Imposed starvation for in vitro maintenance and high regeneration of elite key lime (Citrus aurantifolia)

Preservation of elite key lime (Citrus aurantifolia) genotypes using efficient micropropagation and regeneration protocols is very important for new breeding programmes. The objective of the present study was to develop an efficient protocol for micropropagation and maintenance of elite genotypes of key lime based on slow growth and starvation strategy. To do this, regenerated shoots were starved, and the growth was compromised via avoiding subcultures for 4, 8, 12 and 24 months. The numbers of responsive explants and adventitious shoots per each explant were evaluated in the presence of benzylaminopurine (BAP). The results showed that the 12- and 24-month starved explants treated with 1 mg/l BAP were able to produce four times more shoots than the control. In addition, the influences of horizontal or vertical layering explants over medium were evaluated. The results indicated that all horizontally layered explants significantly produced 25% adventitious shoots more than vertical explants, most likely owing to more contact with the surface medium. Finally, it could be concluded that application of slow growth, starvation strategy and also horizontally layering of the explants could efficiently enhance micropropagation and maintenance of threatened elite key lime genotypes with a low cost.