Callus formation from protoplasts of a sugarbeet cell suspension culture

Protoplasts of sugarbeet (Beta vulgaris L.) were isolated from cell suspension cultures and cultured in modified PGo medium. Conditions required for the efficient division of the protoplasts were investigated.The optimal combination of phytohormones was found to be 1 mg/l NAA, 0.2 mg/l 2,4-D, 0.5 mg/l zeatin. Protoplast division was also considerably stimulated by the addition of 250 mg/l casein hydrolysate, 200 mg/l yeast extract, and 20% v/v conditioned culture medium to the protoplast culture medium. The highest division rate (up to 35% of the protoplasts) was achieved at a density of 4×10⁴- 1×10⁵ protoplasts/ml. From the colonies callus and suspension cultures were readily obtained.Abbreviations BAP 6-Benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA Indole-3-acetic acid - Kin 6-Furfurylaminopurine (kinetin) - NAA -Naphtalenacetic acid - Zea Zeatin     

Callus formation from protoplasts of a maize cell culture

Summary A finely dispersed cell suspension culture from the friable callus of the Black Mexican Sweet line of maize was obtained. Protoplasts from this cell culture, when grown in a simplified medium described here, showed sustained cell divisions and gave rise to callus.Abbreviations 2,4-D 2,4-dichlorophenoxyaceticacid - SDS sodium dodecyl sulfate Cooperative Investigation, United States Department of Agriculture and Institute of Food and Agricultural Sciences, University of Florida, Florida Agricultural Experiment Station Journal Series No. 2453. Mention of a trademark, proprietary, product, or vendor does not constitute a guarantee or warrantly of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable     

Callus formation from protoplasys of Sorghum cell suspension cultures

Cell suspension cultures have been obtained from three cultivars of Sorghum bicolor L. Moench. Protoplasts readily obtained from these cultures underwent sustained cell division and callus formation.     

Callus formation from protoplasts of cell suspension cultures of Rosa 'Paul's Scarlet'

Sustained divisions of protoplast-derived cells obtained from cell suspension cultures of Rosa‘Paul's Scarlet’ leading to colony and callus formation was achieved. The rather low plating efficiencies observed for agar-plated protoplasts were partly due to early arrests in further development of dividing protoplast-derived cells and cell clusters. Successful cultivation of dividing protoplast-derived cells was particularly dependent upon frequent subculturing of the precultures and on an efficient procedure for viable protoplast isolation. Colony formation was largely independent of medium composition.     

Pollination biology and breeding system of Acacia senegal

Phenology, pollination biology and breeding system were investigated in three populations of Acacia senegal located in Delhi and Rajasthan. Flowers emit a mild fragrance and produce a minute quantity of nectar. The stigma is wet non-papillate, cup-shaped and generally accommodates one polyad with 16 pollen grains. The style is solid. The mass effect created by the brush type of blossoms attracts a wide variety of insects, of which the giant Asian honeybee, Apis dorsata , is the effective pollinator. Manual in vivo pollination studies have shown that the species is self-incompatible. Self-incompatibility appears to operate inside the embryo sac. Under natural conditions fruit set is as low as 0.36%. Insufficient pollination is the main cause of low fruit set. Manual xenogamous pollinations substantially improve fruit set to 30%     

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

Summary Routine procedures for the isolation of large numbers of protoplasts from an established cell culture of Zea mays and for the induction of sustained divisions leading to secondary cell cultures have been developed. The critical factors seem to be associated with neither specific enzymatic conditions for the isolation nor specific culture conditions for the protoplasts but with the quality of the culture used for protoplast isolation.     

安徽羽叶报春愈伤组织的诱导和培养

为了寻找大批量快速繁殖安徽羽叶报春(Primula merrilliana)的有效办法,笔者以其叶片、叶轴为外植体,用不同浓度生长调节物质进行愈伤组织诱导实验.结果表明:(1)愈伤组织诱导的最适培养基为:MS 2.0mg/L 6 BA 0.5mg/L～1.0mg/L 2,4-D,叶片、叶柄出愈率均达100%.(2)MS培养基加0.1mg/L 2,4～D和1.0mg/L～1.5mg/L TDZ对叶柄的诱导效果较佳,其诱导率达81%,而且在这种生长调节物质浓度下的愈伤组织可直接分化出不定芽.     

