Teddy E. Morelock

Genotypic Response of Cowpea Vigna unguiculata (L.) to In Vitro Regeneration from Cotyledon Explants

SummaryA plant regeneration system applicable to 17 cowpea genotypes was developed. Cotyledons were initiated on 1/3 MS medium containing 15 to 35 mg N6-benzyladenine (BA) per 1 (66.6 to 155.3 µM) for 5 to 15 d. For shoot regeneration, the explants were transferred to a medium containing 1 mg BA per 1 (4.4 µM). Within 1 wk, shoot formation was visible at the proximal end of the cotyledons. Regeneration percentages (1% to 11%) and the numbers of shoots (4 to 12 per explant) were significantly influenced by genotype. Culture duration and BA concentration in the initiation stage significantly affected regeneration capacity. Explants initiated on media containing 15 mg BA per 1 for 5 d resulted in the highest percentage of explants capable of regeneration. Conversely, the highest number of shoots was obtained from explants initiated on media supplemented with 35 mg BA per 1. Whole plants were obtained on a plant growth regulator-free medium. To our knowledge, this is the first report of plant regeneration from U.S. commercial cowpea cultivars and breeding lines. This system is adaptable to diverse cowpea genotypes and will facilitate cowpea genetic transformation.

Ethylene inhibitors promote in vitro regeneration of cowpea (Vigna unguiculata L.)

SummaryEthylene 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.

Comparison of Colletotrichum orbiculare and Several Allied Colletotrichum spp. for mtDNA RFLPs, Intron RFLP and Sequence Variation, Vegetative Compatibility, and Host Specificity

ABSTRACT Based on spore morphology, appressorium development, sequence similarities of the rDNA, and similarities in amplified restriction fragment length polymorphism (AFLP), it has been proposed that Colletotrichum orbiculare, C. trifolii, C. lindemuthianum, and C. malvarum represent a single phylogenetic species, C. orbiculare. In the current study, the phylogenetic relationship among isolates in the C. orbiculare species complex was reassessed. In all, 72 isolates of C. orbiculare from cultivated cucurbit or weed hosts, C. trifolii from alfalfa, C. lindemuthianum from green bean, and C. malvarum from prickly sida (Sida spinosa) were examined for mitochondrial DNA (mtDNA) restriction fragment length polymorphisms (RFLPs), RFLPs and sequence variation of a 900-bp intron of the glutamine synthetase gene and a 200-bp intron of the glyceraldehyde-3-phosphate dehydrogenase gene, and vegetative compatibility. In addition, host specificity was examined in foliar inoculations on cucurbit, bean, and alfalfa hosts. Inoculations also were conducted on cucumber fruit. Genetically distinct isolates, based on vegetative compatibility, within the species complex (C. orbiculare, C. trifolii, and C. malvarum) had an identical mtDNA haplotype (haplotype A) when examined with each of three different restriction enzymes. Isolates of C. lindemuthianum had a very similar mtDNA haplotype to haplotype A, with a single polymorphism detected with the enzyme HaeIII. The four species represent a phylogenetically closely related group based on a statistical analysis of the 900- and 200-bp intron sequences. However, distinct RFLPs in the 900-bp intron were consistently associated with each species and could be used to qualitatively and quantitatively distinguish each species. Furthermore, each of the species showed distinct host specificity, with isolates of C. orbiculare (from cucurbits), C. lindemuthianum, and C. trifolii being pathogenic only on cucurbits, green bean, and alfalfa, respectively. Consequently, distinct and fixed nucleotide, or genotypic (intron sequences and RFLPs) and phenotypic (host specificity) characteristics can be used to distinguish C. orbiculare, C. lindemuthianum, and C. trifolii from one another; therefore, they should be recognized as distinct species. This species delineation is consistent with the most current species concepts in fungi. More isolates and further characterization is needed to determine whether C. orbiculare from cocklebur and C. malvarum represent distinct species. RFLPs of the 900-bp intron may represent a relatively inexpensive, reliable, and useful diagnostic tool for general species differentiation in the genus Colletotrichum.

