Effect of loss of T-DNA genes on MIA biosynthetic pathway gene regulation and alkaloid accumulation in Catharanthus roseus hairy roots
Genetically transformed root cultures of Rauvolfia serpentina, have been developed following transformation with agropine strains of Agrobacterium rhizogenes. Characterization of Ri transformed root lines on the basis of morphology, karyotype analysis, T-DNA gene integration pattern and ajmalicine content in R. serpentina has been described in the present study. The transformed root lines were morphologically similar and exhibited typical hairy root phenotype. The somatic metaphase plates of the transformed root lines showed 2n = 22 chromosomes which is the diploid chromosome number of the species. The karyotype of both non-transformed and transformed roots showed 20 chromosomes with median to nearly median primary constriction and one pair of chromosomes had two constrictions, one at the median region and the other at the sub terminal region. Based on the pattern of TL and TR gene insertion the Ri transformed root lines can be divided into six types. Complete integration of TL-DNA was noted in 11 root lines induced with strain A. rhizogenes LBA 9402 belonging to Type I and II it is noteworthy that none of the root lines showed presence of aux1 and aux2, which are part of TR-DNA. Variable integration of TL-DNA was characteristic of several R. serpentina root lines. The Ri transformed root lines showed significant variability (p ≤ 0.05) in ajmalicine content (0.004 ± 0.001 to 0.229 ± 0.014 mg g−1 DW) after 4 weeks of culture on solid modified MS medium. Thus, we can conclude that the Ri transformed root lines of R. serpentina are morphologically and cytologically stable however variable pattern of TL and TR-DNA genes integration as well as ajmalicine content was noted among the different root lines. Thus, genetically variant transformed root lines of R. serpentina with high ajmalicine content can be selected for scale up studies.