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.     

草木樨状黄芪甲硫氨酸抗性系的原生质体培养及植株再生

建立了草木樨状黄芪(Astragalus melilotoides Pall．)甲硫氨酸抗性系原生质体再生植株的实验体系。以茎切段诱导的松软愈伤组织为材料,通过酶法分离出大量有活力的原生质体。原生质体经培养持续分裂形成了愈伤组织,并高频率地分化出再生苗。比较了不同培养基、培养方法和培养密度对原生质体分裂和再生的影响。结果表明,原生质体以3×10⁵/mL的植板密度,采用琼脂糖岛法培养在附加1．0mg／L 2,4-二氯苯氧乙酸(2,4-D)、0．5mg／L 6-苄氨基嘌呤(6BA)、500mg／L水解酪蛋白、3％蔗糖、0．3mol／L甘露醇的KM_8p培养基中,可获得最佳效果,其细胞分裂频率达38％左右。原生质体培养后仍然保持对甲硫氨酸的抗性,同时对乙硫氨酸表现交叉抗性。     

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.     

Culture and regeneration of mesophyll-derived protoplasts of sorghum [Sorghum bicolor (L.) Moench]

 A protocol for plant regeneration from mesophyll/protoplasts of sorghum [Sorghum bicolor (L.) Moench] was developed. The yield of intact protoplasts, their subsequent divisions and regeneration were genotype-dependent. The genotype 296B was always more responsive than IS 32266. For 296B, the sixth leaf from 18-day-old plants kept in dark for 2 days before harvesting was found to be the most suitable source of viable protoplasts. The first division was observed 10–12 days after plating, and the second division after 12–14 days. The maximum plating efficiency was 4.8% in 296 B, followed by 2.48% in IS 32266. Microcolonies were visible after 25–30 days, and microcalli after 60–75 days. Whole plants were obtained after 6–8 weeks of culture of microcalli on MS medium containing 0.2 mg l^–1 kinetin and 2 mg l^–1 BAP. The frequency of regeneration in 296B and IS 32266 was 12.80% and 10.58%, respectively. Ten plants transferred to pots in the glasshouse established well. The seeds collected from glasshouse-grown plants were sown in the field where plants were grown to maturity. Received: 7 October 1998 / Revision received: 13 January 1999 / Accepted: 20 January 1999     

Plasma Membrane Redox System in Guard Cell Protoplasts of Pea (Pisum sativum L.)

Guard cell protoplasts (GCP) from leaves of pea (Pisum sativum)were capable of reducing/oxidizing the membrane impermeableelectron carriers, ferricyanide/NADH. The redox activity ofGCP required the presence of both ferricyanide and NADH, althoughsome ferricyanide reduction occurred even in the absence ofNADH. The GCP preferred NADH to NADPH during ferricyanide reductionand the reduction was slow with DCPIP or cytochrome c. A stoichiometryof about 2 existed between moles of ferricyanide reduced andNADH oxidized by GCP. The redox activities of GCP were severaltimes greater than those of mesophyll protoplasts from pea leaves.The ferricyanide reduction or NADH oxidation by GCP was unaffectedby abscisic acid or sodium orthovanadate and fusicoccin indicatingthe non-involvement of plasma membrane ATPase in these redoxreactions.The redox activities were markedly inhibited by chloroquineor 8-hydroxyquinoline. The findings are discussed in relationto the possible regulatory role of a guard cell plasma membraneredox system in stomatal function. Key words: Plasma membrane redox system, mesophyll protoplasts, pea, guard cell protoplasts, stomatal function     

Regeneration of whole plants from protoplasts isolated from tissue-cultured shoot primordia of garlic (Allium sativum L.)

Protoplasts derived from tissue-cultured shoot primordia of garlic (Allium sativum L.) initiated successive cell divisions within 4 days and formed small individual calli (0.2mm in diameter) after 5 weeks of culture on Gamborg's B5 medium supplemented with 0.1% casein hydrolysate, 1mg/1 1-naphthaleneacetic acid and 1mg/1 6-benzylaminopurine. Plating efficiency was roughly 5% at the density of 1x10⁴ protoplasts/ml of medium. Adventitious buds developed from the calli during subsequent subculture on Gamborg's B5 medium supplemented with 40mg/l adenine and 10% coconut milk. When transferred to the same medium without supplements, these buds grew into shoots and rooted. The regenerated garlic plantlets were successfully transferred to the greenhouse and grew into whole plants.     

