Abscisic acid and osmotic induction of synchronous somatic embryo development of sweet potato

Summary Somatic embryos of sweet potato have potential as synthetic seeds. The effects of abscisic acid (ABA) (0,0,0.1, 1.0, 10.0 and 50.0 μM) were examined to improve synchrony and proliferation of somatic embryos. Transferring embryos compared to those cultures transferred at day 0. The development of embryos in suspension culture supplemented with ABA was poor. However, when calli proliferation cultures were in gelled medium and pulsed with 0.1 μM ABA for 14 d, the number of somatic embryos increased. Proembryonic masses cultured in mannitol-containing medium (Y=−1.5 MPa) increased embryo development and synchrony of embryo development. Thus, in this work ABA and mannitol have been shown to improve both the total number and the synchrony of sweet potato somatic embryos.     

Plant regeneration from stem and petiole protoplasts of sweet potato (Ipomoea batatas) and its wild relative,I. lacunosa

A protoplast-to-plant regeneration system has been established for sweet potato (Ipomoea batatas (L.) Lam.) and its wild relative, I. lacunosa L. Viable protoplasts, isolated from preplasmolyzed stems and petioles of in vitro-grown plants, were cultured on liquid MS (Murashige & Skoog 1962) medium that supported cell division and colony formation. Embryogenic calli of sweet potato were induced on agar-solidified MS medium supplemented with 3% (w/v) sucrose, 50 mg l^-1 casamino acids, 0.2–0.5 mg l^-1 2,4-d, 1.0 mg l^-1 kinetin and 1.0 mg l^-1 ABA. On average, 3 plants were regenerated from a single sweet potato callus subcultured on semi-solid MS medium containing 3% (w/v) sucrose, 800 mg l^-1 glutamine, 2.0 mg l^-1 BA or 1.0 mg l^-1 kinetin and 1.0 mg l^-1 GA₃. Embryogenic calli of I. lacunosa L. were initiated on semi-solid MS medium containing 0.2–0.5 mg l^-1 IAA and 1.0–2.0 mg l^-1 BA. An average of 5 plants was regenerated from a single sweet potato callus subcultured on semi-solid MS medium containing 0.5 or 1.0 mg l^-1 GA₃.Abbreviations ABA abscisic acid - BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole acetic acid - MES 2-(N-morpholino)-ethane sulfonic acid - NAA -naphthaleneacetic acid     

Effect of exogenous gibberellic acid,abscisic acid,and benzylaminopurine on epicotyl dormancy of cultured herbaceous peony embryos

Epicotyl dormancy was broken in cultured peony (Paeonia lactiflora Pall.) embryos after topical application of agarose gels containing gibberellic acid, with optimum growth at 1.5 mM gibberellic acid. Addition of 100 M abscisic acid to the medium resulted in complete inhibition of gibberellic acid-stimulated promotion of dormant epicotyls. Epicotyl dormancy was also broken in embryos by culture on media containing 1 or 10 M benzylaminopurine. A highly significant increase in leaf number occurred when embryos were both cultured on medium containing benzylaminopurine and treated topically with gibberellic acid. Anatomical and morphological studies indicated that the increase in shoot growth was due to the development and growth of 1) buds formed at the cotyledonary node, 2) axillary buds, and 3) adventitious meristems originating from subepidermal parenchymatous tissue.Abbreviations ABA abscisic acid - BA N⁶-benzylaminopurine - DMSO dimethyl sulfoxide - GA₃ gibberellic acid - LS Linsmaier and Skoog     

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 M 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.Abbreviations ABA abscisic acid - IBA indolebutyric acid - 2,4-d 2,4-dichlorophenoxyacetic acid - EM embryonal mass     

