Plant regeneration from cultured protoplasts of a glutinous rice

Young embryos of ricy (Oryza sativa L.subsp.japonica var.Guo-xiang No.1) were cultured on MS agar medium(2,4-D 2 mg/l).Calli were formed and subcultured on N6 agar medium (2,4-D 2 mg/l).After selection,the small,grainy and pale yellowish cell clusters with dense cytoplasm were used in protoplast preparation. Isolated protoplasts were cultured in N6 medium (2,4-D 1 mg/l,6-BA 0.2 mg/l)^1* with agarose block culture method.The protoplasts grew,divided and formed calli.After inducing differentiation,the regenerated mature plants were obtained.

The protoplasts isolation,culture and shoots regeneration of broccoli(Brassica oleracea var.italica)

Eight F1-hybrid cultivars of broccoli were studied.We obtained cell division,celled colonies and p-calli in 5 cultivars,roots and shoots regeneration in one cultivar.The leavesof propagated plantlets in vitro were cut into 1—2mm pieces,isolated with an enzyme solutioncontaining 2% cellulase and 1%macerase on a rotary shaker（50 rpm,21℃,3h,2500 lux light）,and purified with a 0.5M sucrose solution.The purified protoplasts were placed on a drop of 1%agarose.2—3 ml liquid medium was added around the agarose drops,and all of the cultures wereincubated at 25℃ under light（4000 lux）for 16 hours.3—5 days after isolation the cell divisionwas found.About 7 days after incubation 4 multicellular colonies were formed.After 3—5 wksome p-calli were developed.When the p-calli were 2—3 mm in diameter it was transferred to asolidified medium.Once they were developed to 1 cm in diameter they were transferred on a re-generation medium.About 5 months after incubation some roots and shoots grown from the calliwere

Plant regeneration from protoplasts of hydroxyprolineresistant cell line in Onobrychis viciaefolia

An efficient protocol for plant regeneration from protoplasts of hydroxyproline(HYP)resistant cell line of Onoblychis viciaefolia was established. In SH medium supplemented with 1 mg/L 2, 4-dichlorophenoxy-acetic acid (2,4-D), 0.5 mg/L kinetin (KT) and 0.2 mg/L naphthalene acetic acid (NAA), the division frequency of protoplastderived cells reached uP to over 60 %, and microcalli were obtained in 5-6 wk. Upon transferring them on agar solidified MS medium plus 2 mg/L indole-3-acetic acid (IAA), shoots were induced. After cultivating them on MS medium with or without IAA, roots were regenerated.Chromosome number of all protoplast-regenerated plants examined were normal (2n=28). The protoplast-derived calli and plants grew vigorously on the medium containing 10 mmol/L HYP.

Plants regenerated from mesophyll protoplasts of white mulberry

Morus alba(white mulberry) mesophyll protoplasts were isolated from leaves of 30-45 day old sterile shoots,with protoplast yields of 2.5 x 107 g-1/F.W. after purification. The protoplasts were cultured in a modified K8P liquid medium containing 0.2 mg/L 2,4-D(2,4- Dichlorophe-noxy acetic acid), 1 mg/L NAA(Naphthyl acetic acid) and 0.5 mg/L BA(6-benzylaminopurine). A low plating density (5 x 104/ml) proved to be favourable to the division of protoplast-derived cells. The first divisioll occurred 4 days after culture, and the division frequency reached 24% at 10 days. A number of cell colonies and microcalli formed in 6 weeks. The microcalli were transferred onto MSB medium with 0.5 mg/L NAA and 0.5 mg/L BA for further proliferation. Shoot formation was initiated when the calli of 3-4 mm in size were transferred onto MSB differentiation medium with 0.1 mg/L NAA and 1 mg/L BA. The frequency of shoot formation was 35%. The shoots of 4-5 cm in height were excised from the callus and rooted on half strength MS medium with 0.5 mg/L IBA and 0.1 mg/L BA. After transplantation into pots, the regenerated plants grew vigorously in the phytotron.

High efficiency of protoplast preparation for artificially cultured Ulva prolifera(Ulvophyceae,Chlorophyta)

Protoplast isolation was relevant for gene manipulation in U lva, and universal protocols have been proposed based on evaluation for various wildly collected species. However, only clonal laboratory cultures were practical for genetic transformation, and whether applicability of such universal protocol existed for these artificial cultures has never been investigated. In this research, samples in different physiological states or developmental stages were tested in U. prolifera. The results proved that the protoplast yields were strongly dependent on the characteristics of samples. Neither F_v/F_m value nor chlorophyll content exhibited an ideal correlation with the protoplast yields. Alternatively, specific growth rate, coupled with developmental stage, could serve as an ef fective combined index to determine the right time for protoplast isolation. According to this instruction, here we reported the highest yields of protoplast((31.5±1.9)×10~6 cells/g f. wt.) in U. prolifera, following comparison between protocols, and further optimizations on enzyme content, incubation period, starting biomass and pretreatment. This specified protocol for artificially cultured clonal samples could meet the need for protoplast-mediated genetic transformation in U. prolifera.

