Majid R. Foolad

Pre‐Sowing Seed Treatment—A Shotgun Approach to Improve Germination, Plant Growth, and Crop Yield Under Saline and Non‐Saline Conditions

Rapid seed germination and stand establishment are critical factors to crop production under salt‐stress conditions. In many crop species, seed germination and early seedling growth are the most sensitive stages to salinity stress. Salinity may delay the onset, reduce the rate, and increase the dispersion of germination events, leading to reductions in plant growth and final crop yield. The adverse effects of salt‐stress can be alleviated by various measures, including seed priming (a.k.a. pre‐sowing seed treatment). The general purpose of seed priming is to partially hydrate the seed to a point where germination processes are begun but not completed. Most priming treatments involve imbibing seed with restricted amounts of water to allow sufficient hydration and advancement of metabolic processes but preventing germination or loss of desiccation tolerance. Treated seeds are usually redried before use, but they would exhibit rapid germination when re‐imbibed under normal or stress conditions. Various seed priming techniques have been developed, including hydropriming (soaking in water), halopriming (soaking in inorganic salt solutions), osmopriming (soaking in solutions of different organic osmotica), thermopriming (treatment of seed with low or high temperatures), solid matrix priming (treatment of seed with solid matrices), and biopriming (hydration using biological compounds). Each treatment has advantages and disadvantages and may have varying effects depending upon plant species, stage of plant development, concentration/dose of priming agent, and incubation period. In this article, we review, evaluate, and compare effects of various methods of seed priming in improving germination of different plant species under saline and non‐saline conditions. We also discuss the known metabolic and ultra‐structural changes that occur during seed priming and subsequent germination. To maximize the utility of various seed priming techniques, factors affecting their efficiency must be examined and potential benefits and drawbacks determined. For example, quality of the seed before treatment, concentration/dose of priming agent, time period for priming, and storage quality of the seed following priming treatment must be carefully determined. Furthermore, such assessments must be based on large‐scale experiments if seed priming is to be used for large‐scale field planting. A better understanding of the metabolic events that take place in the seed during priming and subsequent germination will improve the effective application of this technology. The incorporation of advanced molecular biology techniques in seed research is vital to the understanding and integration of multiple metabolic processes that can lead to enhanced seed germination, and consequently to improved stand establishment and crop yield under saline and non‐saline conditions.

Genome Mapping and Molecular Breeding of Tomato

The cultivated tomato, Lycopersicon esculentum, is the second most consumed vegetable worldwide and a well-studied crop species in terms of genetics, genomics, and breeding. It is one of the earliest crop plants for which a genetic linkage map was constructed, and currently there are several molecular maps based on crosses between the cultivated and various wild species of tomato. The high-density molecular map, developed based on an L. esculentum × L. pennellii cross, includes more than 2200 markers with an average marker distance of less than 1 cM and an average of 750 kbp per cM. Different types of molecular markers such as RFLPs, AFLPs, SSRs, CAPS, RGAs, ESTs, and COSs have been developed and mapped onto the 12 tomato chromosomes. Markers have been used extensively for identification and mapping of genes and QTLs for many biologically and agriculturally important traits and occasionally for germplasm screening, fingerprinting, and marker-assisted breeding. The utility of MAS in tomato breeding has been restricted largely due to limited marker polymorphism within the cultivated species and economical reasons. Also, when used, MAS has been employed mainly for improving simply-inherited traits and not much for improving complex traits. The latter has been due to unavailability of reliable PCR-based markers and problems with linkage drag. Efforts are being made to develop high-throughput markers with greater resolution, including SNPs. The expanding tomato EST database, which currently includes ∼214 000 sequences, the new microarray DNA chips, and the ongoing sequencing project are expected to aid development of more practical markers. Several BAC libraries have been developed that facilitate map-based cloning of genes and QTLs. Sequencing of the euchromatic portions of the tomato genome is paving the way for comparative and functional analysis of important genes and QTLs.

