Explants (cotyledon, cotyledonary node, second node, hypocotyl, epicotyl, and leaf) of cowpea (Vigna unguiculata) genotypes MN13 and Pinkeye Purple Hull were cultured on Murashige and Skoog basal nutrient medium. The medium was supplemented with 1 mg·L–1 benzyladenine (BA) or 1 mg·L–1 benzyladenine plus naphthalene acetic acid (BA + NAA) or 2 mg·L–1 2,4-dichlorophenoxy acetic acid (2,4-D). Cultures were maintained at 22°C for 1 month, after which they were transferred to 1 mg·L–1 BA + NAA. Cotyledons, hypocotyl, epicotyl, and leaf segments produced only calli after subculturing in BA + NAA. The second node and cotyledonary node explants cultured on the BA or BA + NAA followed by subculture on BA + NAA produced calli, shoots, and roots. The plants were then transplanted to promix but later died.
Marceline Egnin and C.S. Prakash
This study aimed to optimize factors for the efficient delivery of foreign genes into sweetpotato using Agrobacterium tumefaciens and develop transgenic plants. Disarmed Agrobacterium C58 carrying a binary vector pBI 121C2H with gusA, nptll, and the nutritional protein asp-l genes was used to cocultivate (4 days) petiole explants of the sweetpotato genotype P1318846-3. Pre-incubation of petioles for 3 days on MS medium with 2,4-D (0.2 mg·liter–1) before infection resulted in higher transformation. Putative transgenic shoots were obtained by transfer of petioles to MS medium with TDZ (0.2 mg·liter–1) and kanamycin (80 to 140 mg·liter–1). The PCR amplification of gusA, nptll, and asp-1 genes in the 37 putative transgenic shoots showed that six plants contained the three genes. However, none of these plants showed histochemical expression of the gusA gene. The introduced gene may have been methylated resulting in the lack of its expression. DNA blot hybridization studies are underway to verify the presence and integration of the transgenes.
A. Raymond Miller and Craig K. Chandler
A protocol was developed for excising and culturing cotyledon explants from mature achenes of strawberry (Fragaria × ananassa Duch.). Cotyledon explants formed callus with multiple shoot buds on agar-solidified Murashige and Skoog media containing several combinations of hormones (1 μm 2,4-D; 10 μm 2,4-D; 1 μm BA + 1 μm 2,4-D; 1 μm BA + 10 μm 2,4-D; 5 μm BA; 5 μm BA + 1 μm 2,4-D; 5 μm BA + 10 μ m 2,4-D; 5 μ m BA + 5 μm NAA; 5 μ m BA + 15 μ m NAA). After three subcultures, only tissues maintained on the medium containing 5 μm BA + 5 μm NAA continued to form shoots. Tissues transferred to other media eventually died (1 μm 2,4-D; 1 μ m BA + 10 μ m 2,4-D; 5 μ m BA; 5 μ m BA + 1 μ m 2,4-D), became unorganized (1 μm BA + 1 μm 2,4-D; 5 μm BA + 10 μm 2,4-D; 5 μm BA + 15 μm NAA), or formed roots (10 μm 2,4-D). Whole plantlets were produced by transferring callus with buds to medium lacking hormones. The rapid regeneration of clonal plantlets from cotyledon explants may be useful for reducing variability in future developmental studies. Chemical names used: N-(phenylmethyl)-1H-purin-6-amine (BA); (2,4-dichlorophenoxy) acetic acid (2,4-D); and 1-naphthaleneacetic acid (NAA).
Timothy K. Broschat and Henry Donselman
Maria Cantor*, Rodica Pop, and Ioana Pop
The Streptocarpus is propagated ease vegetatively from leaf cuttings all year round, but is grown on a very limited scale commercially in Romania. Successful protocol for direct shoot regeneration from in vitro Cape primrose (Streptocarpus × hybridus Voss.) leaf explants has been developed. The ease of tissue culture propagation can promote Streptocarpus production and facilitated the rapid introduction of this new species. Adventitious shoot regeneration was inducted in vitro on MS basal medium, using different concentration of NAA (1, 1.5, 2 mg·L-1) and cyokinin TDZ (0.1, 0.5, 1 mg·L-1). High frequency regeneration was obtained from leaves when cultured in the media supplemented with 1 mg·L-1 NAA plus 0.5 mg·L-1 TDZ and the percent of regeneration resulted is between 70% to 100%. Complete plantlets were acclimatized and successfully transplanted to glasshouse conditions. The total duration of the cycle from leaf explants through complete plantlets was 10 weeks.
