Evaluation of short season soybean cultivars for Rps6 resistance to Phytophthora sojae
Authors:
T. R. Anderson1, R. I. Buzzell1, H. D. Voldeng2, and V. Poysa1
Abstract:
Abstract: Phytophthora root rot caused by Phytophthora sojae (Ps) is an important disease of soybean on poorly drained soil. The resistance gene Rps6, which provides resistance to a number of races, was reported in the cultivar Altona in 1982. A line, 840-7-3 (PI 436.477) has a gene for resistance and has been used in the development of a number of short season cultivars in Canada but the gene for resistance has not been previously identified. Inoculation of the original lines and crosses derived from PI 436.477 with a limited number of Phytophthora races indicated that Rps6 was present in a number of these lines and cultivars either alone or in combination with other genes for resistance to Phytophthora root rot. These lines may be useful sources of the resistance gene Rps6 in future breeding programs.
Introduction: The Rps6 gene for resistance to Phytophthora sojae (Ps) in the soybean cultivar Altona was reported by Athow and Laviolette (1982). Altona resulted from a cross between O-52-903 (PI 194.654) (Rps6) and Flambeau (rps,rps). O-52-903 is from Sweden and another Swedish line, 840-7-3 (PI 436.477), that has been used in soybean breeding, has been resistant to hypocotyl inoculations with Ps in prior trials at the Greenhouse and Processing Crops Research Centre, but it was not clear if the line contained Rps6. Results obtained in 1994-95 from inoculations of the cross 840-7-3 (Rps6?)/HARO 6272 (Rps6,Rps7) with Ps race 1 did not show segregation for resistance and susceptibility so it was concluded that Rps6 was present in 840-7-3. However, the F2 population was small (38 plants) and limited testing was done in the F3 and F4. This study was conducted to identify the gene for resistance in 840-7-3 (PI436.477) and to identify cultivars that may have resistance derived from 840-7-3.
Materials and Methods: Because the number of reported Ps races is increasing and the difficulty of maintaining Ps cultures with the correct virulence formula, it has become increasingly difficult to include all races in the characterization of putative resistance genes. As a compromise, we inoculated the Swedish lines and progeny derived from 840-7-3 with 7 races of Ps to compare their responses. The pedigrees of the 20 cultivars that have 840-7-3 in their parentage and genotype are listed in Table 1. The pedigree and genotype for Cabot and Zephyr is incomplete. The lines and cultivars were inoculated by the hypocotyl method (Anderson and Buzzell, 1992) with isolates B135R3.40, D45R5.10, C20R10.3, D35R17.2, C25R21.6, and B17R31.16 obtained from Dr. A. Dorrance, OARDC, OH., which correspond to Ps races 3, 5, 10, 17, 21, and 31, respectively (Hartman et al., 1999). Rps7 which is found in Harosoy, HARO 1272 (Harosoy 63) and HARO 6272 is susceptible to all of the above races so the presence or absence of this gene could not be verified in any of the soybean lines screened. Results and Conclusions: Expected results were obtained with the control lines/cultivars containing Rps6 (Table 2). Harosoy (rps6, rps1) was susceptible to all races and HARO 1272 (Rps1a, Rps7) was distinguishable from Rps6 by a resistant response with Races 17 and 31. Inoculation of O-52-903 and 840-7-3 resulted in a similar response pattern suggesting both contain Rps6. Of the cultivars evaluated, 5 contained no resistance, 6 contained Rps6 and Rps1a, 4 contained Rps6 and two contained Rps1a based on responses to the Ps races used for screening. Inoculation of 3 cultivars with Races 17 and 31 resulted in a variable response. Results indicate that Rps6 is present in 840-7-3 and some of the progeny derived from this line and that these progeny could be used as a source for Rps6 in breeding programs. Inoculation of Commander resulted in intermediate responses to races 3, 17, 21, and 31; therefore, it was not clear if Rps1a or Rps6 were present. Inoculation of Galaxy and RCAT Bobcat with race 17 resulted in a susceptible response which was not expected if Rps1a were present which suggests the presence of a resistant gene other than Rps1a. The presence of Rps6 in these varieties is assumed but cannot be confirmed.
