This study was initiated to test the embryogenic potential of immature cotyledons (3-5 mm long) of Texas bluebonnet (Lupinus texensis). The embryo initiation medium consisted of B5 salts and vitamins with 3% sucrose and 22.6 μM 2,4-D alone or in combination with 1-15 μM of various cytokinins. Within 15-20 days, globular embryos were formed on the distal end of the cotyledons. Eventually the entire cotyledon surface was covered by embryo-like structures. Addition of cytokinins to the medium did not increase the percentage of cultures which formed embryos. In fact, addition of thidiazuron severely inhibited embryogenesis. Following transfer to an embryo maturation medium (MS medium with 0.38 μM abscisic acid alone or in combination with benzyladenine or zeatin) for 10-14 days, the embryos were placed in MS medium supplemented with GA (2.9 μM) or glutamine (200 mg/liter) with or without activated charcoal (0.5%) for embryo germination and plantlet development. Most of the embryos exhibited precocious germination and well-developed roots but failed to produce normal shoots. Therefore, additional work is needed to improve embryo conversion frequency.
Daksha Sankhla, Tim D. Davis and N. Sankhla
Daksha Sankhla, Tim D. Davis and N. Sankhla
`German Red' is a thermotolerant cultivar of carnation (Dianthus caryophyllus) that blooms almost year-round in Texas. This study was initiated to evaluate the feasibility of inducing somatic embryos for use in gene transfer Studies and rapid mass propagation. Internodal explants, obtained from microshoots of plantlets cultured on MS medium containing 5 μM benzyladenine (BA) and 0.5 μM naphthaleneacetic acid (NAA), were used to initiate callus. Callus formation was induced on MS medium containing 3% sucrose, 0.1% casein hydrolysate and 2,4-D (1-5 μM) alone or in combination with BA (2 or 4 μM) or kinetin (2 or 4 μM). After about 5 weeks, the callus was transferred to either semisolid or liquid MS basal medium with or without kinetin and BA. Within 20-30 days, pro-embryogenic callus masses were observed. The embryos developed from white embryonic tissue and exhibited typical stages of embryogenesis. After 5 weeks, up to 70% of the cultures grown in the liquid medium with or without BA exhibited a profusion of embryo-like structures. Because only a small percentage of these developed into plantlets, more work is needed to enhance conversion frequency.
Tim D. Davis, Daksha Sankhla and Narendra Sankhla
Carnation cultivars `German Red' and `Chabaud' were planted in the field in Dallas, Texas, on 26 May 1994. During the subsequent 3 months, the average daily high temperature was 33C, and the average daily low temperature was 22C. `German Red' plants increased in height and diameter several-fold during this period. In contrast, `Chabaud' did not increase in height or diameter. `German Red' plants began flowering in early August, and by 2 Sept., all of the plants were blooming. None of the `Chabaud' plants produced flowers, and only 50% of the original plants were still alive on 2 Sept. Mean shoot dry weight per plant on 2 Sept. was 71.6 g for `German Red' and only 2.4 g for `Chabaud'. These results document the extraordinary heat tolerance of `German Red' carnation. This plant not only survived the summer, but also grew and began blooming during the hottest time of the year.
Abba Upadhyaya, Tim D. Davis, Daksha Sankhla and N. Sankhla
Both kinetin and BA promoted in vitro shoot formation from hypocotyl explants of Lupinus texensis Hook. placed on Murashige and Skoog (MS) medium. With either cytokinin, shoot formation was best at ≈4.5 μm. Adventitious root formation was observed only on tissue culture-derived shoots placed in MS media containing 5.4 to 54 μM NAA. IAA and IBA, at concentrations ranging from 5 to 55 μm, failed to stimulate rooting. Even at the optimal concentration of NAA, only 14% of the shoots produced roots. Thus, although hypocotyl explants readily produced shoots, adventitious root formation on these shoots occurred with relatively low frequency. Chemical names used: 6-benzylaminopnrine (BA); indole-3-acetic acid (IAA); indole-3-butyric acid (IBA); 6-furfurylaminopurine (kinetin); 1-naphthaleneacetic acid (NAA).
Daksha Sankhla, Tim D. Davis, N. Sankhla and A. Upadhyaya
A variety of Hamelia patens (firebush) explants (nodal and internodal segments, leaf blade pieces, floral buds, shoot tips) were cultured on Murashige and Skoog's revised medium containing various concentrations of 2,4-D and kinetin. Embryogenic callus was produced only from shoot-tip explants placed on media containing 2,4-D or 2,4-D plus kinetin. None of the other explants produced embryogenic callus. Somatic embryogenesis from callus was greatest on media containing both 2, 4-D and kinetin. Direct somatic embryogenesis was observed on the roots of callus-derived primary embryos maintained on media containing 2,4-D or 2,4-D plus kinetin. Conversion of somatic embryos into plantlets only occurred on media containing 2,4-D, kinetin and activated charcoal.
Tim D. Davis, Wayne A. Mackay and Daksha Sankhla
Seeds of Lupinus havardii Wats. (Big Bend bluebonnet), a potential cut flower crop, were subjected to a variety of scarification and temperature treatments. Without scarification, only 10-20% of the seeds germinated within one week. Germination percentages increased sigmoidally as scarification time in concentrated sulfuric acid increased. Nearly 100% germination was obtained within one week after seeds were placed in sulfuric acid for 120 min. Nicking the seed coat with a razor blade also resulted in near 100% germination. Soaking the seed in water for 24 h failed to enhance germination. Soaking the seed in ethanol, methanol, or acetone for 2 h likewise failed to enhance germination. Total germination of scarified seed was >90% between 21 and 33C within 28 h. The most rapid germination occurred within a range of 24-29C. Above or below this range germination was delayed. At 35C, seedling, mortality was observed and total germination was reduced to <50%. Our data indicate that seed of this species requires scarification for optimum germination but the seed can germinate over a relatively wide temperature range.
Tim D. Davis, Daksha Sankhla, N. Sankhla, A. Upadhyaya, J.M. Parsons and S.W. George
Seeds of Aquilegia chrysantha Gray were germinated under a variety of temperature regimes. Germination was nearly 90% under a day/night cycle of 25/20C, but was reduced to ≤ 40% under constant 25C or a 25/10C day/night cycle. With days between 25 and 29C (night = 20C), germination percentage dropped gradually to ≈ 60% with increasing temperature. With days >29C, germination declined dramatically such that no germination occurred at 31C. Neither kinetin (4.6 to 46 μm) nor ethephon (6.9 to 207 μm) was able to reverse the inhibitory effects of 33C days. Our results indicate that germination of A. chrysantha seed is sensitive to temperature and that germination ≈ 75% can be obtained under a 25 to 27C day/20C night regime. Chemical names used: 2-chloroethylphosphonic acid (ethephon); 6-furfurylaminopurine (kinetin).