Plant regeneration via somatic embryogenesis in creeping bentgrass (Agrostis palustris Huds.)

We have established a high-frequency plant regeneration system via somatic embryogenesis from mature seeds of creeping bentgrass (Agrostis palustris Huds). The effects of 2,4-dichlorophenoxyacetic acid (2,4-D), 3.6-dichloroo-anisic acid (dicamba) and 6-benzyladenine (BA) on callus formation and embryogenesis were evaluated. Callus produced on the Murashige and Skoog (MS) (1962) medium containing 2,4-D had low embryogenic potency. In the presence of 30 M dicamba, addition of 2.25 to 9 M BA significantly enhanced embryogenic callus formation over dicamba alone. Optimum frequency of somatic embryogenesis was achieved on MS basal medium containing 30 M dicamba and 2.25 M BA. Over 80% of somatic embryos germinated and formed plantlets on half-strength MS basal medium. These plantlets grew normally in the greenhouse.Abbreviations MS Murashige and Skoog medium - 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzyladenine - dicamba 3, 6-dichloro-o-anisic acid     

In vitro establishment and multiplication of Nymphaea hybrid ‘James Brydon’

Rhizome tips were the most suitable explants for in vitro plant regeneration and multiplication of Nymphaea hybrid James Brydon on Murashige and Skoog medium containing different concentrations and combinations of indole-3-acetic acid, 1-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid, 6-benzyladenine (BA), kinetin, 2-isopentenyladenine (2iP) and gibberellic acid (GA₃). A combination of 2iP, BA, and NAA strongly favored induction of shoot buds and shoot proliferation. Pretreatment of shoot cultures at 8°C for 30 days or with 14.4 or 28.9 M GA₃ for 15 days did not improve shoot multiplication. A 16-h photoperiod with photosynthetic photon flux of 30 mol m^-2 s^-1 was found to be the optimum light condition for shoot growth and multiplication. Multiple shoots produced well developed root systems within 4 weeks after transfer to a plant growth regulator-free medium containing activated charcoal.Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - 2iP 2-isopentenyladenine - MS Murashige and Skoog medium - NAA 1-naphthaleneacetic acid     

Tissue culture and micropropagation of Cuphea ericoides,a potential source of medium-chain fatty acids

Plant rgeneration occurred on leaf-and stem-derived callus of Cuphea ericoides Cham. & Schlechtd obtained in Murashige and Skoog medium supplemented with auxins [indole-3-acetic acid (IAA), -naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-d)] plus cytokinins [6-benzyladenine (BA) or kinetin]. These calluses were subcultured and showed vigorous growth. When subcultured on medium containing 2.22 or 4.44 M BA, the calluses showed profuse regeneration of shoots whereas those subcultured on medium supplemented with 2.69 M NAA or 0.226 M 2,4-d produced numerous roots. Isolated shoots rooted on Murashige and Skoog medium lacking growth regulators or containing 0.54 M NAA or 0.49 M indole-3-butyric acid (IBA). Plantlets were acclimatized to greenhouse conditions.Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige & Skoog medium - NAA 1--naphthaleneacetic acid     

Clonal propagation of Camptotheca acuminata through shoot bud culture

The chinese tree Camptotheca acuminata produces the anti-cancer and anti-retroviral drug camptothecin. Methods were developed for the clonal propagation of this important medicinal plant through shoot bud culture. Shoot buds were excised from 25 to 30 day old seedlings, presoaked for 48 h in three different liquid media containing either BA (2.22–17.4 M), kinetin (2.32–18.58 M), or thidiazuron (0.1–10 M) and were subsequently cultured on semi-solid medium of the same composition. Multiple shoots only developed from the 6-benzyladenine presoaked explants with the maximum number of shoots initiated from buds presoaked in and grown on B5 medium containing 17.4 M 6-benzyladenine. Individual shoots were removed from clusters and rooted on B5 supplemented with indole-3-butyric acid (4.9–19.6 M). The lowest concentration of indole-3-butyric acid (4.9 M) gave the highest percentage of rooting (82%) and the shortest root initiation period (18 d). Over 90% of the in vitro rooted plantlets survived transfer to soil.Abbreviations BA 6-benzyladenine - B5 Gamborg's B5 medium (Gamborg et al., 1968) - CPT camptothecin - 2,4-d 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - kinetin 6-furfurylaminopurine - LS Linsmaier & Skoog medium (Linsmaier & Skoog, 1965) - MS Murashige & Skoog (Murashige & Skoog, 1962) - NAA I-naphthaleneacetic acid - PGR plant growth regulator - TDZ thidiazuron - WPM woody plant medium (Lloyd & McCown, 1981)     