Karen G. Caires, Carol A. Bobisud, Susan P. Martin, and Terry T. Sekioka
Watercress petioles were planted on Murashige and Skoog media containing 30 g/l sucrose and 8.5 g/l agar, as well as concentrations of 10 and 20 uM benzylaminopurine (BAP) in combination with 5 and 25 uM indoleacetic acid (IAA) or 5 and 25 uM naphthaleneacetic acid (NAA). Calli were transferred to the same media or to media with 5 uM IAA and 10 uM BAP. Transfer to media with 5 uM IAA and 10 uM BAP resulted in an increased percentage of explants with shoots in the NAA plus BAP treatments while increasing the number of shoots per explant in the IAA plus BAP treatments. The greatest number of shoots per explant were obtained when explants were grown on media with 25 uM IAA and 20 uM BAP and then transferred to media with 5 uM IAA and 10 uM BAP. Thirty percent of the explants rooted on media with 25 uM NAA and 20 uM BAP followed bv transfer to 5 uM IAA and 10 uM BAP.
Mary W. George and Robert R. Tripepi
Plant Preservative Mixture™ (PPM), a relatively new, broad-spectrum preservative and biocide for use in plant tissue culture, was evaluated as an alternative to the use of conventional antibiotics and fungicides in plant tissue culture. Concentrations of 0.5 to 4.0 mL·L-1 were tested with leaf explants of chrysanthemum (Dendranthem×grandiflora Kitam), European birch (Betula pendula Roth), and rhododendron (Rhododendron catawbiense Michx.). PPM had little effect on the percentage of explants forming shoots and the number of shoots formed per explant in birch and rhododendron, but dramatically reduced both responses in chrysanthemum. Therefore, the effects of PPM must be evaluated for each species of interest prior to use.
Shailaja Sinha and Madhuri Sharon
Somatic embryogenesis from apical meristem, cell elongation zone, and cell differentiation zone of roots of Punica granatum L. var. Ganesh was obtained. The basal medium used was Gamborg's B5. 2, 4-D induced white globular callus in root tip explants, which on further subculture to medium containing 0.5 mg/L BAP, produced somatic embryos from the outer surface of the callus. Direct somatic embryogenesis occurred from all the three zones of the root in presence of 2 mg/L kinetin. BAP induced embryogenic callus in elongation and cell differentiation zone segments of the roots, which, on further subculture onto the same medium, produced somatic embryos. NAA caused rhizogenesis in all the three root segments. Differentiation of somatic embryos into plantlets took place on B5 medium supplemented with 0.01 mg/L NAA + 0.5 mg/l BAP + 2 mg/L kinetin.
Qian Zhang, Jianjun Chen*, and Richard Henny
Pothos (Epipremnum aureum Linden & Andre), a climbing vine with leathery, shiny-surfaced, solid green or variegated heart-shaped leaves, is widely grown as an ornamental tropical foliage plant in hanging baskets or on poles as climbers for interiorscaping. Since pothos easily develops roots from nodes, its propagation is mainly from eye cuttings. Eye cuttings, however, frequently carry diseases from stock plants into production greenhouses. The objectives of this study were to investigate if somatic embryogenesis could be induced from a common cultivar `Golden Pothos' and germinated somatic embryos could be a means of clean propagule production. Using a modified MS medium supplemented with 2 mg·L-1 CPPU or TDZ and 0.2 mg·L-1 NAA or 0.5 mg·L-1 2,4-D, somatic embryos formed directly at cut edges of leaf explants, around petiole and stem explant ends, and along their side surfaces. Most somatic embryos maturated and grew into multiple buds or shoots; some of them developed into whole plants on the original medium. Somatic embryos also germinated and developed into plants on MS medium containing 2 mg·L-1 Zeatin and 0.2 mg·L-1 NAA, MS or 1/2 MS containing 2 mg·L-1 BA with or without 0.2 mg·L-1 NAA. Shoots elongated and roots grew on PGR-free medium. Plantlets grew healthy in shaded greenhouses after transferring to soilless substrates. This study suggests that the established method of somatic embryogenesis can be used to generate disease-free propagules of pothos for production.