Rose-scented geranium
Plant Cell Tiss Organ Cult (2007) 90:215–223 DOI 10.1007/s11240-007-9261-0
ORIGINAL PAPER
Rose-scented geranium (Pelargonium sp.) generated by Agrobacterium rhizogenes mediated Ri-insertion for improved essential oil quality Gauri Saxena Æ Suchitra Banerjee Æ Laiq-ur-Rahman Æ Praveen Chandra Verma Æ G. R. Mallavarapu Æ Sushil Kumar
Received: 20 February 2007 / Accepted: 12 June 2007 / Published online: 7 July 2007 Springer Science+Business Media B.V. 2007
Abstract Transgenic plants of rose-scented geranium (Pelargonium graveolens cv. Hemanti) have been produced from Agrobacterium rhizogenes (strains A4 and LBA9402) mediated hairy root cultures. Amongst the explants tested, leaves were most responsive followed by the petioles and internodal segments, respectively. The A4 strain performed better for all the three explants both in terms of frequency of response and time requirement for hairy root induction. Transgenic shoots could be obtained by spontaneous regeneration without intervening callus phase amongst 16% and 12% root lines of A4 and LBA 9402 origin, G. Saxena (&) Department of Botany, Lucknow University, Lucknow 226001, India e-mail: [email protected] S. Banerjee Central Institute of Medicinal and Aromatic Plants, P.O. CIMAP, Lucknow 226015, India Laiq-ur-Rahman CIMAP Field Station, Puraura, India P. C. Verma National Botanical Research Institute, Rana Pratap Marg, Lucknow 226001, India G. R. Mallavarapu CIMAP Field Station, Bangalore, India S. Kumar National Centre for Plant Genome Research, JNU Campus, P.O. Box 10531, New Delhi 110067, India
respectively, or they were induced in 29% and 22% hairy root lines of A4 and LBA9402 origin, respectively, with different hormonal supplementation. These transgenic plants showed 30% survival as against 90% of their control under the confined environment of glasshouse. The transgenic plants were of similar morphotype having increased branching, higher number of leaves with increased dentations, short and round stature, highly branched root system and absence of leaf wrinkling. These transgenic plants showed opine positive results even after 5 months of their transfer to the glasshouse. The essential oil compositions of 81% of these transgenics were qualitatively similar to that of the wild type parent. However, two transgenic plants (LZ-3 and 14TG) showed increase in concentrations of geraniol and geranyl esters signifying improved oil quality with respect to the citronellol:geraniol ratio. These two oils having better olfactory value represent an improvement over that of the wild type parent from the commercial point of view. Keywords Geranium Pelargonium Agrobacterium rhizogenes Essential oil Abbreviations MS Murashige and Skoog’s medium IBA Indole butyric acid BAP 6-Benzylaminopurine NAA Naphthaleneacetic acid GC Gas chromatography GC/MS Gas chromatography mass spectroscopy
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Introduction Geranium oils extracted from Pelargonium species (scented geraniums) have been used in the perfumery, cosmetic and pharmaceutical industries and also in food industries (Narayana et al. 1986). Due to limited genetic resources, area of cultivation is generally limited to the hilly regions of Southern India. As a result, looking at the high demand of geranium oil, India imports about 100 tons of geranium oil annually (Khanuja et al. 2005). Extension of the area of cultivation of this crop and/or improvement in product yield are feasible alternatives which can solve the above-mentioned constraints for which widening of the genetic base is absolutely essential. But conventional breeding in the case of geranium is very much hampered due to its high degree of male and female sterility (Kubba and Tilney-Basset 1981). Agrobacterium mediated transformation is one of the most efficient techniques to introduce variability due to stable integration and appropriate expression of the limited copy numbers of the transgenes (Finnegan and Elroy 1994; Kumpatla et al. 1998). The concept of using Ri-T-DNA as