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Plant Cell Tiss Organ Cult (2007) 90:215–223DOI 10.1007/s11240-007-9261-0ORIGINAL PAPERRose-scented geranium (Pelargonium sp.) generatedby Agrobacterium rhizogenes mediated Ri-insertionfor improved essential oil qualityGauri Saxena Æ Suchitra Banerjee Æ Laiq-ur-Rahman Æ Praveen Chandra Verma ÆG. R. Mallavarapu Æ Sushil KumarReceived: 20 February 2007 / Accepted: 12 June 2007 / Published online: 7 July 2007Ó Springer Science+Business Media B.V. 2007Abstract Transgenic plants of rose-scented geranium(Pelargonium graveolens cv. Hemanti) have beenproduced from Agrobacterium rhizogenes (strains A 4and LBA9402) mediated hairy root cultures. Amongstthe explants tested, leaves were most responsivefollowed by the petioles and internodal segments,respectively. The A 4 strain performed better for all thethree explants both in terms of frequency of responseand time requirement for hairy root induction. Transgenicshoots could be obtained by spontaneous regenerationwithout intervening callus phase amongst 16%and 12% root lines of A 4 and LBA 9402 origin,G. Saxena (&)Department of Botany, Lucknow University, Lucknow226001, Indiae-mail: gaurigupta72@yahoo.comS. BanerjeeCentral Institute of Medicinal and Aromatic Plants, P.O.CIMAP, Lucknow 226015, IndiaLaiq-ur-RahmanCIMAP Field Station, Puraura, IndiaP. C. VermaNational Botanical Research Institute, Rana Pratap Marg,Lucknow 226001, IndiaG. R. MallavarapuCIMAP Field Station, Bangalore, IndiaS. KumarNational Centre for Plant Genome Research, JNUCampus, P.O. Box 10531, New Delhi 110067, Indiarespectively, or they were induced in 29% and 22%hairy root lines of A 4 and LBA9402 origin, respectively,with different hormonal supplementation. These transgenicplants showed 30% survival as against 90% oftheir control under the confined environment of glasshouse.The transgenic plants were of similar morphotypehaving increased branching, higher number ofleaves with increased dentations, short and roundstature, highly branched root system and absence ofleaf wrinkling. These transgenic plants showed opinepositive results even after 5 months of their transfer tothe glasshouse. The essential oil compositions of 81%of these transgenics were qualitatively similar to that ofthe wild type parent. However, two transgenic plants(LZ-3 and 14TG) showed increase in concentrations ofgeraniol and geranyl esters signifying improved oilquality with respect to the citronellol:geraniol ratio.These two oils having better olfactory value representan improvement over that of the wild type parent fromthe commercial point of view.Keywords Geranium Pelargonium Agrobacterium rhizogenes Essential oilAbbreviationsMS Murashige and Skoog’s mediumIBA Indole butyric acidBAP 6-BenzylaminopurineNAA Naphthaleneacetic acidGC Gas chromatographyGC/MS Gas chromatography mass spectroscopy123

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

Plant Cell Tiss Organ Cult (2007) 90:215–223 219Fig. 1 Generation ofAgrobacterium rhizogenesmediated transgenic plantsin Pelargonium graveolensIndian cultivar Hemanti;A; Hairy root culturemaintained for 2 weeks inhalf strength, hormone freeliquid MS medium; B–C:regeneration of plantletsfrom hairy root culture insemi-solid and liquidculture media, respectively;D–E: in vitro raised rootedcontrol and transformedplants; F: Control (left) andtransformed (right) plantsafter 10 weeks of growth inpots exhibiting increasedbranching and highernumber of leaves in thelatter (· 0.10); G–H: flowerof control and transformed(LZ-3) plant exhibitingaltered morphology (· 3.5)Table 3 Frequency ofregeneration from hairyroots of Pelargoniumgraveolens cv. Hemantiinduced with A 4 and LBA9402 strains ofAgrobacterium rhizogenesGenetic resourceBacterial strain used for hairy rootinductionA 4 LBA 9402Total number of root lines 90 60Total number of root lines that exhibited regeneration 40 20Number of lines showing spontaneous regeneration 14 07Number of lines where regeneration was induced 26 13the plants as compared to that of the control(Fig 1D–F). The number of branches and roots/plants differ significantly as indicated by T-test(Fig. 2). Similar variations had also been reportedin the case of other Pelargonium species (Pellegrineschiet al. 1994; Pellegrineschi and Mariani1996). All the transgenics showed opine positiveresults even after 5 months of transfer to the pot.123

