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.
D. Sankhla, T.D. Davis, N. Sankhla and A. Upadhyaya
This report describes an efficient in vitro regeneration protocol for H. patens (firebush), a heat-tolerant ornamental shrub native to tropical and subtropical America. Shoot cultures were initially established using shoot tips placed on MS-revised medium containing 2.3 μM 2,4-D, 2.3 μM kinetin, and 0.25% polyvinylpyrrolidone. Other types of explants (nodal and internodal segments, leaf pieces, floral buds) did not regenerate shoots when placed on this medium. Two-month-old plantlets derived from the shoot tips were subcultured on MS medium supplemented with 0.5 μM thidiazuron (TDZ), and within 3 to 4 weeks, some callus was produced at the root–shoot junction. When this callus, with a small portion of the root and shoots, was placed on MS medium with 0.05 μM TDZ and 0.01 μM ABA, prolific shoot formation occurred within 3 to 4 weeks followed by root formation. By regular subculturing every 5 to 6 weeks, hundreds of plantlets have been obtained over the past 3 years with no apparent decline in regeneration potential. Addition of activated charcoal (0.5%) to the culture medium has greatly improved growth of the plantlets.
W.A. Mackay, D. Sankhla, T.D Davis and N. Sankhla
Racemes of Big Bend bluebonnet (Lupinus havardii Wats.), a winter annual native to far west Texas with attractive blue flowers, are currently being produced commercially as a specialty cut-flower crop. Our studies indicated that the key determinants of postharvest longevity and performance are flower abscission and flower senescence, both of which can be influenced by ethylene. Therefore, this study was undertaken to evaluate the role of some ethylene biosynthesis inhibitors (aminooxy acetic acid = AOA; cobalt = CO++; salicylic acid = SA) and an ethylene action inhibitor (silver thiosulfate = STS) on flower abscission and flower senescence of bluebonnet racemes. Depending on the concentration used (10 μM - 1 mM), AOA and CO++ exhibited variable effects on flower abscission, flower senescence and vaselife. SA (10-100 μM) slightly delayed senescence but did not affect abscission, while higher levels of SA (500 μM - 2 mM) slightly promoted abscission and also significantly enhanced the senescence of flowers on cut racemes. The effects of SA were found to be pH-dependent. However, STS nearly eliminated flower abscission and enhanced vaselife. The results also demonstrated that the abscission of bluebonnet flowers, in particular, is highly sensitive to ethylene.
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, 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).
Tim D. Davis, Wayne A. Mackay and N. Sankhla
Lupinus havardii (Big Bend bluebonnet) is native to a narrow geographical range along the Rio Grande River in southwest Texas and produces attractive blue flower spikes which have potential as cut flowers. Without any post-harvest treatments, these spikes had an average vaselife in water of about 7 d. During this period, an average of about 13 florets were abscised per spike. When preconditioned for 4 h in 40-80 mg/liter silver thiosulfate (STS), vaselife increased to 11 days and only 1-3 florets were abscised per spike. Post-harvest treatment of the spikes with 25-50 mg/liter oxime ether, a new ethylene inhibitor, surprisingly enhanced floret abscission and shortened vaselife. The basis for this response is not clear. Storage of STS-preconditioned spikes in water at 5C for 72 h only decreased vaselife by about one day compared to unstored controls. Dry post-harvest storage at 5C for 72 h caused severe wilting, but upon rehydration these spikes still had a vase/life of about 8 d. These results indicate mat cut flower spikes of L. havardii have good post-harvest qualities and can be stored for up to 3d without seriously limiting vaselife.