A protocol was developed to make in vitro graft unions among Lycopercicon spp., and regenerates from cultured graft unions were evaluated for chimera formation. Young seedlings were preconditioned for 4 to 6 days in liquid 1/2-strength Murashige & Skoog (MS) basal medium supplemented with 8.9 μM benzyladenine and 1.0 μM indole-3-butyric acid. Preconditioned seedlings exhibited increased biomass and enhanced graft union survival. In particular, survival of cleft grafts increased from 37% to 95% with the seedling preconditioning. When graft unions among different genotypes were excised from apex-to-apex in vitro cleft grafts and plated on MS basal medium supplemented with 9.1 μM zeatin and 3.9 μM ancymidol, as many as 100 plantlets were regenerated from a single graft union. However, no chimeric regenerates were recovered, indicating that asymmetric responses to grafting may be a limiting factor to in vitro chimera formation.
Katherine Kelly Stephenson, John R. Stommel, and Timothy J Ng
John R. Stommel, Mikhail Kozlov, and Robert J. Griesbach
Gordon J. Lightbourn, John R. Stommel, and Robert J. Griesbach
Anthocyanin pigmentation in leaves, flowers, and fruit imparts violet to black color and enhances both ornamental and culinary appeal. Shades of violet to black pigmentation in Capsicum annuum L. are attributed to anthocyanin accumulation. Anthocyanin production is markedly influenced by numerous environmental factors, including temperature and light stress. The objective of this study was to determine the genetic basis for differences in C. annuum anthocyanin content in response to varying environments. Growth experiments conducted under controlled environment conditions demonstrated that anthocyanin concentration was significantly higher in mature leaves in comparison with immature leaves under high light (435 μmol·s−1·m−2) conditions. High (30 °C day/25 °C night) versus low (20 °C day/15 °C night) temperature had no significant effect on anthocyanin concentration regardless of leaf maturity stage. Foliar anthocyanin concentration in plants grown under short days (10 h) with low light intensity (215 μmol·s−1·m−2) was significantly less than under long days (16 h) with low light. Under high light intensity, daylength had no effect on anthocyanin content. Three structural genes [chalcone synthase (Chs), dihydroflavonol reductase (Dfr), anthocyanin synthase (Ans)] and three regulatory genes (Myc, MybA, Wd40) were selected for comparison under inductive and noninductive environmental conditions for anthocyanin accumulation. Expression of Chs, Dfr, and Ans was significantly higher in mature leaves in comparison with younger leaves. Consistent with anthocyanin concentration, temperature had no effect on structural gene expression, whereas light positively influenced expression. Under low light conditions, temperature had no effect on Myc, MybA, and Wd40 expression; whereas under high light conditions, temperature only had an effect on MybA expression. The study of anthocyanin leaf pigmentation in C. annuum under inductive and noninductive environments provides a new approach for elucidating the molecular genetic basis of epistatic gene interactions and the resulting phenotypic plasticity.
Aref A. Abdul-Baki, John R. Stommel, Alley E. Watada, John R. Teasdale, and Ronald D. Morse
Ten cultivars of processing tomatoes (Lycopersicon esculentum Mill.) grown in bare soil or on black polyethylene and hairy vetch (Vicia villosa Roth.) mulches were evaluated for yield, fruit processing quality, and leaf necrosis. Yields were higher, fruit was heavier, and leaf necrosis less in hairy vetch than in bare soil or black polyethylene mulch. With the exception of pH, yield and fruit quality component responses to mulch treatments were not cultivar-dependent. Fruit pH, soluble solids concentration, and color equaled values obtained using bare soil production practices. Percent solids was highest with black polyethylene and lowest in hairy vetch. The hairy vetch mulch delayed fruit maturity compared to the bare soil and black polyethylene. The hairy vetch cultural system has the potential to increase yield of processing tomatoes.
Robert J. Griesbach, Ronald M. Beck, John Hammond, and John R. Stommel
Gene silencing is one of the ways in which gene expression is controlled. The authors have developed a model system to study anthocyanin gene silencing using a recessive mutation in Petunia Juss. (Star mutation) and the ability of certain viruses to reverse the gene silencing mutation. In healthy plants, the star pattern was enhanced (increase in level of gene silencing) under high temperature or light growing conditions. Virus infection did not significantly influence the star pattern when plants were grown under either low-light or low-temperature conditions. Under high-light and -temperature conditions, virus infection reverses silencing, leading to a change in the star pattern. These changes in the star pattern corresponded to changes in gene expression. Viral infection had a greater affect on regulatory gene (Wd40, Myc, and Myb) expression than on structural gene expression (Chs and Ans).
