Leonard M. Pike

Increased calcium in carrots by expression of an Arabidopsis H+/Ca2+ transporter

We demonstrate that carrots expressing the Arabidopsis H+/Ca2+ transporter CAX1 (Cation Exchanger 1) contained up to 50% more calcium (Ca) than plants transformed with control vectors. The CAX1-expressing carrots were fertile, and robust plant growth was seen in the majority of the transgenic plants. CAX1-expressing carrots were crossed to a commercial carrot variety to confirm that the increased Ca accumulation was mediated by CAX1-expression, and the increased Ca content was clearly correlated with the transgene. This study suggests that modulation of ion transporters could be an important means of increasing the Ca content of agriculturally important crops. To our knowledge, this study represents the first attempts to use biotechnology to increase the Ca content of an agriculturally important crop.

Gold color in onions ( Allium cepa): a natural mutation of the chalcone isomerase gene resulting in a premature stop codon

Unusual gold-colored onions were selected from a F3 family originating from a cross between US-type yellow and Brazilian yellow onions. HPLC analysis showed that the gold onions contained a significantly reduced amount of quercetin, the most abundant flavonoid in onions. This result indicated that an early step in the flavonoid biosynthesis pathway might be abnormal in these onions. The expression of flavonoid synthesis genes isolated from onions was examined in gold onions and compared to that in onions of other colors by RT-PCR. The results showed that all genes were transcribed in gold onions as in red onions. In order to identify any critical mutations in flavonoid synthesis genes encoding enzymes involved in early steps of the pathway, the genomic sequence of chalcone isomerase (CHI) was obtained. A premature stop codon and a subsequent single base-pair addition causing a frameshift were identified in the coding region of the CHI gene in the gold onions. Co-segregation of the mutant allele of the CHI gene and the gold phenotype was investigated in the original F2 segregating population. Genotyping of three color groups (red, yellow and gold) of F2 onions revealed perfect co-segregation of the mutant CHI allele with the gold phenotype. All tested gold F2 onions were homozygous for the mutant CHI allele. This perfect co-segregation implies that the presence of a premature stop codon in the gold CHI gene results in an inactive CHI. Inactivation of CHI results in a block in the flavonoid biosynthesis pathway and the accumulation of chalcone derivatives, including a yellow pigment which might be responsible for the gold color in onions.

Inactivation of DFR (Dihydroflavonol 4-reductase) gene transcription results in blockage of anthocyanin production in yellow onions (Allium cepa)

Anthocyanin, one of the flavonoids, is a primary determinant of red color in onions. Inheritance studies indicate that a single gene determines the color difference between yellow and red onions. In order to establish which gene might be responsible for this color difference, full-length cDNAs of five structural genes: chalcone synthase (CHS), flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), and flavonol synthase (FLS) were cloned using degenerate PCR and RACE (Rapid Amplification of cDNA Ends). RT-PCR was carried out for these five genes to examine differential expression between yellow and red colored bulbs. Accumulation of the DFR gene transcript only occurred in red onions. In F3 populations which originated from the cross between yellow and red parents, DFR transcript was detected only in red F3 lines. To design molecular markers for selection of yellow and red DFR alleles, the DFR gene was sequenced from genomic DNA isolated from both types of onions. The genomic DNA sequence revealed the DFR gene consists of six exons and five introns. A PCR-RFLP marker was designed based on 2% polymorphic nucleotide sequence of the DFR gene between yellow and red onions. The co-segregation of markers and red color were observed in F2 segregating populations, supporting the conclusion that color difference in red and yellow onions is likely to be due to the lack of an active DFR gene.

Clonal variations of pungency, sugar content, and bulb weight of onions due to sulphur nutrition

Abstract A greenhouse study was conducted to test the effects of sulphur (S) nutrition on pungency, sugar content, and bulb weight of six cloned lines of ‘TG 1015Y’ onions. Onion bulbs grown under the low S treatment (0.1 meq 1−1 or 2 ppm S) contained 1.9 μmol g−1 fwt. pyruvic acid, while those under the high S treatment (7.7 meq 1−1 or 123 ppm S) contained 5.5 μmol g−1 fwt. There was significant variation in the pyruvic acid content among clones under the high S nutrition, but not under the low S treatment. Total sugar content was higher in the low S treatment than the high S treatment (45.2 vs. 43.1 mg g−1) with a large variation among the clones under both treatments. Bulb weight was significantly less at low S nutrition (311 vs. 504 g) with a great variation among the clones. Total S content in leaf (0.26 vs. 0.85%) and bulb tissues (0.13 vs. 0.43%) were significantly low in the low S treatment and indicated a S deficiency.

A tissue culture technique for the clonal propagation of onion using immature flower buds.

Abstract A tissue culture technique was developed to rapidly obtain large numbers of plants from selected individual plants. Immature flower buds were taken from unopened spathes and cultured on modified Murashige and Skoog (MS) medium containing 0.5 mg l−1 1-naphthaleneacetic acid (NAA) and 5.0 mg l−1 benzylamino purine (BA). About 10% of the flower buds adventitiously gave rise to shoots and 70% developed into deformed flowers which produced a greater number of shoots than those arising adventitiously. This technique provided a ≥ 2-fold increase over previously reported methods and made it possible to produce genetic-cytoplasmic male sterile parental plants in sufficient numbers for commercial hybrid seed production.

