Agrobacterium-mediated genetic transformation system has been developed for two tomato (Lycopersicon esculentum Mill.) varieties, namely Pusa Ruby (PR) and BARI Tomato-3 (BT-3). Prior to the establishment of transformation protocol cotyledonary leaf explants from the two varieties were cultured to obtain genotype independent in vitro regeneration. Healthy multiple shoot regeneration was obtained from the cut ends of cotyledonary leaf segments for both the varieties on MS containing 1.0 mg/l BAP and 0.1 mg/l IAA. The maximum root induction from the regenerated shoots was achieved on half the strength of MS medium supplemented with 0.2 mg/l IAA. The in vitro grown plantlets were successfully transplanted into soil where they flowered and produced fruits identical to those developed by control plants. Transformation ability of cotyledonary leaf explants was tested with Agrobacterium tumefaciens strain LBA4404 harboring binary plasmid pBI121, containing GUS and npt II genes. Transformed cotyledonary leaf explants were found to produce multiple shoots on MS containing 1.0 mg/l BAP and 0.1 mg/l IAA. Selection of the transformed shoots was carried out by gradually increasing the concentration of kanamycin to 200 mg/l since kanamycin resistant gene was used for transformation experiments. Shoots that survived under selection pressure were subjected to rooting. Transformed rooted plantlets were transferred to soil. Stable expression of GUS gene was detected in the various tissues from putatively transformed plantlets using GUS histochemical assay.

Among the in vitro derived different explants such as cotyledonary leaf, hypocotyl, shoot tip and root of two local varieties, namely Singhnath and Kazla (BARI Begun-4) of eggplant (Solanum melongena L.) cotyledonary leaf was found to be the best for multiple shoot regeneration. High frequency direct organogenesis of shoots was achieved from cotyledonary leaf in MS supplemented with 1.0 mg/l BAP and 1.0 mg/l Kn. Anatomical studies using freezing microtome supported the formation of shoots through organogenesis. Proliferation and elongation of such shoots were obtained in hormone free MS. Moreover, the regenerated shoots produced healthy roots when they were cultured on MS without hormonal supplements. Following the formation of roots the in vitro raised plantlets were successfully established in soil. Viable seeds were obtained from the in vitro raised mature plants.

MS with different concentrations and combinations of hormones were used for in vitro multiple shoot regeneration in two varieties of Brassica juncea, namely, BARI Sarisha-11 and BARI Sarisha-16 and one variety of Brassica campestris, Tori-7. The highest percentage of responsive explants towards the regeneration of shoots was obtained on MS with 2.0 mg/l BAP, 0.2 mg/l NAA and 0.5 mg/l Kn in case of BARI Sarisha-11 and BARI Sarisha-16. For Tori-7, 3.0 mg/l BAP and 0.2 mg/l NAA was best for obtaining maximum number of shoots per explant. Among three varieties, BARI Sarisha-11 showed best response in terms of shoot regeneration as well as number of shoot per explant. Days required for induction of shoots was also recorded to be lowest in BARI Sarisha-11. Best root induction in BARI Sarisha-11 and BARI Sarisha-16 was achieved on hormone free MS. After proper hardening the in vitro regenerated plantlets were successfully transplanted into soil. Interestingly some of the in vitro regenerated shoots produced in vitro flowers on regeneration media as well as hormone free MS. Key words: Plant regeneration, Brassica juncea, Brassica campestris D. O. I. 10.3329/ptcb.v21i2.10235 Plant Tissue Cult. & Biotech. 21(2): 127-134, 2011 (December)

Genetic transformation using nodal and internodal segments from three economically important potato (Solanum tuberosum L.) varieties namely, Diamant, Cardinal and Granola was conducted using an Agrobacterium tumefaciens strain LBA4404 harbouring binary plasmid pBI12 containing the GUS and nptII genes. Node and internodal segments were used for direct regeneration as well as regeneration with the intervention of callus. Best responses were obtained for direct regeneration of shoots when the explants were cultured on MS supplemented with 4.0 mg/l BAP +1.0 mg/l IAA, 1.5 mg/l BAP + 0.5 mg/l IAA and 5.0 mg/l BAP +1.0 mg/l IAA in Diamant, Cardinal and Granola, respectively. In Diamant spontaneous in vitro microtuberization was obtained from these proliferated shoots. Further culturing of these in vitro grown green microtubers regenerated a large number of shoots on MS containing 4.0 mg/l BAP +1.0 mg/l IAA. By combining the best treatments, this protocol yielded an average transformation rate of 87% of treared explants. Stable expression of GUS gene was visualized in the various parts of transformed shoots through histochemical assay. Genomic DNA was isolated from transformed shoots and stable integration of the GUS and nptII genes was confirmed by PCR analysis.

Healthy multiple shoot regeneration was observed from petiole-attached cotyledon (PC) explants of CVL-1 on MS containing 0.2 mg/l BAP and 1.0 mg/l IAA. On the other hand, the best response for multiple shoot regeneration in CVE-3 was obtained when the same explants were cultured on MS with 2.5 mg/l BAP and 0.5 mg/l NAA. However, the same explant of D-154 was found to show less responsive compared to other two varieties to produce multiple shoots. Cotyledonary nodal explants of all the three varieties were found to produce maximum number of multiple shoots on MS supplemented with 0.2 mg/l BAP and 1.0 mg/l IAA. Best root induction was observed at the base of the in vitro regenerated shoots on half the strength of MS supplemented with 0.3 mg/l IBA. The in vitro grown plantlets were successfully transplanted into soil. They grew up to maturity, flowered and fruited like the control plants.