Stimulation of root and somatic embryo production in Euonymus europaeus L. by an inhibitor of polyamine biosynthesis

In vitro formation of roots and somatic embryos is obtained from cotyledon explants of a Spindle tree (Euonymus europaeus L.) cultured on two different media: a medium inducing callus formation and the production of roots, and a medium inducing callus formation, root and somatic embryo production. We studied the effects of α-difluoromethylornithine (DFMO), a specific, irreversible inhibitor of ornithine decarboxylase (ODC) on root and somatic embryo production, growth and titers of putrescine in Euonymus explants and explant-derived calli. Early changes in putrescine levels were detected in both cultures before the visible emergence of roots or somatic embryos. DFMO rapidly inhibited putrescine accumulation and growth in non-embryogenic calli and highly stimulated rooting activity. DFMO partially inhibited putrescine accumulation in embryogenic calli. This inhibition had no effects on callus growth but significantly reduced the time of emergence of roots and highly stimulated somatic embryo production. The relationship among putrescine, putrescine metabolism, growth, root and somatic embryo formation is discussed.     

Induction of somatic embryogenesis and embryo development in Rumex acetosella L.

Axillary buds of the dioecious plant Rumex acetosella L. were isolated and cultured in vitro. The callus tissue which developed at the basal parts of the explants displayed a high capacity for shoot formation. This morphogenetic pattern was predominant on Murashige and Skoog (MS) medium supplemented with 2% sucrose, 2.2 mgl^-1 benzylaminopurine and 0.17 mgl^-1 indole-3-acetic acid. Somatic embryogenesis was induced when the osmolality of the medium was increased by adding 6% sucrose instead of 2%, or hexitols in addition to 2% sucrose. Most of the embryogenic calli were formed on the basal parts of leaf laminae and bracts. Development and maturation was strongly promoted by transferring the tissue to a solid or liquid medium lacking benzylaminopurine and indole-3-acetic acid and supplemented with 10 mgl^-1 gibberellic acid. The embryos germinated and developed into normal rosette plants when transferred to vermiculite moistened with hormone-free, half-strength MS salt solution. The histology of successive embryogenic stages is presented.     

Stimulation of somatic embryo formation by mutagens and darkness in culture of immature zygotic embryos of Arabidopsis thaliana (L.) Heynh.

The aim of the study was to evaluate the influence of light conditions,physical state of the induction medium and the mutagenic treatment on theembryogenic ability of Arabidopsis thaliana (L.) immaturezygotic embryos differing in developmental stage. The efficiency of directsomatic embryogenesis (DSE) was analysed in a culture of immature zygoticembryos at an early (ES) and a late (LS) developmental stage. The efficiency ofDSE was scored as a percentage of the explants producing somatic embryos. Theexperiments indicated that the physical state of the induction medium (solid orliquid) did not influence the embryogenic ability of the cultured explants. Inthe cultures on both solid and liquid induction medium, the ES explantsproducedsomatic embryos with a frequency of 25.8–37.3% i.e. 2.5–3-timeslower than LS explants. However, an increase in the embryogenic ability of ESexplants (up to 69.8%) was observed when DSE was induced in darkness. Moreover,the stimulation of DSE efficiency in culture of ES explants was also observedafter mutagenic treatment. The chemical mutagens, MNH and EMS, applied forexplant treatment, both stimulated efficiency of somatic embryo formation inculture of ES explants. The most effective DSE induction was observed when MNHand EMS were applied in doses of 0.125–1.0 mM × 3h and0.05–0.2% × 18h, respectively. In these treatment combinations thefrequency of ES explants forming somatic embryos was found to be about 2 timeshigher than in the control culture.     

Lobeline production by hairy root culture of Lobelia inflata L.

