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Growth and physiological responses of date palm. Phoenix dactylifera L. cv. Barhee, callus to salinity stress were examined. Callus induced from shoot tips of offshoots was cultured on Murashige and Skoog medium supplemented with NaCl at concentrations ranging from 0 to 225 mM, in consective increments of 25 mM. Data obtained after 6 wk of exposure to salt have shown a significant increase in callus proliferation in response to 25 mM NaCl the lowest level tested, beyond which callus weight decreased. At 125 mM NaCl and higher, callus growth was nearly completely inhibited. Physiological studies on callus exposed to salt stress have shown an increase in proline accumulation in response to increased salinity. Proline accumulation was correlated to callus growth inhibition. Furthermore, increasing the concentration of NaCl in the culture medium generally resulted in a steady increase in Na+ and reduction in K+ concentrations. However, at 25 mM NaCl, the only level at which callus growth was significantly enhanced, an increase in K+ content was noted, in comparison to the NaCl free control. In response to increasing external NaCl level, the Na+/K+ ratio increased The Na+/K+ ratio was positively correlated to proline accumulation and hence callus growth inhibition. This study provides, an understanding of the response of date palm callus to salinity, which is important for future studies aimed at developing strategies for selecting and characterizing somaclonal variants tolerant to salt stress.

This study was conducted to examine the effect of biotin and thiamine concentrations on callus growth and somatic embryogenesis of date palm (Phoenix dactylifera L.). Embryogenic callus derived from offshoot tip explants was cultured on hormone-free MS medium containing biotin at 0, 0.1, 1, or 2 mg l−1 combined with thiamine at 0.1, 0.5, 2, or 5 mg l−1. Embryogenic callus weight, number of resultant embryos, and embryo length were significantly influenced by thiamine and biotin concentration. The optimum callus growth treatment consisted of 0.5 mg l−1 thiamine and 2 mg l−1 biotin. This treatment also gave the highest number of embryos. Embryo elongation was greatest at 0.5 or 2 mg l−1 thiamine combined with 1 mg l−1 biotin. Embryos from all treatments germinated and regenerants exhibited normal growth in soil. This study provides an insight into the importance of optimizing various culture medium components to overcome in vitro recalcitrace of date palm.

Ethylene is a plant growth regulator that is known to influence in vitro morphogenesis. This study investigated the effects of three ethylene inhibitors, silver nitrate (AgNO3), 2,5-norbornadiene, and cobalt chloride (CoCl2), on the regeneration of cowpea from cotyledon explants. Significant increases in the percentage of regeneration occurred as a result of adding either 50 µM AgNO3 or 100 µM 2,5-norbornadiene. The number of shoots produced per explant was enhanced by adding 25 µM CoCl2 or 100 µM norbornadiene. Maximum shoot elongation was obtained with 25 µM of either CoCl2 or norbornadiene. The effect of the duration of exposure to AgNO3 was also determined. The greatest percent regeneration was obtained with the addition of 60 µM AgNO3 either to both the initiation and regeneration stages, or to only the regeneration stage. The promotive effects on organogenesis in response to ethylene inhibitors suggests an important role for ethylene in the process of in vitro morphogenesis of cowpea and may contribute to its normally low regeneration frequency.