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.     

Division frequency of pea protoplasts in relation to starch accumulation

Division frequency of alginate-embedded pea (Pisum sativum var. Belman) protoplasts derived from embryonic shoot tips was studied quantitatively by image analysis in relation to starch accumulation and protoplast size. Protoplast divisions were observed from day 4 on and the number of protoplasts undergoing division increased in a stepwise manner to 70% the following days. The starch content increased rapidly during the first 3 days of culture prior to the onset of division and resulted a 4.2-fold increase in the intracellular starch area and a 3.0-fold increase (from 27% to 80%) in the number of protoplasts containing starch. Subsequent periods with rapid increases the number of dividing protoplasts were preceded by further starch accumulation. Dividing protoplasts were 33–60% smaller and contained 8–42% less starch than non-dividing protoplasts. However, calculations showed that, in the dividing protoplasts, the relative area covered by starch was 6–12% higher than in non-dividing protoplasts. These data suggest that starch accumulation precedes division of pea protoplasts.     

Plantlets from mesophyll protoplasts of Solanum xanthocarpum

Young leaves of Solanum xanthocarpum from axenic shoot cultures released viable protoplasts when treated with appropriate enzymes. The protoplasts on culture in modified Murashige and Skoog (1962) medium supplemented with 2,4-dichlorophenoxy-acetic acid (0.5 mg/l), naphtha leneacetic acid (1 mg/l), kinetin (1 mg/l) and organic nutrients of KM (Kao and Michayluk 1975) regenerated to form callus tissue as a result of repeated divisions. Protoplast-derived calli differentiated into shoots on MS medium enriched with kinetin (0.5 mg/l) and rooting could be initiated by transferring the shoot-buds to basal medium.     

Plant regeneration from mesophyll protoplasts of Matthiola incana (L.) R. Br.

Summary A protocol for obtaining regenerated fertile plants from mesophyll protoplasts of three lines of Matthiola incana is described. Protoplasts were isolated from leaves of 21–28 days old Matthiola plants grown in controlled environment. Sustained divisions were achieved when protoplasts were embedded in sodium alginate. Up to 2.0 % of the protoplasts developed into colonies which could be transferred to shoot regeneration media. More than 25 % of the obtained calluses regenerated shoots. About 4 % of these shoots could be rooted and after transfer to soil phenotypically normal plants have been obtained.Abbreviations 2,4-D 2,4-dichlorphenoxyacetic acid - NAA naphthalene acetic acid - IAA indole-3-acetic acid - BAP 6-banzylaminopurine - IPA isopentenyladenine - IPAR isopentenyladenosine - MES (2-[N-morpholino]) ethanesulfonic acid     

Stimulation of respiration by Pb2+ in detached leaves and mitochondria of C3 and C4 plants

The exposure of detached leaves of C₃ plants (pea, barley) and C₄ plant (maize) to 5 m M Pb (NO₃)₂ for 24 h caused a reduction of their photosynthetic activity by 40–60%, whereas the respiratory rate was stimulated by 20–50%. Mitochondria isolated from Pb²⁺-treated pea leaves oxidized substrates (glycine, succinate, malate) at higher rates than mitochondria from control leaves. The respiratory control (RCR) and the ADP/O ratio were not affected. Pb²⁺ caused an increase in ATP content and the ATP/ADP ratio in pea and maize leaves. Rapid fractionation of barley protoplasts incubated at low and high CO₂ conditions, indicated that the increased ATP/ADP ratio in Pb²⁺-treated leaves resulted mainly from the production of mitochondrial ATP. The measurements of membrane potential of mitochondria with a TPP⁺-sensitive electrode further showed that mitochondria isolated from Pb²⁺-treated leaves had at least as high membrane potential as mitochondria from control leaves. The activity of NAD-malate dehydrogenase in the protoplasts from barley leaves treated with Pb²⁺ was 3-fold higher than in protoplasts from control leaves. The activities of photorespiratory enzymes NADH-hydroxypyruvate reductase and glycolate oxidase as well as of NAD-malic enzyme were not affected. The presented data indicate that stimulation of respiration in leaves treated by lead is in a close relationship with activation of malate dehydrogenase and stimulation of the mitochondrial ATP production. Thus, respiration might fulfil a protective role during heavy metal exposure.     

