Number Of Plantlets Research Articles

An improved method for somatic plantlet production in hybrid larch (Larix � leptoeuropaea): Part 2. Control of germination and plantlet development

Germination and plantlet development in somatic embryos of Larix x leptoeuropaea were affected by the duration of the maturation treatment and the concentrations of sucrose and abscisic acid in the maturation media. Extension of the maturation period from 3 weeks to 4 weeks resulted in a significant decrease in germination and plantlet development frequencies. There was no significant effect of abscisic acid concentration on either the number of somatic embryos germinated or the number of plantlets obtained, but it affected the rapidity of the epicotyl development. Sucrose at 0.2 M, applied during maturation, was significantly more beneficial in attaining high germination rates than at 0.1 M. High germination rates (92 and 93%) and plantlet development rates (74 and 80%) were achieved when somatic embryos were matured for a 3-week period on media with either 40 or 60 μM abscisic acid, respectively, and 0.2 M sucrose prior to transfer to the growth regulator-free germination medium. Two acclimatization methods were applied: the first required 10 to 12 weeks and ensured 97% plantlet survival under greenhouse conditions; the second required 2–3 weeks and ensured 86% plantlet survival. This represents the first detailed study of the effects of maturation regimes on the recovery of somatic embryo-derived plants of Larix.

Cytokinin-Enhanced Regeneration of Plants From Microprojectile Bombarded Sugarcane Meristematic Tissue

Sugarcane plants expressing the firefly luciferase gene were regenerated from microprojectile-bombarded meristematic tissue. The concentration and type of cytokinin added to the basal media, both prior to bombardment and during regeneration, significantly affected the number of plantlets produced and the percentage of plantlets expressing luciferase. Maximum shoot initiation from meristematic tissue of sugarcane cultivar Q137 was achieved on media supplemented with N6-benzylaminopurine (BAP). There were differences in the numbers of shoots initiated from different sugarcane cultivars, and some optimisation for each cultivar was required. No luciferase activity was observed in plantlets regenerated in the absence of cytokinin. Sugarcane meristematic tissue preconditioned by exposure to cytokinin appears to be a suitable target tissue for the introduction of genes of agronomic importance into sugarcane. This approach should be readily adaptable to most commercial varieties of sugarcane with minor modification to the cytokinin treatment necessary for optimal regeneration.

In vitro response of Dactylis glomerata leaf segments as affected by growing donor plants in radioactive soil

Sterile plants of Dactylis glomerata L. were grown for 2 months in sterile nonradioactive (control) and radioactive (treatment) soil. The radioactive soil was obtained from a 10-km zone around the Chernobyl Nuclear Power Plant (Pripyat, Ukraine). After exposure, leaf segments were plated on to modified Murashige and Skoog medium containing 2.2 mg 1 −1 2,4-dichlorophenoxyacetic acid. Somatic embryo formation and the average number of plantlets per explant were reduced 1.8 and 2 times, respectively, in the treated plants compared to the control.

Effect of l-proline and l-tryptophan on somatic embryogenesis and plantlet regeneration of rice (Oryza sativa L. cv. Pusa 169)

Here we report our assessment of the promotive effect of proline and tryptophan on the frequency of callusing and regeneration as also on the absolute number of plantlets regenerated per mature seed used for callus initiation. Finally, scanning electron microscopic evidence for regeneration via somatic embryogenesis in seed cultures is also given

Application of image analysis with neural network for plant somatic embryo culture

A method of classifying celery embryos and nonembryos using image analysis with a neural network to decide the time for transfer to the next culture stage in plant somatic embryo culture is presented. Since the image database of celery cells is vast, four key input parameters (area, ratio of length to width, circularity and distance dispersion) were selected. Among these four parameters, use of the first three was found to be satisfactory for classification between embryos and nonembryos. Using the three parameters, the trained neural network was also able to classify globular, heart- and torpedo-shaped embryos at a level comparable with estimation by a human expert. By using the trained neural network, the number of plantlets that would be formed after the second regeneration culture of 14 d could be successfully predicted from number of heart- and torpedo-shaped embryos at the end of the first regeneration stage.