Hairy roots were obtained following inoculation of the stems of Lobelia inflata L. with Agrobacterium rhizogenes strain ATCC 15834. These hairy roots contained agropine and mannopine. In addition, lobeline was detected by HPLC and confirmed by mass spectrometry. Various media were tested for the growth of hairy roots as well as for the content of lobeline in hairy roots. The growth rate of hairy roots cultured in Nitsch and Nitsch's medium was approximately one third of those cultured in other media. The lobeline content of hairy roots (18–54 g/g dry weight) cultured in these media was the same order of magnitude compared with that of roots of L. inflata (24 g/g dry weight) cultivated in pots. The hairy roots cultured in Nitsch and Nitsch's medium were morphologically different from those cultured in other media.Abbreviations MS medium Murashige and Skoog's medium - 1/2 MS medium one-half strength of the standard Murashige and Skoog's medium - B5 medium Gamborg's B5 medium - NN medium Nitsch and Nitsch's medium - FW fresh weight - DW dry weight     

Callus concentration regulates somatic embryo production in orchardgrass suspension cultures

Summary The effects of callus inoculation concentration and culture duration on somatic embryogenesis of orchardgrass,Dactylis glomerata L., were evaluated in suspension cultures of an embryogenic genotype Embryogen-P. Somatic embryo formation was induced in liquid SH medium containing 30 μM dicamba (SH-30 and 1.5% casein hydrolysate; embryo development was in liquid SH medium without plant growth regulators (SH-0); and embryo maturation and germination occurred on solid SH-0 medium. Callus proliferation in SH-30 suspension cultures was greatest when callus was inoculated into the liquid medium at a relatively high concentration of 4% (4 g callus/100 ml medium), but the induction of somatic embryos was highest in this medium if the callus was inoculated at a lower concentration (<2%). In a second experiment, somatic embryo yield was highest when SH-0 development medium was inoculated with suspension culture callus at 0.1% concentration and declined markedly as inoculation concentration increased. Cell concentration is a critical factor in regulating the somatic embryogenesis response in orchardgrass suspension cultures.     

枸杞体细胞胚发生过程中内源多胺代谢动态的研究

完全脱分化的枸杞继代愈伤组织在转入分化培养后第1天就开始启动分化,接着体细胞转变为胚性细胞,继而分裂形成多细胞原胚,球形胚和成熟胚等。与此同时,Put含量迅速上升形成第1个峰值,随后有所下降,但到多细胞原胚期Put含量又上升,并达到最高峰,为对照的6倍,Spd只在胚性细胞分化早期出现,Spm仅在体细胞胚发育晚期存在,外源Put不仅可提高体细胞胚发生频率,而且使3种内源多胺含量均有所提高,加入多胺生物合成抑制剂DFMA后,多胺水平下降,体细胞胚发生几乎完全被抑制。补充外源Put后,多胺的生物合成得到部分恢复,DFMA对体细胞胚发生的抑制效应也被部分解除。结果表明,维持一定量的多胺水平是枸杞体细胞胚发生的必要因素。     

In-vitro regeneration of olive tree by somatic embryogenesis

We induced somatic embryogenesis from the cotyledon segments ofOlea europaea (L) cvs. ‘Chetoui’, ‘Chemleli’, and ‘Arbequina’. Calli were established from all three cultvars on OMc media supplemented with IBA and 2i-R The greatest success was obtained with media that contained zero or low concentrations of growth regulators. High levels of hormones (i.e.,>0.5 mgL^-1 IBA and 2i-P) inhibited embryogenesis. Embryos at different maturation stages were observed with continuously proliferating secondary embryogenesis. Abnormally shaped embryos and teratoma were also noted. Four weeks was the optimal incubation period for inducing embryogenesis on the auxin-containing medium. In addition, 30 to 40 gL^-1 sucrose was more effective than glucose in stimulating the growth and maturation of somatic embryos. Embryogeic efficiency was also higher when multivariate combinations of nitrogen sources (inorganic and organic nitrogen forms) were used. The plantlets that were derived from our germinating somatic embryos were similar to those obtained from axillary buds.     

