SUCROSE LEVEL INFLUENCES MICROPROPAGATION AND GENE DELIVERY INTO LEAVES FROM IN VITRO PROPAGATED HIGHBUSH BLUEBERRY SHOOTS

Authors: CAO, XIAOLING - VISITING SCIENTIST; FORDHAM, INGRID - 1275-11-00; DOUGLASS, LARRY - UNIVERSITY OF MD; Hammerschlag, Freddi

Submitted to: Plant Cell Tissue and Organ Culture
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/27/2003
Publication Date: 5/27/2003
Citation: Cao, X., Fordham, I., Douglass, L., Hammerschlag, F.A. 2003. Sucrose level influences micropropagation and gene delivery into leaves from in vitro propagated highbush blueberry shoots. Plant Cell Tissue And Organ Culture. 75:255-259.

Interpretive Summary: Genes can now be moved from one type of plant to another by artificial gene transfer techniques. This allows scientists to transfer genes for desirable traits into plants that lack these traits. However, there are some requirements for doing this successfully: an efficient and proven method for moving the gene from one type of plant to another; methods to determine which of the treated plants actually have the new gene and the new trait; and a method to increase those plants - such as through tissue culture -so that plants can be provided quickly to growers and researchers. The problem researchers encounter is that sometimes the methods to achieve these separate results conflict with each other. We investigated how to increase the number of blueberry shoots we can get in tissue culture without negatively affecting the success of our gene transfer methods. Since sucrose concentration is critical for getting good shoot proliferation in tissue culture, we tested the effects of sucrose on shoot proliferation of three important blueberry cultivars (Bluecrop, Duke and Georgiagem), and also on the efficiency of transferring genes. We found that the best sucrose concentrations for shoot proliferation reduced our ability to transfer genes into these cultivars. Based on these results, we developed a growth medium with a sucrose concentration that gives us optimal shoot proliferation and gene transfer. These studies will be of value to other scientists who are interested in using gene transfer methods for improving blueberry.

Technical Abstract: In an effort to increase in vitro blueberry (Vaccinium corymbosum L.) shoot production without negatively impacting subsequent genetic engineering experiments, studies were conducted to examine the effects of sucrose concentration in the shoot multiplication medium on in vitro shoot proliferation and on the transfer of an intron-containing B-glucuronidase (GUS) gene into leaf explants from the in vitro propagated shoots. Shoot (> 0.5 cm) production did not increase significantly for 'Bluecrop' when sucrose levels were increased from 15 mM to either 29, 44 or 58 mM, but did increase significantly for Duke when concentrations were increased from 15 mM to 44 mM, and for 'Georgiagem' when sucrose was increased from 15 mM to 58 mM. Four days of cocultivation with Agrobacterium tumefaciens strain EHA105 yielded maximum GUS-expressing leaf zones on leaf explants from shoots cultured on either 15 or 29 mM sucrose. The number of GUS-expressing leaf zones was significantly less on leaf explants from shoots on 58 mM sucrose than from those on 15 mM sucrose for all three cultivars and significantly less on 44 mM compared to 15 mM for cultivars Duke and Georgiagem. These studies suggest that shoot pretreatment conditions need to be considered for optimizing subsequent blueberry genetic engineering experiments. Thus, a blueberry shoot multiplication medium containing 15 to 29 mM sucrose is recommended to support multiplication when multiplication will be followed by gene delivery studies.