220 Plant Cell Tiss Organ Cult (2007) 90:215–223Fig. 2 Comparativeaccount of variousmorphological characters oftransgenic and controlplants of Pelargoniumgraveolens cv. Hemanti8070605040302010Control Plants TransgenicPlants0Plant height (cm)No. of branchesNodes/branchLeaves/branchMorphologicalcharactesrRoots/plantRoot length (cm)One of the 11 transgenic plants, LZ-3, producedflowers in 3 months with two inflorescences, eachbearing four flowers could be seen during the entirecultivation period and the flower size was smallerwith reduced petals and abnormality in their shape(Fig. 1G and H). The small flower size in thetransgenic plant in comparison to control was alsoreported in tobacco (Tepfer 1984). A hundred percentpollen sterility could be recorded through acetocarminestaining technique. The cuttings were usedfor maintaining and further multiplication of transgenicplants for three successive years.All the 11 transgenic plants along with theircontrol were subjected to quantitative and qualitativeanalysis of their essential oils after 5 months oftransfer to the pots. It was observed that all the plantsshowed stable behaviour in this duration. The oilcontents of all the transgenics were comparativelylower than the wild type (Table 4) in contrast to anearlier report (Pellegrineschi et al. 1994). Taking intoconsideration the number of leaves and branches, anoverall increase in the oil yield can be predicted fromthe transgenics since higher herb yield normally has astronger correlation with the oil yield per plant(Saxena et al. 2000). The analysis showed that theoils of nine transgenic plants (A6, 2TG, 5TG, 6TG,10TG, 12TG, 13TG, 15TG and 20TG) had compositionsimilar to that of the control Hemanti type(Table 4) which represents Chinese geranium oil inhaving higher amounts of citronellol and loweramounts of geraniol (Saxena et al. 2000). Theconcentrations of citronellol and geraniol as well astheir esters were found to be at par in thesetransgenics with those of the wild type oil. Amongthese nine transgenics, the oil of A6 was unique inhaving very low concentration of 10-epi-c-eudesmol(0.2%) which signifies better odour value (Teisseire1987). As compared to the oils of the other eighttransgenics and the control, the oil of 6TG was foundto contain lower concentration of isomenthone.Unaltered oil profiles of these transgenics mightrepresent the fact that the insertion sites of Ri TDNAin these plants do not interfere with the secondarymetabolites biosynthetic pathway as is mostly notedin majority of medicinal plants (Jung and Tepfer1987; Banerjee et al. 1998; Zehra et al. 1999).The RAPD analysis of these transgenics alsorevealed similarity at genetic level with that of thewild type in terms of most of the primers used.However, the primer OPT 1 revealed distinct differencesbetween the wild type and A6 in terms of thepolymorphic bands (Fig. 3) which might account forthe distinctiveness of A6 in terms of its unique oilprofile in having very low concentrations of 10-epi-ceudesmol.On the other hand, the difference inpolymorphic bands between the 15TG and controlwith regard to the same primer could not be corelatedwith any biochemical or morphologicalparameters in consideration, however changes inother physiological characters cannot be ruled out.Besides the above nine transgenics, the transgenicsLZ-3 and 14TG were found to differ in their oil123