a vector for regenerating transgenic plants from hairy root cultures has already been in use in a number of plant species due to the fact that roots do not produce chimeras or somaclonal variants (Ooms et al. 1985; Tepfer 1984; Ottaviani et al. 1990; Yamazaki et al. 1997). Genetic transformation systems through Agrobacterium species have been reported for ornamental scented geranium species, in zonal and regal cultivars (Robichon et al. 1995; Boase et al. 1996, 1998), with successful regeneration of whole plants with A. rhizogenes mediated hairy root cultures of scented geraniums (Pellgrineschi et al. 1994; Pellgrineschi and Mariani 1996). Agrobacterium tumefaciens mediated transformation and regeneration of transgenic plants through somatic embryogenesis has also been reported in the case of Pelargonium sp. cultivar Frensham (Krishnaraj et al. 1997), Zonal (Pelargonium X hortorum) and scented geranium (P. capitata) (Hassanein et al. 2005). In continuation of our effort for the genetic improvement of rose-scented geraniums (Saxena et al. 2000), protocol has been established for both spontaneous and induced regeneration of
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whole plants from A. rhizogenes mediated hairy root cultures of the cultivated variety of P. graveolens cv. Hemanti. We felt necessary to evaluate the morphological features and oil characters of as many transformants as possible since the different inserts are independent and each individual transformation event represents a cellular clone (Tempe and Casse-Deebart 1989). The present paper reports successful regeneration studies on transgenic variants of Pelargonium sp. obtained through A. rhizogenes mediated hairy root cultures.
Materials and methods Plant material Leaf, petiole and internodal segments excised from the shoots of in vitro grown plants of Pelargonium graveolens cv. Hemanti (Saxena et al. 2000) were used as explants for the present study.
Bacterial strains Two strains of A. rhizogenes namely A4 and LBA 9402 (Courtesy Dr. David Tepfer, France) were used in the present study. A day-old bacterial suspension grown in liquid YMB medium (Hooykass et al. 1977) at 28 ± 2C on rotary shaker at a speed of 100 rpm was used for experimentation.
Establishment of non-transformed root The non-transformed roots were obtained from the in vitro grown, P. graveolens cv. Hemanti plantlets maintained on a modified Murashige and Skoog’s (MS) half strength medium supplemented with 1 mg/l IBA (Saxena et al. 2000).
Establishment of hairy root cultures Internode, leaf and petiole explants were wounded with sterile needles dipped in bacterial suspension. The inoculated explants were placed on a hormone-free MS
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medium with 3% sucrose. After 24-48 h of inoculation, the explants were transferred to the hormone free MS medium containing 1 mg/ml cephalexine. The hairy roots which emerged from the infected sites were maintained at standard culture conditions of 22 ± 2C temperature and 55–60% relative humidity in liquid half strength hormone free liquid MS medium with 3% sucrose on a rotary shaker at 80 rpm. These hairy root lines were repeatedly subcultured after every 2 weeks. The concentration of antibiotic was gradually reduced and finally withdrawn after six sub-cultures. Regeneration of transgenic plants from hairy root cultures Some of the hairy root lines were regenerated on the same subculture medium while the others had to be transferred to 1/2 MS medium with 1.5% sucrose and various hormonal combinations of BAP (0.25– 0.75 mg/l) and NAA (0.05 and 0.1 mg/l) for promoting regeneration (Table 1). Rooting of the transgenics and acclimatization of transgenic plants In vitro regenerated transgenic shoots of size 1.5– 2.0 cm with 2–3 expanded leaves were excised and transferred to semi-solid hormone free half strength MS medium. The rooted transgenic plantlets were transplanted to pots containing sand and soil in equal ratio and were maintained within the confined Table 1 Shoot regeneration response of Pelargonium graveolens cv. Hemanti hairy roots on semi-solid and liquid MS media containing various combinations of BAP and NAA Phytohormones supplements (mg/l)
