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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
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