PLANTLET REGENERATION FROM PROTOPLASTS ISOLATED FROM CALLUS CULTURES OF IMMATURE INFLORESCENCES OF WHEAT(Triticum aestivum L.)

Wheat (Triticum aestivum L.) is one of the most important cereal crops in the world.Great attention has long been paid to the technique of protoplast culture of this species.Up to now, plantlet regeneration from protoplasts of anther-derived suspension cultures of wheat has been reported for the first time. In the present study, we have obtained numerous embryoids and plantlets from protoplasts of immature inflorescence-derived calli of spring wheat.

CULTURE AND REGENERATION OF POPLAR MESOPHYLL PROTOPLASTS

Populus tomentosa is one special species of poplar growing in North China. Mesophyllprotoplasts were isolated from the axenic shoots and cultured in the modified KM8p liquidmedium. Protoplast-derived cells started to divide after 7 days of culture. The frequencyof cell division reached about 20% in 10 days. The yellowish green calli grew compact andnodular after the hormone concentration of medium was adjusted. Shoot formation occurredwhen the protoplast--derived calli were transferred onto MS medium containing zeatin andIAA or NAA. The shoots rooted readily on 1/2 MS hormone--free medium.

CULTURE OF WHEAT PROTOPLAST ——HIGH FREQUENCY MICROCOLONY FORMATION AND PLANT REGENERATION

Embryogenic calli were induced from the mature seeds of the hexaploid semi-winter wheat. The embryogenic cell line was established, and then the friable calli suitable for suspension were induced by adjusting the content of reduced N and changing the 2,4DAA concentration. Protoplasts were isolated from the suspension cells and cultured(?)in KM8P and some other media. A great number of microcolonies were obtained. By altering media, the microcolonies were promoted to grow further and form compact or nodule-like calli. Intact plants were regenerated through organogenesis and embryogenesis on differentiation media.

‘白天堂’百合原生质体的分离纯化与培养

【目的】探究百合原生质体分离纯化和培养条件,为百合原生质体融合、再生体系建立、转基因育种等研究奠定基础。【方法】以‘白天堂’百合无菌苗叶片为材料,采用纤维素酶、果胶酶和离析酶组合分离原生质体,采取单因素试验法,对影响原生质体的制备及再生条件的主要因素即渗透压、酶解组合、细胞筛和酶解时间进行筛选与优化。【结果】以百合无菌苗叶片在1.0%纤维素酶+0.5%离析酶+0.1%果胶酶+0.6 mol/L甘露醇+10.0 mmol/L CaCl_(2)·2H_(2)O+20.0 mmol/L MES+20.0 mmol/L KCl混合酶解液为最优条件,25℃,25 r/min, pH为5.6黑暗酶解6 h;孔径75μm细胞筛过滤,600 r/min离心5 min收集细胞,原生质体产量可达6.4×10^(5)个/mL,原生质体活力为78.0%;将纯化后密度为1×10^(5)个/mL的原生质体溶液接种在葡萄糖作为唯一碳源的培养基中可以存活,并生长产生细胞壁,但没有启动细胞分裂和诱导形成愈伤组织。【结论】初步建立‘白天堂’百合的高效原生质体分离与纯化体系,为对原生质体进一步操作研究奠定基础。

野生黄花苜蓿叶肉原生质体培养和植株再生

取野生黄花苜蓿15～20天叶龄的叶片，去掉下表皮，用1％纤维素酶、1％半纤维素酶和0．5％果胶酶的混合液游离原生质体。用改良的MS培养基附加2，4－D、BA和NAA等，在黑暗条件下浅层悬浮培养。培养第4天、第7天和第10天，原生质体分别出现第一次、第二次和多次分裂，在此期间数次加液或换液调节渗透压，直至形成1mm左右的大细胞团，然后转移到附加2，4-D和KT等的MS固体培养基，诱导形成愈伤组织，再经4～5次继代培养，分化成为完整的再生植株。

草地早熟禾新格莱德胚性愈伤组织原生质体培养及植株再生的研究

以草地早熟禾品种——新格莱德成熟种子为供试材料,在含3.0 mg/L 2,4-二氯苯氧乙酸（2,4-D）0、.5 mg/L6-苄氨基嘌呤（6-BA）的MB5培养基中进行胚性愈伤组织诱导的培养。并从生长了7-9个月的胚性愈伤组织中分离出原生质体,将该原生质体置于KM8P培养基（含3.0 mg/L 2,4-D、0.5 mg/L 6-BA、100 mg/L水解酪蛋白、100 mg/L水解乳蛋白、1%蔗糖0、.4 mol/L甘露醇）中进行了液体浅层培养。结果表明,新格莱德原生质体在上述KM8P培养基中培养3 d后出现第1次细胞分裂,2-3周后形成小细胞团,此时添加低渗培养液2-3次,小细胞团持续分裂并形成愈伤组织。当愈伤组织块长至3-5 mm时,转入固体培养基MS＋3.0 mg/L 2,4-D＋0.5 mg/L6-BA和MS＋0.5 mg/L萘乙酸（NAA）＋5.0 mg/L 6-BA上进行培养,使其细胞增殖和分化,且逐步形成完整的植株。

