Genetic issues and pitfalls in transgenic plant breeding

Transgene technology provides a powerful tool for developing traits that are otherwise difficult to achieve through conventional breeding. In order to effectively apply the technology to breeding, we need to understand how transgenes behave in plants. Transgenes may or may not follow Mendelian segregation; their expression can be significantly affected by integration positions and structures of the transgenic DNA in host genomes; transgenes may become unstable over generations, genetic background sand environmental conditions; and they may have significantly negative impact on expression of endogenous genes. If not well understood, the sehurdles could become significant barriers in transgenic breeding. This paper reviews some genetic issues and pitfalls that are often encountered in transgenic breeding. Because of the necessity of being brief, transgene expression, silencing, and breeding are the three areas of focuses in this discussion. While molecular mechanisms underlying many of the transgenic phenomena have not been completely understood, some practical ‘rules’ are now available for creating, evaluating and selecting desirable transgenic transformants. It can be certain that with more transgenic plants generated and characterized our knowledge of transgene genetics at both molecular and plant levels will continue to accumulate. This revised version was published online in July 2006 with corrections to the Cover Date.     

Inheritance of anthocyanin content in the ripe berries of a tetraploid × diploid grape cross population

Variation patterns and inheritance of anthocyanin content in the ripe berries of a tetraploid × diploid table grape cross population were investigated in two successive years. The population segregated for three different ploid levels: dipolids, triploids, and tetraploids. A total of 28 different anthocyanins were detected and quantified in the progeny population. Transgressive segregation for the total anthocyanin content was observed in all the three ploid progeny populations. The total anthocyanin content increased as the ploid level increased. The broad sense heritabilities (H²) of the total anthocyanin content were all relatively high, ranging from 0.53 to 0.98, 0.57 to 0.97 and 0.43 to 0.94 in the diploid, triploid and tetraploid population, respectively. Our results suggested that the total anthocyanin content followed an additive inheritance model in this polyploid segregation population. We also observed that the relative contribution of individual anthocyanins to the total anthocyanin content varied significantly among different ploid populations, suggesting that genetic background has important impact on the accumulation of the individual anthocyanin compounds. These results will help develop better breeding strategies in a polyploid table grape breeding program for improving the content of anthocyanins, an important class of polyphenolics possessing antioxidant activities and many other health-related benefits.     

Evidence for common genetic mechanisms controlling the tolerance of sudden salt stress in the tribe Triticeae

Previous studies in several Triticeae species have suggested that salt tolerance is a polygenic trait, but that genes on some chromosomes confer better tolerance to salt stress than others. This suggests an intriguing possibility that there may be a similar basis for salt tolerance in the species of the tribe Triticeae. In this study, chromosomal control of the tolerance to sudden salt stress, measured as the mean rate of leaf elongation in solution cultures with a single increment of 200 mM NaCl, was investigated in the genomes of cultivated barley (Hordeum vulgare L.), rye (Secale cereale L.), and Dasypyrum villosum (L.) Can-dargy by using disomic addition lines of individual pairs of chromosomes or chromosome arms of each of the three species in the ‘Chinese Spring’ wheat genetic background. It was observed that the chromosomes of homoeologous groups 3, 4, and 5 in barley, 5 and 7 in rye, and 4 and 6 in D. villosum carry loci with significant positive effects on salt tolerance. Increased doses of chromosomes of group 2, however, reduce or do not increase the tolerance to salt stress. These results are in agreement with a previous study of the tolerance of this salt stress regime in wheat and wheatgrass Lophopyrum elongatum. A ranking analysis of the chromosomal effects within each genome of the five Triticeae species investigated in this and previous studies revealed that the chromosomes of homoeologous groups 3 and 5 consistently confer large positive effects on the tolerance of sudden salt stress, while the chromosomes of homoeologous group 2 in increased dose have no or negative effects on the tolerance. This strongly suggests that species of the tribe Triticeae share some common genetic mechanisms of tolerance of sudden salt stress. The findings in this study give credence to the proposal that wild relatives can be exploited in the development of wheat cultivars with greater tolerance to salt stress.     

