Three orange-mesocarp derivatives of the xishuangbannan cucumber (Cucumis sativus L. var. xishuangbannanesis Qi et Yuan), P100, P101, and P104; and NPI (P105), an unrelated cucumber (Cucumis sativus L.) reported to have orange flesh, were selected as parents for a diallel experiment to evaluate inheritance of orange cucumber mesocarp pigment over 3 years. Visual color intensity and carotenoid content were closely related. A preponderance of additive genetic effects for cucumber mesocarp pigmentation was observed in grade size 2 fruit (immature fruit used for pickling). Both additive and nonadditive genetic effects were important in grade size 4 fruit (mature). Years and yea× genotype interactions were highly significant for pigmentation of size 2 fruit, indicating the importance of environment on the expression of pigmentation in this size class. In contrast, color development was stable among years for size 4 fruit. P104 exhibited high general combining ability (GCA) estimates for size 4 fruit pigmentation across years, while P101 had high GCA estimates for size 2 fruit. The diallel analysis illustrated high fruit carotene content of parents per se. However, most hybrid combinations of the diallel reduced carotenoid content relative to parents, indicating both dominance for low carotenoid content for both fruit sizes and lack of genetic complementation among parents to enhance fruit color. Genetic control of pigmentation in size 2 fruit appeared to be independent of that for size 4 fruit.
Diverse carrot (Daucus carota L.) inbreds were evaluated as young plants in the greenhouse and mature plants in the field for resistance to the pathogen Alternaria dauci (Kühn) Groves and Skolko. Persistence of leaves after infection was the criterion used to estimate disease damage by the pathogen. Partial resistance was identified. A five-parent diallel evaluated for resistance indicated a preponderance of additive variation with some dominant gene action for resistance. Reciprocal cross differences were significant for certain crosses. Responses of most inbreds were useful in predicting resistance in their hybrids, but exceptions to this trend did occur. Field resistance ratings generally correlated well with resistance ratings obtained in greenhouse, even in grower fields where fungicides were applied.
Carrot tissue cultures, germinating seed, and dry seed were exposed to gamma radiation. Irradiation accelerated germination of carrot seed in the M1 generation at low doses (0.5 and 1 krad), whereas higher doses delayed germination. A high negative correlation was observed between dose and survival of plants after seed irradiation. Plant size and root weight were 20 % to 35% greater than control plants after seeds, but not tissue cultures, were exposed to low doses of gamma irradiation. Higher doses reduced M1 plant size by >50% in germinating seed and tissue culture treatments but less for the dry seed treatment. Seed production decreased while phenotypic variation of M1 plants increased with increasing gamma ray dosage. Root weight and total dissolved solids were highly variable in M2 families. Less variation was observed in total carotene content and none was seen in sugar type (reducing vs. nonreducing sugars). Induced variation in root color and root shape was also observed. Irradiation of germinating seed and tissue cultures yielded more M2 variation than irradiation of dry seed. Putative point mutations were not observed. Unirradiated carrot tissue cultures did not yield significant M2 somaclonal variation. Average root weight of M2 plants increased with increasing gamma ray dosage, especially for the dry seed treatment.
An anatomical comparison of florets from fertile, brown anther partially fertile, petaloid male-sterile, and 3 brown anther male-sterile carrot (Daucus carota L.) lines indicated that most pollen was well-developed and deeply stained in fertile lines. Partially fertile plants contained fertile and sterile anthers in which meiosis had occurred. The 3 lines of brown anther steriles examined failed to enter meiosis. In early development of the fine foliage brown anther sterile, all cells of the anther, including sporogenous cells, hypertrophied. Later, sporogenous tissue collapsed. The anatomical structure of the stamens in petaloid florets was leaf-like and similar to petals.