High frequency somatic embryogenesis in Coffea canephora

Leaf explants of Coffea canephora (P. ex Fr.) produced a friable yellow callus when they were cultured on a conditioning basal medium with 2.2 M 2,4-D, 2.4 M IBA and 9.8 M 2iP for 4 weeks then on an induction basal medium with 4.4 M 2,4-D and 17.8 M BA for 10 weeks. This calus could be maintained by means of regular subcultures or it could give rise to somatic embryos depending on the culture medium. Cytological studies documented somatic embryogenesis and embryo development.Abbreviations BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - 2iP 2-isopentenyladenine - MS Murashige & Skoog medium - NAA -naphthaleneacetic acid - NPR nucleoplasmic ratio - PGR plant growth regulator     

In vitro plant regeneration from leaf-derived callus in ginger (Zingiber officinale Rosc.)

Excised tissues from young leaves of ginger cv. Maran were cultured on revised Murashige and Skoog medium supplemented with various concentrations of growth regulators. The presence of 2, 4-D in the culture medium at 9.0–22.6 M resulted in callus growth. Organogenesis and plantlet formation occurred when the concentration of 2,4-D is reduced to 0.9 M and with the addition of 44.4 M BA into the medium. The rate of plant regeneration increased when the growth regulators are completely removed from the culture medium in the subsequent subcultures. The plantlets developed extensive root systems when they were put in MS liquid medium with 5.4 M of NAA. The establishment of these plantlets in soil is about 80%.Abbreviations BA N⁶-benzyladenine - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Regeneration of plantlets through organogenesis in the Himalayan yellow poppy,Meconopsis paniculata

Plantlet formation through organogenesis in callus cultures of Himalayan yellow poppy,Meconopsis paniculata D.Don (Prain), a threatened taxon of ornamental value, is described. Hypocotyl segments from 3-month-old laboratory-raised seedlings produced callus on agar-solidified Murashige and Skoog medium (MS) containing 10 M -naphthaleneacetic acid and 1 M kinetin. Shoots differentiated best from callus on MS containing 10 M indolebutyric acid (IBA) and 1 M 6-benzyladenine. The regenerated shoots rooted best on MS medium containing 10 M IBA. From seed germination to differentiation of plantlets through the two-step organogenesis process required 28–29 weeks.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - FAA formalin-acetic acid-alcohol - BA 6-benzyladenine - IAA indole-acetic acid - IBA indolebutyric acid - GA₃ gibberellic acid - NAA -naphthaleneacetic acid - RH relative humidity     

Somatic embryogenesis in cultured immature kernels of Pistachio,Pistacia vera L.

Embryogenic tissue was produced from kernels of immature fruits of Pistachio (Pistacia vera L.) cultured in liquid Murashige and Skoog media, supplemented with 200 mgl^–1 casein hydrolysate, 114 M 1-ascorbic acid, and benzylaminopurine. Compact embryogenic masses differentiated directly from the fruit explants after culture for 2 weeks in liquid medium with 8.9 M benzylaminopurine. After transfer of the embryogenic masses into the same medium, but with 4.4 M benzylaminopurine, somatic embryos appeared. Several stages of embryogenesis were present in the cultures. Adventive embryos were readily separated from the friable embryogenic masses by shaking. Separated somatic embryos, germinated on solidified Murashige & Skoog medium without growth regulators, developed into plantlets.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP benzylaminopurine (N⁶-benzyladenine) - EMS embryogenic mass - MS Murashige and Skoog medium (Sigma M-0404) - NAA -naphthalene acetic acid - PGR plant growth regulator - TDZ thidiazuron (1-phenyl-3-(1,2,3, thiadiazol-5-yl)urea) - WP McCown's Woody plant medium (Sigma M6774) - ABA abscisic acid     

Agrobacterium-mediated transformation of hybrid poplar suspension cultures and regeneration of transformed plants