Effect of mutagen on callus induction and in vitro regeneration of soybean cultivar
Authors:
Maya Kumari*1, Shanti R Patil2, and Ramesh N Pudake3
Abstract:
Abstract: The effect of y-irradiation on callus induction and regeneration in soybean cultivar Bragg was studied. It was observed that callus induction was there in all the explants of control. Mean response for callus induction ranged from 47.30% (cotyledon, control) to 25.35% (leaf segments, 25kR). MS+2, 4-D 2mgl-1 gave the best response for both leaf and cotyledon explants. There was significant effect of irradiation as the response of treated explants was low. The response for 20kR was better than 25kR. Mean callus weight ranged from 155mg (cotyledon, 25kR) to 273.23 mg (cotyledon, control). The callus obtained was of embryogenic, rhizogenic, and non-embryogenic morphotypes. Direct organogenesis was obtained from cotyledonary node, nodal segment, and shoot tips from both treated as well as control. Response for bud break was 71.48% in control, 62.32% in 20kR, and 57.20% in 25kR for cotyledonary nodes. The average number of days taken for bud initiation was 4.1 days in control, 5.7 days in 20kR, and 6.1 days in 25kR for cotyledonary nodes followed by nodal segments and shoot tips. Nodal segments gave the highest mean number of buds (9.92 in control, 8.65 in 20kR, and 6.24 in 25kR) followed by cotyledonary node. Mean number of buds obtained from shoot tip was very low (3.65 in control, 3.23 in 20 kR, and 3.05 in 25 kR). Treatment MS+BAP 5mgl-1+IBA 0.1mgl-1 was best. Root induction took place in MS+IBA 0.8mgl-1 or NAA 1.0mg mgl-1 within 15-20 days. Introduction: To achieve desired improvements in soybean, breeders have used different breeding approaches. Most of these conventional approaches have their own limitations. Hence there is a need to supplement conventional breeding with plant biotechnology (Gahukar and Jambhule, 2000). Several techniques in tissue culture have been used effectively for crop improvement. There are reports on in vitro regeneration of soybean (Zheng et al, 1993; Hussian et al, 1996; Gai Junyi et al, 1996, 1997; Thome et al, 1995; Rajasekaran and Pellow, 1997; Settu and Kumari, 1998; and Ubanprasert et al, 1998) but reports on effect of irradiation on callus induction and regeneration in soybean are very scanty. Hence this investigation was attempted with the aim to study the effect of y-irradiation on callus induction and regeneration in soybean cultivar Bragg. Materials and methods: Seeds of soybean cultivar Bragg were given y-rays treatment (20kR and 25kR). 7-10 days old in vitro germinated seedlings of both treated and control were used to collect explants like cotyledon, cotyledonary node, nodal segment, leaf segment, and shoot tips. Different combinations of growth regulators (BAP, IAA, IBA, and 2,4-D) with MS basal medium were used to study their effect on the various explants. Explants inoculated with the culture from germination to callus induction, regeneration, and rooting were maintained at 25 +-2C temperature and a photoperiod of 16 hrs of light and 8 hrs of darkness. Observations were recorded for response on callus induction, number of days to callus induction, callus fresh weight, callus morphology, response to shoot differentiation, days for shoot differentiation, number of shoot buds per culture, response on root differentiation, days for root differentiation (for both direct and indirect pathway of regeneration). The experiment for all aspects was conducted in CRBD and the mean values of 5 aliquots were used in duplex for analysis. Results and Discussion: It was observed that callus induction occurred in all the explants of control. However in treated explants some of the treatment combinations failed to induce callus. Mean response for callus induction ranged from 47.30% (cotyledon, control) to 25.35% (leaf segments, 25kR) (Table.1). MS+2, 4-D 2mgl-1 gave the best response for both leaf and cotyledon explants. There was significant effect of irradiation as the response of treated explants was low. Gahukar and Jambhule, 2000, also found similar type of decrease in callus obtained with increased dose of gamma rays and EMS in sugarcane. The response for 20kR was better than 25kR. Mean callus weight ranged from 155mg (cotyledon, 25kR) to 273.23 mg (cotyledon, control). Similar decrease in callus fresh weight was observed by Reddy et al, 1987, in castor bean and by Singh and Singh, 1993, in sugarcane with the increase in the dose of irradiation. Higher doses of gamma irradiation caused considerable tissue damage, which perhaps in turn leads to reduction in callus fresh weight. The callus obtained was of embryogenic, rhizogenic, and non-embryogenic morphotypes. Rhizogenic callus failed to regenerate into shoot, owing to root differentiation and proliferation. Direct organogenesis was obtained from cotyledonary node, nodal segment, and shoot tips from both treated as well as control. Response for bud break was 71.48% in control, 62.32% in 20kR, and 57.20% in 25kR for cotyledonary nodes (Table.2). The average number of days taken for bud initiation was 4.1 days in control, 5.7 days in 20kR and 6.1 days in 25kR for cotyledonary nodes followed by nodal segments and shoot tips. Treated explants took more days than the controls for bud initiation. Nodal segments gave the highest mean number of buds (9.92 in control, 8.65 in 20kR, and 6.24 in 25kR) followed by cotyledonary node. Mean number of buds obtained from shoot tip was very low (3.65 in control, 3.23 in 20kR, and 3.05 in 25kR). Treatment MS+BAP 5mgl-1+IBA 0.1mgl-1 was best. Root induction took place in MS+IBA 0.8mgl-1 or NAA 1.0mg mgl-1 within 15-20 days. The influence of irradiation was on the lower side far all the aspects studied. This influence was linearly related to increase in concentration of gamma rays dose. Conclusion: Considering the frequency of regeneration both direct and indirect through callus and the effect of irradiation, an alternate method for obtaining additional variants can be followed to generate large quantity of callus for cell suspension culture on specific media, exerting selection pressure to screen out variant cells, which then can be allowed to regenerate to give somaclonal variants.