Maize transposable elements, Activator (Ac) and Ds transformed into several heterologous plant species for transposon tagging of genes. Several genes in Arabidopsis, flax, petunia, tobacco, and tomato have been tagged and cloned by using Ac and Ds. We have double transformed carrot lines, B493 and B7262 with stabilized autonomous Ac and non-autonomous Ds element to develop a two-element based transposon tagging system. PCR and Southern hybridization indicated that Ds element transposed from T-DNA in calli, somatic embryos and transgenic plants. The insertion of Ds element into new sites in carrot genome after excision verified by GUS assay, Southern hybridization and inverse-PCR. Currently, the behavior of non-autonomous Ds element is being studied. Ds induced mutation will be screened in transgenic plants. These initial results demonstrate that the Ac/Ds-based transposon tagging system may work in carrot.
While the carotenoid biosynthetic pathway has been studied several horticultural and agronomic crops, very little information exists for this conserved pathway in carrot, a primary source of dietary carotenoids. Though orange carrots are the most familiar color to Western consumers, yellow, red, and white carrots also exist and have been historically important. Modern carrot breeders are showing renewed interest in these unusual color phenotypes. Beta- and alpha-carotene are the primary pigments in orange carrot roots. Yellow carrots accumulate xanthophylls (oxygenated carotenes), red carrots accumulate lycopene (the precursor to alpha- and beta-carotene), and white carrots accumulate no detectable pigments. Differences between these phenotypes are usually monogenic or oligogenic. Our research has focused on identifying putative genes for carotenoid biosynthetic enzymes in the carrot genome, mapping them, and examining expression patterns in various tissues and carrot root pigment phenotypes. We are using this information to create a carrot pigment biosynthesis function map incorporating biosynthetic enzymes, major carrot color genes, and gene expression information.
Garlic callus derived from young basal plate explants of one genotype was evaluated for regeneration. Callus was initiated on a modified B5 medium supplemented with 2.4-D for 10 weeks and then subcultured on medium with picloram + 2iP for 10 weeks. This friable callus was used in regenerating plants. Callus was transferred to a modified B5 medium with 10 hormonal combinations of auxins and cytokinins in a complete factorial design. Four pieces of callus about 1.0 cm square were transferred to a petri plate with four replications per treatment Callus was subcultured monthly to ensure optimum growth and data was collected after three months in culture. The best regeneration frequency occurred on medium supplemented with picloram + BA. Regenerated lines were then transferred to medium with no hormones for evaluation of single plants. The genetic stability of these regenerated lines was tested using polyacrlyamind gel elcctrophoresis (PAGE) and six isozyme systems. This regeneration system will be very useful in the production of regenerated putative transformed lines from callus.
We evaluated the efficiency of transformation in garlic for promoter activity, osmoticum effect and shaker speed using particle bombardment as the method of gene delivery. Callus was produced from root segments on a modified B-5 medium for four garlic clones. Suspension cultures were then established on a modified B-5 medium + 2,4-D using 6-month-old callus. Cells were collected by vacuum filtration and the Bio-Rad PDS-1000/He system was used to deliver genes. The activities of CaMV 35S, maize Adh1, and rice Act promoters were evaluated for transient expression using the β-glucuronidase (GUS) reporter gene. Osmotic conditioning of cells was performed by adding both mannitol and sorbitol to the medium. Osmoticum effect was evaluated for enhancement of transformation efficiency using GUS. The effect of shaker speed (120, 180 and 240 rpm) on cell type was evaluated for transformation efficiency using GUS. CaMV 35S promoter activity was much higher for garlic than either the maize Adh1 or rice Act promoters. Osmoticum did not enhance promoter activity, but differences in response to osmoticum among garlic clones were observed. Shaker speed did affect cell type, and transformation efficiency was greatly increased at higher shaker speeds. Confirmation of stable transformation and regeneration are in progress.
Current classifications of the genus Daucus are based on morphological and anatomical characteristics. We have used single to low copy nuclear restriction fragment length polymorphisms (nRFLPs) to describe the phylogeny and relationships of eight Daucus species including cultivated carrot (D. carota L.). Parsimony analysis of 247 characters (DNA fragments from 58 probe-enzyme combinations) yielded a tree in which accessions were grouped into three major clades and phenetic analysis using Jaccard's coefficient yielded two major clusters. The phylogenetic relationships from the nuclear RFLP data generally agreed with an earlier morphological classification. Resolution and placement of D. guttatus and D. muricatus were not consistent with the morphological classifications. Molecular variation among carrot inbreds was large.