Endogenous cytokinins in Cocos nucifera L. in vitro cultures obtained from plumular explants

Auxin induces in vitro somatic embryogenesis in coconut plumular explants through callus formation. Embryogenic calli and non-embryogenic calli can be formed from the initial calli. Analysis of endogenous cytokinins showed the occurrence of cytokinins with aromatic and aliphatic side chains. Fourteen aliphatic cytokinins and four aromatic cytokinins were analysed in the three types of calli and all the cytokinins were found in each type, although some in larger proportions than others. The most abundant cytokinins in each type of callus were isopentenyladenine-9-glucoside, zeatin-9-glucoside, zeatin riboside, isopentenyladenine riboside, dihydrozeatin and dihydrozeatin riboside in decreasing order. Total cytokinin content was compared between the three types of calli, and it was found to be lower in embryogenic calli compared to non-embryogenic calli or initial calli. The same pattern was observed for individual cytokinins. When explants were cultured in media containing exogenously added cytokinins, the formation of embryogenic calli in the explants was reduced. When 8-azaadenine (an anticytokinin) was added the formation of embryogenic calli and somatic embryos was increased. These results suggest that the difference in somatic embryo formation capacity observed between embryogenic calli and non-embryogenic calli is related to their endogenous cytokinin contents.     

Localization and identification of phenolic compounds in Theobroma cacao L. somatic embryogenesis

Cocoa breeders and growers continue to face the problem of high heterogeneity between individuals derived from one progeny. Vegetative propagation by somatic embryogenesis could be a way to increase genetic gains in the field. Somatic embryogenesis in cocoa is difficult and this species is considered as recalcitrant. This study was conducted to investigate the phenolic composition of cocoa flowers (the explants used to achieve somatic embryogenesis) and how it changes during the process, by means of histochemistry and conventional chemical techniques. In flowers, all parts contained polyphenolics but their locations were specific to the organ considered. After placing floral explants in vitro, the polyphenolic content was qualitatively modified and maintained in the calli throughout the culture process. Among the new polyphenolics, the three most abundant were isolated and characterized by 1H- and 13C-NMR. They were hydroxycinnamic acid amides: N-trans-caffeoyl-l-DOPA or clovamide, N-trans-p-coumaroyl-l-tyrosine or deoxiclovamide, and N-trans-caffeoyl-l-tyrosine. The same compounds were found also in fresh, unfermented cocoa beans. The synthesis kinetics for these compounds in calli, under different somatic embryogenesis conditions, revealed a higher concentration under non-embryogenic conditions. Given the antioxidant nature of these compounds, they could reflect the stress status of the tissues.     

番茄离体培养过程中器官发生的细胞组织学观察

对番茄下胚轴、子叶、茎段、叶片、叶柄不同类型外植体离体培养中有关细胞启动、分裂、分化以及器官发生作了细胞组织学观察。研究结果表明番茄不同类型外植体在同样的培养条件下,愈伤组织生长表现出明显差异,其中下胚轴、子叶诱导产生愈伤组织时,细胞启动最早,生长最快,其分裂方式基本为无丝分裂,未见有丝分裂,因此我们认为以不定芽方式获得转基因植株时,植株的所有性状变化,是否纯属目的基因所为,应该反复考察,不能忽视不定芽产生过程中的种种变化;下胚轴诱导愈伤组织形成时,细胞不规则的无丝分裂少于子叶,故下胚轴离体培养得到的正常芽的比例高于子叶的;番茄离体培养中不定芽通常发生在愈伤组织的周边区,也可起源于维管组织结节周围的形成层状细胞。不定根则由茎中柱鞘处发生。     

Production of chlorogenic acid and isoorientin hypoglycemic compounds in Cecropia obtusifolia calli and in cell suspension cultures with nitrate deficiency