Ploidy doubling by callus culture of potato dihaploid leaf explants and the variation in regenerated plants

SummarySomatic chromosome doubling of potato dihaploids was achieved by culturing callus from leaf pieces derived from glasshouse and in vitro grown plants. The glasshouse‐grown leaves produced better callus on average but there was no significant difference between the average number of plantlets per callus regenerated from the two types of material.Mixtures of 2x and 4x plants were obtained from callus culture and the proportions of each ploidy type varied with the dihaploid genotype. Leaflet length/breadth ratios were chiefly determined but ploidy but there was variation within ploidy groups. There were also differences in blight and cyst nematode resistance between tetraploids derived from the same dihaploid.

Characterization of factors affecting plantlet regeneration from rice (Oryza sativa L.) callus

Various components of culture media were tested to characterize factors affecting plantlet regeneration from rice (Oryza sativa L.) callus. It was found that plantlet regeneration from rice callus was affected by concentrations of gelling agents, osmoticum, and the combination of hormones in the regeneration medium. High concentrations (4–6 g/l gellan gum, 10–16 g/l agar) of gelling agents promoted regeneration frequency. However, the total number of plantlets decreased with gellan gum concentrations above 4 g/l. Addition of sorbitol (15–75 g/l) promoted plantlet regeneration. However, the addition of mannitol was inhibitory and no regeneration was observed at concentrations above 30 g/l. This difference in the effects on regeneration suggests that sorbitol had another function besides as a osmoticum. High regeneration frequency was obtained with combinations of NAA (0.05–0.5 g/l) and kinetin (0.5–2 mg/l). However, higher concentrations (2 mg/l) of NAA are preferred to increase the total number of regenerated plantlets.

Effects of Auxin, Cytokinin and Nitrogen Concentration on Morphogenesis of Tissue-cultured Shoot Apex of Chinese Yam (Dioscorea opposita Thunb.).

Most shoot apices of Chinese yam (Dioscorea opposita Thunb. cv. Nagaimo) developed into shoots in modified Murashige-Skoog (MS) medium in which the nitrogen level was reduced to one tenth of the recommended concentration and containing 0 to 0.1 mg•liter-1 NAA and 0.1 mg•liter-1 BA. Bases of shoot apices developed friable calli in a medium supplemented with 1.0 mg•liter-1 NAA. The composition of the basal medium had little effect on shoot formation, but morphogenic calli were produced by excised apices on a medium having 1 to 2 mg•liter-1 BA and low concentration of NAA. Morphogenic callus resembled shoot primordium in that adventitious buds were formed on its surface. When morphogenic calli were subcultured on MS medium containing 0 to 0.1 mg•liter-1 BA and 0.01 to 0.1 mg•liter-1 NAA, a large number of plantlets were regenerated.

Frond-bud propagation ofAsplenium nidusL.

Asplenium nidus, a common epiphytic fern, has a short, erect rhizome and a rosette of simple fronds. Propagation is by spores as the plant does not reproduce vegetatively. Plants can be divided into two, four or even eight segments and potted individually to give these numbers of plantlets. A more productive method is described whereby individual fronds are dissected, each with a small piece of stem attached; the pieces are then planted in vermiculite. Up to 33 plantlets per plant and 13 plantlets per frond piece were produced by this method, which is easy and rapid. Sufficient plants are available to meet the limited demands for A. nidus, but the method is useful for multiplying some of the available and attractive cultivars.

Micropropagation of shortleaf, Virginia and loblolly x shortleaf pine hybrids via organogenesis

Protocols were developed for the micropropagation of shortleaf pine (Pinus echinata Mill.), loblolly (P. taeda L.) x shortleaf pine hybrids, and Virginia pine (P. virginiana Mill.). For meristematic tissue induction, modified Gresshoff & Doy (GD) medium with a high concentration of benzyladenine (BA) and short pulse treatment was best for loblolly x shortleaf hybrids whereas a lower concentration of BA and longer pulse treatment was best for shortleaf and Virginia pines. Shoot growth rate for all species was generally slower on Schenk & Hildebrandt medium than on GD medium. Addition of activated charcoal improved shoot growth of shortleaf pine but not of Virginia pine or the loblolly x shortleaf hybrids. Separation of shoots was beneficial before placing in the advanced growth medium. Both GD and Litvay's media produced good advanced shoot growth, especially following the addition of 0.5% activated charcoal. Individual shoot heights of 2–3 cm and 8–12 weeks of age after separation from the cluster were best for rooting. Root induction declined rapidly thereafter. Modified GD medium with 0.5 mg 1-1 α-naphthaleneacetic acid plus 1.0 mg 1-1 3-indolebutyric acid and 20 g 1-1 sucrose was best for root induction for all species except shortleaf pine. Addition of activated charcoal produced better root systems. Too high a light intensity resulted in a lowered frequency of rooting. A large number of plantlets was produced.