紫花苜蓿原生质体培养与植株再生

由和田苜蓿和单选苜蓿叶肉分离原生质体，采用改良的K8P和MS培养基进行液体浅层培养。原生质体经一次分裂，二次分裂和多次分裂，形成小细胞团，并很快长成小愈伤组织。愈伤组织在MS分化培养基上增殖并分化，产生小植株，经MS无激素培养基生根形成完整的再生植株。

红豆杉内生真菌发酵培养基和原生质体制备酶系统的筛选

在对红豆杉内生真菌(Ozoniumsp.)适生碳源、氮源和生长情况研究的基础上,通过正交试验筛选了其发酵培养基和原生质体制备的酶系统;用L9(34)安排了四因素三水平并考虑交互作用的正交试验,对实验结果进行了分析.结果表明,最优的发酵培养基为果糖1%、蔗糖1%、蛋白胨0.2%、酵母粉0.5%、KH2PO40.5%、MgSO4·7H2O0.3%、VB10.001%;分离原生质体的最优酶系统为1.5%溶壁酶+0.5%蜗牛酶+1.5%纤维素酶+1.0%溶菌酶;用此酶系统在30℃条件下酶解3h,原生质体的产量达6.55×107个/mL酶液;经荧光素二醋酸酯(FDA)染色评估原生质体活力,表明该条件下分离的原生质体活力较高,原生质体的再生率为2.56%.该研究为利用生物技术手段改良紫杉醇生产菌奠定了基础.

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。

不结球白菜子叶原生质体培养再生植株

用 1 0g/ 10 0mL纤维素酶 (OnozukaR 10 ) ,0 1g/ 10 0mL果胶酶 (MecerozymeR 10 )及 10mmol/LCaCl2 ·2H2 O ,0 7mmol/LKH2 PO4 和 0 5mol/L甘露醇的混合酶液 ,从 14～ 18d苗龄的不结球白菜子叶上分离出高产率的原生质体。原生质体培养在KM8P1附加 2 ,4 D 0 5mg/L、 6 BA 0 2 5mg/L、NAA 0 5mg/L、葡萄糖 9 0g/ 10 0mL、蔗糖 1 0g/ 10 0mL、半乳糖0 0 3g/ 10 0mL液体培养基上 ,分裂旺盛。形成愈伤组织后经芽诱导和生根培养 ,获得了再生植株。

草莓原生质体的培养和植株再生

通过对已分离提纯的草莓原生质体的培养研究 ,比较得出以KM为培养基 ,以 0 .4mol L葡萄糖和 0 .1mol L蔗糖或仅以 0 .5mol L葡萄糖作碳源 ,采用低溶点琼脂糖包埋的培养方式 ,培养密度采用 1× 10 6个 mL ,原生质体出现分裂和小愈伤组织形成的时间都较早。以MS1 、MS2 、MS3 为培养基、采用分步诱导的方法 ,能顺利诱导产生新植株 。

不同激素条件下大豆原生质体培养和植株再生

以栽培大豆(Glycine max(L.)Merr.)南农86-21、南农86-4、南农73-935为材料,用K8/K8p基本培养基比较了不同原生质体密度和不同激素种类、水平对未成熟子叶原生质体培养的影响,发现它们的差异表现在植板率、产生愈伤组织的速度和频率、愈伤组织的颜色和结构等几方面。对低激素来源南农73-935的愈伤组织通过器官发生诱导分化产生完整植株。通过液/固双层培养能促进分裂后的细胞形成大的愈伤组织。

玉米原生质体培养及可育植株的再生

由玉米获白×莱1029的幼胚诱导愈伤组织,经改造后进行液体悬浮培养,获得了优良的、有再生能力的悬浮细胞系。用其分离原生质体,采用4种方式进行培养,最高原生质体植板率达到1.2％。原生质体再生的小愈伤组织,通过分步诱导分化法,获得了5株完整的绿色植株,经进一步的壮苗培养后,移栽到土壤中全部成活,最终雄穗结籽。后代植株育性正常。

苹果原生质体分离培养及植株再生

以悬浮培养细胞及叶片作为分离原生质体的起始材料 ,对影响苹果原生质体分离和培养的因素进行了系统研究。优化的原生质体培养基为 :改良MT +维生素C 5mg/L +BA0 .5～ 1mg/L +2 ,4 D 0 .2mg/L +蔗糖 0 .6 5mol/L +谷氨酰胺 50 0mg/L +CH 10 0mg/L +ME50 0mg/L ;以葡萄糖代替蔗糖作渗透压调节物质效果好 ,且在培养过程中不需降压 ;适宜的低密度培养 (0 .5× 10 5/mL)可减少褐变。悬浮系原生质体培养 5～ 6d出现第 1次细胞分裂 ,15～ 2 0d形成多细胞团 ,4 0～ 50d形成肉眼可见的小愈伤组织。经培养诱导出不定芽 ,并进一步诱导生根长成完整植株。获得了平邑甜茶、M2 6 、嘎啦 3种基因型的原生质体再生植株。