The antidiabetic properties of Cecropia obtusifolia are attributed to chlorogenic acid (CGA) and isoorientin (ISO) phenolic compounds; both compounds possess hypoglycemic, hypolipidemic, and antioxidant properties. As a potential strategy for an adequate supply of authentic plant raw material, the aim of this study was to establish in vitro conditions for the development of cell suspension cultures that produce these bioactive compounds. Callus cultures of leaf explants from acclimatized tree and in vitro plantlets were set up using different auxin levels; treatments with 2,4-dichlorophenoxyacetic acid (2,4-D) and α-naphthalene acetic acid (NAA) to 8.92 μM with 6-benzylaminopurine (BAP) at 2.22 μM stimulate highest callus production. Seedling cotyledon, hypocotyl, leaf, and stem explants developed calli bearing roots with 2,4-D. With NAA, hypocotyl, cotyledon, and leaf explants developed morphogenic calli; 75% of stem explants formed calli, and the remaining calli developed shoots. Determined CGA concentrations in calli were similar to those detected in the leaves of wild trees, and ISO was not produced. Cell suspension cultures were established from leaf explants friable calli with 8.92 μM 2,4-D in combination with 2.22 μM BAP, employing 4 and 5% inocula in fresh weight; CGA levels were maintained and ISO was produced only at the end of logarithmic growth. On diminishing nitrate content in Murashige and Skoog (MS) medium to 8.0 mM, maximum cell biomasses diminished, CGA production is increased and twice with 16.0 and, instead of CGA production is tripled and quadrupled with 16.0 and 8.0 mM nitrates, respectively, and ISO synthesis was induced earlier and for a longer time period, increasing its levels at the end of culture. Two compounds with ultraviolet spectra similar to those of caffeic and ferulic acids were formed. Our results offer a protocol of cell suspension cultures for C. obtusifolia bioactive production and hypoglycemic property conservation.     

Changes in Formation and Localization of Phenolic Compounds in the Tissues of European and Canadian Yew during Dedifferentiation <Emphasis Type="Italic">In Vitro</Emphasis>

Changes in formation and localizations of phenolic compounds, including flavans, were investigated in the tissues of European and Canadian yew (Taxus baccata L. and T. canadensis Marsh.) during dedifferentiation in vitro. Annual shoots of European yew had the highest capacity for synthesizing these compounds. During the summer growth period, the content of total soluble phenolic compounds and flavans in these shoots was 30–40% higher than in the winter. Cell dedifferentiation and growth in vitro was accompanied by enhanced synthesis of phenolic compounds, including flavans, the change in tissue localization of these compounds, and an increase in the number of cells containing phenolics. Significant accumulation of phenolic compounds in callus cells resulted in necroses following two subcultures in the European and Canadian yew cultures initiated from summer explants, and following seven subcultures of the European yew calli initiated from winter explants. These data allow us to suggest that a high level of phenolic compounds in yew calli could be the reason for their necrosis.     

Plant regeneration from calli of melon (Cucumis melo L., cv. ‘Amarillo Oro’)

Callus cultures from cotyledon and hypocotyl explants of a Spanish cultivar of melon (Amarillo Oro) have been tested for their growth and morphogenic capacity on a series of media with different concentrations of indole-3-acetic acid (IAA) and 6-furfurylaminopurine (kinetin). Melon tissues were able to undergo morphogenesis both via organogenesis and embryogenesis, depending on culture conditions and explant source. Shoot buds were obtained at high rates in cotyledon explants. In response to 1.5 mg/l IAA and 6.0 mg/l kinetin, more than 90% of the calli produced well-developed shoots. Hypocotyls failed to form shoots but formed somatic embryos on auxin containing media while cotyledon explants usually gave abundant shoots but only rarely formed embryos. It was possible to maintain organogenic callus lines for at least 12 months under defined conditions. Plants were recovered from adventitious shoots produced both in cotyledon-derived calli and from organogenic cell lines.     

Changes in jasmonates and 12-oxophytodienoic acid contents of <Emphasis Type="Italic">Medicago sativa</Emphasis> L. during somatic embryogenesis

Jasmonic acid (JA), its methyl ester (MeJA) and the biosynthetic precursor 12-oxophytodienoic acid (OPDA) were detected quantitatively during somatic embryogenesis of Medicago sativa L. Using GC-MS analysis, these compounds were found in initial explants, in calli and in somatic embryos in the nanogram range per gram of fresh weight. In distinct stages of somatic embryogenesis, JA and 12-OPDA accumulated preferentially in cotyledonary embryos. Initial explants exhibited about five-fold higher JA content than OPDA content, whereas in other stages OPDA accumulated predominantly. These data suggest that also in embryogenic tissues OPDA and JA may have individual signalling properties.     

