H. Roger Boerma

Field Evaluation of Soybean Engineered with a Synthetic cry1Ac Transgene for Resistance to Corn Earworm, Soybean Looper, Velvetbean Caterpillar (Lepidoptera: Noctuidae), and Lesser Cornstalk Borer (Lepidoptera: Pyralidae)

Abstract A transgenic line of the soybean ‘Jack’, Glycine max (L.) Merrill, expressing a synthetic cry1Ac gene from Bacillus thuringiensis variety kurstaki (Jack-Bt), was evaluated for resistance to four lepidopteran pests in the field. Jack-Bt and genotypes serving as susceptible and resistant controls were planted in field cages and artificially infested with larvae of corn earworm, Helicoverpa zea (Boddie), and velvetbean caterpillar, Anticarsia gemmatalis (Hübner), in 1996, 1997, and 1998, and also with soybean looper, Pseudoplusia includens (Walker), in 1996. Susceptible controls included Jack (1996–1998), ‘Cobb’ (1996), and Jack-HPH (1996). GatIR 81–296 was used as the resistant control in all 3 yr. Compared with untransformed Jack, Jack-Bt showed three to five times less defoliation from corn earworm and eight to nine times less damage from velvetbean caterpillar. Defoliation of GatIR 81–296 was intermediate between that of Jack and Jack-Bt for corn earworm, and similar to that of Jack for velveltbean caterpillar. Jack-Bt exhibited significant, but lower resistance to soybean looper. Jack-Bt also showed four times greater resistance than Jack to natural infestations of lesser cornstalk borer, Elasmopalpus lignosellus (Zeller), in conventional field plots at two locations in 1998. Data from these experiments suggest that expression of this cry1Ac construct in soybean should provide adequate levels of resistance to several lepidopteran pests under field conditions.

Combining cry1Ac with QTL alleles from PI 229358 to improve soybean resistance to lepidopteran pests

A QTL conditioning corn earworm resistance in soybean PI 229358 and asynthetic Bacillus thuringiensis cry1Ac transgene from therecurrent parent ‘Jack-Bt’ were pyramided intoBC2F3 plants by marker-assisted selection. Segregatingindividuals were genotyped at SSR markers linked to an anitbiosis/antixenosisQTL on linkage group M, and were tested for the presence ofcry1Ac. Marker-assisted selection was used during andafter the two backcrosses to develop a series of BC2F3plants with or without the crylAc transgene and the QTLconditioning for resistance BC2F3 plants that werehomozygous for parental alleles at markers on LG M, and whicheither had or lacked cry1Ac, were assigned to one of fourpossible genotype classes. These plants were used in no-choice, detached leaffeeding bioassays with corn earworm and soybean looper larvae (Lepidoptera:Noctuidae) to evaluate the relative antibiosis in the different genotypeclasses. Resistance was measured as larval weight gain and degree of foliageconsumption. Few larvae of either species survived on leaves expressing theCry1Ac protein. Though not as great as the effect of Cry1Ac, the PI229358-derived LG M QTL also had a detrimental effect on larval weights of bothpest species, and on defoliation by corn earworm, but did not reduce defoliation bysoybean looper. Weights of soybean looper larvae fed foliage from transgenicplants with the PI-derived QTL were lower than those of larvae fed transgenictissue with the corresponding Jack chromosomal segment. This work demonstratesthe usefulness of SSRs for marker-assisted selection in soybean, and shows thatcombining transgene-and QTL-mediated resistance to lepidopteran pests may be aviable strategy for insect control.

A QTL that enhances and broadens Bt insect resistance in soybean.