1/2 MS + 1.5% sucrose
NAA
BAP
Semi-solid
Liquid
0.05
0.25
-NR
-NR
0.05
0.50
-NR
*OR
0.05
0.75
-NR
-NR
0.1
0.25
**OR
**OR
0.1
0.50
***OR
***OR
0.1
0.75
-NR
-NR
* No. of shoots = 1–2 ** No. of shoots = 3–4 *** No. of shoots = 6–8 NR = No response OR = Organogenesis
environment of the glasshouse. The morphological parameters of transformed as well as non-transformed plants were noted after 3 months of their growth. The flowering behaviour and floral morphology was also recorded in comparison to the control plants. The plants were maintained through cuttings and tested for their morphological and biochemical behaviour for three successive years. Detection of opines About 200 mg of fresh hairy roots and non-transformed roots and also 200 mg of leaves each from the transformed and non-transformed plants were separately homogenized with distilled water, centrifuged at 15,000 rpm for 5 min. The supernatant was used for detection of opines according to the protocol established by Morgan et al. (1987). Isolation of essential oils and GC analysis The aerial parts (25 gm, leaf:stem ratio = 1:1) were hydrodistilled for 3 h using mini-Clevenger apparatus for extraction of the essential oils. GC analysis of the oil samples of the transformed plants and the control was carried out on Perkin–Elmer gas chromatograph 8500 using BP-I capillary column (dimethyl poly siloxane) (25 m · 0.5 mm id · 0.25 mm film thickness) with nitrogen as carrier gas at a flow rate of 40 ml/min and 10 psi inlet pressure. Temperature was programmed to increase from 60 to 220C at 5C/min. Compounds were identified by their kovats retention indices (relative to C8–C20 alkanes) (Jenmings and Shibamoto 1980; Adams 1995) peak enrichment on co-infection with standards, and comparison of mass spectral fragments of the peaks in GC/MS with literature values (Jenmings and Shibamoto 1980; Adams 1995). The compounds and their relative concentrations in the oils were identified by electronic integration in GC. RAPD analysis of the transgenic and control plants The DNA from 11 transgenic plants and their control parental cultivar Hemanti were isolated from approximately 1 g fresh leaf tissue from glasshouse grown plants following the protocol given by Khanuja et al. (1998). The PCR amplification reactions and the
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RAPD analysis was done according to the protocol by Shoyama et al. (1997). Two transgenic plants (2TG and 14TG) were analyzed in replicates in order to demonstrate genetic uniformity within the clone.
Results and discussion A transformation frequency from leaf explants was the highest, followed by the petioles and internodal segments (Table 2). However, young leaf petioles did not produce hairy roots in any of the Pelargonium species tested (Pellegrineschi and Mariani 1996). Although transformation frequencies was high with both the bacterial strains used in this study, as has also been reported in lemon-scented geranium (Pellegrineschi et al. 1994). Amongst the two bacterial strains used, A4 proved to be effective than LBA 9402, both in terms of frequency of response and time requirement for hairy-root induction (Table 2). Similar observations had also been reported earlier for diverse plant species (Porter 1991; Rodrigues et al. 1991; Banerjee et al. 1994; Giri 1997; Zehra 1998). In view of the fact that each transformation event is distinct from the other due to different integration sites and copy numbers of Ri T-DNA (Tepfer 1984), 90 independent hairy-root lines of A4 origin and 60 of LBA 9402 origin were selected for regeneration studies (Fig. 1A). Amongst these, 14 regenerants by A4 and seven root lines by LBA 9402 origin showed spontaneous regeneration without the intervening callus phase within 8–10 weeks of subculturing in hormone-free liquid and semi-solid MS medium (Fig. 1B and C; Table 1). Spontaneous regeneration of plants from hairy root cultures has already been reported 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 of the hairy root lines of A4 and LBA 9402 origin,