A method for Agrobacterium-mediated transformation of hybrid poplar (Populus alba x P. grandidentata cv. Crandon) suspension cultures and regeneration of transformed plants is described. Transformants were recovered when suspension cultures were inoculated with Agrobacterium tumefaciens at a density of 10⁷ colony-forming units ml^-1, cocultivated for 48 h, and plated to cellulose acetate filters on Woody Plant Medium containing 4.5 M 2,4-dichlorophenoxyacetic acid and 250 mg l^-1 cefotaxime. Levels of cefotaxime greater than 250 mg l^-1 were unnecessary for control of residual bacteria and inhibited callus growth. Transgenic plants were regenerated by culturing the transformed callus on media containing 0.11 to 27 M thidiazuron. In contrast to thidiazuron, N⁶-benzyladenine had a negative effect on shoot regeneration; the callus became necrotic when we attempted to induce shoots with concentrations of 1.1 to 8.9 M, and growth was inhibited when concentrations of 0.11 or 0.22 M were used to regenerate callus from suspension cultures. Following cocultivation of poplar suspension cultures, we recovered transgenic plants containing the maize transposon Ac, and callus containing an insect toxin gene from Bacillus thuringiensis.Abbreviations BA N⁶-benzyladenine - CIM callus initiation medium - CaMV cauliflower mosaic virus - cfu's colony-forming units - HPT hygromycin phosphotransferase - MS Murashige and Skoog medium (Murashige & Skoog 1962) - NPT-II neomycin phosphotransferase-II - PAR photosynthetically active radiation - PCR polymerase-chain-reaction - TDZ thidiazuron - WPM Woody Plant Medium (Lloyd & McCown 1980) - 2,4-d 2,4-dichlorophenoxyacetic acid     

The in vitro production of an anthocyanin from callus cultures of Oxalis linearis

Callus growth and the production of anthocyanins were sustained on the salts and vitamins of Murashige and Skoog. Callus growth was stimulated at a concentration of 8–32 M -naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-d). Benzyladenine (BA) and zeatin at 8 M inhibited callus growth whereas isopentenyladenine (iP) stimulated callus growth. NAA repressed anthocyanin production with an increase in NAA from 8–32 M. Anthocyanin synthesis was promoted by an increase in 2,4-d from 0.5 to 2 M and decreased thereafter up to a concentration 32 M 2,4-d. A concentration of 8 M BA, thidiazuron and zeatin, respectively stimulated pigment production. Sucrose stimulated callus growth at 60 mM and pigment production at 120–360 mM.Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - iP isopentenyladenine - TZ thidiazuron-N-phenyl-N-1,2,3-thiadiazol-5-yl-urea - Bu-HCl Butanol-2N HCl - BAW Butanol-acetic acid-water     

Somatic embryogenesis and plant regeneration of pepper in liquid media

A protocol was developed for regeneration of pepper (Capsicum annuum var. Ace) through somatic embryogenesis in liquid media. For embryogenic callus formation, mature zygotic embryo explants were used on basal Murashige and Skoog medium with 9.05 M 2,4-dichlorophenoxyacetic acid and 3% sucrose. Embryogenic callus was transferred to liquid basal Murashige and Skoog medium with 4.52 M 2,4-dichlorophenoxyacetic acid and 3% sucrose in order to increase the mass of the embryogenic culture. After pretreatment with potassium citrate, cells were placed into embryo initiation medium with 6 g l^-1 l-proline and a decreased (10 mM) ammonium concentration. Embryos were matured in 1.89 M abscisic acid containing half-strength Murashige and Skoog medium and converted into plants bothin vivo andin vitro at up to a 97% efficiency.     

Induction of microspore embryogenesis in Camellia japonica cv. Elegans

Three methods of microspore culture were tested for the induction of microspore embryogenesis in Camellia japonica L. cv. Elegans. Culture was performed on 17 different media consisting of Murashige and Skoog (MS) and N6 basal media with different combinations of carbon, growth regulators, serine and glutamine. Microspore suspensions plated over solid MS medium containing 4.5 M 2,4-dichlorophenoxyacetic acid and 0.5 M kinetin, with sucrose (MS6) or glucose (MS9) were seen as the best culture conditions for induction of embryogenesis. The development of microspore derived proembryos was obtained in MS medium supplemented with 2.2 M N⁶-benzyladenine (MS10) and reached the highest level when the microspores were cultured in MS6 inducing medium. The development of microspore-derived embryos ceased at the maturation stage.Abbreviations BA N⁶-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid     