In vitro propagation of taro,with spermine,arginine, and ornithine

In vitro growth and multiplication of taro [Colocasia esculenta var. antiquorum cv. Keladi Birah] was improved considerably, when primary shoot apices were cultured on two modifications of Linsmaier and Skoog [1965] medium, containing 5.5 mg 1^–1 naphthaleneacetic acid and 0.2 mg 1^–1 kinetin or 1.85 mg 1^–1 naphthaleneacetic acid and 2 mg 1^–1 kinetin and supplemented with 10^–4 or 10^–3 mol·1^–1 of polyamine spermine or either of the precursors of polyamine putrescine—arginine and ornithine. Plantlets were regenerated directly from primary shoot apices, axillary buds and protocorm-like bodies [PLB]. Frequency of plantlet regeneration, rate of development and growth in height of main plantlets were enhanced by the addition of arginine and ornithine to the media. Secondary plantlet formation from axillary buds and PLB were promoted by spermine and arginine respectively.     

In Vitro Regeneration of Stevia rebaudiana (Bert) from the Nodal Explant

Procedure for micropropagation of Stevia rebaudiana Bertoni, containing stevioside, a natural noncaloric sweetner, has been developed using nodal segments as explant. Higher proliferation of shoots and multiplication was obtained on Murashige and Skoog basal medium (MS) supplemented with 1.0 mg l-1 indoleacetic acid (IAA) plus 10.0 mg l^-1 kinetin and 30.0 mg l-1 adenine sulphate. Sprouting of 90% of the axillary buds was observed within 4 weeks of inoculation, producing >10.0 shoots per explant within 12 weeks. Profuse roots were induced from 90% of the regenerated shoots within 4 weeks of inoculation on half strength MS solid medium supplemented with 1.0 mg l^-1 IAA. High survival rate, > 60%, was obtained when the plantlets were transferred to field conditions. The survival rate of taller plants was always higher. The in vitro regenerated plants were morphologically indistinguishable from the donor plants and leaves were of intense sweet taste upon chewing. The heterogenic nature of S. rebaudiana necessitates establishment of protocol for every genotype independently.     

In vitro clonal propagation of an aromatic medicinal herb Ocimum basilicum L. (sweet basil) by axillary shoot proliferation

Summary An efficient protocol for in vitro propagation of an aromatic and medicinal herb Ocimum basilicum L. (sweet basil) through axillary shoot proliferation from nodal explants, collected from field-grown plants, is described. High frequency bud break and maximum number of axillary shoot formation was induced in the nodal explants on Murashige and Skoog (1962) medium (MS) containing N⁶-benzyladenine (BA). The nodal explants required the presence of BA at a higher concentration (1.0 mg·l⁻¹, 4.4 μM) at the initial stage of bud break; however, further growth and proliferation required transfer to a medium containing BA at a relatively low concentration (0.25 mg·gl⁻¹, 1.1 μM). Gibberellic (GA₃) at 0.4 mg·l⁻¹ (1.2 μM) added to the medium along with BA (1.0 mg·l⁻¹, 4.4 μM) markedly enhanced the frequency of bud break. The shoot clumps that were maintained on the proliferating medium for longer durations, developed inflorescences and flowered in vitro. The shoots formed in vitro were rooted on half-strength MS supplemented with 1.0 mg·l⁻¹ (5.0 μM) indole-3-butyric acid (IBA). Rooted plantlets were successfully acclimated in vermi-compost inside a growth chamber and eventually established in soil. All regenerated plants were identical to the donor plants with respect to vegetative and floral morphology.     