In vitro culture of Cucumis sativus L.

The ability to regenerate plants from leaf explants has been tested for three highly inbred cucumber lines (B, G, S), their reciprocal hybrids, F2 and BC1 generations. The lines differed from each other in their regenerating ability, which was expressed by the percentage of explants regenerating embryoidal callus and mean number of plantlets per plant. Thus, the lines could be classified as frequently (B), intermediately (G) or occasionally regenerating ones (S). There were no reciprocal cross differences in the regeneration. It was found that the intermediately and intensively regenerating lines contain two pairs of dominant genes responsible for plant regeneration, characterized by complementary and probably additive interaction. The frequently regenerating line differed from the intermediately regenerating in the effect of one gene. It is supposed that the above-mentioned genes belong to three different loci. The ability to regenerate plants from leaf expiants had high heritability.

Plant Regeneration of Zoysiagrass from Embryo‐Derived Callus

Tissue culture methods could enhance development of improved cultivars of zoysiagrass (Zoysia japonica Steudel). A tissue culture system for callus induction and plant regeneration of zoysiagrass was developed. Embryos were aseptically excised from seeds and over 90% produced callus. Embryogenic callus was produced on Murashige‐Skoog (MS) agar medium supplemented with 2,4‐D (2,4‐dichlorophenoxy) acetic acid, but not on N6 medium. A 2,4‐D concentration of 1.0 mg L−1 resulted in greater callus production than higher concentrations. A 16‐h photoperiod of fluorescent light (65 μmol photon m−2 s−1) increased callus production over dark conditions. Direct elimination of 2,4‐D and dark callus induction resulted in greater regeneration frequency and a higher number of plantlets per callus than gradual elimination and light callus induction. Normal plantlets were transplanted to soil and grown to maturity in the field, where overwinter survival was exhibited.

Production of pineapple plants in vitro

In vitro culture of pineapple (Ananas comosus) was studied to determine the efficiency of axillary bud culture for rapid propagation of several cultivars. The technique used maximizes the success rate of various steps in the production of pineapple plants. Rapid mass multiplication of plantlets started 9 months after explanting with a significant log phase. The number of plantlets obtained from the culture of a single bud by the thirteenth month ranged from 210 to 380 for 'Perolera'; 300 to 350 for 'PR-1-67'; and 40 to 85 for 'Smooth Cayenne'. The method permits culture of a range of pineapple cultivars. Little morphological variation was observed in young regenerated plants.

Obligatory and period specific requirement of iron for microspore embryogenesis inDatura metel anther cultures

The experiments to assess the role of iron in morphogenesis of microspore-derived plantlets in anther cultures ofDatura metel revealed that even on iron-deficient medium androgenesis is initiated but the induced units fail to grow beyond multicellular stage, if anthers are not transferred to iron-incorporated medium. Another important conclusion drawn from an experiment involving weekly transfer of anthers from nutrient medium lacking both iron source (ferrous sulfate, FeSO4·7H2O, 10−4 M) and chelate (disodium salt of ethylene diamine tetraacetic acid, C10H14N2Na2O8·2H2O, 10−4 M) to that containing both of these or vice-versa, is that iron if present during the initial culture period, in fact affects the plantlet yield adversely. Thus, anthers cultured on iron-deficient medium, for initial one or two weeks and then transferred to iron-containing medium, yielded higher number of plantlets than those developed from anthers cultured continuously on complete basal medium having both iron and chelate.

Ovary slice culture of Lilium formosanum Wallace.

A new technique of ovary culture was applied in Lilium, resulting in the formation of a lot number of plantlets in vitro. Ovaries of Lilium formosanum WALLACE, sampled 20 days after self-pollination, were sliced into disks 2mm thick. They were inoculated on 32 kinds of modified MS agar media. The arrangement of those media was based on the L 32 table of orthogonal arrays, and 15 medium elements with 2 levels each, including pH, sucrose, major salts, minor salts, vitamins and several hormones, were combined with one another. Ovules grew well and became recognizable as seeds 40 or 50 days after inoculation. They germinated successively in the test tubes. Higher pH value (6.3) and sucrose concentration (8%) gave better results than lower ones, and the use of IAA (1 ppm) inhibited seed maturation. Standard concentration of the major salts was preferable to a lower one, while the inhibitory effect of hormones tended to decrease 170 days after inoculation.