A system for micropropagation of Epimedium grandiflorum Morr. from rachis explants was developed. Explants were cultured onto Murashige and Skoog (MS) basal salts medium supplemented with (per L) 100 mg myo-inositol, 2 mg pyridoxine-HCl, 2 mg nicotinic acid, 0.40 mg thiamine-HCl, 30 g sucrose, and 2 g Phytagel. The medium also contained 2,4-dichlorophenoxyacetic acid (2,4-D) at 0.1, 0.2, or 0.25 mg/L (0.5, 0.9, or 1.1 µM) combined with either N6-benzyladenine (BA) or 2-isopentenyl adenine (2ip) at 2.5, 5, or 10 mg/L (11.1, 22.2, or 44.4 µM BA or 12.3, 24.6, or 49.2 µM 2iP). Cultures were maintained at a 16-h photoperiod (40 µmol/m2/s) and 23±2° C. Callogenesis preceded shoot regeneration. Callus formation increased with higher 2,4-D concentrations. The highest percent regeneration, 83% of explants, was obtained on 10 mg BA per L (44.4 µM) combined with 0.25 mg 2,4-D per L (1.1 µM). The maximum number of shoots, 15 per explant, was obtained from explants cultured on a medium containing 0.1 mg 2,4-D per L (0.45 µM) combined with 2.5 mg BA per L (11.1 µM). Maximum shoot length, 0.4 cm, was obtained on 5 mg BA per L (22.2 µM) combined with 0.2 mg 2,4-D per L (0.9 µM). To produce whole plants, shoots were separated and rooted on hormone-free medium containing 1 g activated charcoal per L. Rachises provided an excellent source of explants for Epimedium micropropagation and proved suitable for callus production.

Transgenic spinach (Spinacia oleracea L.) plants expressing cucumber mosaic virus (CMV) coat protein genes were regenerated. Leaf and hypocotyl explants from two spinach cultivars, ‘Fall Green’ and ‘High Pack,’ were transformed with Agrobacterium tumefaciens strain LBA 4404 carrying the binary vector pBI121 in which the β-glucuronidase (GUS) gene was replaced with the coat protein gene of CMV isolate SP103 or SP104. Calluses were induced from both cultivars at rates of 10-27% on MS selective medium containing 86 µM kanamycin, 237 µM carbenicillin and 209 µM cefotaxime. After 12 wk (three transfers), putatively transformed calluses were transferred to MS medium without kanamycin to promote callus proliferation. Shoots were regenerated at the rates of 23-29% for ‘High Pack’ and less than 0.5% for ‘Fall Green’. Approximately 80% of the regenerated shoots from both cultivars were subsequently induced to establish whole plants. Regenerated plants were screened for transgene incorporation using a polymerase chain reaction (PCR) method using the same primer pair originally used to amplify the CMV coat protein genes for insertion into pBI121. Based on the PCR screening, approximately 12% of the regenerated spinach plants contained a CMV coat protein gene. Southern blot analysis confirmed the presence of viral coat protein genes in the plant genomes. Western blot analysis indicated that the CMV coat protein genes were expressed in transgenic spinach plants.

Shoot multiplication was induced in cowpea, cv. Georgia-21, from shoot tip explants. Shoot tips, 5 mm long, were isolated from in vitro-grown seedlings and cultured on MS medium containing N6-benzyladenine (BA) at 1, 2.5, or 5 mg/liter (4.4, 11.1, or 22.2 µM) or 6-furfurylaminopurine (kinetin) at 1, 2.5, or 5 mg/liter (4.6, 11.6, or 23.2 µM) combined with 2,4-dichlorophenoxyacetic acid (2,4-D) at 0.01, 0.1, or 0.5 mg/liter (0.05, 0.5, or 2.3 µM) or naphthaleneacetic acid (NAA) at 0.01, 0.1, or 0.5 mg/liter (0.05, 0.5, or 2.7 µM). Cultures were maintained at a 12-h photoperiod (40 µmol·m−2·s−1) and 23 ± 2° C. Treatments with BA induced greater shoot proliferation than those with kinetin. The highest number of shoots was produced on 5 mg (22.2 µM) BA per liter in combination with NAA or 2,4-D at 0.01 mg/liter (0.05 µM). Callus proliferated from the basal ends of shoot pieces in all treatments. The cultures also formed roots in the presence of kinetin, but not on BA-containing medium. To produce whole plants, the shoots were separated and rooted on 0.1 mg (0.5 µM) NAA per liter. Resulting plants grew normally under greenhouse conditions. Shoot tips provide an excellent explant source for cowpea micropropagation and can be used for callus induction.