茅苍术愈伤组织诱导及其细胞悬浮培养研究

采用正交试验法研究了不同的植物生长调节剂对茅苍术叶柄、叶片和根茎愈伤组织诱导的影响,结果表明,不同外植体在各自的最佳培养条件下,叶柄、叶片和根茎愈伤组织的诱导率分别为99.0%、83.5%和71.5%,以叶柄的培养效果最好,其中2,4-D对茅苍术愈伤组织的诱导具有极显著的效果,在各种植物生长调节剂组合中,诱导叶柄愈伤组织形成的最佳组合为0.4mg·L-1NAA、4.0mg·L-12,4-D和0.4mg·L-1KT,培养20d左右,诱导率达到99.0%。此外,将茅苍术叶柄细胞悬浮培养至18d时,细胞量、多糖和苍术素的含量均达到最大值,分别为9.07g·L-1、15.68mg·L-1和19.62ug·L-1。     

Callus induction and culture from explants of Erysimum scoparium in a growth regulator-free medium

Calli from cotyledon, hypocotyl and radicle explants of Erysimum scoparium (Brassicaceae) were induced and cultured using a MS medium without growth regulators. Calli obtained from cotyledon or hypocotyl explants showed a higher growth rate while radicle-derived calli exhibited poor growth. Cotyledon-derived calli were compact and green-colored while hypocotyl- and radicle-derived calli were friable and with creamy-green pigmentation. Histological analysis was carried out during culture and the development of abundant vascular elements and meristematic nodules were found. Subculture on a growth regulator-free medium during 10 months did not ecrease callus growth.     

Callus production from photoautotrophic soybean cell culture protoplasts

Summary Protoplasts were prepared from a photoautotrophic (PA) cell line of Glycine max (soybean). A yield of 75 to 90% after two to three hours digestion in a mixture of 1% Cellulase R10, 0.2% Pectolyase Y23 and 2% Driselase was obtained. Cell division and colony formation occurred from approximately 18% of the plated protoplasts. The cultured protoplasts were as sensitive to the herbicide atrazine, a photosynthetic inhibitor, as the original PA cells under the same conditions. Protoplasts and cells of a heterotrophic (HT) soybean culture were not as sensitive to atrazine. The isolated protoplasts retained the PA characteristics of the parental culture in the callus and cell suspension cultures obtained from the protoplasts. The chromosome numbers in the parental cell line and in cells derived from the isolated protoplasts (both PA and HT) were found to be largely (99%) the normal diploid number of 40.Abbreviations BA Benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - HT Heterotrophic - MES 2-(morpholino) ethane sulfonic acid - NAA Naphthaleneacetic acid - PA Photoautotrophic - PCM Protoplast culture medium     

Rhizobial Populations in Soils from Natural Acacia senegal and Acacia nilotica Forests in Mauritania and the Senegal River Valley

Eighty-two strains of rhizobia were isolated from soils taken from several sites in Mauritania and Senegal. These soil samples were collected from natural stands of Acacia nilotica and Acacia senegal. The soils from Mauritania were less rich in native rhizobia than the soils from Senegal. The strains were characterized using polymerase chain reaction–restriction fragment length polymorphism and by sequencing the rDNA 16S–23S intergenic spacer region (IGS). They were sorted into seven IGS groups. These groups were not associated with the geographical origin of the strains or with the host-plant species at the site where the soils were collected. Most of the strains were in three of the IGS groups (I, IV, and V). One representative strain from each IGS group was sequenced and showed that the strains were from the genus Mesorhizobium. IGS groups I, IV, and VI were close to the species M. plurifarium (AF34563), IGS groups IIand III were close to the species Mesorhizobium sp. (AF510360), IGS group V was close to the species Mesorhizobium sp. (AF510366), and IGS group VII was close to Mesorhizobium sp. (AF510346).     