Alfalfa embryo production in airlift vessels via direct somatic embryogenesis

A procedure for the development of alfalfa (Medicago falcata L.) somatic embryos to the torpedo stage in air-lift vessels is described. Embryos were initiated from chopped leaf explants and were formed by direct somatic embryogensis. The system produced a high number of torpedo stage embryos. The effect of various inoculation densities on embryo development was studied. A procedure for the development and maturation of embryos in aerated liquid media was established. The rate of conversion of the torpedo stage embryos formed in the vessels was 83%.Abbreviations ABA abscisic acid - B5 Gamborgs B5 medium (Gamborg et al. 1968) - COT cotyledon embryo state - 2,4-d 2,4-dichlorophenoxyacetic acid - FW fresh weight - ID internal diameter - MS Murashige and Skoog medium (Murashige & Skoog 1962) - PEG polyethylene glycol - POLY polyembryos - VVM volume of gas/volume of bioreactor     

Somatic embryogenesis in pumpkin (Cucurbita pepo L.): control of somatic embryo development by nitrogen compounds

Embryogenic cultures of pumpkin (Cucurbita pepo L.) were initiated from mechanically wounded mature zygotic embryos on 2,4-D-containing MS medium, and on hormone-free, semisolid modified MS medium containing NH4Cl as the sole source of nitrogen. The habituated line was derived from the embryogenic tissue induced with 2,4-D and maintained on medium without growth regulators. Sustained subculturing of the three embryogenic lines on a medium with NH4Cl as the sole source of nitrogen enabled the establishment of highly uniform cultures in which no further development into mature embryo stages occurred. The tissue consisting of proembryogenic globules or globular stage embryos was maintained, without decline, for over six years. Globular embryos proceeded to maturity when a combination of reduced (NH4) and unreduced (NO3) forms of nitrogen was provided in the medium. Different nitrogen sources in the medium caused changes of medium pH during subculture in the pH range of 4.0-6.5. The tissue growth and embryo development were blocked on medium with pH adjusted and stabilized at 4.0 or at 3.2.     

The effects of reduced nitrogenous compounds suggests that glutamine synthetase activity is involved in the development of somatic embryos in carrot

The addition of l-glutamine, -alanine or l-glutamic acid strongly stimulates somatic embryo formation in carrot, not only in the number of somatic embryos formed but also with respect to their development. The effects of the amino acids on somatic embryogenesis were stronger than that of ammonium ion. In particular, l-glutamine strongly stimulated the development of somatic embryos. To clarify the different effects of amino acids and ammonium ion, the activity of glutamine synthetase (GS; EC 6.3.1.2), a key enzyme involved in nitrogen assimilation, was measured. Its activity decreased during the later stages of embryo development.Abbreviations -Ala -alanine - Glu l-glutamic acid - Gln l-glutamine - 2,4-D 2, 4-dichlorophenoxyacetic acid - -GHA l-glutamic acid -monohydroxamate - GS glutamine synthetase - MS medium Murashige & Skoog (1962) medium - MS-NH₄ medium MS medium without NH₄NO₃ - MS+NH₄ medium MS-NH₄ medium with 10 mM NH₄Cl - MS+ala medium MS-NH₄ medium with 10 mM -alanine - MS+GLU medium MS-NH₄ medium with 10 mM l-glutamic acid - MS+GLN medium MS-NH₄ medium with 10 mM l-glutamine - NIR nitrite reductase - NR nitrate reductase     

Plant regeneration via somatic embryogenesis in cotton

An efficient in vitro plant regeneration system characterized by rapid and continuous production of somatic embryos using leaf and stem explants of abnormal seedling as an explant have been developed in Gossypium hirsutum L. Embryogenic callus and somatic embryos have been obtained directly from the explants of cotton abnormal seedlings. Plant growth regulators influenced the induction of cotton somatic embryogenesis. The optimal medium for direct somatic embryogenesis was modified MS medium supplemented with 0.1 mg l^-1 ZT and 2 g l^-1 activated carbon. On this medium, an average of 28.0 and 28.1 matured somatic embryos formed from per leaf and stem explants respectively. The highest frequency of somatic embryogenesis was 100%. The somatic embryos were converted into normal plantlets when cultured on modified MS medium supplemented with 0.1 mg l^-1 ZT. Upon transfer to soil, plants grew well and appeared normal. Plants could be regenerated within 60–80 days. The system of cotton somatic embryogenesis and plant regeneration described here will facilitate the application of plant tissue culture and genetic engineering on cotton genetic improvement. This revised version was published online in June 2006 with corrections to the Cover Date.     

