Reducing etiolation-like effect and flowering in an in vitro micropropagated Trifolium resupinatum elite genotype

There is an ever-increasing demand for high quality forage legumes, essential to feed livestock. Micropropagation may be useful to preserve elite allelic compositions in allogamous and auto-incompatible forage legume species, as is the case of Trifoliumresupinatum L. (Persian clover). Etiolation-like phenotypes in in vitro cultured plants, with long weak stems and sparse leaves, together with flowering, compromises the long term establishment and maintenance of explants and the adequate development of plants when transferred to ex vitro. Aiming to develop a solution to limit the impact of in vitro etiolation-like effects, flowering and plant viability, we used a very prone to etiolate elite T. resupinatum genotype maintained through in vitro propagation for over 1 year. Stem segments were subcultured in Murashige and Skoog (MS) supplemented with 2.22, 4.56 or 9.12 µM of zeatin; with 4.44 or 8.88 µM of benzyladenine or with 2.89 or 14.44 µM of gibberellic acid. Gibberellic acid supplementation was detrimental and ultimately led to the death of the explants. Supplementation with 2.22 µM zeatin produce no significant differences from control; however higher concentrations of zeatin, as well as of benzyladenine, diminished the longer internodes and increased the development of shoots with more stout stems. Although rooting was prevented by cytokinins, it was successfully induced when explants were transferred to MS media without plant growth regulators. The 9.12 µM zeatin condition was the most favorable since it reversed the etiolation-like phenotype, inhibited in vitro flowering and improved acclimation from 28.7 to 90 % in the FTTr07.13 T. resupinatum genotype.