Four cowpea [Vigna unguiculata (L). Walp] genotypes; IT 82E-18, IT 82E-16, Pinkeye Purple Hull, and Coronet were tested for somatic embryo formation and embryogenesis. Explants were 3-week-old cotyledons from which the embryonic axes were removed. Cotyledons were cultured in eight media combinations representing modifications of two media, one containing Murashige and Skoog Basal salt with B5 vitamins (MSB), 500 mg/L casein-hydrolysate (CS), 500 mg/L sodium chloride, 3% sucrose, 0.7% agar, 2mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg/L benzylamino purine, and the other containing (MSB), 3% sucrose, 40 mg/L 2-4-D and 0.2% gellan gum. After 1 month, 40% to 100% of explants produced calli and few produced shoots. Subcultured shoots in MS with 0.1 mg/L indole-3-butyric acid (IBA) or with IBA and 0.5mg/L kinetin (KT) failed to produced roots. The only green cotyledonary stage embryo was produced on this latter medium. Subculture of calli in MSB containing CS, mannitol, sucrose, agar, indoleacetic acid, and KT produced cream-colored globular embryos, roots, and a few leaves.
Bridget M. Lamp, Joseph H. Connell, Roger A. Duncan, Mario Viveros, and Vito S. Polito
Scanning electron microscopy was used to examine almond [Prunus dulcis (Mill.) D.A. Webb (syn. Prunus amygdalus Batsch, Amygdalus communis L.)] flower bud development for three cultivars (Nonpareil, Carmel, and Butte) from four California locations (which span the range of almond production in California) for 2 years, and for `Nonpareil' in a single location for a third year. The objectives were to document timing of floral developmental events and to better understand the extent of variation that exists within and among cultivars, locations, and years. Results indicated that the time of floral initiation relative to hull split varied among cultivars. Median time for floral initiation in `Nonpareil' was more than 3 weeks after the onset of hull split. For `Butte' and `Carmel', median time of floral initiation preceded the onset of hull split. Extensive variation in the timing of bud development events within a cultivar was apparent. Timing of developmental events varied among locations, but no patterns emerged consistent with the north to south range which spanned 4°15' latitude and 520 km. Among years, development occurred earliest in 1997, a relatively warm year, and was delayed in 1998 and 1999, relatively cool years. Results indicate an earlier onset of floral initiation than reported in the classical literature on the subject.
Yeh-Jin Ahn and Grace Qianhong Chen
hypocotyl tissues were used as explants ( Ahn et al., 2007 ). In other plant species, there are a number of studies reporting the beneficial effect of the dark preconditioning of explants on the subsequent plant regeneration through organogenesis such as
S. Sorvari, S. Ulvinen, T. Hietaranta, and H. Hiirsalmi
The effect of preculturing in vitro plantlets of two strawberry (Fragaria ×ananassa Duch.) cultivars grown on micropropagation medium with and without hormones on regenerating shoots from leaf disks was examined. Preculturing stock plants on micropropagation medium with hormones (BAP at 0.5 mg·liter-1 + IBA at 0.5 mg·liter-1 GA, at 0.2 mg·liter-1) promoted shoot regeneration in the two cultivars tested. Using hormone-containing micropropagation medium for preculture, the highest mean regeneration rate of 9.9 shoots per total number of leaf disks was obtained for the Finnish cultivar Hiku on modified Murashige and Skoog (MS) regeneration medium supplemented with (in mg·liter-1) 2000 KNO3, 400 casein hydrolysate (CH), 3 BAP, and 0.1 IBA. For the Norwegian cultivar Jonsok, the highest mean regeneration rate of 12.8 shoots per total number of leaf disks was obtained on modified MS regeneration medium with (in mg·liter-1) 600 CH, 3 BAP, and 0.1 IBA. Chemical names used: 6-benzylaminopurine (BAP); 3-indolebutyric acid (IBA); gibberellic acid (GA).
F. Takeda, B. C. Strik, and J. R. Clark
Western trailing blackberries (e.g., `Boysen' and `Marion') are grown in Oregon. USDA-released semi-erect thornless blackberries (e.g., `Chester Thornless') and erect, thorny blackberries (e.g., `Cherokee') from Arkansas are grown across the United States from the mid-Atlantic coast region to Oregon. Flower bud development in several blackberry cultivars growing at three sites (Arkansas, Oregon, and West Virginia) was studied. In buds of `Boysen' and `Marion' blackberries from Oregon, sepal primordia were first observed in September and November, respectively. Further floral bud development continued into January. Sepal development in `Cherokee' buds occurred in November in Oregon and in December in Arkansas. At all subsequent sampling dates, the development was more advanced in Oregon than in Arkansas. Buds of `Chester Thornless' blackberry from all three sites remained undifferentiated until spring. Preliminary findings indicated that the time of flower bud initiation varied considerably among the cultivars examined. The results suggest that floral bud development in blackberry, once initiated, is continuous, but periods of low temperature can arrest bud development.
