Polyamine Levels in Relation to Growth and Somatic Embryogenesis in Tissue 6Medicago Sativa L.

Polyamine levels in petiole-derived tissue cultures of two highlyregenerable Medicago sativa L. genotypes were determined duringthe embryo induction and embryo differentiation phases of somaticembryogenesis. Putrescine levels increased 27–32-foldduring the 10 d on 2, 4-D- and kinetin-containing embryo inductionmedium and fell sharply following transfer to growth substance-freeembryo differentiation medium. The rapid increase in putrescinecontent occurred during a period of relatively little growthwhile the decline coincided with the initiation of rapid tissuegrowth on embryo differentiation medium. The addition of putativeinhibitors of putrescine or spermidine biosynthesis to the embryoinduction medium led to reduced levels of polyamines, particularlyof putrescine, in cultures of both genotypes but subsequentsomatic embryo formation was inhibited in cultures of one genotypeonly. It was concluded that the pronounced change in putrescinemetabolism was not specifically associated with embryogenesis,but appeared to be related generally to re-programming of cellsinto a new pattern of in vitro development. Key words: Medicago sativa, polyamines, somatic embryogenesis     

Requirement of Ethylene for Growth of Callus and Somatic Embryogenesis in Medicago sativa L.

The role of ethylene in the growth of callus and somatic embryogenesisin Medicago sativa was examined. The application of 2,5-norbornadiene,a competitive inhibitor of ethylene action, during a 10 d inductionperiod to medium containing 2,4-D and kinetin inhibited thegrowth of callus but did not affect somatic embryogenesis, nordid it affect ethylene production during the induction stage.The exposure of tissue, incubated on differentiation medium,without hormones, to an atmosphere of 2,5-norbornadiene, inhibitedboth growth and embryo maturation and stimulated pigmentation.The inhibition of embryo maturation was observed even in thepresence of norbornadiene at a concentration which did not affectgrowth of tissue. It is suggested that the action of endogenous ethylene is necessaryfor the growth of the callus and embryo maturation. Key words: Medicago sativa, ethylene, callus growth, somatic embryogenesis     

Inhibition of Somatic Embryogenesis in Tissue 2Medicago sativa by Aminoethoxyvinylglycine, Amino-oxyacetic acid, 2, 4-Dinitrophenol and Salicylic Acid at Concentrations which do not Inhibit Ethylene Biosynthesis and Growth

Meijer, E. G. M. and Brown, D. C. W. 1988. Inhibition of somaticembryogenesis in tissue cultures of Medicago sativa by aminoethoxyvinylglycine,amino-oxyacetic acid, 2, 4-dinitrophenol and salicylic acidat concentrations which do not inhibit ethylene biosynthesisand growth. J. exp. Bot. 39: 263–270. The effects of aminoethoxyvinyglycine (AVG), amino-oxyaceticacid (AOA), 2, 4-dinitrophenol (DNP) and salicylic acid (SA)on ethylene production, tissue proliferation and somatic embryo-genesisin a recently developed rapid in vitro regeneration system ofMedicago sativa L. were examined. Contrary to numerous publications,AVG, AOA and DNP did not affect the rate of ethylene biosynthesis,while SA even caused an increase in ethylene production. Allfour compounds were, however, potent inhibitors of somatic embryoformation in the M. sativa tissue cultures, even at concentrationswhich did not affect tissue growth. Generally, a 5-d exposureto the inhibitors reduced the number and quality of somaticembryos obtained. It is suggested that the inhibitors may notreach the site of action of enzymes involved in ethylene biosynthesisand may possibly block other biosynthetic pathways which areof crucial importance to somatic embryo development. The resultsindicate that a delicate differentiation process like somaticembryogenesis is very sensitive to metabolic perturbances. Theresults are also discussed in the light of other known effectsof these four compounds on higher plants. Key words: Ethylene, Medicago sativa, somatic embryogenesis, tissue culture     

Endogenous ethylene in indirect somatic embryogenesis of <Emphasis Type="Italic">Medicago sativa</Emphasis> L.