Production of solasodine by calli from different parts of Solanum eleganifolium Cav. plants

Callus tissues from different explants (hypocotyl, cotyledon, root, leaf and fruit) of Solanum eleagnifolium Cav. were cultured on a modified Murashige-Skoog medium, with 1 mg.1^–1 2,4-D as the sole growth regulator. The presence of the alkaloid solasodine was determined by spectrophotometric and TLC methods. Its concentration ranged from 1.00 to 2.15 mg.g^–1 DW. The calli from different explants showed a direct association between the solasodine production and their growth, although they have a different production rate. It was also observed that about the seventh week of culture the metabolite concentration decreased in all cases.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - DW dry weight - TLC thin layer chromatography     

The influence of salicylic acid elicitation of shoots, callus, and cell suspension cultures on production of naphtodianthrones and phenylpropanoids in Hypericum perforatum L.

Hypericum perforatum is a well known medicinal plant. The main pharmacological properties are due to the presence of naphtodianthrones such as hypericin and pseudohypericin. Unfortunately the levels of these compounds vary under different environmental conditions. Elicitation of in vitro cultures is a useful approach to enhance and extend production of desirable products. Therefore, the effects of salicylic acid were characterized on different explants of H. perforatum L. (cells, calli and shoots) cultured in vitro. It appears at first that salicylic acid did not affect growth and development of these explants. In addition, the production of both hypericin and pseudohypericin has doubled in elicited cell suspension cultures but not in the two other cultures. Furthermore, phenylpropanoids that are among the most frequently observed metabolites affected upon treatment of in vitro culture material with elicitors, were produced and the enzymatic activities of phenylalanine ammonia lyase and of chalcone isomerase were stimulated upon elicitation. These effects were dependant of the type of in vitro culture, the concentration of salicylic acid and the duration post-elicitation. The H. perforatum cells were globally more sensitive to salicylic acid elicitation when maintained in an undifferentiated state and particularly in cell suspension cultures. In the absence of glands considered as the sites of naphtodianthrones biosynthesis, cells and calli were capable of producing these compounds. This implies that salicylic acid could act at biosynthesis level but not for the accumulation of both hypericin and pseudohypericin. Consequently, the regulation of this process is more complex than cited in the literature involving the responsibility of only Hyp-1 gene, encoding a hypericin biosynthetic enzyme, cloned and characterized from H. perforatum.     

Cyclic somatic embryogenesis and efficient plant regeneration from callus of safflower

Efficient plant regeneration through somatic embryogenesis was established for safflower (Carthamus tinctorius L.) cv. NARI-6. Embryogenic calli were induced from 10 to 17-d-old cotyledon and leaf explants from in vitro seedlings. High frequency (94.3 %) embryogenic callus was obtained from cotyledon explants cultured on Murashige and Skoog’s germination (MSG) basal medium supplemented with thidiazuron, 2-isopentenyladenine and indole-3-butyric acid. Primary, secondary and cyclic somatic embryos were formed from embryogenic calli in a different media free of plant growth regulators, however, 100 % cyclic somatic embryogenesis was obtained from cotyledon derived embryogenic calli cultured on MSG. Somatic embryos matured and germinated in quarter-strength MSG medium supplemented with gibberellic acid. Cotyledons with root poles or non root poles were converted to normal plantlets and produced adventitious roots in rooting medium. Rooted plants were acclimatized and successfully transferred to the field.     

Dynamics of morphological and anatomical changes in leaf tissues of an interspecific hybrid of oil palm during acquisition and development of somatic embryogenesis

Oil palm is an economically important plant species due to its high oil production per unit area. Large-scale clonal propagation of the species’s elite specimens is only possible through somatic embryogenesis, although methodology is partially still unknown and insufficiently understood. Current study characterizes in morphological and anatomical terms the acquisition and development stages of somatic embryogenesis of the oil palm’s immature leaves. The respective embryogenic stages were analyzed and characterized: immature leaves (initial explants); leaves with calli formation; leaves which failed to respond to calli formation; leaves with formation of root structures; primary calli; primary calli with differentiation of embryogenic calli; embryogenic calli; pro-embryogenic calli; calli with differentiated somatic embryos; somatic embryos at globular and torpedo stage; and mature fruit zygotic embryos. Cell masses emerged after approximately 60 days of cultivation through the proliferation of cells associated to initial explants´ vascular bundles. Consequently, the formation of two different types of calli was identified, namely, primary and embryogenic, respectively consisting partially and completely of meristematic cell clusters. After 420 days of cultivation, the propagules formed somatic embryos with no connection to source tissues, initially composed (globular stage) of a very characteristic ground meristem and protoderm. After 480 days of cultivation, as the cultures matured (torpedo stage), procambial strands, a structural characteristic also observed in mature zygotic embryos, were reported. The results provide an in-depth understanding of somatic embryogenesis of immature leaves of oil palm. Further, current analysis develops morphological markers at different stages of development obtained during the process.     

