Highly Efficient Transformation and Regeneration of Transgenic Aspen Plants Through Shoot-Bud Formation in Root Culture, and Transformation of Pinus Halepensis

Abstract

The natural capacity of aspen (Populus tremula) roots for shoot-bud regeneration was harnessed to establish a highly efficient transformation and regeneration procedure which does not require pre-selection with antibiotics. The procedure involved the use of wild type Agrobacterium rhizogenes and uidA (GUS) as the a reporter gene. High transient GUS expression was found in inoculated aspen stem segments, which then formed GUS-expressing adventitious roots. The roots proliferated in liquid culture and formed adventitious shoot buds expressing GUS activity. Kanamycin resistance, resulting from the expression of the nptII gene in the binary plasmid, was confirmed in transformed plants. Specific phenotypic changes in several transgenic aspen plants were monitored. The same A. rhizogenes strain was used for transformation of Pinus halepensis embryos, seedlings and adventitious shoots. Mature embryos were highly susceptible to the Agrobacterium, as detected by transient GUS expression in the radicle. Stable transformation was evidenced by the regeneration of GUS-expressing roots and calli from infected P. halepensis seedlings and adventitious shoots. PCR and Southern blot analyses confirmed the transgenic nature of aspen plants and transformed P. halepensis tissues.