This study was conducted to establish and optimize a regeneration system for adapted U.S. rice genotypes including three commercial rice cultivars (LaGrue, Katy, and Alan) and two Arkansas breeding lines. Factors evaluated in the study were genotype, sugar type, and phytohormone concentration. The system consisted of two phases, callus induction and plant regeneration. In the callus induction phase, mature caryopses were cultured on MS medium containing either 1% sucrose combined with 3% sorbitol or 4% sucrose alone, and 0.5 to 4 mg·L−1 (2.26 to 18.10 μM) 2,4-D with or without 0.5mg·L−1) (2.32 μM) kinetin. In the plant regeneration phase, callus was transferred to 2,4-D-free MS medium containing 0 or 2 mg·L−1 (9.29 μM) kinetin combined with 0 or 0.1 mg·L−1 (0.54 μM) NAA. Callus induction commenced within a week, independent of the treatments. Callus growth and plant regeneration, however, were significantly influenced by interactions among experimental factors. Generally, the greatest callus growth and plant regeneration were obtained with 0.5 mg·L−1 (2.26 μM) 2,4-D and decreased with increasing 2,4-D concentrations. Kinetin enhanced callus growth only when combined with 0.5 mg·L−1 (2.26 μM) 2,4-D, and 4% sucrose. Inducing callus on kinetin-containing medium generally enhanced regeneration capacity in the presence of sucrose but not with a sucrose/sorbitol combination. Media containing sucrose alone generally supported more callus proliferation, but the sucrose/sorbitol combination improved regeneration of some cultivars. NAA and kinetin had little effect on regeneration.

A micropropagation system was developed forAcacia mearnsii De Wild., which is the principal source of the world’s tanbark and an excellent firewood. Shoot tips 5-mm long from 3-wk-old seedlings germinated in vitro served as explants. The seeds were germinated on hormone-free MS medium and the shoot tips were cultured on three-fourth-strength MS medium supplemented with combinations of auxins [indole-3-butyric acid (IBA) andα-naphthaleneacetic acid (NAA)] and cytokinins [kinetin and benzylaminopurine (BAP)]. Cultures were maintained at 25° ± 5° C and exposed to 12-h photoperiods of cool-white fluorescent light (70 µEm−2·s−1). Multiple shoot formation was promoted by BAP at 2 mg · liter−1 (8.87µM) and higher combined with or without 0.01 mg · liter−1 (0.049µM) IBA. Cytokinins at concentrations of less than 1 mg · liter−1 combined with 0.01 to 0.1 mg · liter−1 auxin inhibited multiple shoot formation and promoted rooting of the shoot tip explants. Shoot multiplication cultures were maintained by transferring segments of multiple-shoot clusters onto a medium containing 2 mg · liter−1 BAP and 0.01 mg · liter−1 IBA. Although higher levels of BAP promoted more multiple shoot formation, this BAP level allowed shoot elongation as well as multiplication. In-vitro-produced shoots were induced to root on a range of NAA concentrations (0.0 to 0.8 mg · liter−1[4.3µM]) supplemented to half- or full-strength MS medium. The highest frequency of root proliferation was on half-strength MS medium supplemented with 0.6 mg · liter−1 (3.22µM) NAA. Plantlets survived in potting soil and exhibited normal growth under greenhouse conditions.

A system for the regeneration of spinach (Spinacia oleracea L.) from mature dry seed explants has been established. The response of two commercial spinach cultivars, ‘Grandstand’ and ‘Baker’, was examined. Callus proliferation was most prominent on MS medium supplemented with 9.3 μM of 6-furfurylaminopurine (kinetin) and 3.39 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Adventitious shoot formation was observed within 8 wk after callus was transferred onto regeneration medium. Shoot regeneration was best from callus induced on 9.3 μM kinetin and 4.56 μM 2,4-D. The regeneration medium contained 9.3 μM kinetin, 0.045 μM 2,4-D, and 2.89 μM gibberellic acid (GA3). Shoots were rooted on hormone-free medium, and plants grown in a greenhouse showed normal phenotype. This system is beneficial in rapid propagation of spinach plants, particularly when only a limited number of seeds are available.