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 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     

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 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.     

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％。     

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.     

小麦胚性悬浮系与原生质体植株再生

普通小麦(Triticum aestivum)昌乐5号(冬性)胚性悬浮细胞系的组成成分对原生质体再生频率发生影响,此种悬浮系是由混合型愈伤组织建立起来的,建成的悬浮系中含有2—3mm的小愈伤组织和分散好的几十至上百个细胞的细胞团。分别用悬浮系中的小愈伤组织和细胞团分离原生质体进行培养。结果表明．由小愈伤组织来源的原生质体再生植株的频率显著高于由细胞团分离的原生质体的再生频率。培养基中不同成分对原生质体分裂的影响也作丁研究。     

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 from Cell and Protoplast Cultures of Triticum aestivum-Haynaldia villosa Hybrid

Starting materials used in these experiments were taken from Triticum aestivum-Hay-naldia villosa hybrid embryo-derived callus, which had been maintained for nearly two years. To establish suspension cultures, the callus was subcultured till its compact texture became friable, and then shaken in a liquid medium. Upon transferring the suspended cells onto a semisolid medium, high frequency of plant regeneration was achieved.     

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.     

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.     

Plant Regeneration from Supersweet Maize Protoplasts Derived from Gametophytic Cell Suspensions

Plant regeneration from protoplasts isolated from haploid cell suspensions of commercial supersweet maize (SS 7700) was achieved and the plants were survival after transfer into soil in pots. Protoplast plating efficiency obtained from feeder layer system was 130 folds higher as compared with conventional liquid culture method, the composition of protoplast culture medium, the pore size of supportive membrane filter and the relationship between protoplasts and feeder cells were critical for callus formation. An enriched medium containing vitamins, organic acids, amino-acids and other organic substances such as coconut water could extremely improve callus formation. Filters with pore size within the range of 0.22–8.0 μm in diameter was useful. Filters with smaller pore size of 0.04 μm or larger 11 μm appeared to decrease the frequency of protocolony formation. The feeder cells which belong to the same species (Zea mays) as protoplasts greatly increased protoplast plating efficiencies as compared to those of feeder cells belonging to other species such as Avena nuda and Nicotiana tabacum. Among 11 protoplast-regenerated plants examined, 10 plants were haploid and one plant was diploid.     

Plant Regeneration from Protoplasts of Ligusticurn wallichii Franch

The hypocotyls of the embryoid derived plantlets of Ligusticum wallichii Franch were used for protoplast preparation. Protoplasts were obtained with the enzyme mixture containing 1.5% Onozuka R-10, 0.3% Macerozyme R-10, 0.5% Snailase, 5 mmol/l CaCl2, 1 mmol/l KH2PO4, 0.6 mol/l manitol, at pH 5.6–5.8 and 27℃. Protoplasts were cultured in a modified MS liquid medium containing 1 mg/l 2,4-D + 0.5 mg/l 6- BA. The first divisions were found after twelve days, and the dividing cells formed cell colonies of 0.5–1 mm after about fourty days. When they were transferred to MS agar medium (with half quantity of macronutrients) supplemented with 2,4-D (0.5mg/l) and 6-BA(0.5mg/l), they grew into calli. At last, on the medium without any phytohormones, the growing calli differentiated embryoids which developed into plantlets with many green leaves and roots.     

大赖草染色体组构成的生化分析

本文通过对不同来源大赖草及其可能供体物种的EST、GOT、SOD同工酶及HMW-GS的分析, 发现不同来源大赖草的同工酶及蛋白图谱呈一定差异,但其中为不同来源大赖草所共有的一些谱带, 同时也存在于具N染色体组物种的分析图谱中,而在具J染色体组的物种中却没有特异性分布,推断大赖草中含有N染色体组,但可能不含J染色体组。 Abstract:The zymogram phenotypes of Esterase(EST),Glutamate oxalocate transminase(GOT),Superoxide dismutase(SOD)and the banding patterns of high molecular weight glutenin subunits(HMW-GS) were determined for different Leymus racemosus accessions and their putative donor species,It was found that although the zymogram phenotypes and the HMW-GS banding patterns appeared polymorphism,common bands for different Lr accessions were found in the species with N genome,but not in those with J genome,suggesting that the Psathyrostachys species with N genome must be phylogenetically closer to Lr,while the Thinopyrum species with J genome are farther to it.     

