Doubling Acclimatization Survival of Micropropagated American Chestnuts with Darkness and Shortened Rooting Induction Time

Abstract

One of the most difficult processes of micropropagation is rooting and acclimatizing in vitro shoot cultures, especially for hardwood tree species. As more transgenic lines of potentially blight-resistant American chestnut ( Castanea dentata ) are developed, we expect to produce thousands of tiny shoots to be rooted, transferred to potting mix, and grown to a large enough size for planting outdoors. Many shoots are lost during rooting and acclimatization, so pinpointing factors that enhance survival is extremely important. Five factors were examined in relation to acclimatization success — light intensity, light color, time in rooting medium, temperature, and presence of activated charcoal. The percentage of plantlets surviving from rooting initiation to 16 weeks in the growth chamber was increased from 33 to 67% by rooting the shoots in darkness instead of on a light bench. The best combination of rooting factors was to place shoots in rooting medium containing activated charcoal in complete darkness for only four days at 25C. This combination of factors increased plantlet survival from approximately 33% using the original rooting protocol to 73%. Finding that American chestnut plantlets have better acclimatization survival after being placed in rooting medium for only four days should enhance many laboratory practices. Shortening the time in rooting medium and including a period in darkness will increase the survival of novel transgenic American chestnut lines, allowing them to be planted in field trials more quickly.