An investigation on the cause of recalcitrance to genetic transformation in soybean,glycline max (L.) merrill

Abstract

Genetic transformation offers great opportunities for rapidly introducing, selecting or inducing desired characteristics in various leguminous plants for breeding purposes. But, this technique remains aloof for soybean improvement due to challenges such as genotype specificity, inefficient regeneration protocols and the rapid loss of viability in seeds required to develop explants. However, the rate of seed deterioration and its influence on in-vitro plant genetic transformation differs according to the age, storage duration and moisture content of the seeds used. The moisture status of the seeds is usually high during harvesting and deterioration (loss of viability) starts to occur when seeds are stored under ambient conditions for long periods. This seed deterioration also results in a phenomenon called “recalcitrance”, which is predominantly realised in soybean. In the present study, selected soybean genotypes were analysed for: (i) the efficiency of germination using seeds stored for 0, 3, 6 and 9-months under ambient conditions (ii) the effect of seed storage on in-vitro multiple shoot induction, (iii) the competency of the selected soybean genotypes on callus induction and Agrobacterium-mediated genetic transformation and (iv) the evaluation of protein profiles of the genotypes following co-cultivation of cotyledonary node explants with A. tumefaciens. The results obtained in this study showed that, seed stored for more than 3-months had reduced rates of germination, seedling development and in-vitro shoot multiplication. In particular, seed stored for 9-months showed a significant drop in seed germination, and less than 50% overall seed germination (Dundee-42%, LS678- 49%, TGx140-2F-44% and TGx1835-10E-48%) except for LS677 and Peking with 52 and 55%, respectively. The efficiency of multiple shoot induction also decreased with the prolonged seed storage, with all genotypes recording overall decline from about 96% to 40% regeneration efficiency over this period. The mean number of induced shoots decreased from more than 10.5 to 4.2 shoots per explant, for each genotype. The results obtained clearly indicated that efficient in-vitro shoot induction depended largely on seed storage duration, viability and significantly differed according to genotype. Following the evaluation for callus induction and Agrobacterium-mediated genetic transformation frequencies, the results indicated that the responses were genotype specific. This trend was consecutively observed in all soybean cultivars used (LS677, LS678, Dundee, Peking, TGx1740-2F and TGx1835-10E). Furthermore, the responses of the genotypes were also dependent on the culture media composition,especially, plant growth regulators and antibiotics. Amongst the cultivars used, Peking demonstrated the highest callus induction capacity (more than 70%) on MS-A and the mean number of shoots induced (1.65) using cotyledonary explants co-cultivated with Agrobacterium. This was followed by LS677 (1.42 shoots), LS678 (1.40 shoots), Dundee (1.30 shoots), TGx1835-10E (0.80 shoots) and TGx1740-2F (0.75 shoots), respectively. These genotypes also demonstrated low yields of proteins, extracted using a TCA buffer, and separated by means of two-dimensional polyacrylamide gel electrophoresis. The one-dimensional and two-dimensional profiles of proteins extracted from explants infected with Agrobacterium differed significantly to those expressed without co-cultivation of cotyledonary nodes with bacteria. These observations suggested that, the infection and co-cultivation of explants with Agrobacterium may have caused the expression of new proteins. Newly expressed proteins could also be found to either promote or inhibit transgene integration and expression on the cotyledonary node explants transformed with Agrobacterium tumefaciens for trait improvement. This study has clearly demonstrated that soybean production is confronted with a myriad of stress factors, including seed storage and quality problems due to unfavourable storage duration and weather conditions, amongst others. Thus, soybean seeds used for germination, callus induction, multiple shoot induction and genetic improvement should be harvested at R8 stage after reaching physiological maturity (with 20-35% seed moisture content) to avoid any mechanical damage, shattering or loss of seed viability.