Ethylene biosynthesis during different phases of somatic embryogenesis in Medicago sativa L. cv. Rangelander using two regeneration protocols, RPI and RPII, was studied. The highest ethylene production was detected during callus growth on induction medium in both regeneration protocols. Significantly less ethylene was produced by embryogenic suspension than by callus (RPII). Developing embryos synthesized higher amounts of ethylene than mature embryos. Production of ethylene was strongly limited by the availability of 1-aminocyclopropane-1-carboxylic acid and also by ACC-oxidase activity. However, removal of ethylene from culture vessels’ atmosphere using KMnO₄ or HgClO₄ had no significant effect on callus growth, somatic embryo induction and development. Reducing of ethylene biosynthesis by aminoethoxyvinylglycine substantially decreased somatic embryo production and adversely affected their development, indicating ethylene requirement during proliferation and differentiation but not induction.     

The Use of Single Somatic Embryo Culture in Propagating and Regenerating Lucerne (Medicago sativa L.)

Embryogenic cultures of lucerne (Medicago sativa L.) cv. Robothave been established and propagated on medium containing yeastextract. These cultures consisted of unorganized callus tissuebearing embryogenic centres which increased in size during subculture,yielding new regenerated somatic embryos at the end of each20-d subculture. A development in the propagation of the embryogenic cultureswas the establishment of single embryo culture in hormone-freemedium where, in selected cases, the process of recurrent somaticembryogenesis (RSE) took place on the hypocotyl of explantedembryos. The process was independent of supporting callus tissueand occurred on simple defined medium. Single embryos underwenteither plantlet development or continued RSE on the hypocotyl.One third of the regenerated plantlets showed RSE after thetwo to three trifoliate leaf stage. In these cases shoot developmentstopped and only somatic embryo production took place. In vitrocloning of regenerated plantlets allowed us to reproduce eachparticular genotype before transplantation into soil. Lucerne (alfalfa), Medicago sativa L., somatic embryogenesis, single embryo culture     

A novel system for rapid high frequency somatic embryogenesis in Medicago sativa

A novel, genotype dependent system for rapid high frequency somatic embryogenesis in Medicago sativa L. was developed in which the first embryos are visible as early as 15 days after the explant (hypocotyl, petiole, leaf) is put into culture. The simplest method involves culture of the explants on a single Murashige and Skoog (MS) medium supplemented with 2 g l−¹ casein hydrolysate, 9 μ M 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.2 μ M kinetin. An efficient two-step, two-medium system was developed to allow separation of the induction and differentiation phases. The explants are cultured on MS with 22.6 μ M 2,4-D and 4.7 μ M kinetin (induction medium) for 10 days and then on basal MS for 20 days. Embryo yields and embryo conversion to plantlets were strongly dependent on the 2,4-D and kinetin concentrations in the induction medium. Both petiole and leaf explants were highly embryogenic and very little callus proliferation occurred when this method was used. Selected clones from three ssp. falcata -based M. sativa cultivars showed a response very similar to the highly regenerable falcata clone F1.1, but it was not possible to produce large numbers of somatic embryos in tissue cultures of cv. Regen S, which is used in most M. sativa tissue culture research, with this procedure. These results suggest that there are two distinct developmental pathways for somatic embryogenesis in M. sativa , with Regen S cultures requiring extensive dedifferentiation during a prolonged callus phase, while the genotypes described in this report have no such requirement.     

Plant regeneration from suspension culture and mesophyll protoplasts of Medicago sativa L.

A system was established for achieving plant regeneration from mesophyll protoplasts and cotyledon-derived cell suspension cultures of alfalfa, Medicago sativa L. Peeled leaflets or cells from 6-day-old cell suspensions were incubated in an enzyme mixture containing 1% Driselase, 1% Rhozyme, 0.1% Cellulase and 72 gl^-1 mannitol at pH 5.8 for 2–16 h to liberate protoplasts. A complex Kao medium supported cell division and colony formation, whereas a high auxin/low cytokinin treatment on Schenk and Hildebrandt medium followed by culture on growth regulator-free Blaydes or Linsmaier and Skoog medium resulted in somatic embryo formation. Of the three varieties tested. Citation, Answer and Regen S, the latter two produced embryos from which plants could be regenerated.     

