Isolation of morphovariants through plant regeneration in Agrobacterium rhizogenes induced hairy root cultures of Plumbago rosea L.

In vitro raised shoots of Plumbago rosea L. were infected with A4 strain of Agrobacterium rhizogenes to initiate hairy root formation, which produced 3.0+/-0.33 hairy roots per incision on explants in 20 d incubation. Southern blot analysis confirmed the integration of T-DNA into the genome of the roots. The hairy roots were cultured on MS agar medium supplemented with 2.0 mg/L BAP to induce the formation of shoots (3.2+/-0.24) of 0.2-0.4 cm length in 7-8 wks. Isolated shoots were multiplied through sub culturing in the presence of 0.5 mg/L BAP and the resultant shoots were subjected to combined elongation (3.29+/-0.16 cm) and rooting (12.6+/-0.57) in a medium supplemented with 0.1 mg/L IBA. The rooted plants were invariably abnormal with short internodes and wrinkled leaves showing 34.5% establishment in 2 months after transplantation in polybags and rearing under 75% sunlight in a shade house. Out of 38 plants transferred to the field, 20 (52.6%) survived and grew over 10-month period to reveal variations in morphological and growth characters. The growth of all the hairy root-derived plants was slow to varied extent, 13 among them retaining actively growing hairy roots and shoots having short internodes and expanded leaves, one with emerging normal shoot and a tuberous root in the midst of the abnormal shoot and hairy roots and the rest with hairy roots as well as 1-2 normal roots and abnormal shoots having wrinkled leaves. The biomass production ability of the transformed plants contributed by foliage and root characters was poor compared to plants raised from the nodal explants of normal shoot cultures. Southern blot analysis of DNA further confirmed the presence of bacterial T-DNA in these established plants in the field after 10 months. The demonstrated hairy root regeneration system including field establishment of the plants may be useful for scoring new variations especially in non-seed setting P. rosea.