John R. Stommel, Marie E. Tousignant, Thanda Wai, and Jacobus M. Kaper
Viral satellite RNA associated with cucumber mosaic virus (CMV) is know to modulate CMV symptomology. Virulent CMV associated RNA 5 (CARNA 5) satellites may intensify crop disease. Naturally occurring variants of these satellites, however, attenuate CMV symptoms. Satellite transgenic tomato plants expressing the S-CARNA 5 or 1-CARNA 5 ameliorating forms of the satellite were evaluated under simulated CMV epidemic conditions in USDA–APHIS approved field trials. Trials conducted at Beltsville, Md., in 1994 and 1995 demonstrated that CMV can be effectively controlled under field conditions in satellite transgenic plants. Yields of transgenic lines infected with CMV were 50%–65% greater than that of non-transgenic infected controls. Yields of noninfected transgenic lines ranged from 5% greater than, to 33% less than, noninfected nontransgenic controls. Expression of CARNA 5 in inoculated transgenic plants greatly reduced CMV foliar symptoms and virus titers when compared to inoculated control plants. Levels of CARNA 5 were detected at varying levels in infected transgenic plants throughout the growing season. Virus or satellite was not detected in samples collected from tomato border plants and weeds growing inside and outside a nonhost crop border surrounding the test plot. Field tests conducted in 1996 will evaluate transgenic tomato plants with a double construct coding for the CMV coat protein gene and 1-CARNA 5 satellite.
John R. Stommel, Judith A. Abbott*, David Francis, and Mary J. Camp
Tomato fruit firmness is a key quality component of tomatoes produced for processing applications. Fruit firmness is generally considered a quantitatively inherited trait. Pericarp firmness of modern tomato cultivars is believed to be derived from a fairly narrow genetic background and is the result of the cumulative effort of numerous breeders over many years. Despite inferior phenotypes, wild species contain loci that can substantially increase tomato fruit quality. In the current study, inheritance of fruit firmness in firm and ultra-firm processing tomato germplasm developed from transgressive segregants of interspecific Lycopersicon esculentum × L. hirsutum and intraspecific L. esculentum crosses was characterized. Large-fruited breeding lines that varied in fruit firmness from soft to firm were identified for genetic analyses. A six-parent diallel of these advanced breeding lines was developed for field trials over multiple locations. Fruit firmness in the resulting 36 lines was determined by measuring fruit elastic properties during fruit puncture and compression. Following loading for compression, stress relaxation was recorded for 15 s. A three-parameter model was used to fit the relaxation curves. There was little correlation between firmness (maximum force) and the three relaxation parameters, i.e., firmness measured the elastic component and the relaxation parameters measured the viscous portions of the texture. General and specific combining ability for firmness derived from the respective genetic backgrounds was determined. Genetic variance components for fruit firmness were estimated using a diallel analysis and narrow sense heritability was measured using parent-offspring regression.
Carmina Gisbert, Judith M. Dumm, Jaime Prohens, Santiago Vilanova, and John R. Stommel
We identified a single plant in a grow out of the eggplant (Solanum melongena L.) variety ‘Black Beauty’ bearing green fruit. ‘Black Beauty’ normally produces violet/black pigmented fruit attributed to anthocyanin accumulation. We selected the green-fruited true-breeding genotype E13GB42 from the S2 generation obtained from selfing of the S0 green-fruited color mutant. Characterization of 12 simple sequence repeat (SSR) markers, eight fruit morphological attributes and fruit yield support E13GB42 arising as a spontaneous mutant of ‘Black Beauty’. With the exception of fruit calyx prickliness, E13GB42 was not significantly different from ‘Black Beauty’ for fruit morphological attributes and yield. E13GB42 exhibited an SSR marker profile identical to that of ‘Black Beauty’ but polymorphic with that of eight violet/black-fruited modern eggplant hybrids, older open-pollinated varieties and landraces. Transcript levels of key anthocyanin biosynthetic genes (Chs, Dfr, and Ans) and regulatory genes (Myb C, Myc, and Wd) were significantly lower in the green-fruited E13GB42 mutant in comparison with the black-fruited variety ‘Black Beauty’ at various stages of fruit development ranging from small post-anthesis fruit to full-size marketable fruit. Progeny obtained from selfing of the original mutant and reciprocal crosses with ‘Black Beauty’ produced violet, green, and green with violet striped color classes that together were not compatible with one or two gene inheritance models, suggesting that the mutation responsible for the E13GB42 phenotype influences multiple genetic factors that control fruit pigmentation.