Recent advances in genetics of salt tolerance in tomato

Salinity is an important environmental constraint to crop productivity in arid and semi-arid regions of the world. Most crop plants, including tomato, Lycopersicon esculentum Mill., are sensitive to salinity throughout the ontogeny of the plant. Despite considerable research on salinity in plants, there are only a few instances where salt-tolerant cultivars have been developed. This is due in part to the complexity of the trait. A plants response to salt stress is modulated by many physiological and agronomical characteristics, which may be controlled by the actions of several to many genes whose expressions are influenced by various environmental factors. In addition, salinity tolerance is a developmentally regulated, stage-specific phenomenon; tolerance at one stage of plant development is often not correlated with tolerance at other stages. Specific ontogenic stages should be evaluated separately for the assessment of tolerance and the identification, characterization, and utilization of useful genetic components. In tomato, genetic resources for salt tolerance have been identified largely within the related wild species, and considerable efforts have been made to characterize the genetic controls of tolerance at various developmental stages. For example, the inheritance of several tolerance-related traits has been determined and quantitative trait loci (QTLs) associated with tolerance at individual developmental stages have been identified and characterized. It has been determined that at each stage salt tolerance is largely controlled by a few QTLs with major effects and several QTLs with smaller effects. Different QTLs have been identified at different developmental stages, suggesting the absence of genetic relationships among stages in tolerance to salinity. Furthermore, it has been determined that in addition to QTLs which are population specific, several QTLs for salt tolerance are conserved across populations and species. Research is currently underway to develop tomatoes with improved salt tolerance throughout the ontogeny of the plant by pyramiding QTLs through marker-assisted selection (MAS). Transgenic approaches also have been employed to gain a better understanding of the genetics of salt tolerance and to develop tomatoes with improved tolerance. For example, transgenic tomatoes with overexpression of a single-gene-controlled vacuolar Na+/H+ antiport protein, transferred from Arabidopsis thaliana, have exhibited a high level of salt tolerance under greenhouse conditions. Although transgenic plants are yet to be examined for field salt tolerance and salt-tolerant tomatoes are yet to be developed by MAS, the recent genetic advances suggest a good prospect for developing commercial cultivars of tomato with enhanced salt tolerance in near future.

The Physiological, Biochemical and Molecular Roles of Brassinosteroids and Salicylic Acid in Plant Processes and Salt Tolerance

Plant hormones regulate plant growth and development by affecting an array of cellular, physiological, and developmental processes, including, but not limited to, cell division and elongation, stomatal regulation, photosynthesis, transpiration, ion uptake and transport, initiation of leaf, flower and fruit development, and senescence. Environmental factors such as salinity, drought, and extreme temperatures may cause a reduction in plant growth and productivity by altering the endogenous levels of plant hormones, sensitivity to plant hormones, and/or signaling pathways. Molecular and physiological studies have determined that plant hormones and abiotic stresses have interactive effects on a number of basic biochemical and physiological processes, leading to reduced plant growth and development. Various strategies have been considered or employed to maximize plant growth and productivity under environmental stresses such as salt-stress. A fundamental approach is to develop salt-tolerant plants through genetic means. Breeding for salt tolerance, however, is a long-term endeavor with its own complexities and inherent difficulties. The success of this approach depends, among others, on the availability of genetic sources of tolerance and reliable screening techniques, identification and successful transfer of genetic components of tolerance to desired genetic backgrounds, and development of elite breeding lines and cultivars with salt tolerance and other desirable agricultural characteristics. Such extensive processes have delayed development of successful salt-tolerant cultivars in most crop species. An alternative and technically simpler approach is to induce salt tolerance through exogenous application of certain plant growth–regulating compounds. This approach has gained significant interest during the past decade, when a wealth of new knowledge has become available on the beneficial roles of the six classes of plant hormones (auxins, gibberellins, cytokinins, abscisic acid, ethylene, and brassinosteroids) as well as several other plant growth–regulating substances (jasmonates, salicylates, polyamines, triacontanol, ascorbic acid, and tocopherols) on plant stress tolerance. Among these, brassinosteroids (BRs) and salicylic acid (SA) have been studied most extensively. Both BRs and SA are ubiquitous in the plant kingdom, affecting plant growth and development in many different ways, and are known to improve plant stress tolerance. In this article, we review and discuss the current knowledge and possible applications of BRs and SA that could be used to mitigate the harmful effects of salt-stress in plants. We also discuss the roles of exogenous applications of BRs and SA in the regulation of various biochemical and physiological processes leading to improved salt tolerance in plants.

Molecular organization of a gene in barley which encodes a protein similar to aspartic protease and its specific expression in nucellar cells during degeneration

The nucellar cells of barley undergo progressive degeneration after ovule fertilization. This degeneration is a characteristic of programmed cell death. Increasing evidence has indicated that proteases are important regulators of programmed cell death in animals. We have cloned and characterized a barley gene which encodes an aspartic protease-like protein and is specifically expressed in nucellar cells during degeneration. The gene contains eight exons and seven introns and encodes a polypeptide of 410 amino acid residues. The deduced polypeptide is characterized by having two aspartic protease catalytic site motifs, the Asp-Thr-Gly-Ser in the N-terminal and Asp-Ser-Gly-Ser in the C-terminal region, and two other regions nearly identical to two regions of plant aspartic proteases. However, it shares <20% overall sequence identity with the known plant aspartic proteases, and does not contain a ‘prosequence’ or a ‘plant-specific insert’ which are characteristics of plant aspartic proteases. We have named this aspartic protease-like protein ‘nucellin’. In northern analyses nucellin transcripts were most abundant in ovaries 3–4 days after pollination, but only marginally detectable before pollination or 10 days after pollination. RNA in situ hybridization showed that before pollination the nucellin gene was expressed at a very low level only in a cluster of nucellar cells close to the embryo sac at the chalazal end, but after pollination it was highly expressed in most nucellar cells surrounding the entire embryo sac. Furthermore, no nucellin transcripts were detectable in anther, leaf, or root tissue. The temporal and spatial pattern of the nucellin gene expression is synchronal with nucellar cell degeneration and thus, nucellin may be involved with nucellar cell death.