Elicitor-mediated induction of enzymes of lignin biosynthesis and formation of lignin-like material in a cell suspension culture of spruce (Picea abies)

Incubations of photomixotrophic suspension culture cells of spruce (Picea abies) (L.) (Karst) with an autoclaved cell wall preparation of Rhizosphaera kalkhoffii as elicitor led to a rapid increase of the activity of a number of enzymes involved in lignin biosynthesis. l-phenylalanine ammonia-lyase (EC 4.3.1.5) was induced about 10-fold, feruloyl-Coenzyme A reductase (ED 1.2.1.44) 4-fold, cinnamyl alcohol dehydrogenase (NADP⁺) (EC 1.1.1.195) 2-fold and peroxidase (EC 1.11.1.7) about 1.5-fold. The induction of the enzymes, with the exception of the peroxidase, was transient, showing maximal activity within 3 days after elicitation. Extracellular peroxidase activity, determined in the culture medium, rapidly decreased on initiation of elicitation.Concomitant with the increase of activity of the enzymes of lignin synthesis was a rapid clouding of the culture medium. Phloroglucinol-HCl staining revealed the presence of lignin-like material in the medium and also in the cells. The IR-spectrum of this material was identical with the IR-spectrum of authentic spruce lignin.Abbreviations PAL l-phenylalanine ammonia-lyase - FCR feruloyl-Coenzyme A reductase - CAD cinnamyl alcohol dehydrogenase - POD peroxidase     

Properties of peroxidases from tobacco cell suspension culture

An enzyme preparation from suspension cultured tobacco cells oxidized IAA only in the presence of added cofactors, Mn²⁺ and 2,4-dichlorophenol, and showed two pH optima for the oxidation at pH 4·5 and 5·5. Effects of various phenolic compounds and metal ions on IAA oxidase activity were examined. The properties of seven peroxidase fractions separated by column chromatography on DEAE-cellulose and CM-Sephadex, were compared. The peroxidases were different in relative activity toward o-dianisidine and guaiacol. All the peroxidases catalysed IAA oxidation in the presence of added cofactors. The pH optima for guaiacol peroxidation were very similar among the seven isozymes, but the optima for IAA oxidation were different. The anionic and neutral fractions showed pH optima near pH 5·5, but the cationic isozymes showed optima near pH 4·5. With guaiacol as hydrogen donor, an anionic peroxidase (A-1) and a cationic peroxidase (C-4) were very different in H₂O₂ concentration requirements for their activity. Peroxidase A-1 was active at a wide range of H₂O₂ concentrations, while peroxidase C-4 showed a more restricted H₂O₂ requirement. Gel filtration and polyacrylamide gel studies indicated that the three cationic peroxidases have the same molecular weight.     

Callus induction and plantlet formation through culture of isolated microspores of eggplant (Solanum melongena L.)

Morphogenic calli were obtained efficiently from ab initio cultures of isolated microspores in eggplant. Initial culture of freshly isolated microspores in sucrose-free medium at high temperature (35°C) for 3 d was a prerequisite for callus induction. The microspores were re-cultured in modified NLN medium containing 2% sucrose and phytohormones (NAA 0.5 mg l^–1, BA 0.5 mg l^–1) in the dark. After 4 weeks of re-culture, small calli derived from microspores were transferred to MS medium containing 4 mg l^–1 zeatin and 0.2 mg l^–1 IAA for shoot regeneration. The ploidy of 12 randomly selected regenerants was assessed by chromosome counts in root tips. Only one of the regenerants was haploid, 7 were diploid, 3 were triploid and one was tetraploid. The diploids set seeds after self-pollination and showed no segregation for morphological traits in the progeny, suggesting that they were spontaneously doubled haploids.Abbreviations BA 6-benzylaminopurine - NAA 1-naphthaleneacetic acid - IAA indole-3-acetic acid - DAPI 4,6-diamidino-2-phenylindole dihydrochloride     

Embryogenesis from microinjected single cells in a carrot cell suspension culture

A micrroinjection method was established for intact single cells with cell walls using a carrot suspension culture system in which selected single cells differentiate to embryos at high frequency. A solution of a fluorescent dye, Lucifer Yellow CH, was microinjected into those single cells, using an inverted microscope and a hydraulic micro-manipulator. In order to hold cells with cell walls and to overcome their turgor pressure, certain modification to conventional microinjection methods for protoplasts were necessary. The microinjected cells could divide and differentiate to embryos at a frequency of about 50%.     

