of cotton protoplasts.     

Plant Regeneration from Protoplast of Trititrigia (Triticum sect. trititrigia Maekey)

Trititrigia is the intergeneric hybrid which is from the hybridization between Triticum durum Desf. and Elytrigia intermedium (Host) Nevski. Protoplasts of Trititrigia were isolated from the embryogenic cell suspension derived from immature inflorescence-induced calli of the hybrid F1. The first division occured 48 hr after plating in modified KM8p culture medium. The plating efficiency of protoplasts was 2% and 12.14% when they were cultured in liquid medium and agarose solidified medium, respectively. Clusters grew vigorously under these conditions. Fresh medium with decreased osmoticum was added 20–30 days after plating. When protoplast-derived calli, 2–4 mm in the size, were transferred step by step to different differentiation media, embryoids, green spots emerged and numerous plants regenerated eventually.     

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 Sainfoin(Onobrychis viciaefolia)Protoplasts Derived from Cell Suspensions of Hypocotyl

The protoplasts were isolated from cell suspension cultures of hypocotyl (Onobrychis viciaefolia) cullured continuously for 3–4 months, and were cultured in modified Wguid Ⅴ- KM medium. The first division of the regenerated cell occurred after 24 h. culture. Small calli could be seen with naked eyes in 4 weeks. The calli which were propagated to 2–4 mm long in diameter in the (Ⅳ) medium were transferred onto differentiation medium and shoots appeared after 2–3 weeks. The differentiated shoots formed their roots on 1/2 MS supplamented with NAA 1.0mg/1 and grew into plantlets.     

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.     

甘蔗原生质体的植株再生

从甘蔗（Ｓａｃｃｈａｒｕｍ ｏｆｆｉｃｉｎａｒｕｍ Ｌ．）嫩叶外植体诱导愈伤组织,经继代培养后,挑选胚性愈伤组织,转入ＭＳ３ 液体培养基,进行悬浮培养。当培养物分离出小粒状的细胞团,细胞变得小而圆时,用于分离原生质体。原生质体以琼脂糖固化的培养方式培养于ＭＲＰ１ 培养基中。由原生质体再生的愈伤组织有两种类型。挑选粒状、坚实的再生愈伤组织转移到Ｎ６ 分化培养基上,“新台糖１ 号”再生的愈伤组织,在含有ＫＴ ０．５ ｍ ｇ／Ｌ的培养基中,分化出绿芽并长成完整的植株。而“粤糖５７－４２３”和“ＵＳ６６－５６－９”再生的愈伤组织,在加有０．１％ 的活性炭的培养基中,前者分化出白化苗,后者分化出根     

Regeneration Of Plantlets from Solanum nigrum L. Mesophyll Protoplasts

Using the “EA3-867” cellulase prepared in our laboratory, viable mesophyll protoplasts were isolated from Solanum nigrum L. The protoplasts grew and divided when cultivated in banging drops and thin liquid layer of NT medium, and calli formed. After transfering the calli on Dudits and MS solid medium (both supplemented with zeatin 1 mg/L, NAA 0.5 mg/L), regenerated plantlets had been obtained. The effects of different inositol amounts in NT medium on the growth of protoplasts were compared. The percentage of 1st and 2nd cell division after 5–8 days culture and the number of cell clusters increased in NT medium containing 250 mg/L inositol.