Rose-scented geranium - CIMAP Staff - Central Institute of Medicinal ...

218 Plant Cell Tiss Organ Cult (2007) 90:215–223RAPD analysis was done according to the protocol byShoyama et al. (1997). Two transgenic plants (2TGand 14TG) were analyzed in replicates in order todemonstrate genetic uniformity within the clone.Results and discussionA transformation frequency from leaf explants wasthe highest, followed by the petioles and internodalsegments (Table 2). However, young leaf petioles didnot produce hairy roots in any of the Pelargoniumspecies tested (Pellegrineschi and Mariani 1996).Although transformation frequencies was high withboth the bacterial strains used in this study, as hasalso been reported in lemon-scented geranium (Pellegrineschiet al. 1994). Amongst the two bacterialstrains used, A 4 proved to be effective than LBA9402, both in terms of frequency of response and timerequirement for hairy-root induction (Table 2). Similarobservations had also been reported earlier fordiverse plant species (Porter 1991; Rodrigues et al.1991; Banerjee et al. 1994; Giri 1997; Zehra 1998).In view of the fact that each transformation eventis distinct from the other due to different integrationsites and copy numbers of Ri T-DNA (Tepfer 1984),90 independent hairy-root lines of A 4 origin and 60 ofLBA 9402 origin were selected for regenerationstudies (Fig. 1A). Amongst these, 14 regenerants byA 4 and seven root lines by LBA 9402 origin showedspontaneous regeneration without the interveningcallus phase within 8–10 weeks of subculturing inhormone-free liquid and semi-solid MS medium(Fig. 1B and C; Table 1). Spontaneous regenerationof plants from hairy root cultures has already beenreported in Pelargonium (Pellegrineschi et al. 1994;Pellegrineschi and Mariani 1996; Banerjee et al.1997; Perez-Morphe-Balch and Ochoa-Alejo 1987).Direct organogenesis was observed in 26 and 13 ofthe hairy root lines of A 4 and LBA 9402 origin,respectively (Table 3), within 6–8 weeks of culturewith different hormonal combinations.Only two of the five regeneration media testedwith varying concentrations of NAA and BAPresponded both in their semi-solid and liquid state.A maximum of 6–8 shoots per root explant wasproduced after 7 weeks of culture containing 0.5 mg/lBAP and 0.1 mg/l NAA (Table 1). These concentrationswere lower than those as reported in regalPelargonium (Pelargonium · domesticum Dubonnet)(Boase et al. 1998). Various combinations of differenthormones including NAA, BAP, zeatin as well assupplementary vitamin source and PVP-10 have beenused for inducing direct and indirect regeneration oftransgenic shoots through transformation with A.tumefaciens in leaf discs of Zonal (Pelargonium· hortorum) and scented (P. capitatum)geranium (Hassanein et al. 2005).It is pertinent to mention here, that in addition todifferent hormonal combinations, reduction in theconcentrations of sucrose as well as that of othernutrients to half appears to be an essential prerequisiteof the present study in order to optimize the shootregeneration efficiency from hairy root cultureswhich has not been considered earlier (Boase et al.1998).All the transgenic plantlets of the present study,irrespective of their spontaneous or hormone-mediatedorigin, exhibited spontaneous rhizogenesis upontransfer to hormone-free half strength MS mediumwithin 2 weeks of transfer (Fig. 1E).A total of 11 transgenic plants out of the total36 tested were successfully transferred to theglasshouse. The leaves of all transformed plantswere darker in colour, more dentated and leafwrinkling was absent in all of them as has also beenreported earlier (Pellegrineschi et al. 1994).The transformants also had highly branched rootsystems, shorter internodes, increased number ofleaves and branches and short and round stature ofTable 2 Susceptibility ofdifferent explants ofPelargonium graveolens cv.Hemanti to A 4 and LBA9402 strains ofAgrobacterium rhizogenesBacterial strainsTransformation frequencyInternode Petiole LeafA 4 50 ± 3.2 71.3 ± 3.3 100 ± 0.0LBA 9402 20 ± 1.9 50 ± 2.6 82 ± 4.1123