A genetic map of Prunus based on an interspecific cross between peach and almond

A genetic linkage map of Prunus has been constructed using an interspecific F2 population generated from self-pollinating a single F1 plant from a cross between a dwarf peach selection (54P455) and an almond cultivar ‘Padre’. Mendelian segregations were observed for 118 markers including 1 morphological (dw), 6 isozymes, 12 plum genomic, 14 almond genomic and 75 peach mesocarp specific cDNA markers. One hundred and seven markers were mapped to 9 different linkage groups covering about 800 cM map distance, and 11 markers remained unlinked. Three loci identified by three cDNA clones, PC8, PC5 and PC68.1, were tightly linked to the dw locus in linkage group 5. Segregation distortion was observed for approximately one-third of the markers, perhaps due to the interspecific nature and the reproductive (i.e. self-incompatibility) differences between peach and almond. This map will be used for adding other markers and genes controlling important traits, identifying the genomic locations and genetic characterizing of the economically important genes in the genus Prunus, as well as for markerassisted selection in breeding populations. Of particular interest are the genes controlling tree growth and form, and fruit ripening and mesocarp development in peach and almond.

Gene Expression Profiling of Plants under Salt Stress

Soil salinity is among the leading environmental stresses affecting global agriculture, causing billions of dollars in crop damages every year. Regardless of the cause, ion toxicity, water deficit, or nutritional imbalance, high salinity in the root zone severely impedes normal plant growth and development, resulting in reduced crop productivity or crop failure. Development of salt-tolerant cultivars is an attractive and economical approach to solving this problem. Although several salt-tolerant plant genotypes have been developed through transgenic approaches, often they have failed or exhibited limited success under field saline conditions. This is due to several reasons, including the fact that plant growth and development under saline conditions in the field is often influenced by cumulative effects of multiple environmental stresses and genetic factors, which may not have been considered during the development of salt-tolerant transgenic plants. Adoption of inappropriate screening techniques or selection criteria may also lead to selection of genotypes that may not be stress tolerant in a real sense. In most plant species, salt tolerance is a genetically complex trait, often modulated by multiple biosynthetic and signaling pathways. Cross-talks among various stress-controlling pathways have been observed under salt stress, many of which are regulated by transcription factors. Thus, a comprehensive knowledge of the up- and downregulating genes under salt-stress is necessary, which would provide a better understanding of the interactions among pathways in response to salt stress. Attaining such knowledge is a good step toward successful development of salt-tolerant crop cultivars. To this end, DNA microarray technology has been employed to study expression profiles in different plant species and at varying developmental stages in response to salt stress. As a result, large-scale gene expression profiles under salt stress are now available for many plant species, including Arabidopsis, rice, barley, and ice plant. Examinations of such gene expression profiles will help understand the complex regulatory pathways affecting plant salt tolerance and potentially functional characterization of unknown genes, which may be good candidates for developing plants with field salt tolerance. In this article, we review and discuss the current knowledge of plant salt tolerance and the extent to which expression profiling has helped, or will help, a better understanding of the genetic basis of plant salt tolerance. We also discuss possible approaches to improving plant salt tolerance using various tools of biotechnology.

Mapping salt-tolerance genes in tomato (Lycopersicon esculentum) using trait-based marker analysis

