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

Authors: Sakhanokho, Hamidou; Babiker, Ebrahiem; Smith, Barbara; DRACKETT, PATRICIA - Mississippi State University

Submitted to: Plant Cell Tissue and Organ Culture
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/12/2019
Publication Date: 2/9/2019
Citation: Sakhanokho, H.F., Babiker, E.M., Smith, B.J., Drackett, P.R. 2019. High-frequency somatic embryogenesis, nuclear DNA estimation, and genome size stability of micropropagated milkweed (Asclepias spp.) plants. Plant Cell Tissue and Organ Culture. 137:149-156.

Interpretive Summary: Milkweeds (Asclepias spp. L) are considered pests by most farmers. In addition to competing with crops for nutrients, most milkweed species are toxic to livestock such as sheep and cattle because the leaves and stems of the plants contain toxins which are harmful to animals once ingested. Milkweeds have been disappearing at alarming rates. The extensive use of glyphosate-tolerant (Roundup Ready) soybean and corn as well as herbicide applications in roadside vegetation control are often cited as the reasons the widespread losses of milkweed. These milkweed losses pose serious risks to monarch butterflies whose populations have also been declining as monarch larvae feed solely on milkweed leaves. In addition to being critical to the survival of monarch butterflies, milkweeds provide food or shelter for diverse other insects. Also, milkweeds have been used for other purposes, such as source of fiber, food, medicine, nematicide, and insecticide. Modern day industrial applications include the use of milkweed floss-fiber for the manufacture of hypoallergenic pillows and floss-insulated high-end winter jackets and comforters. To reverse these milkweed losses which could potentially resulting in the extinction of monarch butterflies, serious efforts towards large-scale habitat restoration are necessary. These large-scale restoration projects require efficient and dependable propagation methods. For this reason, we developed a tissue culture method capable of mass-producing plants in three milkweed species, namely Asclepias latifolia (broadleaf milkweed), A. speciosa (showy milkweed), and A. subverticillata (horsetail milkweed).

Technical 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 1962) with Gamborg’s (1968) vitamins, 1.5 µM 2,4-D, 2.3µM 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. 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.