Plant Cell Tiss Organ Cult (2007) 90:215–223 221Table 4 Oil content and percent concentration (±SD) of the terpenoid constituents in essential oils of transgenic plants and the wild type Pelargonium graveolens cv. HemantiCompound Control A-6 2TG 5TG 6TG 10TG 12TG I3TG 15TG 2OTG LZ-3 14TGOil content (%) 0.15 ± 0.01 0.11 ± 0.01 0.10 ± 0.06 0.06 ± 0.001 0.10 ± 0.01 0.09 ± 0.005 0.11 ± 0.005 0.05 ± 0.001 0.10 ± 0.04 0.10 ± 0.017 0.11 ± 0.010 0.11 ± 0.010Linalool 0.50 ± 0.04 1.00 ± 0.02 0.50 ± 0.02 0.30 ± 0.004 0.60 ± 0.008 0.50 ± 0.01 0.60 ± 0.016 0.40 ± 0.020 0.30 ± 0.005 0.90 ± 0.01 1.60 ± 0.04 0.90 ± 0.010Rose oxides (cis + trans) 2.50 ± 0.09 2.60 ± 0.14 1.60 ± 0.06 1.90 ± 0.07 0.70 ± 0.015 1.60 ± 0.09 1.80 ± 0.07 2.60 ± 0.16 2.40 ± 0.07 2.30 ± 0.02 1.60 ± 0.04 trIsomenthone 9.60 ± 0.40 10.9 ± 0.13 8.20 ± 0.11 9.50 ± 0.68 4.0 ± 0.28 8.00 ± 0.31 9.30 ± 0.37 9.40 ± 0.51 10.3 ± 0.49 9.50 ± 0.36 7.90 ± 0.33 1.80 ± 0.12Citronellol 47.0 ± 0.65 46.1 ± 0.45 45.6 ± 0.27 47.0 ± 0.62 54.4 ± 0.69 48.3 ± 0.78 52.4 ± 0.32 46.3 ± 0.66 46.9 ± 0.74 52.7 ± 0.55 35.7 ± 0.30 33.3 ± 0.88Geraniol 1.10 ± 0.11 0.70 ± 0.004 1.20 ± 0.06 0.50 ± 0.08 1.90 ± 0.09 0.80 ± 0.14 1.40 ± 0.06 0.40 ± 0.12 0.50 ± 0.08 2.00 ± 0.10 9.60 ± 0.40 7.90 ± 0.746- Caryophyllene 2.00 ± 0.09 2.10 ± 0.09 2.30 ± 0.12 2.50 ± 0.21 2.20 ± 0.05 2.10 ± 0.06 1.70 ± 0.03 2.30 ± 0.16 2.40 ± 0.10 1.60 ± 0.08 1.30 ± 0.06 2.00 ± 0.05Guaia-6,9- diene 2.70 ± 0.12 2.20 ± 0.09 1.40 ± 0.11 1.80 ± 0.06 1.40 ± 0.07 2.50 ± 0.10 2.00 ± 0.01 2.30 ± 0.08 1.80 ± 0.06 1.70 ± 0.54 0.30 ± 0.02 0.30 ± 0.01610-epi-Y-Eudesmol 3.80 ± 0.15 0.20 ± 0.01 4.40 ± 0.14 3.00 ± 0.21 5.00 ± 0.07 4.20 ± 0.05 4.20 ± 0.09 4.20 ± 0.08 3.00 ± 0.12 3.00 ± 0.14 6.60 ± 0.15 10.3 ± 0.27Citronellyl esters 20.4 ± 0.43 23.8 ± 0.49 23.2 ± 0.81 22.8 ± 0.72 19.7 ± 0.41 19.6 ± 0.96 17.0 ± 0.69 21.6 ± 0.62 22.7 ± 0.78 17.5 ± 1.16 12.9 ± 0.65 14.9 ± 0.88Geranyl esters 2.20 ± 0.09 1.50 ± 0.06 1.90 ± 0.04 1.50 ± 0.03 2.60 ± 0.09 1.80 ± 0.12 2.30 ± 0.12 1.10 ± 0.05 1.80 ± 0.03 2.00 ± 0.16 9.00 ± 0.19 14.8 ± 0.73tr, traceFig. 3 PCR profiles of DNA isolated from 11 transgenic plantsand their parent Pelargonium graveolens cv. Hemanti amplifiedwith primer OPT1 5 0 GGGCCACTCA3 0 and resolved on0.8% agarose gel. Samples in gel lanes (from right to left) areas follows: Lane 1: marker k DNA digested with HindIII ; Lane2: wild type Pelargonium graveolens cv. Hemanti; Lanes 3,4and 5: 2TG (in replicates); Lane 6: 5TG; Lane 7: 6TG; Lane 8:LZ-3; Lanes 9 and 12: 14TG (in replicate); Lane 10: 10TG;Lane 11: 12TG; Lane 12: 14TG; Lane 13: 13TG; Lane 14:15TG; Lane 15: 20TG; Lane 16: A-6profiles from that of the control oil (Table 4). Thesetwo oils had comparatively lower concentration ofcitronellol (33.3% and 35.7%) and higher concentrationof geraniol (6.6% and 10.3%) than the control oilwhere citronellol was 47% and geraniol was 2.1%.Since the citronellol:geraniol ratio in geranium oilsdetermines their odour value (Lawrence 1984) thesetwo oils represent better quality oils resemblingBipuli type (Rao et al. 1999). The oils of these twotransgenic plants also possess relatively higheramounts of linalool, geranyl esters and 10-epi-ceudesmolas compared to that of the control. On theother hand, the concentrations of 6, 9-guaiadiene andthe esters of citronellol were found to be low in thesetwo oils as compared to the oil of the control. Further,the concentrations of isomenthone and rose oxideswere quite low (1.8% and trace, respectively) in oneof these two oils (14TG) as compared to the oil ofcontrol (9.6% and 2.5%, respectively). At the geneticlevel these two transgenics resembled each other withregard to OPT 1 primer while distinct differencescould be noted between them from the others alongwith the wild type control with regards to sameprimer (Fig. 3) which further substantiates theirtotally altered oil profile in terms of the majorterpenoid constituents. On the basis of overallanalysis, it can be stated that since the increase ingeraniol and geranyl esters concentration signifiesimproved olfactory value (Rao et al. 1999), the oils ofthese two transgenics definitely represent an improvementover the control from the commercial point of123

222 Plant Cell Tiss Organ Cult (2007) 90:215–223view. Reduction in citronellol concentration andincrease in geraniol concentration had earlier beennoted in three transgenic plants of lemon-geranium ofhairy root origin (Pellegrineschi et al. 1994).The present study once again highlights thesuccess of hairy root transformation strategy for thecreation of new fragrances through the easiest andquickest method of establishment of transgenic plantsfrom hairy root cultures.Acknowledgements The authors are thankful to theDepartment of Biotechnology, Government of India for thefinancial support and the Regional SophisticationInstrumentation Facility, CDRI, Lucknow for GC-MS of theoils. Thanks are also due to S. Sharma and S.A. Hasan for theirhelp in manuscript preparation and A.P. Dhiman forphotography.ReferencesAdams RP (1995) Identification of essential oils by gas chromatographymass spectroscopy. 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