Somatic embryogenesis and plant regeneration in cultured immature inflorescences of Setaria italica

Young inflorescence explants of Setaria italica in culture showed high capacity for regenerating plantlets through somatic embryogenesis. Embryogenic callus formation was initiated from the explants cultured on Murashige and Skoog's medium with 2 mg/l 2,4-D and 0.2-0.5 mg/l KT or BAP, but it was better for the maintenance of embryogenic growth to subculture the calli on the medium with 2,4-D and KT/BAP and on the medium with 2 mg/l 2iPA and 0.2 mg/l NAA alternately. A number of plantlets were regenerated when embryogenic calli were transferred onto the same basic medium but with 2 mg/l BAP and 0.5 mg/l NAA. Plant regeneration capacity has been maintained in some embryogenic calli during fourteen months of subculture.

Regulatory factors forin vitro multiplication of sandalwood tree (Santalum album Linn.) I. Shoot bud regeneration and somatic embryogenesis in hypocotyl cultures

Large number of plantlets were obtained in hypocotyl cultures ofSantalum album either through direct differentiation of shoot buds and their subsequent induction into rooted plants or through callus induction and somatic embryogenesis. Factors which triggered both these morphogenetic processes were investigated.

Efeito do estádio de desenvolvimento do fruto sobre a qualidade da semente do cultivar Patrícia de videira

Durante a maturação do fruto do cultivar Patrícia de videira, podem-se identificar três diferentes estádios de desenvolvimento, com base, principalmente, na coloração do fruto. Neste trabalho, coletaram-se sementes de cada estádio, armazenando-as por diferentes períodos, sendo a qualidade examinada após a colheita e no armazenamento, visando detectar o ponto de menor índice de dormência e máxima germinação. O estádio de maturação do fruto afetou-lhes a viabilidade: as sementes dos frutos maduros sempre apresentaram os maiores valores de viabilidade, ao contrário daquelas de frutos imaturos. Durante o armazenamento, houve diminuição da viabilidade nos três tipos de sementes testados, sendo as de frutos imaturos as mais afetadas. Em sementes de frutos maduros, ocorreu redução da dormência durante o armazenamento. Antes e depois do armazenamento, as sementes dos frutos maduros e meio maduros emergiram mais rápido e houve desenvolvimento de maior número de plântulas, com maior peso de matéria seca, comparativamente às sementes do fruto imaturo. A máxima germinação e vigor da semente são alcançados em sua maturidade fisiológica, aspecto esse a ser considerado antes da colheita, para que se obtenham sementes de alta qualidade.

In vitro Culture of Cucumis sativus I. Regeneration of Plantlets From Callus Formed by Leaf Expiants

Summary Cucumber leaf expiants from three different cultivar varieties regenerate a various number of plantlets (the highest number was 220 from one expiant) after 10–12 weeks of culture. The primary media used contained 1.2 mg · l -1 2,4,5-T and 0.8 mg · l -1 6-BAP or 0.4 mg · l -1 2,4-D and 0.8 mg · l -1 2iP. The secondary media contained reduced amounts of auxin or none and the same cytokinin concentration. Media containing 2,4,5-T gave characteristic gel-like callus composed of single cells and multicellular aggregates.

Adventitious Bud Formation From Bulb-scale Expiants of Lilium speciosum Thunb. in vitro. Effects of Wounding, TIBA, and Temperature

Effects of tissue wounding, TIBA, and temperature on the process of adventitious bud formation in vitro from bulb-scale expiants of Lilium speciosum Thunb., cv. Rubrum nr. 10, were studied. Removal of the abaxial epidermis from the expiants led to an increase in the number of plantlets formed at the adaxial surface and to suppression of the normally basipetal polarity of the regeneration process. It is concluded that the actual wounding itself was responsible for these effects and that wounding must have given rise to a transmittable stimulus. When incorporated into the nutrient medium, TIBA had an effect similar to that of wounding: the number of plantlets per expiant increased and the polarity was suppressed. However, at concentrations higher than 2.10-6M, TIBA inhibited the regeneration process. Within the range of 15°–25°C, a rise of the temperature led to more plantlets per expiant and to (partial) suppression of basipetal polarity. The results are discussed with special attention to the similarity in the action of these three factors.