Table 2 Susceptibility of different explants of Pelargonium graveolens cv. Hemanti to A4 and LBA 9402 strains of Agrobacterium rhizogenes
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Bacterial strains
respectively (Table 3), within 6–8 weeks of culture with different hormonal combinations. Only two of the five regeneration media tested with varying concentrations of NAA and BAP responded both in their semi-solid and liquid state. A maximum of 6–8 shoots per root explant was produced after 7 weeks of culture containing 0.5 mg/l BAP and 0.1 mg/l NAA (Table 1). These concentrations were lower than those as reported in regal Pelargonium (Pelargonium · domesticum Dubonnet) (Boase et al. 1998). Various combinations of different hormones including NAA, BAP, zeatin as well as supplementary vitamin source and PVP-10 have been used for inducing direct and indirect regeneration of transgenic 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 to different hormonal combinations, reduction in the concentrations of sucrose as well as that of other nutrients to half appears to be an essential prerequisite of the present study in order to optimize the shoot regeneration efficiency from hairy root cultures which has not been considered earlier (Boase et al. 1998). All the transgenic plantlets of the present study, irrespective of their spontaneous or hormone-mediated origin, exhibited spontaneous rhizogenesis upon transfer to hormone-free half strength MS medium within 2 weeks of transfer (Fig. 1E). A total of 11 transgenic plants out of the total 36 tested were successfully transferred to the glasshouse. The leaves of all transformed plants were darker in colour, more dentated and leaf wrinkling was absent in all of them as has also been reported earlier (Pellegrineschi et al. 1994). The transformants also had highly branched root systems, shorter internodes, increased number of leaves and branches and short and round stature of
Transformation frequency Internode
Petiole
Leaf
A4
50 ± 3.2
71.3 ± 3.3
100 ± 0.0
LBA 9402
20 ± 1.9
50 ± 2.6
82 ± 4.1
Plant Cell Tiss Organ Cult (2007) 90:215–223
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Fig. 1 Generation of Agrobacterium rhizogenes mediated transgenic plants in Pelargonium graveolens Indian cultivar Hemanti; A; Hairy root culture maintained for 2 weeks in half strength, hormone free liquid MS medium; B–C: regeneration of plantlets from hairy root culture in semi-solid and liquid culture media, respectively; D–E: in vitro raised rooted control and transformed plants; F: Control (left) and transformed (right) plants after 10 weeks of growth in pots exhibiting increased branching and higher number of leaves in the latter (· 0.10); G–H: flower of control and transformed (LZ-3) plant exhibiting altered morphology (· 3.5)
Table 3 Frequency of regeneration from hairy roots of Pelargonium graveolens cv. Hemanti induced with A4 and LBA 9402 strains of Agrobacterium rhizogenes
Genetic resource
Bacterial strain used for hairy root induction
Total number of root lines
A4
LBA 9402
90
60
Total number of root lines that exhibited regeneration 40
20
Number of lines showing spontaneous regeneration
14
07
Number of lines where regeneration was induced
26
13
the 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 reported
in the case of other Pelargonium species (Pellegrineschi et al. 1994; Pellegrineschi and Mariani 1996). All the transgenics showed opine positive results even after 5 months of transfer to the pot.
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220 Fig. 2 Comparative account of various morphological characters of transgenic and control plants of Pelargonium graveolens cv. Hemanti
Plant Cell Tiss Organ Cult (2007) 90:215–223 80 Contr ol Plants
70
Transgenic Plants
60 50 40 30 20 10
) (c m en gt h Ro ot l
Ro ot s/ pl an t
Le av es /b ra nc h
No de s/ br an ch
of b No .
