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Wendy Wagoner, J. Stamp, H. Matthews, J. Kellogg, and R. Bestwick

Ethylene is a known causal factor in the decay and senescence of fruits and vegetables. The aim of the present study was to incorporate a gene for control of ethylene biosynthesis in order to prevent or delay the senescence of the cauliflower curds. We first developed a reproducible transformation system using marker genes for beta glucuronidase (GUS) and antibiotic resistance. Brassica oleraceae L. var. botrytis was transformed by inoculating hypocotyl explants with the Agrobacterium tumefaciens strains C58 or EHA101 containing plasmids pAG5110, pAG5420, or pAG5520. The plasmid pAG5110 contains the genes for neomycin phosphotransferase II (NPTII) and GUS. The plasmids pAG5420 and pAG5520 contain a functional gene for S-adenosylmethionine hydrolase (SAMase) under an ethylene or wound inducible promoter, respectively. Hypocotyl explants were screened on regeneration medium with kanamycin for selection of transformants. Shoot regeneration occured within 4-6 weeks and morphologically normal plants developed within 3-4 months. The transgenic nature of the plants was confirmed by histochemical GUS assay, an ELISA based NPTII assay and Southern blot analysis. Transgenic plants outplanted in the greenhouse are being evaluated and selfed to study expression and inheritance pattern of the introduced trait.

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K.M. Batal, D.M. Granberry, and B.G. Mullinix Jr.

The effects of three rates of N, Mg, and B on cauliflower (Brassica oleracea, Botrytis group) yield, average curd mass, and hollow stem disorder were evaluated on sandy and clay loam soils. Cultivars White Empress and Stovepipe were tested on the sandy loam soil and `White Empress' was tested on the clay loam soil. Maximum mean curd mass and maximum yields were obtained with the highest N rates (269 and 381 kg·ha-1) applied to sandy loam and clay loam soils, respectively. Yield response to increased N rates varied with cultivar. Increasing Mg from 22.5 to 90 kg·ha-1 did not affect yield or curd mass on clay loam soil, but increased yield and mean curd mass on sandy loam soil. The Mg effect on curd mass was influenced by N and B rates. On both soil types, the higher Mg and B rates reduced the incidence of hollow stem, but the Mg effect was influenced by N applications. On clay loam soil, increasing B from 2.2 to 8.8 kg·ha-1 reduced hollow stem but had no effect on yield or curd mass. On sandy loam soil, B at 4.4 kg·ha-1 maximized yield and curd mass, but the hollow stem disorder continued to decrease as B rates were increased from 2.2 to 8.8 kg·ha-1.

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A.G. Taylor, D.B. Churchill, S.S. Lee, D.M. Bilsland, and T.M. Cooper

Color sorting was performed to upgrade seed quality by removal of fluorescent coated seeds. The fluorescent coating was attributed to sinapine leakage from nonviable seeds. Nine seedlots, three seedlots each of cabbage (Brassica oleracea L. Capitata group), broccoli, and cauliflower (B. oleracea L. Botrytis group) were custom coated. Seed samples were pretreated before coating with or without 1.0% NaOCl for 10 minutes to enhance leakage. All samples revealed a percentage of seeds with fluorescence. The light emission from selected fluorescent and nonfluorescent coated seeds was quantified by fiber-optic spectrophotometry. Fluorescence was expressed from 400 to 560 nm, with peak emission being from 430 to 450 nm. These data confirmed our visual interpretation of blue-green fluorescence. The ratio of light emission from fluorescent compared to nonfluorescent coated seeds ranged from 4.5 to 7.0 for all samples and averaged 5.7. An ultraviolet (UV) color sorter was employed to separate fluorescent (reject) from nonfluorescent (accept) coated seeds. The percentage of nonfluorescent coated seeds (averaged over seedlot and NaOCl pretreatment) before and after sorting was 89.5% and 95.9%, respectively. Therefore, color sorting was able to remove a high percentage of fluorescent coated seeds with an average loss (rejection of nonfluorescent coated seeds) of 6%. An increase in the percent germination was recorded in eight of the nine seedlots following color sorting, and the greatest improvement was obtained with seedlots of medium quality. Germination of three medium quality lots was increased by 10 to 15 percentage points. The average increase in germination with or without NaOCl pretreatment was 8.2 and 5.5 percentage points, respectively. In conclusion, the germination of Brassica seedlots could be improved by separating (removing) fluorescent from nonfluorescent coated seeds. UV color sorting technology was employed to demonstrate that seed conditioning could be conducted on a commercial basis to upgrade seed quality.