Apical Dominance, Water Deficit and Axillary Inflorescence Growth in Zea mays: The Role of Abscisic Acid

In the sweet corn cultivar, Iochief, an episode of water deficitduring early tassel development results in a subsequent promotionof the growth of the lower axillary inflorescences. This responseis also produced by the application of abscisic acid (ABA) atthis period of growth to well-watered plants, and the hypothesisthat the response to water deficit was due to an increase inendogenous ABA concentration was examined. The ABA contentsof the tassel, leaf and axillary inflorescences were found toincrease during water stress, the increase in the tassel andaxillary buds being most rapid in the first 2 days of waterdeficit. This increase in free ABA content was followed after4 days of water deficit by a progressive increase in the concentrationof ‘bound’ ABA in the tissues. There was littleincrease in free ABA concentration after 4 days water deficit;this paralleled the subsequent growth response of the axillaryinflonscences which also was unaffected by prolonging the epidoseof water deficit beyond 4 days. In order to establish whether the response of the axillary inflorescencesto ABA was dependent upon the presence of the tassel, ABA wasapplied to watered plants with or without the developing tassel.As had been previously found with water stress, removing thetassel inhibited the response of the plant to applied ABA. Zea mays, apical dominance, water stress, inflorescence growth, abscisic acid     

The effect of abscisic acid on root and shoot growth of cauliflower plants

Addition of abscisic acid (ABA) to the nutrient solution increased the root to shoot ratio of hydroponically-grown cauliflower plants by reducing the dry weight of the shoot and increasing that of the root. At concentrations higher than 10^–7 M, ABA increased root branching and root hair formation. Root extension was inhibited in plants kept continuously in solutions containing high ABA concentrations but following removal from the ABA solution root elongation was increased in comparison with plants given no ABA treatment. This elongation was greatest in plants with increased root branching caused by higher ABA concentrations.     

Effects of Abscisic Acid on Phenolic Content and Lignin Biosynthesis in Tobacco Tissue Culture

A significant depression of callus growth resulted from low concentrations of abscisic acid (ABA) added to the medium recommended by Linsmaier and Skoog. Low concentrations also decreased the chlorogenic acid and lignin content of the callus, and generally decreased amounts of scopolin and scopoletin in the tissue. Gibberellic acid (GA₃) stimulated callus growth in a low concentration (0.1 mg/1) and inhibited growth at a high concentration (10.0 mg/1). Both levels of GA₃ increased scopoletin accumulation in tobacco callus. A high concentration of GA₃ increased the accumulation of scopolin and chlorogenic acids, whereas a low concentration decreased the amounts of these two phenolic compounds. In comparison with the control, lignin synthesis was stimulated by a low GA₃ concentration, but a high GA₃ concentration did not have a significant effect. Both low and high concentrations of GA₃ overcame ABA inhibition of growth and lignin synthesis, and partially reversed ABA inhibition of scopoletin production. However, GA₃ did not reverse the inhibitory effect of ABA on scopolin production. The low concentration of GA₃ overcame the inhibition of chlorogenic acid production resulting from a 0.01 mg/1 concentration of ABA, but this was the only reversal of chlorogenic acid inhibition resulting from addition of GA₃ to the medium.     

Fluoranthene influences endogenous abscisic acid level and primary photosynthetic processes in pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.) plants in vitro

The influence of increasing concentrations (0.1, 1.0 and 5.0 mg l⁻¹) of fluoranthene (FLT) on growth, endogenous abscisic acid (ABA) level and primary photosynthetic processes in 21-day-old pea plants (Pisum sativum L.) in vitro was investigated. Murashige and Skoog’s (MS) medium, with or without FLT, was enriched with indole-3-acetic acid (IAA; 0.1 mg l⁻¹) or a combination of IAA (0.1 mg l⁻¹) plus N⁶-benzyladenine (BA; 0.1 mg l⁻¹). The level of endogenous ABA significantly increased with increasing FLT concentrations in the presence of both IAA and IAA plus BA. An increased level of endogenous ABA was observed in plants treated with IAA alone. The growth of shoot, callus and the content of photosynthetic pigments (chlorophyll a and b, carotenoids), in both IAA- and IAA plus BA-treated plants, were significantly stimulated by FLT at its lowest concentration (0.1 mg l⁻¹) assayed in this study. However, FLT at higher concentrations (1.0 and 5.0 mg l⁻¹) significantly inhibited all these parameters. Chlorophyll fluorescence imaging showed that FLT only at the highest concentration (5.0 mg l⁻¹) in the presence of IAA (0.1 mg l⁻¹) significantly increased F₀, but decreased F_V/F_M and Φ_II.     