Plant regeneration from mesophyll protoplasts of Agrobacterium rhizogenes-transformed Astragalus melilotoides

Plant regeneration from mesophyll protoplasts of Agrobacterium rhizogenes-transformed Astragalus melilotoides Pall. was here developed. The protoplasts were isolated directly from the leaves of the hairy root-induced plants. The highest yield of protoplasts was obtained from fully expanded leaves of young plants. Their viability was up to 72 ± 2.3 %. The highest division frequency (32.4 ± 0.13 %) and sustained divisions were obtained in Durand, Potrykus and Donn (DPD) medium supplemented with 2.0 mg dm⁻³ 2,4-dichlorophenoxyacetic acid, 0.2 mg dm⁻³ 6-benzylaminopurine, 0.3 M mannitol, 2 % sucrose and 500 mg dm⁻³ casein hydrolysate at the plating density of 3.0 × 10⁵ cm⁻³. The frequency of shoot differentiation from protocalli reached to 91.75 ± 3.1 %. Opine synthesis and polymerase chain reaction analysis confirmed that T-DNA still existed in the protoplast regenerated plants.     

Plants regenerated from mesophyll protoplasts of white mulberry

INTRODUCTIONProtoplastcultureis0neofthen1ostrapidlydevel0pingareasinp1anttissueculture,becauseofitsimportancei11plantgeneticmanipulation.However,sofar,thereareonlyafewforesttreespeciesinwhichplantregenerationfr0mprotoplastshaJsbeensuccessful,namelyLiriode…     

Callus formation from stem protoplasts of corn (Zea mays L.)

Summary The first successful culture, with sustained divisions, of protoplasts from intact plants of Zea mays is described. The method involves the use of a hanging microdrop array technique which permits the testing of very large numbers of different culture media and hormone variations. Several different phytohormone combinations were found to allow sustained divisions in protoplasts isolated from stem tissue of corn plants, suggesting the importance of the source of the protoplasts rather than specific medium conditions. In some cases more than 5% of the protoplasts divided, giving macroscopic calluses within 35 days.     

鹰嘴紫云英甲硫氨酸抗性系原生质体培养及植株再生

本研究建立了鹰嘴紫云英(AstragaluscicerL.)甲硫氨酸抗性系原生质体再生植株的实验体系。以茎切段诱导的松软愈伤组织为材料,通过酶法游离出大量有活力的原生质体。原生质体经培养持续细胞分裂形成了愈伤组织,并分化出再生苗。比较了不同培养基、培养密度对原生质体形成细胞分裂和再生的影响。结果表明,原生质体以2×105个/ml的植板密度,在附加2.0mg/L2,4-二氯苯氧乙酸(2,4-D)、0.2mg/L6-苄氨基嘌呤(6-BA)、200mg/L水解酪蛋白、2%蔗糖和0.3mol/L甘露醇DPD培养基中培养后,其分裂频率达38.3%。原生质体培养形成的愈伤组织仍具有对甲硫氨酸的抗性。转移到附加10mg/LKT、0.5mg/LNAA的MS分化培养基上,获得大量的再生苗。     

Light-induced conversion of nicotinamide adenine dinucleotide to nicotinamide adenine dinucleotide phosphate in higher plant leaves

Light-induced conversion of NAD to NADP was investigated in higher plants. Upon illumination, conversion of NAD to NADP was observed in intact leaves of wheat and pea following incubation in the dark. This conversion was also observed in mesophyll protoplasts of wheat leaves when they were isolated in the dark or isolated in light and then preincubated in the dark. Chloroplasts isolated from wheat protoplasts prepared in the dark carried out the conversion. The conversion in the mechanically isolated spinach chloroplasts was observed only when they were isolated in the dark from leaves preincubated in darkness.     