Genotype-specific normalization of soybean somatic embryogenesis through the use of an ethylene inhibitor

The effect of ethylene on somatic embryogenesis from cotyledons of soybean (Glycine max) cultivars `Bragg', `IAS-5', and `RS-7' was studied through the application of silver nitrate or aminoethoxyvinylglycine. The addition of these chemicals to the induction medium had no effect on embryo induction, in spite of aminoethoxyvinylglycine having decreased ethylene production and silver nitrate enhancing it. However, subsequent histodif-ferentiation and conversion capacity of somatic embryos was affected by treatments applied to the induction medium. The effects of ethylene on embryo histodifferentiation and conversion were genotype-specific. Cultivars `IAS-5' and `RS-7' produced high frequencies of dicotyledonous embryos and had high conversion rates. These were also the least affected by alterations in ethylene production. For `Bragg', which has a low regeneration capacity, the use of aminoethoxyvinylglycine led to a significant improvement in the frequency of normal embryo formation as well as in the frequency of conversion into plants. The results suggest that the use of ethylene inhibitors during the induction process may facilitate plant recovery from soybean genotypes, such as `Bragg', which have a low regeneration capacity. Received: 8 October 1996 / Revision received: 6 May 1997 / Accepted: 3 June 1997     

The influence of the jasmonates and abscisic acid on callus growth and somatic embryogenesis in Medicago sativa L. tissue culture

The jasmonates as well as abscisic acid were found to be inhibitors of callus growth and somatic embryogenesis in Medicago sativa L. tissue cultures. An exposure to these inhibitors during the induction as well as the differentiation stage reduced the number of somatic embryos obtained. The jasmonates showed to be less active in the inhibition of callus growth and somatic embryo production than abscisic acid.     

Induction of somatic embryogenesis from different explants of shoot cultures derived from young <Emphasis Type="Italic">Quercus alba</Emphasis> trees

The induction of somatic embryogenesis from shoot apices and leaf explants of shoot cultures derived from 6- to 7-year-old white oak (Quercus alba L.) trees is reported in this study. Embryogenic response was obtained in two out of the three genotypes evaluated with embryo induction frequencies up to 50.7% for WOQ-1 and 3.4% for WOQ-5 genotypes. The embryogenic explants formed translucent nodular structures and cotyledonary-stage somatic embryos, which developed from callus tissue, indicating an indirect embryogenesis process. An efficient procedure was developed for WOQ-1 material on the basis of the most appropriate leaf developmental stage. Growing leaves excised from two nodes below the shoot apex showed the highest embryogenic induction index. These leaves contain cells in an undifferentiated state, as shown by the presence of precursor cells of stomata, absence of intercellular spaces and low starch content in the mesophyll cells. Nodular structures and/or somatic embryos began to arise 7–8 weeks after culture initiation, although most emerged after 9–12 weeks in culture. The sequence of application of media for somatic embryo induction was optimized with a two-step procedure consisting of culturing the explants in medium supplemented with 21.48 μM NAA and 2.22 μM BA for 8 weeks and transfer of explants into plant growth regulator-free medium for another 12 weeks. Clonal embryogenic lines were established and maintained by secondary embryogenesis. Embryo germination (30%) and plantlet conversion (16.6%) were achieved after cold storage for 2 months.     

Studies on the Growth in Culture of Plant Cells: VIII. THE PRODUCTION OF ETHYLENE BY SUSPENSION CULTURES OF ACER PSEUDOPLATANUS, L.

Sycamore cell suspension cultures in a synthetic medium releaseethylene; during a 24-day incubation period a single culture(initial volume 70 ml) produces c. 4 µ moles. There isa very sharp peak of ethylene production between day 10 andday 14 of culture; at the peak of production c. 2 nmoles ethyleneare released per million cells in 24 h. Evidence is presentedthat 2,4-D enhances ethylene production independently of itseffects on culture growth. Under the standard conditions of culture (250-ml Erlenmeyerflasks closed with aluminium foil and containing 70 ml cellsuspension) the concentration of ethylene in the gas phase ofthe cultures rises above 10 ppm. No evidence was obtained thatthis ethylene is inhibitory to culture growth or that a criticallevel of ethylene is necessary to initiate cell division incultures at a critically low cell density. The low rate of ethylene release by stationary phase culturesis temporarily enhanced by the addition of various solutes andfurther depressed by dilution with water.     

