S04 - Session O4 - Molecular insights to understand micropropagation - Studies of adventitious rooting in a tobacco mutant (rac) impaired in rooting

Authors: Margareta Welander *, Li-Hua Zhu

Adventitious root formation is of great importance for vegetative propagation and thus vital for horticultural industry. It is well known that auxin plays a crucial role in root initiation and several auxin induced genes have been isolated mainly from Arabidopsis hypocotyls. The rol B gene from Agrobacterium rhizogenes has proven to significantly stimulate adventitious rooting in several species. The rac mutant of Nicotiana tabacum L.Cv.Xanthii is impaired in rooting. The objective of the present study was to determine if the rol B gene introduced to rac could restore the rooting ability. The rac and wild- type shoots were multiplied in vitro on MS shoot multiplication medium containing 3% sucrose and 0.13 µM BAP. The Agrobacterium strain C58C1 holding the binary vector pCMB-B:GUS harboring nptII , rolB and gus genes was used for transformation. Two transformed clones were obtained confirmed by PCR. Unfortunately, the rooting ability was not restored. The rac can only be propagated in vitro due to lack of roots. To obtain seeds from rac for further studies on rooting we grafted rac shoots onto wild type seedlings. Grafted plants were planted in soil to obtained flowers. Fruit setting and seed production was poor in the rac. Selfing of both wild-type and rac was performed. Seeds from both wild-type and rac were surface sterilized and sown on Petri dishes with MS medium. Roots from seedlings penetrate seed coat after 3 days. After 6 days elongated roots were covered with root hair. Roots from rac penetrate seed coat first after 6 days. At day 8 root growth ceased. The root tip was swollen and covered with root hair. Since it has been shown that the root tip in the rac contains 14 times more auxin we were interested to investigate if the rac was impaired in some PIN genes responsible for lateral auxin transport from the root meristem. We performed western analysis with various PIN antibodies using protein extracts from petioles and leaves of wild-type and rac . Both PIN 2 and 3 gave good signals. Next will be immunolocalization of PINs in the root.