Plant regeneration from callus and protoplast cultures of Helianthus giganteus L.

Summary Methods of plant regeneration from callus and protoplasts of Helianthus giganteus L. are described. Embryogenic callus was obtained from leaf explants and plants were regenerated from these calli on MS media with different combinations of benzyladenine and naphtaleneacetic acid. Leaf protoplasts isolated from in vitro grown plants formed somatic embryos when cultured in agarose solidified droplets of V-KM medium containing benzyladenine and naphtaleneacetic acid. Embryos developed into plantlets on media with reduced auxin contents. Regenerated plants were successfully planted in soil.Abbreviations BA benzyladenine - IAA indoleacetic acid - MS Murashige and Skoog medium - NAA naphtaleneacetic acid - V-KM protoplast culture medium of Binding and Nehls     

Regeneration of coconut (Cocos nucifera L.) from plumule explants through somatic embryogenesis

A protocol was developed for coconut regeneration using plumules from mature zygotic embryos as explants, and media with the synthetic growth regulators 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine. Evidence for the regeneration process from these tissues occurring through somatic embryogenesis is presented. The somatic embryos were capable of germination, subsequent development into plantlets and successful transfer to the nursery. The yields were larger, nearly twofold for calli and over tenfold for calli bearing somatic embryos, than those previously reported with inflorescence explants. The present protocol thus represents an improvement in time and yield over previous protocols. Even though plumule explants are not the ideal tissue source due to possible genetic heterogeneity, the improvements made here may be applicable to tissues from mature plants. In addition, micropropagation of coconut using plumules is potentially useful when they are obtained from fruit produced from selected parents of outstanding performance, such as those resistant to diseases. Received: 19 March 1997 /Revision received: 11 September 1997 / Accepted: 4 October 1997     

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.     

爪哇三七组织培养植株再生

本文报道了爪哇三七通过组织培养再生植株的方法。爪哇三七的叶片,叶柄或茎段外植体分别接种于6种分化培养基上,均难以直接分化。在附加1mgL~(12),4-D和0.1mgL~(-1)KT的B_5培养基上的叶片形成无色疏松的愈伤组织转接于MS附加1mgL~(-1)BAP和0.1mg L~(-1)NAA培养基上,5天后开始形成绿色球胚状结构,继而形成不定芽或丛生芽。这些不定芽或丛生芽在锈根培养基中迅速长成根系发达的完整植株。此外,本试验比较了不同浓度及不同组合的激素对爪哇三七外植体的脱分化、再分化的影响。并讨论了此项工作对柑桔裂皮病类病毒(简称CEV)的复制及致病机理研究的意义。     

In vitro plantlet regeneration from cotyledons of the tree-legume Leucaena leucocephala

Two plant regeneration methods applicable to Leucaenaleucocephala were developed. In the first method, involvingorganogenesis via callus formation, cotyledon, hypocotyl and root segments wereinitiated on MS medium containing different concentrations ofN⁶-benzyladenine (BA), 2,4-dichlorophenoxyacetic acid (2,4-D), andnaphthaleneacetic acid (NAA). Both compact (type I) and friable (type II) calliwere obtained from the cotyledon and hypocotyl explants treated with differentconcentrations of the growth regulators. Shoots were generated only from thefriable calli formed from the cotyledon explants. The calli formed from thehypocotyl explants did not generate shoots and the root explants died withoutforming callus. Cotyledon explants from 3–4 day old seedlings showedmaximum callus induction compared to those from older seedlings. In a secondmethod involving direct organogenesis, excised cotyledons were cultured on 1/2MS medium containing 10–35 mg l^–1N⁶-benzyladenine (BA) for 7–14 days. Transfer of thecotyledonsto regeneration medium containing low BA resulted in callus formation andsubsequent shoot regeneration from the base of the excised cotyledon explants,with up to 100% frequency. Regenerated shoots rooted best on a basal mediumcontaining no growth regulators.     