Richard L. Bell
Discs of cambial tissue were excised from actively growing shoots of `Bartlett' pear, and explanted directly on regeneration induction media. The basal medium was 1/2 strength MS macro-nutrients, MS micro-nutrients and organics, 8 g/l agar, and 30 g/l sucrose. Phytohormone treatments consisted of a factorial design of NAA (0 and 5μM) and TDZ (1, 2, 3, 4, and 5μM). After 4 weeks incubation in the dark, the explants were transferred to auxin-free media with identical concentrations of TDZ. There was an absolute requirement for auxin in the induction medium, as all discs on auxin-free initial media died without callusing. Maximum shoot regeneration 4 weeks after transfer to expression media was obtained with an initial medium containing 5μM NAA and 3μM TDZ, from which 30% of the explants produced one or more adventitious shoots. This rate of regeneration is similar to that obtained in some experiments with in vitro leaf explants, and provides an alternative system for regeneration of pear.
Sharon Bates, John E. Preece, John YOPP, and Robert Trigiano
Dissected white ash seeds were placed on a MS basal medium containing 10 μM TDZ and 1 μM 2,4-D. Adventitious buds formed directly and indirectly on cotyledons and hypocotyls that were in contact with the medium. Histological observations after 7 days from initiation indicated early divisional events originated directly in subepidermal layers on adaxial portions of the cotyledons. As these cells divided, the growth ruptured the epidermis. Bud-like structures were seen at 3 weeks. After transfer to a secondary medium containing 3 μM TDZ, 1 μM BA, and 1 μM IBA, some of adventitious buds elongated. Efforts (gibberellin, etiolation, ABA, and silver nitrate treatments) to increase the number of elongated buds have been unsuccessful. Excised adventitious shoots were rooted under mist and established in the greenhouse.
Nancy Santana-Buzzy, Adriana Canto-Flick, Felipe Barahona-Pérez, María del Carmen Montalvo-Peniche, Patricia Yolanda Zapata-Castillo, Anabel Solís-Ruiz, Amílcar Zaldívar-Collí, Omar Gutiérrez-Alonso, and María de Lourdes Miranda-Ham
To induce multiple shoots from habanero pepper (Capsicum chinense Jacq.), nodes and stem segments were cultivated in MS medium supplemented with varying concentrations of kinetin, benzyladenine, and thidiazuron. The effect of the age of the explant in the medium on shoot formation and their latter development into plants was assessed. Ethylene concentration was measured along the experiments. Thidiazuron was the key growth regulator in the process, which at 3.4 μm induced seven to eight shoots that developed into healthy plants per explant. Plantlets in nonventilated vessels, where ethylene concentration was 0.25 ± 0.1102 μL·L–1, showed early defoliation and the formation of calli on the leaves and stems.
Sharon A. Bates, John E. Preece, and John H. Yopp
Dissected white ash seeds were placed on an agar-solidified MS medium with 10 μM TDZ and 1 μM 2,4-D (shoot initiation medium). After 4 weeks, explants were transferred to shoot elongation medium (3 μM TDZ, 1 μM BA, and 1 μM IBA) solidified with 0.7% Sigma agar, 0.525% agar + 0.05% gelrite, 0.35% agar + 0.1% gelrite, 0.175% agar + 0.15% gelrite, 0.2% gelrite, or no gelling agent (liquid medium). After 12 weeks in vitro, shoot growth and number were suppressed in cultures containing 0.2% gelrite (9.3 mm and 0.7 shoots) and in cultures containing no gelling agent (6.9 mm and 0.7 shoots). There were no differences in shoot growth and number in cultures containing 0.7% Sigma agar (2.2 mm and 16.5 shoots), 0.525% agar + 0.05% gelrite (2.6 mm and 18.7 shoots), 0.35% agar + 0.1% gelrite (1.6 mm and 17.4 shoots), and 0.175% agar + 0.15% gelrite (2.0 mm and 20.4 shoots). The most vitrification occurred in cultures on medium with the lowest amount of agar, gelrite only, and liquid medium.
Ahmed A. Obeidy and M.A.L. Smith
The regenerative capacity of mature pecan [Carya illinoinensis (Wangenh.) K. Koch] embryonic tissues was demonstrated after pretreating mature nuts to eliminate associated endogenous contaminants. Cultured cotyledon segments were induced to form adventitious roots in a medium with 50 μm NAA. A regeneration medium with 20 μm BA and 5 μm IBA stimulated prolific axillary shoot production from the embryonic axis without causing cotyledon abscission. Cotyledon retention was essential for shoot initiation and long-term development. Eighty-five percent of the shoots emerging from embryonic axes formed at the cotyledonary nodes. Thirty percent of the microshoots rooted on an auxin-free medium after preculture in a medium with 20 μm IBA. TDZ (25 μm) stimulated callus production from the cotyledonary nodes and radicles. Adventitious buds emerged on the callus surface and internally in callus. Chemical names used: a -naphthaleneacetic acid (NAA); 6-benzylaminopurine (BA); indole-3-butyric acid (IBA); N-phenyl-N'-1,2,3-thidiazol-5-ylurea (TDZ).