Correlative Inhibition of Lateral Bud Growth in Pisum sativum L. and Phaseolus vulgaris L.: Studies of the Role of Abscisic Acid

The possibility has been investigated that abscisic acid (ABA)might act as a correlative inhibitor of lateral bud growth inPisum sativum and Phaseolus vulgaris. Application of ABA insmall quantities (2µg) to axillary buds on decapitatedplants of P. sativum caused appreciable inhibition of theirgrowth, and induced a compensatory growth of the bud on an adjacentnode. Application of this same quantity of ABA to axillary budson decapitated plants of Phaseolus vulgaris was without effect,but a high concentration in lanolin (1 mg g^–1) did substantiallyreduce bud outgrowth. Endogenous ABA-like substances in Phaseolusvulgaris, detected by bioassay and electron capture g.l.c.,were present in similar concentrations in shoot tips, lateralbuds on intact plants and lateral buds on plants decapitated24 h earlier. The effects of applied ABA suggested that it might be involvedin the mechanism of correlative inhibition in Pisum sativum,but it was not possible to test this hypothesis by determiningendogenous ABA levels in axillary buds because of their smallsize. The evidence presented here suggests that ABA is not acorrelative inhibitor in Phaseolus vulgaris even though at highconcentration it can inhibit the growth of axillary buds.     

Organogenesis in callus derived from stem and leaf tissues of apple and cherry rootstocks

Callus formation from stem internodes of the apple rootstocks M.9, M.25, M.26, M.27 and the cherry rootstock Colt, and from pith of Nicotiana tabacum cv. Wisconsin 38 was initiated on 4 -naphthaleneacetic acid (NAA)-based media (2.0–10.0 mg1^-1). Transfer of callus to corresponding media lacking NAA allowed regeneration of shoots from callus of M.25, M.27, Colt and tobacco but not of M.9 and M.26. With M.25 phloroglucinol (PG) depressed regeneration from 30 to 10% and no regeneration was observed in cultures grown in the presence of casein hydrolysate (CH) and glutathione (GSH).Organogenesis was also obtained from leaf discs of M.27 employing 6-benzyl-aminopurine (BAP) at 5.0mg 1^-1 and 2,4-dichlorophenoxy acetic acid (2,4-D) at 0.1 mg1^-1. The regenerated shoots have been multiplied and rooted.Organogenesis also occurred in M.26 from small (1–2mm), green, compact embryoid-like structures derived from stem and leaf surfaces of excised axillary shoots. These structures differentiated into shoots at a low frequency (< 1%) on media containing BAP (1.0mg1^-1) and indole-3-butyric acid (IBA) (0.1 mg1^-1) and could also be micropropagated by subsequent axillary shoot proliferation.     

Rapid micropropagation of Woodfordia fruticosa (L.) Kurz (Lythraceae), a rare medicinal plant

A rapid propagation method comprising initiation of in vitro shoot tip culture from field-grown flowering plants and reculture of the nodal segments of regenerated shoots in Schenk and Hildebrandt (1972) medium was developed for Woodfordia fruticosa (L.) Kurz., a rare medicinal shrub. A medium supplement of 6-benzylaminopurine (0.2 mg.l^–1) induced high frequency (88%) development of axillary shoot buds (3.2) in 4–5 weeks. Subculture of the explants with multiple new shoots in fresh medium for 30 days yielded an even larger number (9.7) of shoots. Highest multiplication (26–35 shoots) was recorded when using culture initiation media with 0.5 mg.l^–1 each of BAP and NAA followed by subculture in 0.2 mg.l^–1 BAP. The shoot multiplication rate was further accelerated by reculturing 0.4–0.6 cm nodal segments of regenerated shoots in media with 1.0 mg.l^–1 BAP. Shoot cuttings (3.5–7.0 cm) were rooted in 0.2 mg.l^–1 IAA. Regenerated plants displayed uniform morphological, growth and flowering characteristics.Abbreviations BAP 6-benzylaminopurine - NAA naphthaleneacetic acid - IAA Indole-3-acetic acid - IBA indole-3-butyric acid - SH Schenk and Hildebrandt (1972) medium     

