High-frequency somatic embryogenesis, nuclear DNA estimation of milkweed species (Asclepias latifolia, A. speciosa, and A. subverticillata), and genome size stability of regenerants

Abstract

A high-frequency somatic embryogenesis was developed for three Asclepias species, A. latifolia (broadleaf milkweed), A. speciosa (showy milkweed), and A. subverticillata (horsetail milkweed) using gibberellic (GA3) and the amino acid l-proline. A somatic embryo initiation medium consisting of MS salts (Murashige and Skoog, in Physiol Plant 15:473–497, 1962) with Gamborg’s (1968) vitamins, 1.5 µM 2,4-D, 2.3 µM kinetin, and 2% (w/v) sucrose supplemented with various concentrations of l-proline (0, 8.7, or 17.4 mM) combined with various of concentrations of GA3 (0, 2.9, or 5.8 µM), resulting in nine different media (MWM0–MWM8). All media produced callus, but no embryos were obtained on the control medium which contained no l-proline or GA3. Once calli produced somatic embryos, they were transferred to a medium referred to as somatic embryo conversion medium or SECM, which contained MS salts with Gamborg’s vitamins (Gamborg et al., Exp Cell Res 50:151–158, 1968), 2.3 µM kinetin, 2.9 mM GA3, 1.5% (w/v) sucrose, 8 g/L. The conversion percentage of somatic embryos into plants was high for all media, in particular for MWM2 (17.4 mM l-proline + 0 µM GA3) with conversion rates of 90.2, 93.4, and 97% for A. latifolia, A. speciosa, and A. subverticillata, respectively. Flow cytometry was used to estimate the nuclear DNA content of both seed-derived and in vitro grown plants. The 2C-DNA values of all three species were 0.92 pg, which did not differ from the values of in vitro grown plants, thus verifying that the regeneration system produces genetically stable plants.