Direct somatic embryogenesis through thin cell layer culture in Panax ginseng

Direct somatic embryos were differentiated on cotyledon transverse Thin Cell Layers (tTCLs) of Panax ginseng after 9 weeks in the Murashige and Skoog basal (MS) medium containing 2,4-d (5M). When MS medium containing 2,4-d (5M) was used for seedling pretreatment and for tTCLs culture, somatic embryos were observed 2 weeks earlier, i.e. after 7 weeks of culture. On the tTCLs from seedlings pretreated with 2,4-d (5M) combined with benzyladenine and zeatin at 0.1 M (BZ), somatic embryos were observed after 6 weeks of culture and the percentage of embryogenesis was higher (62%) than when 2,4-d was used alone for pretreatment (40%). Similar results were also obtained from pretreatment with combinations of 2,4-d (5M) and thidiazuron (TDZ) (0.01, 0.1M). When a combination of 2,4-d (5M) and BZ (0.1M) was used both for seedling pretreatment and for tTCLs culture, both somatic embryos and shoots were observed after only 3 weeks. As the concentration of BZ increased, the percentage of somatic embryogenesis decreased but the percentage of organogenesis increased. Similar responses were obtained with a combination of 2,4-d (5M) and TDZ (0.01M). On the medium containing both NAA (0.3M) and BZ (1M), globular- and heart- stage embryos developed after 4 weeks of culture into cotyledonary-staged embryos which remained dormant after a short elongation of the embryo axis. The importance of seedling pretreatment by growth substances in enhancing somatic embryogenesis is reported.Abbreviations BA 6-benzyladenine - BZ combination of BA and zeatin - 2,4-d 2,4-dichlorophenoxyacetic acid - MS medium Murashige and Skoog basal medium - NAA a-naphthaleneacetic acid - TDZ thidiazuron - tTCLs transverse thin cell layers - TCL longitudinal thin cell layer     

In vitro somatic embryogenesis ofDalbergia sissoo Roxb. —a multipurpose timber-yielding tree

Somatic embryogenesis was achieved in callus cultures dervied from 40-day-old semimature zygotic embryos ofDalbergia sissoo on semi-solid Murashige and Skoog (MS) salts and vitamins supplemented with 0.46–1.16 M kinetin, 6.78–9.04 M 2,4-dichlorophenoxy acetic acid (2,4-D) and 30 g/1 sucrose. Somatic embryos proliferated rapidly by secondary somatic embryogenesis after transfer to half-strength basal MS medium supplemented with 0.46-1.16 M kinetin and 6.78–9.04 M 2,4-D with 2% (w/v) sucrose. The light-green somatic embryos germinated on half-strength MS salts and vitamins supplemented with 0.5 mg/1 abscisic acid and 2% (w/v) sucrose. The developmental stages of somatic embryogenesis were studied by light and scanning electron microscopy.Abbreviations ABA Abscisic acid - BA 6-benzyladenine - Kn kinetin - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - MS Murashige and Skoog (1962) basal medium     

Micropropagation of safed musli (Chlorophytum borivilianum), a rare Indian medicinal herb

In vitro clonal multiplication of safed musli (Chlorophytum borivilianum Sant. et. Fernand.), a rare Indian medicinal herb, has been achieved on Murashige and Skoog's (MS) medium supplemented with 22.2 M benzyladenine using young shoot bases as explants. Shoots multiplied at a rate of four-fold every 3 weeks. All shoots rooted when transferred to MS medium with 3/4-strength inorganic and organic constituents and 9.8 M indolebutyric acid and 67% of the micropropagated plants were successfully established in pots. Such plants produced normal fasciculated storage roots as in wild plants.Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid - IBA indolebutyric acid - MS Murashige and Skoog (1962) - NAA -naphthaleneacetic acid     

Establishment of fast-growing callus and root cultures of Cephalotaxus harringtonia

Summary Callus cultures were established from Cephalotaxus harringtonia (Japanese plumyew) stem expiants cultured on Murashige and Skoog medium supplemented with 4.5 M 2,4-dichlorophenoxyacetic acid and 0.05 M 6-furfurylaminopurine. The inclusion of 4.9 M 6-(,-dimethylallylamino) purine as the sole hormone significantly increased the growth rate of the callus. Organogenesis giving rise to both shoots and roots occurred upon transfer of the callus onto a hormonefree medium. Vitrification was common on all regenerated shoots cultured on Gelrite-containing medium. Regenerated roots were excised and established in McCown's woody plant medium. Doubling the phosphate and nitrate levels in the medium increased the growth of these root cultures.Abbreviations MS Murashige and Skoog basal medium - B5 Gamborg's B5 basal salt medium - WP McCown's woody plant basal salt medium - 2,4-D 2,4-dichlorophenoxyacetic acid - Kinetin 6-furfurylamino-purine - 2iP 6-(,-dimethylallylamino) purine - IBA indole-3-butyric acid     