Genotype effects on proliferative embryogenesis and plant regeneration of soybean

Summary Proliferative somatic embryogenesis is a regeneration system suitable for mass propagation and genetic transformation of soybean [Glycine max (L.) Merr.]. The objective of this study was to examine genotypic effects on induction and maintenance of proliferative embryogenic cultures, and on yield, germination, and conversion of mature somatic embryos. Somatic embryos were induced from eight genotypes by explanting 100 immature cotyledons per genotype on induction medium. Differences in frequency of induction were observed among genotypes. However, this step was not limiting for plant regeneration because induction frequency in the least responding genotype was sufficient to initiate and maintain proliferative embryogenic cultures. Six genotypes selected for further study were used to initiate embryogenic cultures in liquid medium. Cultures were evaluated for propagation of globular-stage tissue in liquid medium, yield of cotyledon-stage somatic embryos on differentiation medium, and plant recovery of cotyledon-stage embryos. Genotypes also differed for weight and volume increase of embryogenic tissue in liquid cultures, for yield of cotyledon-stage embryos on differentiation medium, and for plant recovery from cotyledon-stage embryos. Rigorous selection for a proliferative culture phenotype consisting of nodular, compact, green spheres increased embryo yield over that of unselected cultures, but did not affect the relative ranking of genotypes. In summary, the genotypes used in this study differed at each stage of plant regeneration from proliferative embryogenic cultures, but genotypic effects were partially overcome by protocol modifications.     

Origin and development of secondary somatic embryos in transformed embryogenic cultures of <Emphasis Type="Italic">Medicago sativa</Emphasis>

Non-transformed and transformed embryogenic cultures of alfalfa (Medicago sativa L. cv. Zaječarska 83), long-term maintained on growth regulator-free medium, were histologically analyzed. In all examined cultures, somatic embryos at various stages of development were observed and secondary embryos were formed in the cotyledonary, hypocotylary and radicular region of the primary embryos. Detailed histological analysis of the torpedo shape somatic embryo revealed that secondary somatic embryos arose directly from single epidermal cells of hypocotylary axis after an unequal periclinal division. Bipolar proembryos were composed of one smaller cytoplasm rich cell and one larger more vacuolated cell. Further cell division pattern was similar for both non-transformed and transformed embryos. However, multicellular origin of secondary embryos in a direct process and even from callus can not be excluded.     

Increased ethylene and decreased phenolic compounds stimulate somatic embryo regeneration in leaf thin section cultures ofDoritaenopsis hybrid

The effects of endogenous levels of ethylene and phenolic compounds on somatic embryogenesis, medium-browning, and peroxidase activity were evaluated in thin section cultures ofDoritaenopsis. Cultures were maintained for 8 weeks with four different treatments: i) thick leaf segment culture, ii) thin leaf section culture, iii) thin leaf section culture with ventilation, or iv) thin leaf section culture after expiants were first washed. Expiants cultured in closed vessels produced a larger number of somatic embryos than those reared in the ventilated vessels. This enhanced formation confirmed the greater involvement of accumulated ethylene under non-ventilated conditions, because wound-induced tissues from thin leaf sections normally release high level of ethylene. When expiants were washed in the liquid medium and inoculated on the same solid medium, somatic embryo production was 1.7 and 18.5 times higher than in the thin section cultures and thick segment cultures, respectively. Reducing the level of phenolics in expiants at the initial stage of culturing apparently stimulated this embryo regeneration.     

High frequency somatic embryogenesis in peanut (Arachis hypogaea L.) using mature,dry seed

Peanut (Arachis hypogaea L.) somatic embryos were produced from the embryo axes of mature, dry seeds of cultivar GK-7. Percent embryogenic explants ranged from 88–100% using 10–40 mg/1 of 2,4-D in the induction medium. Neither 2,4-D concentration nor photoperiod during the induction period had a large effect on percent embryogenesis, mean number of embryos per explant, or embryo morphology. However, embryos obtained from cultures grown in the dark were easier to remove from the explant than those under a 16-h photoperiod. Somatic embryos developed on the epicotyl portion of the embryo axis, primarily on the young, expanding leaves. A survey of 14 genotypes indicated that genotype had a large influence on embryogenic capacity, with all genotypes being embryogenic to some extent. The ability to recover somatic embryos from axes of harvested, stored seeds represents significant advantages for the establishment of peanut embryogenic cultures, including the use of simple sterilization procedures and a constant source of explant tissue.Abbreviations B5 medium of Gamborget al. (1968) - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) salts medium     

