Using Oxygen Bioavailability and Somatic Embryogenesis Techniques for Regeneration of Old Seeds of Common Bean (Phaseolus vulgaris L.)

Abstract

The deterioration of the viability of seeds of old varieties (heirloom varieties) by exposure to long storage periods is one of the main problems facing plant breeding programmes; Thus, oxygen bioavailability and somatic embryogenesis techniques can overcome this problem to some extent. The goals of this study were to: 1) define the optimal oxygen bioavailability concentration for increasing the germination rates of old and waterlogged seeds; 2) define the relationship between the concentration of bioavailable oxygen in the liquid ingested by the germinating seed and seed vigor; and 3) use somatic embryogenesis techniques as a method for recovering plant germplasm from old or improperly stored seeds for use in plant breeding programs. This study was conducted in the tissue culture laboratory of the Potato and Vegetatively Propagated Vegetables Department, Horticulture Research Institute, Agricultural Research Centre, Egypt. This research was conducted to regenerate perennial seeds (8 years) of common bean (Phaseolus vulgaris L.) variety Giza 6 (local variety) and compared with 1 year old seeds as control treatment of the same cultivar. This study established that prolonged hypoxia throughout a protracted period of storage can inhibit germination. Hypoxia during the impregnation and germination of bean seeds can be overcome by supplying the seeds with hydrogen peroxide (20 mM) throughout the germination process. Catalase enzyme (10.5 units/mL) is needed to convert hydrogen peroxide into oxygen. Also, to release oxygen from hydrogen peroxide, other catalytic agents, such as transition metal ions like Fe+2, Cu+2, and Mn+2, can be used at the following concentrations: 0.001M, 0.001M, and 0.0001M, respectively. A protective layer of CaSO4 (0.5 mM) was applied since aged seeds have very fragile biological membranes. The test of tetrazolium gave results for some seeds that it non-viable or some parts of the seed are damaged or defective; However, 2,4-D at a concentration of 5 mg/l + supplements (0.1 µM GA3/L + 2 mg kintien/L + 1.0 mM glutamine) was able to produce somatic embryos of the old seeds tissues for common bean cv. Giza 6 (a local variety). The study of the morphology and induction of somatic embryogenesis was done used scanning electron microscopy. In its early phases, the callus appeared as a scattering of elongated cells. And after being transferred to the DCR medium, the cells became tightly packed together, and globular-shaped embryos began to emerge between the tightly packed together cells.