Influence of explant source, plant growth regulators and culture environment on culture initiation and establishment of Quercus robur L. in vitro

Suitable cytokinin supplements and culture environments havebeen determined for the initiation and establishment of shootcultures of Quercus robur seedling tissue. Initiation of axillaryshoot development from nodal explants required culture mediumsupplemented with BA (6-benzylamminopurine). The greatest numbersof stem segments for culture proliferation were obtained using1.0 mg I^-1 BA after 56 d culture. The frequency of shoot developmentand subsequent formation of multiple shoots at initiation wasinfluenced by the position of the nodal explant in the seedlingshoot, incubation temperature and daylength. Explants from basaland apical regions, which contained multiple axillary buds,produced the lowest frequencies of axillary shoot developmentand multiple shoot formation, many remained quiescent. Axillaryshoot development was greatest in single nodal explants excisedfrom the midstem positions, elongated regions of the shoot wherenodes were formerly associated with a leaf. Higher temperaturesstimulated shoot formation with greater numbers of stem segmentsfor culture multiplication being obtained from nodal explantsincubated at 25C. Axillary shoot development was promoted innodal explants maintained under daylengths of 16 h or more.Stem segments cut from axillary shoots which developed fromnodal explants were used to establish shoot multiplication cultureson medium supplemented with 0.4 mg I^-1 BA. Shoot formation fromstem segments was greater at higher incubation temperaturesof 25C and 30C. Multiplication coefficients for stem segmentsincreased after one subculture. Key words: Quercus robur, oak, micropropagation, cytokinin, temperature, daylength, rest, quiescence     

Somatic embryogenesis and plant regeneration in Quercus acutissima

Immature embryos of Quercus acutissima were collected weekly beginning 5 weeks post-fertilization and cultured on modified MS(Murashige and Skoog) medium containing 1,000 mg/l glutamine and 5 mM proline with different combinations of IBA(0.5–10.0 mg/l) and BA(0 or 1.0 mg/l) in light. The highest percentage of embryogenic cultures occurred on the medium containing 0.5 mg/l IBA or 1.0 mg/l BA and 0.5 mg/l IBA. Four weeks after initiation, the embryogenic cultures were transferred to MS medium without plant growth regulators and cultured for 4 weeks. The somatic embryos were then transferred to germination medium. The best germination results were achieved from WPM(Woody Plant Medium) containing 0.1 mg/l BA. Plantlets from somatic embryos were incubated on WPM supplemented with 0.2 mg/l BA for 4 weeks and plantlets with well developed shoots and roots were transplanted to perlite and peat moss(11, v/v) mixtures and placed in a culture room. After being hardened off for 8 weeks, they were transferred outdoors where they grew.Abbreviation BA N⁶-benzyladenine - IBA indole-3-butyric acid - GA₃ gibberellic acid - ABA abscisic acid - MS Murashige & Skoog Medium - WPM Woody Plant medium     

Mutation induced by ethylmethanesulphonate (EMS), in vitro screening for salt tolerance and plant regeneration of sweet potato (<Emphasis Type="Italic">Ipomoea</Emphasis><Emphasis Type="Italic">batatas</Emphasis> L.)