Tests of Linkage between Necrotic Root Locus Rn1 and Homozygous Chromosome Translocation KS172-11-3
Authors:
Reid G. Palmer
Abstract:
Introduction: Twenty linkage groups have been identified in soybeans that are designated as the classical genetic linkage (CGL) groups. Several linkage groups have only two loci, and two linkage groups have nine loci. Six translocation (interchange) genetic stocks have been cytologically characterized in soybean (Mahama et al., 1999). These chromosome translocations have been used to place gene order for mutants of CLG 6 and 8. In fact, these translocations indicated that linkage groups 6 and 8 were the same linkage group (same chromosome) (Mahama and Palmer, 2003). Thus linkage group 6 has been merged with linkage group 8, and is no longer considered a separate linkage group. A small F2 population (48 plants) suggested linkage of KS172-11-3 (homozygous chromosome translocation) with necrotic root, Rn1 locus (A. A. Mahama, unpublished results). One of the KS172-11-3 chromosome translocation breakpoints was linked to mutants on CLG 8 (Mahama and Palmer, 2003), which is molecular linkage group (MLG) F. The location of the other breakpoint is not known. Our objective was to test for linkage between the necrotic root locus (Rn1) and chromosome translocation KS172-11-3. Materials and Methods Plant materials The KS172-11-3 chromosome translocation line was crossed as female parent with grafted plants of T328 (rnl, rn1) (normal rootstock, necrotic root scion) as male parent. The F1 plants were about 50% pollen and ovule sterile (semisterile) which indicated successful hybridizations. Necrotic root mutant plants are usually lethal when field-grown. Thus about half of the F2 seed from self-pollination of each F1 plant was planted in the field (summer, 2006). The homozygous recessive necrotic root plants died. The surviving F2 plants were individually identified and pollen fertility/sterility determined by I2KI staining. If all the pollen gains from a plant were well-stained, the plant was classified fertile. This means that the fertile F2 plants were either homozygous normal chromosomes or homozygous translocated chromosomes. An F2 plant with about equal numbers of well-stained pollen and aborted pollen grains was considered heterozygous for the chromosome translocation. All the F2 plants were threshed individually and 20 F3 seed were germinated in a growth chamber. After eight days, the seedling roots were examined. The genotype of the F2 plants was determined to be homozygous dominant for normal root (Rn1, Rn1) or heterozygous (Rn1, rn1) based upon the segregation of normal and necrotic root phenotypes. The remainder of the F2 seed from self-pollination of each F1 plant was germinated in a growth chamber. Only the necrotic root seedlings were saved and transplanted to pots in the USDA greenhouse (summer, 2006). These F2 plants were individually identified and pollen fertility/sterility determined by I2KI staining, as was done with the non-necrotic root field grown plants. Based upon pollen grain staining phenotypes, the necrotic root plants were classified as either completely fertile or heterozygous for the chromosome translocation; ie, about 50% pollen and ovule sterile. Results Linkage test A total of 221 field-grown non-necrotic F2 plants were classified for pollen/sterility by I2KI staining. There were 107 fertile: 114 semisterile plants which was a good fit to the expected 1:1 ratio; 2 = 0.22, P = 0.64. In the USDA greenhouse, 74 necrotic root plants gave 35 fertile: 39 semisterile plants. This was a good fit to the expected 1:1 ratio, 2 = 0.22, P = 0.64. The combined field and greenhouse data gave a good fit to the expected 1:2:1:2:1:1 ratio; 2 = 0.70, P = 0.98 for the combined segregation of normal and necrotic root and for the chromosome translocation. Unexpectedly, seven greenhouse-grown necrotic root F2 plants were highly male sterile, as determined by I2KI staining. Repeated sampling of these seven plants on different days, gave pollen sterility values between 50% to near 100%. A few selfed seed were harvested from each of the seven plants grown in the summer of 2006, in the USDA greenhouse. These seeds were planted in January of 2007, in the USDA greenhouse and the plants flowered in April, 2007. All seven plants had both fertile pollen progeny and semisterile pollen progeny. Discussion Linkage test The rn1 locus was not linked to either of the breakpoints in chromosome translocation KS172-11-3. The rn1 locus is on MLG G (R. G. Palmer et al., submitted). The KS172-11-3 showed linkage with mutants on CLG 8 (Mahama and Palmer, 2003), which is MLG F. Additional linkage studies with KS172-11-3 are necessary to determine the other chromosome involved in the translocation. The seven necrotic root greenhouse F2 plants that had varying levels of sterility greater than 50% were all heterozygous chromosome translocation plants; ie, semisterile plants. Also only 7 of the 39 semisterile necrotic root plants expressed this higher level of pollen sterility and only on certain days. The necrotic root plants are weak plants. Thus it was not possible to collect floral buds to check meiosis. The pollen grain morphology of the plants with very high levels of sterility was similar to pollen morphology from homozygous asynaptic and desynaptic soybean mutants. An explanation for this observation awaits more detailed studies.