Effective strategies are needed to manage insect resistance to Bacillus thuringiensis (Bt) proteins expressed in transgenic crops. To evaluate a multiple resistance gene pyramiding strategy, eight soybean (Glycine max) lines possessing factorial combinations of two quantitative trait loci (QTLs) from plant introduction (PI) 229358 and a synthetic Bt cry1Ac gene were developed using marker-assisted selection with simple sequence repeat markers. Field studies were conducted in 2000 and 2001 to evaluate resistance to corn earworm (Helicoverpa zea) and soybean looper (Pseudoplusia includens), and detached leaf bioassays were used to test antibiosis resistance to Bt-resistant and Bt-susceptible strains of tobacco budworm (TBW; Heliothis virescens). Based on defoliation in the field and larval weight gain on detached leaves, lines carrying a combination of cry1Ac and the PI 229358 allele at a QTL on linkage group M were significantly more resistant to the lepidopteran pests, including the Bt-resistant TBW strain, than were the other lines. This is the first report of a complementary additive effect between a Bt transgene and a plant insect resistance QTL with an uncharacterized mode of action that was introgressed using marker-assisted selection.

Genotype-specific optimization of plant regeneration from somatic embryos of soybean

Abstract Proliferative embryogenic suspension cultures were established from immature cotyledon explants of Hartz ‘H7190’, a cultivar adapted to growing conditions in the southern USA. Germination frequency of somatic embryos of H7190 was low relative to that of PI 417138, a genotype known to have a high germination capacity. Of those H7190 somatic embryos that germinated, the quality of germinants was poor and no plants were recovered. To optimise germination and plant recovery of H7190 somatic embryos, the effect of relative humidity during desiccation treatment was tested. Desiccation in an atmosphere of 85% relative humidity resulted in higher germination frequencies and higher quality germinants than those in other treatments. The optimal desiccation treatment for somatic embryos of H7190 did not improve germination of somatic embryos of PI 417138, ‘Peking’ or ‘Century’, indicating that no current protocol is optimal for plant recovery from all soybean genotypes.

Discovery and utilization of QTLs for insect resistance in soybean

Insect resistance in soybean has been an objective in numerous breeding programs, but efforts to develop high yielding cultivars with insect resistance have been unsuccessful. Three Japanese plant introductions, PIs 171451, 227687 and 229358, have been the primary sources of insect resistance alleles, but a combination of quantitative inheritance of resistance and poor agronomic performance has hindered progress. Linkage drag caused by co-introgression of undesirable agronomic trait alleles linked to the resistance quantitative trait loci (QTLs) is a persistent problem. Molecular marker studies have helped to elucidate the numbers, effects and interactions of insect resistance QTLs in the Japanese PIs, and markers are now being used in breeding programs to facilitate transfer of resistance alleles while minimizing linkage drag. Molecular markers also make it possible to evaluate QTLs independently and together in different genetic backgrounds, and in combination with transgenes from Bacillus thuringiensis.

Identification of a Second Asian Soybean Rust Resistance Gene in Hyuuga Soybean

ABSTRACT Asian soybean rust (ASR) is an economically significant disease caused by the fungus Phakopsora pachyrhizi. The soybean genes Rpp3 and Rpp?(Hyuuga) confer resistance to specific isolates of the pathogen. Both genes map to chromosome 6 (Gm06) (linkage group [LG] C2). We recently identified 12 additional soybean accessions that harbor ASR resistance mapping to Gm06, within 5 centimorgans of Rpp3 and Rpp?(Hyuuga). To further characterize genotypes with resistance on Gm06, we used a set of eight P. pachyrhizi isolates collected from geographically diverse areas to inoculate plants and evaluate them for differential phenotypic responses. Three isolates elicited different responses from soybean accessions PI 462312 (Ankur) (Rpp3) and PI 506764 (Hyuuga) (Rpp?[Hyuuga]). In all, 11 of the new accessions yielded responses identical to either PI 462312 or Hyuuga and 1 of the new accessions, PI 417089B (Kuro daizu), differed from all others. Additional screening of Hyuuga-derived recombinant inbred lines indicated that Hyuuga carries two resistance genes, one at the Rpp3 locus on Gm06 and a second, unlinked ASR resistance gene mapping to Gm03 (LG-N) near Rpp5. These findings reveal a natural case of gene pyramiding for ASR resistance in Hyuuga and underscore the importance of utilizing multiple isolates of P. pachyrhizi when screening for ASR resistance.

