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- Author or Editor: Mark Farnham x
Broccoli (Brassica oleracea L. Italica group) breeders are increasingly using anther or microspore culture to produce dihaploid (diploid), homozygous lines for use in making hybrids. During the process of anther culture and subsequent plant regeneration, wherein embryos develop from microspores and plants are regenerated from the embryos, polyploidization occurs and diploid regenerants can result. However, polyploidization may not occur at all, or it may involve a tripling or quadrupling of the chromosome complement, instead of a doubling. Thus, populations may contain haploids, triploids, or tetraploids, in addition to diploids. In two cycles (1994-95 and 1995-96) of anther culture, regenerated populations from different broccoli hybrid sources were evaluated using flow cytometry to facilitate efficient identification of diploids vs. haploids, tetraploids, or others and to determine if anther donor genotype has an effect on the frequency of different ploidy levels among regenerants. In the first cycle, five broccoli hybrids had anther-derived populations in which ≈33% were haploid, 55% diploid, 37% tetraploid, and 5% aneuploid or abherent types. The hybrid, `Marathon', was different; it's regenerants were 78% diploid and only 15% tetraploid. In the second cycle, anther-derived populations had a significantly different makeup with a most hybrids giving 30% to 40% diploids and 50% to 60% tetraploids. However, consistent with the previous cycle, `Marathon' gave significantly more diploids (68%) and fewer tetraploids (25%) than other hybrids. These results indicate that anther donor genotype affects ploidy frequency among regenerants. Genotypes producing a high frequency (>60%) of diploids may be relatively uncommon.
Collard (Brassica oleracea L. var. acephala) is an important vegetable the southeastern U. S. There are few (about 10) commercial cultivars, half being open-pollinating (OP) lines, the remainder more recent F1 hybrids. There is a potential untapped B. oleracea germplasm pool in the form of collard landraces perpetuated by southeastern gardeners and farmers. To determine the amount of genetic variation among cultivars and also whether landraces represent unique genotypes, ten cultivars and eight lines or landraces were evaluated using RAPD analysis. Decamer primers were used to amplify total genomic DNA and to differentiate collard lines and other B. oleracea crop cultivars. Additionally, individuals of an OP collard cultivar and a land-race were analyzed to evaluate intra-line variation. Virtually all primers detected polymorphic bands among lines although some identified considerably more variants. Intra-line analysis indicated that OP lines are genetically broad-based populations. Many unique RAPD markers were identified in landraces indicating that the lines represent unique genotypes and that further line collection is warranted.
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.
Using anther culture to generate doubled-haploid (DH) homozygous lines for use as parents in F1 hybrid crosses has become a common practice in breeding broccoli (Brassica oleracea L. Italica Group). During anther culture and subsequent embryogenesis and plant regeneration, polyploidization of microspore-derived embryos may not occur or it may occur accompanied by a doubling, tripling, quadrupling, octupling, or irregular polyploidization of the genome. Thus regenerants from the process can be haploids, diploids, triploids, tetraploids, octaploids, or aneuploids. The objectives of this research were to 1) conduct repeat cycles of broccoli anther culture using a group of F1 hybrids as anther donors and develop populations of regenerants; 2) analyze resulting populations using DNA flow cytometry and determine the influence of F1 source on frequency of different ploidy levels among regenerants; and 3) compare seed set in broccoli inbreds developed in a traditional selfing program compared to seed set in DH broccoli derived from anther culture. In two cycles (1994 and 1995) of anther culture, anther-derived populations of regenerants were developed using the F1 hybrids `Marathon', `Everest', `High Sierra', and `Futura' as sources of anthers. In 1994, `Everest', `High Sierra', and `Futura' yielded populations that included 2% to 7% haploids, 53% to 56% diploids, 32% to 38% tetraploids, and 5% to 6% other types. `Marathon'-derived regenerants were 5% haploid, 78% diploid, 15% tetraploid, and 2% other, showing significantly more diploids. In 1995, `Marathon' regenerants again included significantly more diploids and fewer tetraploids than those derived from other F1 sources, confirming that the genotype of the anther source affects the frequency of a particular ploidy level among regenerants derived from culture. In manual self-pollinations of 1994 regenerants, only diploids and rare tetraploids set seed. When plants that set no seed were discounted, seed production following manual self pollinations of 1995 regenerants was not significantly different from that of traditional inbreds derived from the same F1 sources.
