Diane Luth

Comparisons of genetic and morphological distance with heterosis between Medicago sativa subsp. sativa and subsp. falcata

Biomass yield heterosis has been shown to exist between Medicago sativasubsp. sativa and Medica gosativa subsp. falcata. The objective of this study was to gain a better understanding of what morphological and genetic factors were most highly correlated with total biomass yield heterosis. We calculated genetic distances among nine sativa and five falcate genotypes based on amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) DNA markers. Genetic distance did not correlate with specific combining ability (SCA) or mid-parent heterosis. In contrast, a morphological distance matrix based on seventeen agronomic and forage quality traits was significantly correlated with heterosis; the agronomic traits of maturity, midseason regrowth, and autumn regrowth showed strong association with heterosis. Heterosis was also correlated with subspecies. We suggest that in many cases progeny heterosis can be accounted for by the interaction of genes controlling morphologically divergent traits between the parents. In other cases, progeny heterosis could also be due to divergence between the parents at particular genetic loci that do not control field-level phenotypic differences. Genetic distanceper se between parental genotypes, based on neutral molecular markers, however, does not reflect the potential of individual genotypes to produce heterosis in their progeny.

Transgenic grapefruit plants obtained by Agrobacterium tumefaciens-mediated transformation

The economically important citrus crop plant, grapefruit, was genetically transformed, which has not been previously reported. Seedling epicotyl tissue was infected with Agrobacterium tumefaciens carrying a T-DNA vector plasmid containing genes for the scorable marker, β-glucuronidase (GUS) and the selectable marker NPTII. Shoots that regenerated from epicotyl segments from nucellar seedlings in the presence of kanamycin were analyzed. Histochemical staining verified expression of the GUS reporter gene in 43.5% of the regenerated shoots examined; however, the majority were chimeric for GUS expression while 11.9% stained solid blue. Transgenic plants were recovered from regenerated shoots by direct rooting using a simple protocol. The presence of the transgene was confirmed in 25 regenerated grapefruit plants by histochemical staining, PCR and Southern hybridization.

High‐throughput linkage analysis of Mutator insertion sites in maize

Insertional mutagenesis is a cornerstone of functional genomics. High-copy transposable element systems such as Mutator (Mu) in maize (Zea mays) afford the advantage of high forward mutation rates but pose a challenge for identifying the particular element responsible for a given mutation. Several large mutant collections have been generated in Mu-active genetic stocks, but current methods limit the ability to rapidly identify the causal Mu insertions. Here we present a method to rapidly assay Mu insertions that are genetically linked to a mutation of interest. The method combines elements of MuTAIL (thermal asymmetrically interlaced) and amplification of insertion mutagenized sites (AIMS) protocols and is applicable to the analysis of single mutants or to high-throughput analyses of mutant collections. Briefly, genomic DNA is digested with a restriction enzyme and adapters are ligated. Polymerase chain reaction is performed with TAIL cycling parameters, using a fluorescently labeled Mu primer, which results in the preferential amplification and labeling of Mu-containing genomic fragments. Products from a segregating line are analyzed on a capillary sequencer. To recover a fragment of interest, PCR products are cloned and sequenced. Sequences with lengths matching the size of a band that co-segregates with the mutant phenotype represent candidate linked insertion sites, which are then confirmed by PCR. We demonstrate the utility of the method by identifying Mu insertion sites linked to seed-lethal mutations with a preliminary success rate of nearly 50%.

Increasing seed size and quality by manipulating BIG SEEDS1 in legume species

Significance One of the most important agronomic traits in crop breeding is yield, which includes increased seed size and weight in grain crops and leaf biomass in forage crops. In this work, we demonstrate that a transcription regulator encoded by the BIG SEEDS1 (BS1) gene from the model legume Medicago truncatula, negatively regulates primary cell proliferation in plants. The deletion of this gene in M. truncatula and down-regulation of its orthologs in soybean (Glycine max) lead to significant increases in the size of plant organs, including leaf and seed. Understanding the BS1 gene function and its regulatory mechanism offers an opportunity for increasing plant yield in legumes and other grain crops. Plant organs, such as seeds, are primary sources of food for both humans and animals. Seed size is one of the major agronomic traits that have been selected in crop plants during their domestication. Legume seeds are a major source of dietary proteins and oils. Here, we report a conserved role for the BIG SEEDS1 (BS1) gene in the control of seed size and weight in the model legume Medicago truncatula and the grain legume soybean (Glycine max). BS1 encodes a plant-specific transcription regulator and plays a key role in the control of the size of plant organs, including seeds, seed pods, and leaves, through a regulatory module that targets primary cell proliferation. Importantly, down-regulation of BS1 orthologs in soybean by an artificial microRNA significantly increased soybean seed size, weight, and amino acid content. Our results provide a strategy for the increase in yield and seed quality in legumes.

Comparison of transformation frequency using the bar gene regulated by the CaMV 35S or NOS promoter in Agrobacterium-mediated soybean (Glycine max L.) transformation

In Agrobacterium tumefaciens-mediated plant transformation, the promoter chosen to drive a selectable marker gene has an effect on transformation frequency. The objective of this work was to compare the effect on soybean transformation of different promoter and regulator combinations driving a selectable marker gene using mature seeds as explants. Two commonly used promoters, double cauliflower mosaic virus 35S (CaMV 35S) and nopaline synthase (NOS), and one regulator, tobacco etch virus (TEV) translational enhancer, were tested. Four different promoter/enhancer combinations were constructed to drive a bialaphos resistance gene (bar) and used for multiple independent transformation experiments. Herbicide application and PCR analysis were used to confirm the inheritance of the bar gene in the T1 generation. Quantitative real-time PCR (qRT-PCR) was used to estimate transgene copy number in T1 transgenic events. The average transformation frequency from combining NOS with the TEV enhancer (3.5% across 12 replications) was significantly higher than the frequencies obtained from the double CaMV 35S without an enhancer (1.6% across 16 replications), double CaMV 35S with an enhancer (1.4% across 8 replications), and NOS without an enhancer (1.0% across 12 replications). The bar transcript levels in T1 transgenic leaf tissue did not correlate with transformation frequencies achieved by the different constructs. No significant differences were identified between constructs in the average transgene copy number across events. These data show that a selectable marker system comprised of the bar gene regulated by the NOS promoter in combination with the TEV enhancer is preferred in Agrobacterium-mediated soybean transformation.

Soybean [ Glycine max (L.) Merr.]

In this chapter we describe an Agrobacterium tumefaciens transformation method of soybean that utilizes mature half seeds and regeneration from the cotyledonary node region. This method results in fertile transformed soybean plants and transgenic seed in approximately 9 months. Using mature half seeds as starting material has proven to be a reliable method that does not require additional wounding for infection to occur. We have continued to make improvements in the protocol, resulting in an efficient plant regeneration system.