Isolation and culture of protoplasts from mesophyll tissue of the legume Cyamopsis tetragonoloba L.

Protoplasts of Cyamopsis tetragonoloba were isolated from leaves of in vitro grown plants. The yield of the protoplasts, their viability and subsequent divisions were greatly influenced by the pH of the media used for isolation and culture of protoplasts. Sustained divisions of the cultured protoplasts were best supported by a modified Kao and Michayluk (1975) nutrient medium containing glucose (0.4 M), NAA (4 mgl^–1), 2,4-D (1 mgl^–1) and KIN (2 mgl^–1 ). The protoplast derived cells developed calli on transfer to Murashige and Skoog (1962) medium supplemented with 1 mgl^–1 each of 2,4-D, NAA and KIN.     

Regeneration of Mesophyll Protoplasts Isolated from Dihaploid Clones of Solanum tuberosum

Protoplasts have been isolated from leaves of shoot cultures of six dihaploid clones of Solanum tuberosum L. (2n = 2x = 24). In the KM medium (Kao and Michayluk 1975), sustained cell divisions were obtained in up to 50% of the plated protoplasts of four clones, whereas only a few divisions occurred in the other two clones. The first mitosis appeared 2–8 days after plating, dependent on the clones. In the clones showing sustained cell divisions, a protoplast titre of about 5 × 10³ per ml turned out to be optimal. The culture conditions for protoplasts of one of the poorly growing clones, clone H² 140, have been improved using modified KM media, plating at a concentration of as high as 5 × 10⁴ cells per ml, and subsequent diluting at intervals 5 days. The dilutions were carried out with media containing 0.25% agar. Up to 60% of the plated protoplasts underwent divisions within 10 days under these conditions. After about 15 days, the regenerants were transferred onto media inducing organogenesis. Shoots and roots were formed on modified media MS (Murashige and Skoog 1962) and B5 (Gamborg et al. 1968). Plants have been regenerated in four of the investigated clones. Countings of chromosomes revealed a satisfactory stability of the karyotype in shoot culture and protoplast regeneration.     

Plant regeneration from protoplasts of soybean (Glycine max L.)

Protoplasts were isolated from immature cotyledons of six cultivars of Glycine max L. and cultured in the KP8 liquid medium supplemented with 0.2 mg/L 2,4-D, 1 mg/L NAA and 0.5 mg/L ZT. The protoplasts started to divide after 3–5 days of culture. Sustained divisions resulted in mass production of cell colonies and small calli in 6 weeks. The calli further grew to 2–3 mm on the gelritesolidified K8 medium and were transferred onto the MSB medium with 1 mg/L 2,4-D and 0.25 mg/L BA, to obtain compact and nodular calli. Shoot formation was initiated on MSB medium with 0.15 mg/L NAA, and BA, KT and ZT, 0.5 mg/L of each, with or without 500 mg/L CH. It was followed by plant regeneration. So far, 87 plants have been regenerated from 4 cultivars, and normal seeds were obtained from them after transplanting into pots.Abbreviation IAA indol-3-acetic acid - NAA naphthalene acetic acid - 2,4-D 2,4-dichlorophenoxy acetic acid - KT kinetin - BA 6-benzyladenine - ZT zeatin - CH casein hydrolysate     

Isolation and culture of protoplasts from cotyledons of Pinus coulteri D. Don.

A protocol was developed for the isolation and culture of protoplasts from the cotyledons of seedlings of Pinus coulteri D. Don. Incubation of cotyledon pieces in a mixture consisting of cellulase Onozuka R10 2%, Pectolyase Y-23 0.1%, mannitol 10%, CaCl₂ 500 mg/l and other macro and micro-nutrients yielded viable protoplasts. After 24 hours of culture in a complex nutrient medium, the protoplasts regenerated new cell walls and the first divisions were observed within 7–10 days. Small cell colonies were formed within 15–20 days, but these started to accumulate phenolics and no further growth of the colonies was observed.