John R. Stommel, Judith A. Abbott, T. Austin Campbell, and David Francis
Fruit firmness is a key quality component of tomatoes (Lycopersicon esculentum Mill.) for fresh-market and processed product applications. We characterized inheritance of firmness in processing tomato germplasm developed from interspecific L. esculentum Mill. × L. cheesmanii f. minor (Hook. f.) C.H. Mull. and intraspecific L. esculentum crosses. Although firmness is a key quality attribute of tomato, there is no standard method for measuring it. We measured the elastic portion of firmness by compression (compression Fmax) and puncture (puncture Fmax), and the viscoelastic portion by force-relaxation. The experimental design incorporated six genotypes in a complete 6 × 6 diallel. Compression Fmax and force measurements recorded at 0.5, 1.0, 5.0, and 10.0 seconds of relaxation were strongly related to each other, while relaxation parameters (A, B, C) describing relaxation curve shape were generally independent. Compression Fmax, relaxation curve parameter A, and puncture Fmax were significantly different among hybrids. Significant differences between Maryland and Ohio environments were evident for compression Fmax and relaxation curve parameter A. The patterns of firmness means differed among firmness measurement methods, namely for compression Fmax and puncture Fmax, indicating that they measure different aspects of tomato fruit firmness. Soft-fruited parents generally exerted a negative effect on compression Fmax, whereas firm-fruited parents most often exerted a positive effect on compression Fmax. The force required for fruit compression best approximated subjective assessment of fruit firmness. Force required for fruit puncture was subject to a significant environmental × hybrid influence in the genotypes evaluated. Shape of the force relaxation curve (i.e., parameter A) was not predictive of relative fruit firmness. General combining ability (GCA) and specific combining ability were both significant with GCA being the principal source of genetic variation. In agreement with combining ability estimates, narrow-sense heritability estimates for compression Fmax and puncture Fmax were relatively high.
John R. Stommel, Gordon J. Lightbourn, Brenda S. Winkel, and Robert J. Griesbach
Anthocyanin structural gene transcription requires the expression of at least one member of each of three transcription factor families: MYC, MYB, and WD40. These transcription factors form a complex that binds to structural gene promoters, thereby modulating gene expression. Capsicum annuum L. (pepper) displays a wide spectrum of tissue-specific anthocyanin pigmentation, making it a useful model for the study of anthocyanin accumulation. To determine the genetic basis for tissue-specific pigmentation, we used real-time polymerase chain reaction to evaluate the expression of anthocyanin biosynthetic (Chs, Dfr, and Ans) and regulatory (Myc, MybA, and Wd) genes in flower, fruit, and foliar tissue from pigmented and nonpigmented C. annuum genotypes. No differences were observed in expression of the Wd gene among these tissues. However, in all cases, biosynthetic gene transcript levels were significantly higher in anthocyanin-pigmented tissue than in nonpigmented tissues. MybA and Myc transcript levels were also substantially higher in anthocyanin-pigmented floral and fruit tissues. Our results demonstrate that differential expression of C. annuum MybA as well as Myc occurs coincident with anthocyanin accumulation in C. annuum flower and fruit tissues. In contrast to the situation in flowers and fruit, differential expression of MybA and Myc was not observed in foliar tissue, suggesting that different mechanisms contribute to the regulation of anthocyanin biosynthesis in different parts of the C. annuum plant. Cloning and sequencing of MybA genomic and cDNA clones revealed two introns of 249 and 441 bp between the R2R3 domains. Whereas the Myb R2R3 domains were conserved between C. annuum and Petunia ×hybrida Vilm., the sequence of the non-R2R3 domains was not conserved, with very little homology in these related Solanaceous species.