Identification of the fourth allele of the ANS (anthocyanidin synthase) gene and its effect on red color intensity in onions ( Allium cepa )

Bulb color in onions (Allium cepa) is an important trait, and homogenous red coloration is desirable in red onion cultivars. The gene encoding anthocyanin synthase (ANS) is required for anthocyanin biosynthesis in onions. We have previously described three different alleles of the ANS gene. Here we report identification of the fourth allele of ANS, ANS-h1, found in a dark red doubled haploid line. ANS-h1 is similar to a non-functional allele found in Brazilian yellow cultivars except that it has several point mutations and indels throughout the promoter and coding regions, none of which are predicted to inactivate enzymatic activity. F2 and backcross populations originating from the crosses between wild-type (ANS-L) allele-containing red and pink (ANS-p) allele-containing white or yellow parents show a discrete segregation ratio of 3 red to 1 light pink, indicating that the wild-type allele is completely dominant over the pink allele. In contrast, segregating populations derived from the crosses between ANS-h1 allele-containing red and the same white or yellow parents show a gradient of red intensity from light pink to dark red, suggesting that other genetic factors may affect expression of ANS-h1. A newly developed PCR-based marker and two previously developed markers for allelic selection of the ANS gene were used to examine allele composition in fifty-six breeding lines and commercial cultivars. Most lines are heterogeneous for the ANS gene with two or three alleles detected. The frequency of the pink allele is low in red breeding lines, but it is predominant in white and yellow lines.

The basic color factor, the C locus, encodes a regulatory gene controlling transcription of chalcone synthase genes in onions (Allium cepa)

In onions (Allium cepa), a variety of bulb colors exist ranging from white, yellow, to red, with different intermediate shades. In order to identify the function of the basic color factor, the C locus, which is required for any color production, a candidate gene approach was attempted utilizing anthocyanin synthesis pathway genes. RT-PCR was carried out to examine differential expression of the genes involved in the anthocyanin synthesis pathway among four different bulb colors: recessive white, dominant white, yellow, and red. The transcription of two homologous chalcone synthase (CHS) genes (CHS-A and CHS-B) was significantly reduced in both dominant and recessive white onions. The reduced transcription of CHS genes was also observed in white, but not yellow, F2 plants originating from the cross between white and yellow onions. Single nucleotide polymorphism (SNP) markers tagging parental alleles of CHS genes were utilized to determine whether the reduced transcription of CHS genes was caused by mutations in CHS genes or other regulatory genes. In the F2 populations originating from the cross between recessive white and yellow or red parents, the SNP markers tagging two parental alleles of the CHS-A and CHS-B genes did not co-segregate with the genotype of the C locus. These results suggest that the basic color factor is likely to be a regulatory gene controlling CHS gene transcription.

Application of Genetic Bit Analysis (GBATM) for allelic selection in plant breeding

Genetic Bit Analysis (GBA) is a relatively new technique developed to score single-nucleotide polymorphisms among alleles. It is currently being used as a genetic diagnostic tool in human paternity tests as well as in pedigree analysis of farm animals. GBA relies on the polymerase chain reaction and enzyme-linked colorimetry to distinguish differences among genotypes. The identification of a single-nucleotide polymorphism which distinguished the plastome of cytoplasmic male-sterile onion varieties from the plastome of fertile lines provided a model system for testing the utility of GBA in plants. Our results demonstrate that GBA permits rapid and accurate allele determination in onion breeding lines, resulting in accurate prediction of sterility at the seedling stage. The use of 96-well microtiter plates and automated liquid-handlers allowed semi-automation of Genetic Bit Analysis.

Heritability of thrips resistance in the ‘IPA-3’ onion cultivar in South Texas

A two season study was conducted to compare the onion cultivars TG1015Y and IPA-3 for resistance to thrips in South Texas. Narrow and broad sense heritabilities were estimated from populations developed from the cross, ‘IPA-3’ × ‘TG1015Y’. Parents, F1, F2, and the respective backcross populations were evaluated for thrips numbers at the Texas Agricultural Experiment Station, Weslaco, TX. ‘IPA-3’ had significantly fewer thrips than ‘TG1015Y’ in both seasons. Yield was significantly different only in the 1996–97 season. The heritability of thrips resistance in this study was very low. Depending on the methods of estimation h2 was 5.3% and 4.0%, and H2 was 4.1% and 8.0%. These results suggest that greater genetic gains for thrips resistance in onion can be achieved by selection on a family basis rather than using single plant selection.

Internal CO2 concentrations in onion bulbs at different storage temperatures and in response to sealing of the neck and base

Abstract Internal CO 2 concentrations were measured in onion ( Allium cepa L. cv. TG 1015Y) bulbs stored at 1, 7, 13, 20, 27, or 34°C for 12 weeks and their relationships with shoot growth and respiration rates were investigated. Maximum shoot growth was observed at 13 and 20°C. Respiration rates were greatest at 13 and 20°C for 8 weeks, then linearly increased with storage temperatures after 12 weeks. Internal CO 2 concentrations ranging from 2 to 5% increased with increasing storage temperatures, while internal gas volume decreased. Bicarbonate concentrations in outer scales ranged from 130 to 190 μ M and increased with increasing storage temperatures. The centre scale tissues contained 11–17% CO 2 , which paralleled respiration rates. Sealing the neck area of onion bulbs stored at 1 or 27°C significantly increased internal CO 2 concentrations, but had no effect on inhibiting shoot growth. Internal CO 2 concentrations appeared to be regulated by gas exchange rates through the neck area and/or elevated HCO 3 − concentrations in outer scales. Elevated internal CO 2 concentrations or high levels in centre scale tissues did not appear to be a primary reason for inhibited shoot growth at high storage temperatures. There seems to be thermo-dormancy controlling shoot growth and respiration in onion bulbs.