The discovery that broccoli (Brassicaoleracea L., Italica Group) sprouts contain high levels of sulforaphane, a constituent that may provide chemoprotection against certain carcinogens, has stimulated much interest in seed production of this crop. Studies were undertaken to determine the potential for producing broccoli seed using self-compatible selections from open-pollinated (OP) populations or doubled-haploid (DH) programs. In all outdoor and greenhouse trials, three OP selections and seven DH lines produced selfed seed, but seed weight per plant and number per plant varied significantly among the entries. In all environments there were individuals with relatively high (i.e., >3 g/plant) production that were significantly different from low (i.e., <2 g/plant) producers. The relative productivity of some lines varied greatly between experiments, which indicates that seed production of particular genotypes is affected differently by environmental conditions. This indicates the importance of identifying lines that are high producers of selfed seed across different environments. Two OP cultivar-derived lines (USVL102 and USVL104) and two DH lines (USVL062 and USVL093) were identified that consistently produced relatively high yields in greenhouse and screen cage trials. These lines are good candidates for evaluating seed production in field tests and as possible sources of seed for sprouting.
Wirestem, caused by Rhizoctonia solani, is a destructive disease of B. oleracea cole crops and is distributed worldwide. Effective means of wirestem control include soil fumigation and soil treatment with pentachloronitrobenzene, which are increasingly expensive and environmentally undesirable. As a consequence, alternative methods of wirestem control are needed. Thus, we conducted controlled-environment and field experiments to develop methodology to study host-plant resistance and possibly biocontrol agents as potential wirestem control alternatives. Seedlings of 12 cultivars (three each of cauliflower, broccoli, cabbage, and collard) at the four- to five-leaf stage were transplanted to trays in a growth room or into field plots and covered with soil infested with R. solani AG-4 sclerotia. Disease progression (percent of plants healthy, diseased, and dead) was observed every 3 days for 2 weeks in the controlled environments and for 3 weeks in field trials. At the end of two studies, plants were dug with roots intact and rated for disease using a 1 to 10 scale. In all trials, percent healthy plants stabilized at about 2 weeks after inoculation. Incidence of wirestem disease varied among experiments ranging from 70% to 100% diseased, dead plants in controlled environments, and from 51% to 88% and 33% 65% in the two respective field studies. Disease rating was always negatively and significantly correlated with percent healthy plants. Although a genotype × environment interaction was observed, some cultivars (i.e., `Snowcone' and `Snowcrown' cauliflowers) were always severely diseased, while others (i.e., `Viking' broccoli and `Blue Max' collard) were consistently among the least diseased.
Downy mildew, caused by Peronospora parasitica (Pers. ex Fr.), is one of the most economically important diseases in broccoli (Brassica oleracea L. Italica group). Previous studies reported that resistance to downy mildew in broccoli depends on plant age and that seedling resistance appears to be independent of mature-plant resistance. The objectives of our studies were to evaluate resistance and susceptibility of USDA broccoli inbreds to downy mildew and to investigate the interaction between the host and pathogen at two plant stages with single or double inoculation. Multiple screening tests at both cotyledon and three-expanded leaf stages using 38 entries, including USDA inbreds and commercial hybrids, were conducted in randomized complete-block designs. In these tests, every leaf of each plant was thoroughly sprayed with P. parasitica isolate PP1 at a concentration of 10,000 sporangia per ml at both stages. Ratings for downy mildew reaction phenotype were made at 9 days postinoculation on a 0-9 scale of increasing disease severity. We found significant phenotypic variation to infection among broccoli entries. We observed three general phenotypes: 1) resistance at both stages; 2) susceptible at cotyledon stage combined with resistance at three-expanded leaf stage; and 3) susceptibility at both stages. Additionally, inoculation at the cotyledon stage had no effect on inoculation at the three-expanded leaf stage.