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).
A. Raymond Miller and Craig K. Chandler
Mihoko Tamura, Ryutaro Tao, and Akira Sugiura
Chiwon W. Lee, Joel T. Nichols, Lijuan Wang, and Shanqiang Ke
Excised leaf sections of lance coreopsis cultured on Murashige Skoog (MS) medium produced adventitious shoots in response to BA. When the combinations of 0, 0.5, 1, or 2 μm NAA with 0, 5, 10, 20, or 40 μm BA were tested, shoots were induced by any of the four BA concentrations used in the medium, regardless of the presence of NAA. The average number of shoots formed per leaf section ranged from 1.4 to 4.3 seven weeks after culture initiation. Roots were induced at the base of individual shoots on the same regeneration medium when cultures were kept longer than 7 weeks. The rooted plants were transferred successfully into soil. The regenerated plants had the same growth and flowering characteristics as the seed-grown plants. Chemical names used: benzyladenine (BA); naphthaleneacetic acid (NAA).
P. Gercheva, R.H. Zimmerman, L.D. Owens, C. Berry, and F.A. Hammerschlag
Shoot regeneration from apple (Malus domestica Borkh.) leaf explants following particle bombardment at various acceleration pressures was studied. Basal leaf segments of micropropagated `Royal Gala' apple were bombarded with 1 μm gold particles, accelerated using helium pressures of 4.5, 6.2, 7.6, 9.3, or 13.8 MPa (650–2000 psi), and cultured on shoot regeneration medium consisting of N6 salts supplemented with 10 μM TDZ for 5, 10, or 20 days in darkness. Bombarded and control explants exhibited 63% to 100% shoot regeneration. With a 5-day dark period, average shoot production per explant ranged from 6.1 to 14; bombardments of 4.5 and 6.2 MPa significantly increased shoot production over the controls. With a 10-day dark period, average shoot production per explant ranged from 9.1 to 22 following bombardment at 9.3 and 6.2 MPa, respectively. Following bombardment at 6.2 MPa, 75% of the explants produced more than 20 regenerants per explant. With a 20-day dark period, average shoot production per explant ranged from 8.9 to 19 following bombardment at 13.8 MPa and following no bombardment, respectively. Shoot production per explant was significantly less than the controls following bombardments ranging from 6.2 to 13.8 MPa. Shoot production was highest per explant with particle bombardment at 6.2 MPa followed by incubation in darkness for 10 days. Chemical name used: thidiazuron (TDZ).
Giovanni Iapichino, Tony H.H. Chen, and Leslie H. Fuchigami
An efficient adventitious shoot production protocol has been developed for Rhododendron laetum × aurigeranum. Shoot tips taken from greenhouse-grown plants were cultured on Anderson's medium supplemented with 74 μM 2iP. Axillary shoots were excised and cultured on medium containing 23 μM IAA and 74 μM 2iP. After 6 months, brown callus developed at the cut surfaces of the shoot-tip explants. This callus produced many adventitious shoots (up to 70 per explant). Clusters of adventitious shoots were divided, subculture, and continued to proliferate shoots. An estimated 1600-fold increase in the number of shoots could be readily achieved in 6 months. In vitro rooting of adventitious shoots was accomplished in 4 weeks. Seventy-three percent of shoots rooted on 1/4 strength Anderson's medium supplemented with 28 μm IAA. Plantlet survival was 100%3 weeks after transfer to soil. Chemical names used: 1-H-indole-3-acetic acid (MA); N-(3 -methy1-2-butenyl) -1H-purine-6 amine (2iP).
Carole H. Saravitz, Frank A. Blazich, and Henry V. Amerson
Cotyledons and hypocotyls of Fraser fir [Abies fraseri (Pursh) Poir.] were excised from seeds treated with H2 O2 for 9 days and placed on bud induction medium containing 10 mg BA/liter and 0.01 mg NAA/liter or medium without growth regulators. Although adventitious buds did not develop, cotyledons exposed to growth regulators responded differently than cotyledons placed on medium lacking growth regulators. Cotyledons and hypocotyls responded similarly to growth regulators during the initial phase in culture, but cell divisions ceased in cotyledons, thus preventing meristemoid and subsequent bud development. After 3 days on medium containing growth regulators cell divisions were localized in epidermal and subjacent layers of hypocotyls, whereas similar cell divisions were' not observed in hypocotyls placed on medium without growth regulators. Cell clusters consisting of two to five cells (promeristemoids) were present after 7 days on hypocotyls placed on bud induction medium. In hypocotyls placed on medium without growth regulators, stomata continued to develop and cells within the cortex became vacuolated during the first 2 weeks in culture. All explants were transferred to secondary medium after 3 weeks. Cell clusters continued to enlarge into meristemoids on hypocotyls initially placed on bud induction medium. Gradually, meristemoids developed into buds and cataphylls were observed covering bud meristems. Chemical names used: N -(phenylmethyl)-1 H -purine-6-amine (BA), 1-naphthaleneacetic acid (NAA).
