Protoplast isolation systems in blueberry (Vaccinium corymbosum) and mortiño (Vaccinium floribundum): a novel and efficient approach

Abstract

The Ericales encompass a diverse group of plants, including commercially significant species such as persimmon, blueberries, kiwifruits, Brazil nuts, argan, and azalea. However, to date, no research has been conducted on the isolation of protoplasts in blueberry species such as Vaccinium corymbosum L. and Vaccinium floribundum Kunth (mortiño). The use of protoplasts for in vitro propagation has become a powerful tool to overcome sexual incompatibility barriers between plant species or genera and to transfer genes for resistance to diseases, pests, herbicides, and other stress factors, thereby enabling the production of a large number of high-quality hybrid plants. Consequently, the isolation of a substantial quantity of protoplasts and the establishment of an efficient regeneration protocols are essential prerequisites for the successful advancement of modern botany. Nonetheless, the establishment of efficient protoplast-based systems remain a challenge for numerous crop plants. The present work outlines the state-of-the-art of protoplast isolation systems within the Ericales order. Furthermore, we have successfully established the first and highly efficient system for the isolation of blueberry protoplasts from leaves employing an enzymatic solution comprising 1% cellulase, 1.5% macerozyme, and 0.3% pectinase at incubation for 28 h resulting in a yield of 5.95 × 104 protoplasts (FW). Additionally, callus induction was achieved in mortiño leaf explants by using semi-solid Woody Plant Medium supplemented with 2.5 mg/L of 2,4-Dichlorophenoxyacetic acid. Subsequently, we developed a highly efficient system for the isolation of mortiño protoplast, employing an enzymatic solution containing 2.5% cellulase, 3% macerozyme, and 0.3% pectinase atincubation for 5 hours resulting in a yield of 1.05 × 105 protoplasts (FW). Here, we have reported for the first time a highly efficient system for protoplast isolation in blueberries, making a significant milestone in the advancement of new plant breeding technologies for these species and other related crops.