Somatic embryogenesis and plant regeneration from immature embryos of saw palmetto,an important landscape and medicinal plant

Somatic embryogenesis and plant regeneration from immature zygotic embryos was achieved for saw palmetto (Serenoa repens (Bartr.) Small). Embryos, isolated from immature fruit of native-grown plants, were cultured on Murashige and Skoog medium plus 0.15% (w/v) activated charcoal and supplemented with 452 M 2,4-dichlorophenoxyacetic acid (2,4-D) and 14.7 M N⁶-(2-isopentenyl)adenine (2iP). Clusters of somatic embryos developed from all immature zygotic embryos 5 weeks after culture initiation. After 12 weeks, explants were transferred to the same medium with the amount of 2,4-D reduced to 90.4 M which resulted in somatic embryo proliferation. Somatic embryos were then transferred to the basal medium containing 0.9, 9 M thidiazuron (TDZ), or no growth regulator for conversion into plantlets. The 9 M TDZ treatment was ineffective for plant regeneration. However, 12% of the embryos subcultured on 0.9 M TDZ were able to produce complete plantlets. Shoot production was obtained from 35% of the embryos subcultured in the absence of growth regulators. Rooting (100%) was achieved when these shoots were transferred onto medium containing 22.2 M -naphthaleneacetic acid (NAA).     

Thidiazuron stimulates shoot organogenesis and somatic embryogenesis in white ash (Fraxinus americana L.)

Immature and mature nonstratified seeds of white ash (Fraxinus americana L.) were dissected transversely and 2/3 of each seed was placed onto agar-solidified Murashige and Skoog medium. Adventitious buds, shoots, and somatic embryos formed on callus, cotyledons, and hypocotyls of the resulting seedlings. Shoot organogenesis was induced on explants cultured on medium with 10 M thidiazuron but not on explants on media with benzyladenine (BA) or isopentenyladenine. Not all seed sources were equally capable of shoot organogenesis and embryogenesis. Atypical of adventitious regeneration of other woody plants, mature seed explants of white ash were more organogenic with shoots that elongated better than explants from immature seeds. Somatic embryogenesis was observed in cultures where mature seeds were first cultured for 4 weeks on a medium containing 10 M adenine 2,4-dichlorophenoxyacetic acid in combination with 0.1 and 1.0 M thidiazuron, followed by transfer to a medium containing 0.05 M 6-benzyladenine and 0.5 M naphthaleneacetic acid. Adventitious shoots and epicotyls from both seedlings and germinated somatic embryos were rooted under intermittent mist and acclimatized to the greenhouse.Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - IBA indolebutyric acid - 2iP isopentenyladenine - NAA naphthaleneacetic acid - TDZ thidiazuron-N-phenyl-N-1,2,3-thiadiazol-5-ylurea - WPM woody plant medium     

Axillary shoot induction and plant regeneration in Plantago ovata Forssk

Axillary shoot induction and plant regeneration were obtained in Plantago ovata. The optimum medium for inducing axillary shoots was Murashige & Skoog (MS) medium [5] supplemented with 4.6 M kinetin and 0.05 M NAA. Rooting of shoots was best on half-strength MS medium containing 5.0 M IBA and 0.05 M kinetin. The regenerated plants were similar to the control plants in karyotypic and phenotypic details.     

Rapid plant regeneration of pea using thidiazuron

A simple and rapid pea regeneration procedure was developed. An average of up to 20 shoots formed from hypocotyl explants of cvs. Sugar Ann and Patriot cultured on Murashige and Skoog basal medium supplemented with 0.5 or 1.0 M thidiazuron (N-phenyl-N-1,2,3-thiadiazol-5-ylurea). Hypocotyls of Puget and Sugar Daddy did not respond. Regenerated shoots rooted rapidly when cultured on Murashige and Skoog basal medium containing either 2.0 M -naphthaleneacetic acid or 1.0–2.0 M indole-3-butyric acid. Seeds were harvested from regenerated plants after only 9–11 weeks.Abbreviations BAP 6-benzyladenine - 2,4-d 2,4-dichlorophenoxy acetic acid - GA₃ gibberellic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) medium - NAA -naphthaleneacetic acid - TDZ thidiazuron (N-phenyl-N-1,2,3-thiadiazol-5-ylurea)