Variation in nicotine content of tobacco callus cultures

Plants ofNicotiana tabacum L. cv. Burley 21 which showed no difference in nicotine content were used to establish callus cultures. Cultures were initiated from different plants and from different leaves within each plant. The nicotine content of the calli was determined, and the results subjected to an analysis of variance. Differences between plants and differences within plants significantly affected the nicotine content of the cultures. The differences between plants were transmitted sexually and asexually, providing evidence that they are genetically determined. No such differences in nicotine content were found between the plants from which the cultures were established, indicating that nicotine production in vitro involves additional genes to those which are needed for nicotine production in the plant. The differences within plants were further investigated by establishing callus cultures from pith explants taken from different parts of the stem. Explants from apical pith tissue gave calli having far more nicotine and more roots than cultures derived from basal pith explants. This results may reflect the proximity of the apical pith explants to the site of auxin synthesis in the stem apex. Callus cultures derived from pith explants showed greater growth and nicotine production than those derived from leaf explants when the calli were induced on Murashige-Skoog medium containing -naphthalene acetic acid. This result is in conflict with the widely held belief that explants from different parts of the plant give cultures with similar yields of species-specific compounds.Abbreviations HN High nicotine - LN low nicotine     

Production of aposporous gametophytes and calli from Pteris vittata L. pinnae strips cultured in vitro

Murashige and Skoog's modified medium in 1% Difco Bacto-agar supplemented with sugar alcohols (sorbsitol, mannitol), growth regulators (1-naphthalenacetic acid, 2,4-dichlorophenoxyacetic acid, benzyladenine, kinetin) and sugars (fructose, glucose, sucrose) induced aposporous gametophytes from pinnae of Pteris vittata cultured in vitro at lower concentrations of all the mentioned components. Aposporous gametophytes and vegetative calli were produced at higher concentrations. The calli regenerated sporophytes when cultured on MS medium without growth regulators. The gametophytes grew vegetatively on MS medium but produced sporophytes when transferred into 0.1 strength MS medium. This is the first report of simultaneous production of calli and gametophytes from fern explants.     

Genetic transformation of sweet potato by particle bombardment

Transient and stable expression of foreign genes has been achieved in sweet potato using the particle bombardment system of gene delivery. Callus and root isolates of two genotypes (Jewel and TIS-70357) with positive signs of transformation have been recovered. Tungsten microcarriers coated with plasmid DNA (pBI 221 containing the gusA gene) were accelerated at high velocity using a biolistic device into sweet potato target tissues. Histochemical examination of bombarded leaf and petiole explants revealed that most had cells expressing the gusA gene. When explants were cultured, calli and roots developed in most bombarded tissues. Similar results but with a lower frequency of transformation were observed when the plasmid pBI 121 (with gusA and antibiotic resistance npt II genes) was employed and bombarded explants cultured on an antibiotic selection medium. Subcultured roots and calli were positive for gusA expression when tested even after one year of in vitro culture, and thus the expression of the foreign gene is fairly stable. The particle bombardment approach of gene delivery appears to have a potential for generating transgenic sweet potatoes with useful agronomic traits.Abbreviations BA 6-benzylaminopurine - CaMV cauliflower mosaic virus - 2,4-D 2, 4-dichlorophenoxyacetic acid - GUS ß glucuronidase - NAA naphthaleneaceticacid - nos nopaline synthase gene - NPT II neomycin phosphotransferase II - MS Murashige and Skoog (1962) - MS-CP MS cell proliferation medium     

In vitro culture and plant regeneration of large flowered purslane

Culture conditions were established for callus induction from a range of Portulaca grandiflora Hook tissues. Rapidly growing calli were obtained on Murashige and Skoog medium with stem-, leaf- and sepal-derived explants. Plant regeneration via organogenesis was explant-origin dependent with hypocotyl tissues giving the highest shooting frequency. Light conditions, pH and carbon source had a pronounced effect on the percentage of explants regenerating buds and the number of buds formed. It was possible to establish stable regenerated plants in the glasshouse.Abbreviations BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IAA indoleacetic acid - MS Murashige and Skoog (1962) medium