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L. Agus Sukamto

Hypocotyl sections of cauliflower were cultured in horizontal, upright or inverted positions on Murashige and Skoog medium with different concentrations of 6-benzylaminopurine (BAP) and α-naphthylacetic acid (NAA). Inverted position gave the highest differentiated score and greatest fresh weight of the two month-old tissues, followed by upright and horizontal positions respectively. Combination BAP and NAA caused synergistic effect on tissues. The best treatment for getting callus was combination of 5 mg/liter BAP, 2 mg/liter NAA and upright position, whereas for getting tissue differentiation was combination of 5 mg/liter BAP, 1 mg/liter NAA and inverted position.

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Mary C. Christey and Elizabeth D. Earle

Peduncle explants from 12 Brassica oleracea L. lines representing five varieties [broccoli (italica), cabbage (capitata), cauliflower (botrytis), Chinese broccoli (alboglabra), and rapid-cycling B. oleracea] readily regenerated shoots in vitro. Average regeneration rates of more than 75% were obtained for most lines, with up to 35 shoots per explant. Shoots were visible within 7 to 10 days. Initial regeneration was polarized, occurring mainly from the basal end of explants. Linsmaier-Skoog-based medium containing 1 mg BA/liter was suitable for shoot regeneration from all 12 lines tested. Plants were rooted on hormone-free medium and transferred to soil. Chemical name used: benzyladenine (BA).

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V. Esensee, D.I. Leskovar, and A.K. Boales

Nonomura and Benson (1992) reported that foliar applications of dilute solutions of methanol caused growth and yield increases and reduced water use in several crops. The request from commercial growers for explicit information regarding this report prompted our experiments using the same procedures. Growth of cantaloupe, pepper, cabbage, cauliflower and onion seedlings and mature plants were evaluated in the laboratory and greenhouse in 1993 and in the field in 1993 and 1994. Treatments of 0%, 10%, 20%, 30%, or 40% methanol (v/v water) with 0.1% surfactant generally did not cause significant growth differences. Stem diameters or lengths, shoot fresh and dry weights, or root fresh and dry weights of seedlings were unaffected as a result of methanol treatment. In the field, cabbage head weight was slightly higher after methanol application only in 1993, whereas cantaloupe fruit weight and number were significantly lower in 1993, but not in 1994.

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Mark W. Farnham

A collection of collard (Brassica oleracea L., Acephala group) germplasm, including 13 cultivars or breeding lines and 5 landraces, was evaluated using randomly amplified polymorphic DNA (RAPD) markers and compared to representatives of kale (Acephala group), cabbage (Capitata group), broccoli (Italica group), Brussels sprouts (Gemmifera group), and cauliflower (Botrytis group). Objectives were to assess genetic variation and relationships among collard and other crop entries, evaluate intrapopulation variation of open-pollinated (OP) collard lines, and determine the potential of collard landraces to provide new B. oleracea genes. Two hundred nine RAPD bands were scored from 18 oligonucleotide decamer primers when collard and other B. oleracea entries were compared. Of these, 147 (70%) were polymorphic and 29 were specific to collard. Similarity indices between collard entries were computed from RAPD data and these ranged from 0.75 to 0.99 with an average of 0.83. Collard entries were most closely related to cabbage (similarity index = 0.83) and Brussels sprouts entries (index = 0.80). Analysis of individuals of an OP cultivar and landrace indicated that intrapopulation genetic variance accounts for as much variation as that observed between populations. RAPD analysis identified collard landraces as unique genotypes and showed them to be sources of unique DNA markers. The systematic collection of collard landraces should enhance diversity of the B. oleracea germplasm pool and provide genes for future crop improvement.