Pl
an th
ei gh t(
cm
)
ra nc he s
0
Morphological characters
One of the 11 transgenic plants, LZ-3, produced flowers in 3 months with two inflorescences, each bearing four flowers could be seen during the entire cultivation period and the flower size was smaller with reduced petals and abnormality in their shape (Fig. 1G and H). The small flower size in the transgenic plant in comparison to control was also reported in tobacco (Tepfer 1984). A hundred percent pollen sterility could be recorded through acetocarmine staining technique. The cuttings were used for maintaining and further multiplication of transgenic plants for three successive years. All the 11 transgenic plants along with their control were subjected to quantitative and qualitative analysis of their essential oils after 5 months of transfer to the pots. It was observed that all the plants showed stable behaviour in this duration. The oil contents of all the transgenics were comparatively lower than the wild type (Table 4) in contrast to an earlier report (Pellegrineschi et al. 1994). Taking into consideration the number of leaves and branches, an overall increase in the oil yield can be predicted from the transgenics since higher herb yield normally has a stronger correlation with the oil yield per plant (Saxena et al. 2000). The analysis showed that the oils of nine transgenic plants (A6, 2TG, 5TG, 6TG, 10TG, 12TG, 13TG, 15TG and 20TG) had composition similar to that of the control Hemanti type (Table 4) which represents Chinese geranium oil in having higher amounts of citronellol and lower amounts of geraniol (Saxena et al. 2000). The
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concentrations of citronellol and geraniol as well as their esters were found to be at par in these transgenics with those of the wild type oil. Among these nine transgenics, the oil of A6 was unique in having very low concentration of 10-epi-c-eudesmol (0.2%) which signifies better odour value (Teisseire 1987). As compared to the oils of the other eight transgenics and the control, the oil of 6TG was found to contain lower concentration of isomenthone. Unaltered oil profiles of these transgenics might represent the fact that the insertion sites of Ri TDNA in these plants do not interfere with the secondary metabolites biosynthetic pathway as is mostly noted in majority of medicinal plants (Jung and Tepfer 1987; Banerjee et al. 1998; Zehra et al. 1999). The RAPD analysis of these transgenics also revealed similarity at genetic level with that of the wild type in terms of most of the primers used. However, the primer OPT 1 revealed distinct differences between the wild type and A6 in terms of the polymorphic bands (Fig. 3) which might account for the distinctiveness of A6 in terms of its unique oil profile in having very low concentrations of 10-epi-ceudesmol. On the other hand, the difference in polymorphic bands between the 15TG and control with regard to the same primer could not be corelated with any biochemical or morphological parameters in consideration, however changes in other physiological characters cannot be ruled out. Besides the above nine transgenics, the transgenics LZ-3 and 14TG were found to differ in their oil
Fig. 3 PCR profiles of DNA isolated from 11 transgenic plants and their parent Pelargonium graveolens cv. Hemanti amplified with primer OPT1 50 GGGCCACTCA30 and resolved on 0.8% agarose gel. Samples in gel lanes (from right to left) are as follows: Lane 1: marker k DNA digested with HindIII ; Lane 2: wild type Pelargonium graveolens cv. Hemanti; Lanes 3,4 and 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-6
1.80 ± 0.12 2.60 ± 0.09 1.90 ± 0.04 1.50 ± 0.03 2.20 ± 0.09 1.50 ± 0.06 Geranyl esters
tr, trace
14.9 ± 0.88
1.80 ± 0.03
9.00 ± 0.19
14.8 ± 0.73
221
2.30 ± 0.12
1.10 ± 0.05
2.00 ± 0.16
10.3 ± 0.27
12.9 ± 0.65 22.7 ± 0.78 23.2 ± 0.81 22.8 ± 0.72 20.4 ± 0.43 23.8 ± 0.49 Citronellyl esters
