Effect of explant orientation, pH, solidifying agent and wounding on initiation of soybean somatic embryos

Abstract

Several methods have been developed to obtain somatic embryos of soybean. We report here a new procedure that results in high frequency somatic embryo initiation in a short period of time. Somatic embryos were induced from immature cotyledons of the cultivars “Jack,” “Thorne,” “Resnik,” and “Chapman.” Immature cotyledons were cultured on a medium containing MS salts, B5 vitamins, 6% sucrose, and 40 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). Culture modifications included: orientation of the explants (adaxial or abaxial side of the cotyledon in contact with the medium), adjustment of medium pH (5.7 or 7.0), wounding of cotyledons with scalpel blades, inclusion of ethylene modulators, and use of Noble agar or Gelrite™ as the solidifying agent. The treatment that resulted in the highest embryo induction across the cultivars consisted of abaxial side of the explant facing the medium, pH 7.0 and 0.2% Gelrite™. “Jack” was the most responsive cultivar showing the first embryos as early as 14 d after culture. After 21 d, an average of 44 embryos per cotyledon was obtained with this cultivar. The inclusion of silver nitrate (AgNO3) in the culture medium did not enhance the number of primary somatic embryos induced per cotyledon, but the addition of 15 µM AgNO3 did result in a faster production of secondary embryos using the cultivar “Jack.” Wounding of the explants with a scalpel resulted in an earlier induction of somatic embryos. Embryo initials were first observed after only 7 d. Histological examination of cultured cotyledons indicated that the somatic embryos originated from the subepidermal tissues and were of multicellular origin. This somatic embryo induction procedure could be useful for direct transformation work and permits the production of embryogenic tissue within 2 wk.