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A.G. Taylor, D.H. Paine, and C.A. Paine

The purpose of this project was to study factors that influence the leakage rate and develop methods to enhance leakage of sinapine from Brassica seeds. Six seedlots (two seedlots of one cultivar each of cabbage (Brassica oleracea L., Capitata group), broccoli, and cauliflower (B. oleracea L., Botrytis group) were studied. Leakage was quantified spectrophotometrically by measuring the absorbance of the soak water at 330 nm. The onset of sinapine leakage was determined from single seeds by the visual presence (yellow soak water) caused by soaking seeds in a biological buffer adjusted to pH 10. The leakage pattern from heat-killed seeds of all seedlots was sigmoidal with a distinct lag phase followed by a rapid efflux and final slower rate. The duration of the lag phase and the total amount of sinapine leaked after 24 hours was not the same for all seedlots after adjusting for seed count, seed weight, or sinapine content. Therefore, another factor was responsible for differences measured in leakage. Embryos or seeds with cracked testas leaked faster than intact seeds, and the leakage pattern without testa integrity was biphasic. From these studies, we conclude that the testa was a major factor regulating sinapine leakage. Pretreating heat killed seeds, with up to 1.0% NaOCl for 10 minutes, accelerated the onset of leakage. The time for 50 percent of the seeds to leak (T50) decreased as NaOCl concentration increased. Leakage uniformity, as measured by the standard deviation of the distribution, generally increased as NaOCl concentration increased. The sigmoidal leakage pattern from heat-killed Brassica seeds may be attributed to seedcoat cracking associated with imbibitional swelling. A NaOCl pretreatment may have increased the permeability of the testa and, thereby, enhanced the leakage rate.

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Wendy J. Wagoner, Jill A. Kellogg, Richard K. Bestwick, and James A Stamp

Broccoli and cauliflower are among the most regeneratively intractable genotypes found in the brassicaceae. To develop a method for transfer of the gene encoding S-adenosylmethionine hydrolase (SAMase) into inbred broccoli and cauliflower germplasm, we investigated the morphogenic competence and Agrobacterium susceptibility of a wide range of tissues of varied source. Appropriately controlled expression of the SAMase gene should, theoretically, reduce the plant's capacity for ethylene biosynthesis and extend the post harvest shelf life of the flower head.

Through examination of the in vitro response of a wide range of tissues we identified procedures which support caulogenesis from 100% of explants, each producing more than 30 shoots which readily convert to plantlets. Studies with several wild type and disarmed Agrobacterium strains, and utilization of the binary vector system and appropriate marker and reporter genes, led to the identification of methods for high frequency T-DNA transfer to explant tissues and the flow frequency of transgenic plants containing SAMase gene.

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Denise V. Duclos and Thomas N. Björkman

Brassica oleracea species differ in the developmental stage of their reproductive meristems at harvest. The stage that characterizes each variety depends on its genetic makeup, environment and the interaction between them. We tested a model of arrest in B. oleracea to determine functional redundancy among the paralogous genes CAL, AP1a, AP1c, FULa, FULb, FULc, and FULd; and to resolve the immediate effect of temperature on gene expression in meristems whose developmental fate is temperature regulated. By varying temperature during reproductive development, three stages of arrest were obtained: inflorescence meristem (cauliflower), floral meristem (intermediate) and floral bud (broccoli), the latter initiated by low temperature. Gene expression was measured by quantitative real time PCR (qRT-PCR). The LFY/TFL1 ratio increased as the reproductive development advanced, mainly due to decreased TFL1 expression; influenced by a dramatic increase in AP1c toward floral bud formation. The expression patterns of the FUL paralogs indicate different roles in reproductive development. FULa was more abundant in the floral primordia, while FULb, FULc, and FULd were associated with earlier arrest at the inflorescence meristem stage. The high expression of FULc and FULd at all stages of arrest differs from their homolog in Arabidopsis. High temperature reduced AP1 and LFY expression but the meristem did not revert from reproductive to vegetative. Floral bud formation in plants recessive for AP1a and CAL reaffirm that functional redundancy among some of these genes can complement the mutations. Varying temperature alone, at a fixed developmental stage, caused little variation in the expression of genes studied, causing small significant differences in TFL1 and AP1c.