火炬松胚性细胞悬浮培养物的生长参数变化

以火炬松（PinustaedaL.）成熟合子胚来源的胚性愈伤组织为材料建立了胚性细胞悬浮系,测定了其培养物的鲜重、干重、细胞体积和胚数及培养液中的pH值、电导率和蔗糖浓度等生长参数在培养过程中的变化动态。结果表明,在培养周期内,培养液中的pH值、电导率和蔗糖浓度的逐步降低与培养物的鲜重、干重、细胞体积和胚胎数的逐步增加保持一致性。在培养至18—21d,pH值、电导率和蔗糖浓度均接近或降到最低点,而胚数及细胞体积的增长都达到最高点。     

Scaled-up proliferation and regeneration of Nerine in liquid cultures Part II. Ontogeny of somatic embryos and bulblet regeneration

The ontogeny of somatic embryos was followed in liquid cultured Nerine tissue. Proliferating, nodular meristematic clusters were maintained in bubble bioreactors in a medium supplemented with 0.25 M 1-naphthalene acetic acid (NAA), 10 M 6-benzyladenine (BA) and 8.7 M Paclobutrazol (PAC). Regeneration of plantlets from this tissue was limited. Omission of PAC from the medium induced proembryogenic clusters. Embryo development and maturation were enhanced in flask cultures by substituting N⁶-(isopentenyl) adenine (2iP) for BA and elevating the sucrose concentration in the medium to 6%. High rates of embryo germination occurred in a growth regulator-free, low (3%) sucrose medium. Bulblet-bearing plantlets developed on agar-solidified, auxin-supplemented media. Recurrent embryogenesis occurred in long term growth regulator-free, or high sucrose media. The potential of using the somatic embryogenesis pathway for micropropagation of Nerines is described.     

Disturbance of ethylene biosynthesis and perception during somatic embryogenesis in <Emphasis Type="Italic">Medicago sativa</Emphasis> L. reduces embryos’ ability to regenerate

Effects of non-specific ethylene biosynthesis inhibitors: salicylic acid (SA) and aminoethoxyvinylglycine (AVG), and of specific inhibitors of ethylene binding to receptors: 1-methylcyclopropene (1-MCP) and 2,5-norbornadiene (NBD) applied during proliferation and differentiation phases of indirect somatic embryogenesis (SE) of Medicago sativa L. cv. Rangelander on embryogenic suspension growth, embryo production, development, and ability to germinate and convert were studied. Application of SA and AVG alone or together at concentrations from 1 to 500 μM in B₅g liquid medium during the proliferation phase had an inhibitory effect on ethylene production and embryogenic suspension growth. Additionally, it caused a drastic reduction in production of embryos and their development on BOi2Y solid differentiation medium. The inhibitory effect of SA was more visible than that of AVG. In addition, disturbance of ethylene biosynthesis during the proliferation phase of SE resulted in diminished lateral germination and conversion of cotyledonary embryos on MS solid medium. Moreover, blocking of ethylene receptors by 1-MCP during the proliferation phase also inhibited ethylene production and embryogenic suspension growth and reduced embryo production during differentiation. MCP almost completely inhibited development of cotyledonary embryos. At the same time, development of more embryos was arrested at the globular stage, and the number of abnormal embryos almost doubled. Similarly, addition of 1-MCP or NBD to the ambient atmosphere during the differentiation phase evidently arrested the development of embryos and, consequently, their ability to germinate and convert on MS regeneration medium. All the results presented above demonstrated that not only ethylene biosynthesis, but also ethylene action is involved in the control of individual phases of SE in Medicago sativa L. cv. Rangelander. And what is more, disturbance of these processes during distinct phases of SE adversely affects vigor of the somatic embryos obtained.     

BA enhances the germination of oil palm somatic embryos derived from embryogenic suspension cultures

Embryogenic suspension cultures of oil palm ( Elaeis guineensis Jacq.) allow mass propagation of somatic embryos; however regeneration rates are low. Histological observations have revealed that shoot development might be limited by the absence of a caulinary meristem. The addition of 6-benzyladenine during development was found to induce shoot apex differentiation and thus increased germination rates, by up to 70%. However, multiple shoot formation was a consequence of a longer period of cytokinin supply during the development of the embryo. In contrast, a short period of culture on medium with 6-benzyladenine at the begining of embryo development was found to result in single shoot production.