The germination responsiveness of an F2 population derived from the cross Lycopersicon esculentum (UCT5) x L. pennellii (LA716) was evaluated for salt tolerance at two stress levels, 150 mM NaCl + 15 mM CaCl2 and 200 mM NaCl + 20 mM CaCl2. Individuals were selected at both tails of the response distribution. The salt-tolerant and salt-sensitive individuals were genotyped at 16 isozyme loci located on 9 of the 12 tomato chromosomes. In addition, an unselected (control) F2 population was genotyped at the same marker loci, and gene frequencies were estimated in both selected and unselected populations. Trait-based marker analysis was effective in identifying genomic locations (quantitative trait loci, QTLs) affecting salt tolerance in the tomato. Three genomic locations marked by Est-3 on chromosome 1, Prx-7 on chromosome 3, and 6Pgdh-2 and Pgi-1 on chromosome 12 showed significant positive effects, while 2 locations associated with Got-2 on chromosome 7 and Aps-2 on chromosome 8 showed significant negative effects. The identification of genomic locations with both positive and negative effects on this trait suggests the likelihood of recovering transgressive segregants in progeny derived from these parental lines. Similar genomic locations were identified when selection was made either for salt tolerance or salt sensitivity and at both salt-stress treatments. Comparable results were obtained in uni- and bidirectional selection experiments. However, when marker allele gene frequencies in a control population are unknown, bidirectional selection may be more efficient than unidirectional selection in identifying marker-QTL associations. Results from this study are discussed in relationship to the use of molecular markers in developing salt-tolerant tomatoes.

Genetics, Genomics and Breeding of Late Blight and Early Blight Resistance in Tomato

Late blight (LB), caused by the oomycete Phytophthora infestans, and early blight (EB), caused by the fungi Alternaria solani and A. tomatophila, are two common and destructive foliar diseases of the cultivated tomato (Solanum lycopersicum) and potato (Solanum tuberosum) in the United States and elsewhere in the world. While LB can infect and devastate tomato plants at any developmental stages, EB infection is usually associated with plant physiological maturity and fruit load where older senescing plants exhibit greater susceptibility and a heavy fruit set enhances the disease. At present, cultural practices and heavy use of fungicides are the most common measures for controlling LB and EB. Genetic resources for resistance have been identified for both diseases, largely within the tomato wild species, in particular the red-fruited species S. pimpinellifolium and the green-fruited species S. habrochaites. A few race-specific major resistance genes (e.g., Ph-1, Ph-2 and Ph-3) and several race-nonspecific resistance QTLs have been reported for LB. Ph-3 is a strong resistance gene and has been incorporated into many breeding lines of fresh market and processing tomato. However, new P. infestans isolates have been identified which overcome Ph-3 resistance. Recently, a new resistance gene (Ph-5) has been identified, which confers resistance to several pathogen isolates including those overcoming the previous resistance genes. Advanced breeding lines including Ph-5 alone and in combinations with Ph-2 and Ph-3 are being developed. Genetic controls of EB resistance have been studied and advanced breeding lines and cultivars with improved resistance have been developed through traditional breeding. Additionally, QTLs for EB resistance have been identified, which can be utilized for marker-assisted resistance breeding. Currently, new inbred lines and cultivars of tomato with good levels of EB resistance and competitive yield performance are being developed at the Pennsylvania State University. This review will focus on the current knowledge of both LB and EB with respect to the causal pathogens, host resistances, and genetics and breeding progresses.

Marker-Assisted Selection in Tomato Breeding

The cultivated tomato, Solanum lycopersicum L., is the second most consumed vegetable crop after potato and unquestionably the most popular garden crop in the world. There are more varieties of tomato sold worldwide than any other vegetable crop. Most of the commercial cultivars of tomato have been developed through phenotypic selection and traditional breeding. However, with the advent of molecular markers and marker-assisted selection (MAS) technology, tomato genetics and breeding research has entered into a new and exciting era. Molecular markers have been used extensively for genetic mapping as well as identification and characterization of genes and QTLs for many agriculturally important traits in tomato, including disease and insect resistance, abiotic stress tolerance, and flower- and fruit-related characteristics. The technology also has been utilized for marker-assisted breeding for several economically important traits, in particular disease resistance. However, the extent to which MAS has been employed in public and private tomato breeding programs has not been clearly determined. The objectives of this study were to review the publically-available molecular markers for major disease resistance traits in tomato and assess their current and potential use in public and private tomato breeding programs. A review of the literature indicated that although markers have been identified for most disease resistance traits in tomato, not all of them have been verified or are readily applicable in breeding programs. For example, many markers are not validated across tomato genotypes or are not polymorphic within tomato breeding populations, thus greatly reducing their utility in crop improvement programs. However, there seems to be a considerable use of markers, particularly in the private sector, for various purposes, including testing hybrid purity, screening breeding populations for disease resistance, and marker assisted backcross breeding. Here we provide a summary of molecular markers available for major disease resistance traits in tomato and discuss their actual use in tomato breeding programs. It appears that many of the available markers may need to be further refined or examined for trait association and presence of polymorphism in breeding populations. However, with the recent advances in tomato genome and transcriptome sequencing, it is becoming increasingly possible to develop new and more informative PCR-based markers, including single nucleotide polymorphisms (SNPs), to further facilitate the use of markers in tomato breeding. It is also expected that more markers will become available via the emerging technology of genotyping by sequencing (GBS).