Induction and Development of Somatic Embryos in In Vitro Cultures of Medicago sativa L. (Alfalfa)
Board Location: #76
Discipline: Biological Sciences
Subcategory: plant science
Session: 2
Laila Harris-Smith - Fort Valley State University
Co-Author(s): Suma Basak, Fort Valley State University, Fort Valley, Ga Sarwan Dhir, Fort Valley State Univeristy, Fort Valley, Ga Terri Brearley, Fort Valley State University, Fort Valley, Ga
Alfalfa (Medicago sativa L.) is a cultivated perennial forage legume valued for its nutritional and medicinal benefits.The goal of this study was to find chemicals to expedite the growing process.This study explored the effects of explant types, basal media, and varying concentrations of cefotaxime, carbenicillin, and activated charcoal on callus induction, somatic embryogenesis, and plant regeneration in alfalfa. Embryogenic callus formation (96.3%) was most efficiently achieved from leaf explants on Gamborg’s B5 (B5H) medium supplemented with 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 0.9 μM kinetin, 6.65 g glutamine, 0.8 g serine, 0.004 g adenine, and 0.08 g L-glutathione, incubated in the dark for three-week compared to other explant types (hypocotyl, petiole, and root) and basal media (MS and 1/2 MS). Somatic embryogenesis and shoot regeneration were influenced by cefotaxime (0–1000 mg/L), carbenicillin (0–700 mg/L), and activated charcoal (0–4% w/v). Optimal induction and maturation of somatic embryos, with globular structures reaching 93.8%, 86.4%, and 85.2%, occurred in media containing 500 mg/L cefotaxime, 450 mg/L carbenicillin, and 1.0% activated charcoal after three to four weeks of dark incubation. Higher concentrations of these additives impeded embryo development. Somatic embryos cultured on media with 500 mg/L cefotaxime, 450 mg/L carbenicillin, and 1.0% activated charcoal successfully regenerated into normal plantlets, with regeneration rates ranging from 65.4-79.8% on MS basal medium. However, concentrations exceeding 500 mg/L cefotaxime, 450 mg/L carbenicillin, and 2.0% activated charcoal led to abnormal somatic embryos and plantlets. Healthy plantlets were acclimatized on MS medium containing 3% sucrose and 0.03% GELRITE®, then transplanted into soil cups. After one week in a growth chamber, the plants were hardened and transferred to maturity in the field. This research will be put towards the regeneration protocol that will be helpful to produce multiple cultivars of alfalfa. This developed protocol will be used for Agrobacterium-mediated stable transformation or gene editing.
Funder Acknowledgement(s): National Science Foundation National Institute of Food and Agriculture (NIFA)(GEOX-5223- 336135)
Faculty Advisor: Sarwan Dhir, dhirs0@fvsu.edu
Role: For this study, I explored one explant type to see how well it could develop into calluses and eventually turn into Alfalfa plants. Over the course of three month, I did multiple trials of cutting Alfalfa leaves and putting them in B5H medium. I would continuously monitor them and transfer them into new medium until they reach final full-growth stage