Establishment and characteristics of a cell suspension culture from a moss,Atrichum undulatum

A green cell suspension culture was established from callus tissue obtained from a moss,Atrichum undulatum. This suspension culture was subcultured every ten days in Murashige and Skoog's liquid medium (MS) containing 10^?6 M 2, 4-D and 4% glucose. The suspended cells grew vigorously with the passage of subculture, maintaining small clusters made up of a few cells. In this suspension culture, round spore-like cells released from the cell clusters were found. The yield of protoplasts from the suspension culture was significantly higher than that of the conventional method using moss protonemata developed directly from spores.     

Callus production,suspension culture and in vitro alkaloid yields of Ephedra

The effects of a range of plant growth regulators on callus production in various Ephedra species were examined. Species examined were E. andina, E. distachya, E. equisitina, E. fragilis var, camplyopoda, E. gerardiana, E. intermedia, E. major ssp procera, E. minima and E. saxatilis. All species produced callus on modified MS medium supplemented with 0.25 M kinetin and 5.0 M 2,4-dichlorophenoxyacetic acid or 1-naphthaleneacetic acid. Neither indole-3-acetic acid nor 3-indolebutyric acid induced significant callus formation but the latter maintained growth of established callus cultures in several species. Suspension cultures of several species were established in MS medium supplemented with 0.25 M kinetin and 5.0 M 2,4-dichlorophenoxyacetic acid or 1-naphthaleneacetic acid. Sustained fresh weight doubling times of 70±7h were recorded for cell suspension cultures of E. andina grown in a semi-continous air-lift bubble bioreactor and a minimum doubling time of 56 h was recorded for E. andina in batch culture. It also proved possible to immobilise E. andina batch cultures in sodium alginate beads.Neither parent plants or in vitro cultures of E. distachya, E. fragilis or E. saxatilis produced alkaloids. Trace quantities of 1-ephedrine and trace-0.14% dwt d-pseudoephedrine were produced by in vitro cultures of other species. The ability to produce alkaloid diminished to zero with successive subcultures.Abbreviations Eph 1-ephedrine - Peph d-pseudoephedrine - RGR relative growth rate - KIN kinetin - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IBA 3-in-dolebutyric acid - IAA indole-3-acetic acid     

Callus and cell suspension cultures of Rudgea jasminoides,a tropical woody Rubiaceae

Rudgea jasminoides is a woody Rubiaceae that produces phytoalexins in response to fungal inoculation, the response being dependent of the seasonal conditions. With the aim of studying phytoalexin induction under controlled conditions, callus cultures were established from petiole explants of R. jasminoides on a modified basal MS medium supplemented with picloram alone or in combination with kinetin. The highest frequency of callus formation was observed in solid medium containing 2.22 M kinetin and 2.07 M picloram. Development of fast-growing friable white callus was achieved in the absence of kinetin, in cultures supplemented only with 8.28 M picloram. Cell suspension cultures were established from this friable callus by transferring pieces directly to the same medium without agar. Preliminary experiments revealed that cell suspension cultures of R. jasminoides represent a useful system to analyse induced defensive metabolites produced by this Rubiaceae species.     

Promotion of respiration by auxin in the induction of cell division in suspension culture

Auxins (IAA, NAA, p-CPA) caused 20–30% promotion of respiration of auxin-dependent cell cultures ofNicotiana tabacum, Glycine max, Taraxacum mongolica, andAtriplex sp. and had small if any effect on respiration of auxin-independent cell cultures ofRubus sp. andScorzonera hispanica. Antiauxin (p-CPIBA) did not affect the respiration. The auxin effect on the respiration of tobacco cells was revealed 10 min after its addition to the suspension and reached a maximum value in 60 min. This stimulation preceded the induction of cell division by auxin. Mitochondria isolated from auxin-treated tobacco cells had greater oxidative and phosphorylative activity than mitochondria from untreated cells. However, isolated mitochondria did not respond to auxin. The inhibitors of respiration (cyanide, monoiodoacetate, malonate, and 2,4-dinitrophenol) eliminated auxin effect on the respiration and cell division. It is concluded that the promotion of respiration is a common event for the auxin effects both on cell extension and on cell division. This promotion is necessary for the induction of cell division and is exerted via direct activation of mitochondriain situ.