Efficient in vitro regeneration of Leucaena leucocephala through direct organogenesis from cotyledonary node explants

The present study reports a reliable regeneration protocol for the commercially important genotype K-8 using hypocotyl and cotyledonary node explants derived from four-day-old aseptically germinated seedlings. Hypocotyl explants cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of 6-benzylaminopurine (BAP) or kinetin (Kn) produced friable callus but failed to regenerate shoots. In contrast, cotyledonary node explants showed direct organogenesis with 100% regeneration frequency on BAP-containing media. Maximum shoot multiplication (17.13 shoots per explant) was achieved on MS medium supplemented with 11.11 µM BAP, while higher concentrations reduced shoot length and number. Kinetin induced fewer shoots compared to BAP. Efficient rooting (100%) was obtained on half-strength MS medium supplemented with 2.5 µM indole-3-butyric acid (IBA). The regenerated plantlets were successfully acclimatized with a survival rate of approximately 90%. This protocol provides a simple and efficient regeneration system suitable for large-scale propagation and future genetic transformation studies in L. leucocephala.