IN VITRO SHOOT TIP MULTIPLICATION OF COWPEA VIGNA UNGUICULATA (L.) WALP.

SummaryShoot 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.

Genotype-dependent response of spinach cultivars to in vitro callus induction and plant regeneration

Abstract Callus was induced from leaf explants of four spinach ( Spinacia oleracea L.) cultivars on Murashige-Skoog (MS) medium supplemented with 2 mg 1 −1 6-(furfurylamino)purine (kinetin) and 0.1, 0.5 or 1 mg 1 −1 2,4-dichlorophenoxyacetic acid (2,4-D) either in darkness or exposed to light (50 μE m −2 s −1 ). Callus production was cultivar dependent. Maximum callus proliferation was obtained with 0.5 mg 1 −1 2,4-D, which also gave rise to the highest percentage of shoot formation. Callus induced in darkness exhibited higher regeneration capacity than callus induced in the light. Regeneration capacity of the callus varied among cultivars and was enhanced by the addition of ascorbic acid. Gibberellic acid was required for shoot regeneration. Complete plants were obtained by inducing the shoots to root with 1 mg 1 −1 indole butyric acid (IBA).

In vitro seed production from sex-modified male spinach plants regenerated from callus cultures☆

Abstract Shoots regenerated from leaf-derived callus of spinach (Spinacia oleracea L. cultivar ‘High Pack’) were induced to flower by exposure to 14-h photoperiods (50 μmol m−2 s−1) and day/night temperatures of 20° C 16° C . Leaves from male and female plants served as sources for explants. Shoots regenerated from callus were induced to flower on a hormone-free medium. The majority of regenerated plants exhibited sex corresponding to their respective explant donors; a proportion of regenerants of male origin, however, shifted to monoecious. These sex-modified plantlets developed viable seeds in vitro. This phenomenon can be useful in producing seeds from elite selected male plants.

In vitro plant regeneration of spinach from mature seed-derived callus

SummaryA 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.

Abstract 5685: The effects of spinach and two of its components, spinacetin and patuletin, on the treatment of prostate cancer

Prostate cancer possesses long latency periods and is responsive to dietary mediators, making it a target for phytochemoprevention. It has been reported that spinach consumption can reduce the incidence of prostate cancer leading our lab to look at structures of compounds present in spinach. We chose to study spinacetin and patuletin, two novel isoflavones found in spinach. We hypothesized that these spinach compounds would inhibit prostate cancer in vitro in the mouse prostate cancer cell line, TRAMP-C2, and that a spinach-containing diet would reduce cancer incidence in our TRAMP mouse model. Methods: We first isolated a crude spinacetin-containing fraction and patuletin-containing fraction from whole spinach leaves using HPLC column purification. A further purified extract was also isolated for each compound. Using these extracts we determined the effect of the spinach compounds on growth of TRAMP-C2 cells, and the effect that they have on the concentration of Gli1, an indicator of hedgehog signaling, as measured by RT-PCR and a Gli1-luciferase reporter assay. We also tested ground dried spinach at 0.2% and 2% incorporated into a casein-based diet fed to B6/FVB TRAMP mice, and then evaluated the development of prostate cancer histologically. Results: The extracts, each containing 0.1% spinacetin or patuletin, both inhibit prostate cancer cell growth in vitro, with the patuletin extract inhibiting approximately 30% and the spinacetin extract inhibiting nearly 50%. In addition, the purified compounds each inhibited Gli1 expression in both TRAMP-C2 (as measured by RTPCR) and Shh Light II cells (as measured by luciferase assay), with patuletin having an effect at 300nM and spinacetin at 1μM. The 2% spinach diet led to a 50% decrease in the incidence of well-differentiated carcinoma, but had no apparent effect on the more aggressive poorly-differentiated carcinoma. In conclusion, spinach and its compounds are capable of inhibiting prostate cancer growth and incidence in in vitro and in vivo mouse models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5685.