Kinetic studies of embryo development and nutrient utilization in an alfalfa direct somatic embryogenic system

A method for direct somatic embryogenesis in alfalfa (Medicago falcata) is described. The time course in the development phase has been followed for fresh weight, cell density, pH, sugar uptake and embryo number and type. The method of disrupting the explant material has also been shown to influence subsequent embryo formation.     

Plant regeneration of Iris pallida Lam. and Iris germanica L. via somatic embryogenesis from leaves,apices and young flowers

Irones are violet-scented ketonic compounds contained in the rhizome of certain species of iris. As cultivation of the iris tends to decrease, a selection program has been initiated to find the best performing clones in terms of growth and yield. Parallel to this selection, in vitro regeneration studies have been carried out in order to multiply interesting clones. A method of rapid multiplication by somatic embryogenesis associated with multibudding was developed. Callus was obtained from leaf bases, flower pieces or rhizome apices; the best explants were flower pieces. The induction media used to obtain embryogenic callus were Murashige & Skoog (1962) media. Assays with adding of proline in these media have showed that it could double the yield of embryogenic callus. The embryogenic expression medium was the Knudson's orchid agar (Knudson 1946) medium. Conformity of the plants obtained was checked by comparing their chemotypes with those of the mother plants.Abbreviations AIB indolyl butyric acid - BAP 6-benzylaminopurine - Kin 6-furfurylaminopurine - KN Knudson's orchid Agar - MS Murashige & Skoog medium - NAA naphthaleneacetic acid - N6 Chu et al. medium - Pro proline - 2,4-D dichlorophenoxyacetic acid     

Induction of somatic embryos in pea,Pisum sativum L.

Using low concentrations of picloram (0.06 mg/l), embryoids were formed on the surface of leaf-derived callus of pea, Pisum sativum L. (c.v. Dippes Gelbe Victoria) upon transfer to liquid medium. After some days in culture, embryoids spontaneously separated from the calli, and developed into torpedo-shaped embryos, which were transferred to solid medium. In a second series of experiments, embryos were also formed by mutant 489C and a genetic line of Pisum arvense, which additionally exhibited embryogenesis also from epicotyl-derived callus. Some of the embryos showed root formation, but no shoot morphogenesis occurred. In a limited number of cases, an additional root was formed in the apparent shoot apical region after 2–5 days.     

Developmental regulation of polyamine metabolism in growth and differentiation of carrot culture

Polyamine levels and the activities of two polyamine biosynthetic enzymes, arginine decarboxylase (EC 4.1.1.19) and S-adenosylmethionine decarboxylase (EC 4.1.1.50), were determined during somatic embryogenesis of carrot (Daucus carota L.) cell cultures. Embryogenic cultures showed severalfold increases in polyamine levels over nondifferentiating controls. A mutant cell line that failed to form embryos but grew at the same rate as the wild-type line also failed to show increases in polyamine levels, thus providing evidence that this increased polyamine content was in fact associated with the development of embryos. Furthermore, inhibition of these increases in polyamines caused by drugs inhibited embryogenesis and the effect was reversible with spermidine. The activities of arginine decarboxylase and Sadenosylmethionine decarboxylase were found to be suppressed by auxin; however, the specific effects differed between exogenous 2,4-dichlorophenoxyacetic acid and endogenous indole-3-acetic acid. The results indicate that increased polyamine levels are required for cellular differentiation and development occurring during somatic embryogenesis in carrot cell cultures.Abbreviations ADC arginine decarboxylase - 2,4-D 2,4-dichlorophenoxyacetic acid - DFMA difluoromethylarginine - DCHAS dicyclohexylammonium sulfate - SAMDC S-adenosylmethionine decarboxylase     