Testing transgenes for insect resistance using Arabidopsis

One possible strategy to delay the selection of resistant insect populations is the pyramiding of multiple resistance genes into a single cultivar. However, the transformation of most major crops remains prohibitively expensive if a large number of transgene combinations are to be evaluated. Arabidopsis thaliana is a potentially good plant for such preliminary evaluations. We determined that four major agricultural pests, Spodoptera exigua, Helicoverpa zea, Pseudoplusia includens, and Heliothis virescens grew as well when feeding on ‘Landsberg Erecta’ Arabidopsis as they did on plants of ‘Cobb’ soybean. Landsberg Erecta was then transformed with either a synthetic Bacillus thuringiensis cryIA(c) gene, or the cowpea trypsin inhibitor gene. Transformed plants were crossed to produce plants transgenic for both genes. Following quantification of transgene expression, the four caterpillar species were allowed to feed on wild-type plants, plants expressing either cryIA(c) or the cowpea trypsin inhibitor gene, or plants expressing both. Both genes reduced growth of the species tested, but cryIA(c) was more effective in controlling caterpillar growth than the cowpea trypsin inhibitor gene. The resistance of plants with both transgenes was lower than that of plants expressing the cryIA(c) gene alone, but higher than that of plants expressing the only the CpTI gene. This could be due to a lower concentration of Cry protein in the hemizygous F1 plants. Thus, if the cowpea trypsin inhibitor had any potentiation effect on cryIA(c), this effect was less than the cryIA(c) copy number effect. Alternatively, expression of the trypsin inhibitor gene could be antagonistic to the function of the cryIA(c) gene. Either way, these results suggest that the combined use of these two genes may not be effective.

An R package for SNP marker-based parent-offspring tests

BackgroundWith the advancement of genotyping technologies, whole genome and high-density SNP markers have been widely used for genotyping of mapping populations and for characterization of germplasm lines in many crops. Before conducting SNP data analysis, it is necessary to check the individuals to ensure the integrity of lines for further data analysis.ResultsWe have developed an R package to conduct a parent-offspring test of individuals which are genotyped with a fixed set of SNP markers for further genetic studies. The program uses monomorphic SNP loci between parents and their progeny genotypes to calculate the similarity between each offspring and their parents. Based on the similarity of parents and individual offspring, the users can determine the threshold level for the individuals to be included for further data analysis. We used an F5-derived soybean population of ‘5601T’ x PI 157440 that was genotyped with 1,536 SNPs to illustrate the procedure and its application.ConclusionsThe R package ‘ParentOffspring’ coupled with the available SNP genotyping platforms could be used to detect the possible variants in a specific cross, as well as the potential errors in sample handling and genotyping processes. It can be used in any crop which is genotyped with a fixed set of SNP markers.

Evaluation of pest vulnerability of 'Benning' soybean value added and insect resistant near isogenic lines.

ABSTRACT Crop enhancement with value added traits may affect vulnerability to insects, and evaluating the susceptibility levels of the various value added traits in elite germplasm would aid in developing integrated pest management strategies. During 2007–2008, five ‘Benning’ soybean (Glycine max (L.) Merr) lines with different value added nutritional traits and four insect resistant quantitative trait loci (QTL) lines were evaluated in an effort to determine their pest vulnerability under artificial and natural insect pest populations. The lines showed variable susceptibility to lepidopterous insect pests classified as defoliators and stem feeders in replicated greenhouse and field tests. The study was carried out in Athens and Midville, GA. The green cloverworm (Hypena scabra (F.)) was the most common lepidopteran defoliator occurring in the fields. Other caterpillar pests found included the soybean looper (Pseudoplusia includens (Walker)), the bollworm (Helicoverpa zea (Boddie)), and the velvetbean caterpillar (Anticarsia gemmatalis (Hübner)). Data indicated that there was no significantly increased pest susceptibility among the value added cultivais with improved nutritional qualities, with the insect resistant quantitative trait loci lines Benning M and Benning MGH consistently being less susceptible to lepidopterous (Noctuidae) leaf injury.