Salt tolerant cultivars of sweet potato (Ipomoea batatas L.) can be obtained from induced mutation. The objective of the present study was to induce mutation for salt tolerance using ethylmethanesulphonate (EMS) in calli of sweet potato, followed by cell line selection and subsequent plant regeneration. Calli initiated from leaf explants were treated with 0.5% EMS for 0, 1, 1.5, 2, 2.5 and 3 h, followed by rinsing with sterile distilled water for four times. Preliminary experiments showed that 200 mM NaCl could be used as selection pressure. Salt tolerant calli were sub-cultured on medium supplemented with 200 mM NaCl for selection of mutant cell lines and this process repeated 5 times (20 days each). The selected calli were transferred onto somatic embryo formation medium, which was Murashige and Skoog (MS) medium supplemented with 4 mg l⁻¹ abscisic acid (ABA), 10 mg l⁻¹ gibberellic acid (GA). After 15 days, somatic embryos were transferred onto MS medium supplemented with 0.05 mg l⁻¹ ABA, 0.2 mg l⁻¹ zeatin (ZT) for regeneration. Plants designated as ML1, ML2 and ML3 were regenerated from the somatic embryos formed by calli treated with 0.5% EMS for 2 and 2.5 h. After propagation, salt tolerance of these mutants was investigated. Data suggested the mutants were more salt tolerant than control plants.     

In vitro propagation of Halesia carolina L. and the influence of explantation timing on initial shoot proliferation

Halesia carolina L., a small, ornamentally valuable tree, is difficult to propagate due to the complexity of seed propagation and the unavailability of propagules for conventional vegetative propagation. A micropropagation system was developed to facilitate easy propagation of this species. Actively growing shoot tips achieved optimum shoot proliferation from axillary buds when placed on Woody Plant Medium supplemented with 1.0 to 2.5 mg/l benzyladenine. The addition of 0.1 mg/l naphthaleneacetic acid had little effect on culture performance. Murashige and Skoog medium was incapable of supporting vigorous shoot proliferation. Non-sterile rooting conditions provided better rooting and subsequent plantlet growth, when compared to an in vitro rooting method. The seasonal fluctuations in the stock plant dramatically affected the shoot proliferating potential of the explants in vitro. Rapidly elongating shoots formed shoot proliferating cultures more slowly than explants taken either before or after the rapid elongation phase.     

Plant regeneration from leaf protoplasts of Solanum torvum

A protocol to obtain regenerated plants from protoplasts of Solanum torvum Sw a wild species of eggplant resistant to Verticillium wilt is reported. Leaf protoplasts were enzymatically isolated from six-week old seedlings grown in a controlled environment chamber. Protoplasts were plated on modified KM medium (0.4 M glucose)+(mg/l): 1.0 p-chlorophenoxyacetic acid (CPA)+1.0 naphthaleneacetic acid (NAA)+0.5 6-benzylaminopurine (BAP) and 0.02 abscisic acid (ABA). The protoplast density was 5×10⁴ per ml with 5 ml placed in each of two quadrants in X-dishes (100×15 mm). The reservoir medium was modified KM+(mg/l): 0.1 NAA+0.5 BAP+0.1 M sucrose+0.1 M mannitol+0.6% washed agar+1% activated charcoal. Dishes were initially placed in the dark at 27°C. Protoplast division was initiated in 1–2 weeks and 4 weeks later p-calli were 1–3 mm. Plating efficiency was 11% when measured at 3 weeks. Six-week old p-calli were transferred individually onto Whatman No. 1 filter paper layered on modified KM (0.15 M sucrose)+mg/l: 2.0 indoleacetic acid (IAA)+2.0 zeatin+0.5% washed agar for 2 weeks. Subsequently, shoots occurred within 4 weeks at 70% efficiency on MS+30 g/l sucrose+2 mg/l zeatin. Shoots were rooted on half strength MS+10 g/l sucrose.Abbreviations ABA abscisic acid - BAP 6-benzylaminopurine - CPA p-chlorophenoxyacetic acid - IAA indoleacetic acid - KM Kao and Michayluk - MS Murashige and Skoog - NAA naphthaleneacetic acid - 2ip 6-dimethylallyamino purine Michigan Agricultural Experiment Station Journal Article No. 12167