Contrasting pattern of somatic and zygotic embryo development in alfalfa (Medicago sativa L.) as revealed by scanning electron microscopy

Scanning electron microscopy has been used to investigate the morphological changes occurring during the development of alfalfa somatic embryos. Embryos were initiated from callus, transferred to suspension culture and matured on solid agar medium. This developmental pattern was compared to that of zygotic embryos developing in ovulo. Somatic embryos begin as distinct pro-embryos within the callus tissue pieces placed in suspension culture. They become globular and heart-shaped while on solid agar medium and then undergo cotyledon elongation and maturation. Somatic embryos develop comparatively slower at early stages of development and faster at the later stages than zygotic embryos. They lack a well-defined suspensor and have a very rough, poorly-differentiated epidermis, the first layer of which is lost after pro-embryo formation. The cotyledons of somatic embryos are multiple and poorlydeveloped; there appears to be a correlation between the amount of surface roughness of the developing embryo and the extent to which polycotyledony occurs.     

Some features of somatic embryo maturation of algerian fir

Summary Maturation of Abies numidica De Lann. somatic embryos was tested on media with abseisic acid and various maltose and polyethylene glycol-4000 concentrations. The effect of basal medium and subculture period on maturation was also examined. The maturation of somatic embryos was promoted by polyethylene glycol-4000, at 7.5–10%. Three to 6% maltose significantly enhanced the yield of mature embryos. The most effective somatic embryo maturation occurred when embryogenictissue was transferred to maturation medium after 14–21 d cultivation on proliferation medium. The ability for A. numidica cultures to form cotyledonary somatic embryos was assessed over a period of 3 yr. Plantlets germinated on half-strength SH (Schenk and Hildebrandt) medium with charcoal and survived transfer to the soil.     

Morphological and histological changes during somatic embryo formation from coconut plumule explants

Summary Studies on the development of protocols for the clonal propagation, through somatic embryogenesis, of coconut have been reported for the past three decades, mostly using inflorescence explants, but with low reproducibility and efficiency. Recent improvements in these respects have been achieved using plumular explants. Here, we report a developmental study of embryogenesis in plumule explants using histological techniques in order to extend our understanding of this process. Coconut plumule explants consisted of the shoot meristem including leaf primordia. At day 15 of culture, the explants did not show any apparent growth; however, a transverse section showed noticeable growth of the plumular leaves forming a ring around the inner leaves and the shoot meristem, which did not show any apparent growth. At day 30, the shoot meristem started to grow and the plumular leaves continued growing., At day 45, the explants were still compact and white in color, but showed partial dedifferentiation and meristematic cell proliferation leading to the development of callus structures with a translucent appearance. After 60 d, these meristematic cells evolved into nodular structures. At day 75, the nodular structures became pearly globular structures on the surface of translucent structures, from which somatic embryos eventually formed and presented well-developed root and caulinar meristems. These results allow better insights and an integrated view into the somatic embryogenesis process in coconut plumule explants, which could be helpful for future studies that eventually could lead us to improved control of the process and greater efficiency of somatic embryo and plantlet formation.     

Enhancement of production and regeneration of embryogenic type II callus in Zea mays L. by AgNO3

In order to evaluate the impact of ethylene in maize tissue culture, silver nitrate has been used as an inhibitor of ethylene action. Type II callus initiation rate was improved when immature embryos were cultured on a modified Murashige & Skoog medium containing various concentrations of silver nitrate (5, 10, 20 mgl^-1). Regeneration ability of calli initiated and maintained in presence of silver nitrate was enhanced. No modification of callus growth rate neither of ethylene production has been detected.     

Solasodine glycoside production by hairy root cultures of Physalis minima Linn.

Hairy root cultures of Physalis minima L. were developed using Agrobacterium rhizogenes, strain ATCC 15834 mediated transformation and grown in half strength of Murashige and Skoog medium containing 8% (w/v) sucrose. Media supplementation with 1 mg naphthalenacetic acid l(-1) and 1 mg benzyladenine increased solasodine glycoside up to 900 g dry wt, which was 20 times higher than that in the native root.