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.     

Somatic Embryogenesis and Plant Regeneration from Protoplasts of Cowpea (Vigna sinensis)

Immature cotyledons of cowpea (Vigna sinensis Endlo) were used for protoplast isolation. Enzyme solution for protoplast isolation contained 40% cellulase Onozuka R-10,0.30% Macerozyme R-10 and 2% hemicellulase. The purified protoplasts were cultured in Bs,MS or KM8p liquid medium in dark (25℃) at a density of 1 × 105–5 × 105/ml. The protoplasts started cell division in 3–5 days . Sustained cell divisions resulted ill formation of cell clusters and small calli,with cell division frequency reaching 23%–28% in MS medium . Calli of 2 mm in size were transferred onto MSB (MS salts+B5 vitamins) medium with 2 mg/L 2,4-D, 0. 5mg /L BA forfurther growth. Embryogenic calli appeared on this medium. After passage to fresh medium with the same composition, the embryogenic calli were transferred into MSB liquid medium to establish suspension culture. When the suspended calli were transferred back onto MSB agar medium with 0. 1 mg /L IAA, 0.5mg/L KT, 5% mannitol (cultured in light,2000 lx,12h/d), a lot of adventitious roots formed in 7–10 days, and then somatic embryos formed from the protoplast derived calli. But only a few embryoids developed further into the cotyledonary stage ,and the others died at globular, heart-shaped, or torpeto stage . Finally, some cotyledonary embryoids germinated and developed into plantlets or shoots with leaves.     

油菜游离细胞培养及易再生的体细胞无性系的建立

以芥菜型油菜(Brcssica juncea)下胚轴来源的愈伤组织为材料,进行悬浮细胞振荡培养,得到游离的单细胞。这些细胞作浅层液体静置培养,可获得高频率的愈伤组织。在附加有水解乳蛋白200毫克/升、BA 2—3毫克/升及GA,0.1—1毫克/升的MS培养基上,这些愈伤组织分化了芽。分化的芽在无激素或附加有0.01—0.1毫克/升IAA或NAA的MS培养基上长出根,从而发育成完整植株。当对游离细胞获得的再生植株的茎切段愈伤组织,再经上述程序进行细胞培养时,发现愈伤组织诱导率及愈伤组织分化频率均远远高于原供体,从而建立了B.juncea易再生的体细胞无性系。     

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 Protoplasts of Suspension Cells of Saposhnilkovia divaricata (Turcz.) Schischk

Embryogenic calli were produced from the segments of the young roots, hypocotyls or petioles of test-tube seedlings on MS agar medium containing 1 mg/1 2,4-D. When shaken in the MS liquid medium, the calli formed cell suspension with many embryogenic cell clumps.Using the enzyme mixture: Onozuka R-10 1.5%+MacerozymeR-10 0.3%+Snailase 0.5%+CaCl2 5 mmol/l + Mannitol 0.6 mol/1 (pH=5.8), protoplasts were obtained from the cell clumps which had been subcultured for three to' seven days. When cultivated, the protoplasts grew and began to divide after four days, and formed cell clumps about l—2 mm within fifty days. Protoplast-derived calli were formed from the cell clumps on the MS agar medium with 0.5 mg/l 2,4-D. When transferred onto the MS agar medium containing 0.1 mg/1 6-BA or 0.1 mg/1 2,4-D and 0.5 mg/1 6-BA, the calli differentiated into embryoids. On the MS agar medium without phytohormone, the embryoids grew into plantlets.