J.M. Van Eck and S.L. Kitto
Plant regeneration from callus cultures of mint depended on expiant source, genotype, and culture medium components. Mature embryos, seedling and flower parts, as well as chilled or desiccated immature embryos of peppermint (Mentha piperita L.) and spearmint (Mentha spicata L.) were cultured on a Murashige-Skoog medium containing various combinations of growth factors. Shoots regenerated from callus that developed either on mature peppermint embryos cultured on medium that contained BA at 0.5 mg·liter-1 and NAA at 0.5 mg·liter-1 or on immature peppermint embryos (chilled at SC for 0.6 day or nonchilled) cultured on basal medium containing BA at 1 mg·liter-1 and TIBA at 1 mg·liter-1 Shoots were proliferated, rooted, and acclimated. with 100% survival under greenhouse conditions. Chemical names used: N-(phenylmethyl) -1H-purin-6-amine (BA); 1-naphthaleneacetic acid (NAA); 2,3,5-triiodobenzoic acid (TIBA).
Peggy Ozias-Akins and Srini Perera
One cm segments from adventitious roots of sweet potato (Ipomoea batatas (L.) Lam.) will regenerate shoots when cultured on Murashige and Skoog salts and vitamins plus either sucrose (1-3%) or fructose (1-6%). The best source for adventitious roots is sweet potato shoot cultures maintained in Magenta vessels. A low concentration of cytokinin (0.02 mg/liter) promotes shoot formation. Higher levels of cytokinin (0.1-0.5 mg/liter) encourage callus growth. The maximum average number of shoots formed per root segment attained thus far is 0.5. Attempts are being made to increase the frequency of shoot formation. Regeneration of shoots from roots also may be a useful method for obtaining plants from protoplasts of sweet potato. Protoplasts can be isolated from mesophyll tissue and petioles of in vitro grown plants. Plating efficiency of up to 12% routinely can be obtained. Shoot formation directly from callus is sporadic; root formation is more frequent.
Ann M. Chanon and R. Daniel Lineberger
The genus Betula contains many important forest and ornamental species and a method of rapid clonal propagation of superior genotypes is needed. Thidiazuron (TDZ) is a potent synthetic plant growth regulator with cytokinin-like activity. TDZ was used to differentiate shoots after long term exposure to dichlorophenoxyacetic acid (2.4-D) as part of a larger study on clonal fidelity. Birch calli were cultured on Woody Plant Medium supplemented with 10-5 M 2,4-D for up to 30 weeks. The calli were transferred to media containing TDZ at concentrations of 10-6 to 10-9M. Most of the tissue which had not been exposed to 2.4-D differentiated shoots five weeks after being exposed to 10-6M TDZ. Increasing the of time exposure to 2.4-D or decreasing the concentration of TDZ delayed differentiation. Calli exposed to 2.4-D for more than 18 weeks rarely differentiated shoots regardless of the concentration of TDZ used.
Mohamed F. Mohamed, Dermot P. Coyne, and Paul E. Read
Plant regeneration has been achieved in two common bean lines from pedicel-derived callus that was separated from the explant and maintained through successive subcultures. Callus was induced either on B5 or MS medium containing 2% sucrose and enriched with 0.5 or 1.0 mg thidiaznron/liter alone or plus various concentrations of indoleacetic acid. The presence of 0.07 or 0.14 g ascorbic acid/liter in the maintenance media prolonged the maintenance time. Up to 40 shoot primordia were observed in 4-week-old cultures obtained from 40 to 50 mg callus tissues on shoot-induction medium containing 1-mg benzyladenine/liter. These shoot primordia developed two to five excisable shoots (>0.5 cm) on medium with 0.1-mg BA/liter. A histological study confirmed the organogenic nature of regeneration from the callus tissues. The R2 line from a selected variant plant showed stable expression of increased plant height and earlier maturity. Chemical names used: ascorbic acid, N- (phenylmethyl)-1H-pnrin-6-amine [benzyl-adenine, BA], 1H-indole-3-acetic acid (IAA), N- phenyl-N'-1,2,3-thiadiazol-5-ylurea [thidiazuron, TDZ].