Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 137, Issue 1, pp 149–156 | Cite as

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

  • Hamidou F. SakhanokhoEmail author
  • Ebrahiem M. Babiker
  • Barbara J. Smith
  • Patricia R. Drackett
Original Article


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.


Broadleaf milkweed Genetic stability Horsetail milkweed Monarch butterflies Showy milkweed Somaclonal variation 



Gibberellic acid A3



KNOX genes

Knotted1-like homeobox genes


Murashige and Skoog’s medium (1962)


Milkweed medium


2,4-dichlorophenoxyacetic acid



The authors are grateful to Denise Hardy and Robin Hayes for technical assistance. This work received the financial support of USDA-ARS CRIS project 6062-21000-009-00D. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture. USDA-ARS Equal Employment Opportunity (EEO) Policy mandates equal opportunity for all persons and prohibits discrimination in all aspects of the agency’s personnel policies, practices, and operations.

Author Contributions

HFS conceived, conducted experiments, and analyzed data. EMB contributed to the conception and writing of the manuscript. BJS helped organize and edit the manuscript. PRD helped select Asclepias species and write the manuscript.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.


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Copyright information

© This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply 2019

Authors and Affiliations

  1. 1.Thad Cochran Southern Horticultural LaboratoryUnited States Department of Agriculture, Agricultural Research ServicePoplarvilleUSA
  2. 2.The Crosby ArboretumMississippi State University Extension ServicePicayuneUSA

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