Characterization of polyphenolic metabolites in the seeds of Vitis germplasm

The composition and content of polyphenols in the seeds of 91 grape accessions from 17 Vitis species were characterized. Eleven compounds, including 2 gallic derivatives, 3 monomeric flavan-3-ols, 3 flavonols, resveratrol, and procyanidin B1 and B2, were identified via HPLC-MS and quantified by HPLC-DAD. In addition, seventeen dimeric and trimeric flavan-3-ols were also quantified. Tremendous variation was observed both among and within species for these compounds. Monomeric flavan-3-ols were the most abundant polyphenols in seeds, followed by dimeric and trimeric flavan-3-ols, which collectively accounted for more than 96% of the total polyphenols. V. palmata, V. vinifera, and V. vulpina had significantly higher content of total polyphenols than other species. A number of Vitis accessions with high content of various types of seed polyphenols were identified, and they can serve as potential germplasm for improving the composition and content of seed polyphenols in cultivated grapes.     

Increased phloridzin content associated with russeting in apple (<Emphasis Type="Italic">Malus domestica</Emphasis> (Suckow) Borkh.) fruit

Phloridzin is a phenolic compound unique to apple (Malus domestica (Suckow) Borkh.) and its wild relatives. Since its discovery, phloridzin has been researched for its nutraceutical properties, including anti-diabetic, anti-cancer, and antioxidant activities, making phloridzin a potential target for nutritional improvement in new apple cultivars. However, phloridzin accumulates at significantly lower concentrations in fruit than in vegetative tissues and seeds. In ‘Golden Delicious’ and its sports, we observed higher phloridzin content in peels of sports with a cuticle disorder termed russet. In russeted apples, the smooth, waxy fruit cuticle is partially or entirely replaced by a corky layer, induced through environmental and genetic effects. To understand the variation of phloridzin content and fruit russet in apple fruit, we surveyed 108 accessions with variation in russeting from the USDA-ARS Malus germplasm collection in Geneva, NY. Russeting in apple fruit ranged from 0 to 100%, and phloridzin content ranged from 24.3 to 825.0 μg/g in peels. Mean phloridzin content varied significantly between russeting groups; in groups with light (0–5%), medium–high (70–80%), and high (90–100%) russeting mean phloridzin content was 115.2, 591.2, and 378.8 μg/g, respectively. We observed that genetic factors and russeting are strong predictors of phloridzin content in peels, but not fruit flesh or leaves. Conversely, other peel phenolics are negatively associated with russeting. We observed variable phloridzin content related to russet incidence during fruit development in ‘Golden Delicious’ (low to medium russet) and its sports, ‘Empress Spur’ (low russet), ‘Razor’ (complete russet), and ‘Sergeant Russet’ (medium to high russet).     

Genetic diversity of dihydrochalcone content in <Emphasis Type="Italic">Malus</Emphasis> germplasm

Dihydrochalcones, beneficial phenolic compounds, are abundant in Malus Mill. species, particularly in vegetative tissues and seeds. Phloridzin (phloretin 2′-O-glucoside) is the primary dihydrochalcone in most Malus species including cultivated apple, Malus?×?domestica Borkh. A few species contain sieboldin (3-hydroxyphloretin 4′-O-glucoside) or trilobatin (phloretin 4′-O-glucoside) in place of phloridzin, and interspecific hybrids may contain combinations of phloridzin, sieboldin, and trilobatin. Proposed health benefits of phloridzin include anti-cancer, antioxidant, and anti-diabetic properties, suggesting the potential to breed apples for nutritional improvement. Sieboldin and trilobatin are being investigated for nutritional value and unique chemical properties. Although some of the biosynthetic steps of dihydrochalcones are known, little is known about the extent of variation within Malus germplasm. This research explores the genetic diversity of leaf dihydrochalcone content and composition in Malus germplasm. Dihydrochalcone content was measured using high performance liquid chromatography (HPLC) from leaf samples of 377 accessions, representing 50 species and interspecific hybrids from the USDA-Agricultural Research Service (ARS) National Plant Germplasm System Malus collection. Within the accessions sampled, 284 accessions contained phloridzin as the primary dihydrochalcone, one had only trilobatin, two had phloridzin and trilobatin, 36 had sieboldin and trilobatin, and 54 had all three. Leaf phloridzin content ranged from 17.3 to 113.7 mg/g with a heritability of 0.76 across all accessions. Beyond the potential of dihydrochalcones for breeding purposes, dihydrochalcone composition may be indicative of hybridization or species misclassification.