The Y2 locus conditions α- and β-carotene accumulation in the xylem (core) of carrot roots. The dominant allele suppresses carotene, but not xanthophyll accumulation, resulting in yellow-cored roots. Individuals homozygous for the recessive allele are rich in carotenes and are therefore orange-cored. Increased consumer interest in high carotene produce requires improved understanding of carotene biosynthesis and color development and more-efficient breeding techniques. We examined 103 F2 individuals generated from inbred populations differing in core carotene content. Bulked segregant analysis identified AFLP bands putatively linked to Y2. Linkage was confirmed for some bands by mapping. Linked bands were excised from gels, re-amplified, cloned into pGEM vectors, and sequenced. Cloned fragments and sequence information were used to characterize larger genomic regions to identify codominant markers. Currently we are developing codominant, PCR-based markers that can be used to rapidly genotype individuals in breeding programs, to characterize wild, feral, and cultivated populations for diversity and evolution studies, and to examine the role of Y2 in carotene accumulation.
James M. Bradeen and Philipp Simon
Yuan-Yeu Yau and Philipp Simon
The β-fructofuranosidases (invertases) cleave sucrose and related sugars into fructose and glucose. The enzyme is present in most plant tissues in multiple forms. Carrots contain an insoluble β-fructofuranosidase, which is ionically bound to the cell wall and soluble β-fructofuranosidases. The Rs locus in carrot conditions the accumulation of sucrose, fructose, and glucose. The inbred B493 is homozygous for the recessive allele (rs/rs) and accumulates high levels of sucrose, while most carrots accumulate glucose and fructose (Rs/Rs). Increased consumer interest in sweeter carrots require greater understanding of sucrose metabolism and its biochemistry. We established gene transformation systems for carrots using Agrobacterium-mediated and particle bombardment-mediated methods. Intact soluble invertase cDNA was synthesized from B493 and other carrots as measured by RT-PCR. The cDNA fragment was cloned into pBI121 and into a sequencing vector. B493 callus was transformed by Agrobacterium containing the pBI121 vector with invertase driven by the 35S promoter. Invertase expression was compared in rs/rs and Rs/Rs transformed carrots (with invertase overexpression) and non-transformed carrots. These results improve our understanding of the role of soluble invertase in sucrose metabolism of carrot.
Ahmet Ipek and Philipp W. Simon
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.
J. Michele Myers and Philipp W. Simon
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.
Philipp W. Simon and James O. Strandberg
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.
Bassam Al-Safadi and Philipp W. Simon
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.
Brian J. Just* and Philipp W. Simon
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.
J. Michele Myers and Philipp W. Simon
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.
Jack E. Staub, Philipp Simon, and Irwin Goldman
Meryem Ipek, Ahmet Ipek, and Philipp W. Simon
Garlic (Allium sativum L.) is an asexually propagated crop that displays much morphological diversity. Studies which have assessed garlic diversity with isozymes and randomly amplified polymorphic DNA (RAPD) markers generally agreed with the morphological observations but sometimes failed to discriminate clones. To discriminate among closely related garlic clones in more detail, we introduced amplified fragment-length polymorphism (AFLPs) to evaluate the genetic diversity and phenetic relatedness of 45 garlic clones and three A. longicuspis clones and we compared AFLP results with RAPD markers and isozymes. Three AFLP primer combinations generated a total of 183 polymorphic fragments. Although similarities between the clusters were low (≥0.30), some clones within the clusters were very similar (>0.95) with AFLP analysis. Sixteen clones represented only six different banding patterns, within which they shared 100% polymorphic AFLPs and RAPD markers, and likely are duplicates. In agreement with the results of other investigators, A. longicuspis and A. sativum clones were clustered together with no clear separation, suggesting these species are not genetically or specifically distinct. The topology of AFLP, RAPD, and isozyme dendrograms were similar, but RAPD and isozyme dendrograms reflected less and much less polymorphism, respectively. Comparison of unweighted pair group method with arithmetic averaging (UPGMA) dendrograms of AFLP, RAPD, and isozyme cluster analyses using the Mantel test indicated a correlation of 0.96, 0.55, and 0.57 between AFLP and RAPD, AFLP and isozyme, and RAPD and isozyme, respectively. Polymorphic AFLPs are abundant in garlic and demonstrated genetic diversity among closely related clones which could not be differentiated with RAPD markers and isozymes. Therefore, AFLP is an additional tool for fingerprinting and detailed assessment of genetic relationships in garlic.