19.7 ± 0.41
19.6 ± 0.96
17.0 ± 0.69
21.6 ± 0.62
17.5 ± 1.16
0.30 ± 0.016
6.60 ± 0.15 3.00 ± 0.12 4.40 ± 0.14 3.00 ± 0.21 3.80 ± 0.15 0.20 ± 0.01 10-epi-Y-Eudesmol
5.00 ± 0.07
4.20 ± 0.05
4.20 ± 0.09
4.20 ± 0.08
3.00 ± 0.14
2.00 ± 0.05
0.30 ± 0.02 1.80 ± 0.06 1.40 ± 0.11 1.80 ± 0.06 2.70 ± 0.12 2.20 ± 0.09 Guaia-6,9- diene
1.40 ± 0.07
2.50 ± 0.10
2.00 ± 0.01
2.30 ± 0.08
1.70 ± 0.54
7.90 ± 0.74
1.30 ± 0.06 1.60 ± 0.08 2.40 ± 0.10 2.30 ± 0.12 2.50 ± 0.21 2.00 ± 0.09 2.10 ± 0.09 6- Caryophyllene
2.20 ± 0.05
2.10 ± 0.06
1.70 ± 0.03
2.30 ± 0.16
9.60 ± 0.40
33.3 ± 0.88 35.7 ± 0.30
0.50 ± 0.08
2.00 ± 0.10
46.9 ± 0.74
0.40 ± 0.12 1.40 ± 0.06 0.80 ± 0.14
48.3 ± 0.78 54.4 ± 0.69
1.10 ± 0.11 0.70 ± 0.004 1.20 ± 0.06 0.50 ± 0.08 Geraniol
45.6 ± 0.27 47.0 ± 0.62 47.0 ± 0.65 46.1 ± 0.45 Citronellol
1.90 ± 0.09
52.4 ± 0.32
46.3 ± 0.66
52.7 ± 0.55
tr
1.80 ± 0.12 7.90 ± 0.33 9.50 ± 0.36 10.3 ± 0.49 8.20 ± 0.11 9.50 ± 0.68 9.60 ± 0.40 10.9 ± 0.13 Isomenthone
4.0 ± 0.28
8.00 ± 0.31
9.30 ± 0.37
9.40 ± 0.51
1.60 ± 0.04
0.90 ± 0.010 1.60 ± 0.04
2.30 ± 0.02 2.40 ± 0.07 2.60 ± 0.16 1.80 ± 0.07 0.70 ± 0.015 1.60 ± 0.09 1.60 ± 0.06 1.90 ± 0.07 Rose oxides (cis + trans) 2.50 ± 0.09 2.60 ± 0.14
0.10 ± 0.017 0.11 ± 0.010 0.11 ± 0.010
0.60 ± 0.016 0.40 ± 0.020 0.30 ± 0.005 0.90 ± 0.01
0.09 ± 0.005 0.11 ± 0.005 0.05 ± 0.001 0.10 ± 0.04
Linalool
0.10 ± 0.06 0.06 ± 0.001 0.10 ± 0.01 0.15 ± 0.01 0.11 ± 0.01
0.50 ± 0.04 1.00 ± 0.02
Oil content (%)
0.50 ± 0.02 0.30 ± 0.004 0.60 ± 0.008 0.50 ± 0.01
14TG LZ-3 2OTG 15TG I3TG 12TG 10TG 6TG 5TG 2TG A-6 Control Compound
Table 4 Oil content and percent concentration (±SD) of the terpenoid constituents in essential oils of transgenic plants and the wild type Pelargonium graveolens cv. Hemanti
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profiles from that of the control oil (Table 4). These two oils had comparatively lower concentration of citronellol (33.3% and 35.7%) and higher concentration of geraniol (6.6% and 10.3%) than the control oil where citronellol was 47% and geraniol was 2.1%. Since the citronellol:geraniol ratio in geranium oils determines their odour value (Lawrence 1984) these two oils represent better quality oils resembling Bipuli type (Rao et al. 1999). The oils of these two transgenic plants also possess relatively higher amounts of linalool, geranyl esters and 10-epi-ceudesmol as compared to that of the control. On the other hand, the concentrations of 6, 9-guaiadiene and the esters of citronellol were found to be low in these two oils as compared to the oil of the control. Further, the concentrations of isomenthone and rose oxides were quite low (1.8% and trace, respectively) in one of these two oils (14TG) as compared to the oil of control (9.6% and 2.5%, respectively). At the genetic level these two transgenics resembled each other with regard to OPT 1 primer while distinct differences could be noted between them from the others along with the wild type control with regards to same primer (Fig. 3) which further substantiates their totally altered oil profile in terms of the major terpenoid constituents. On the basis of overall analysis, it can be stated that since the increase in geraniol and geranyl esters concentration signifies improved olfactory value (Rao et al. 1999), the oils of these two transgenics definitely represent an improvement over the control from the commercial point of
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view. Reduction in citronellol concentration and increase in geraniol concentration had earlier been noted in three transgenic plants of lemon-geranium of hairy root origin (Pellegrineschi et al. 1994). The present study once again highlights the success of hairy root transformation strategy for the creation of new fragrances through the easiest and quickest method of establishment of transgenic plants from hairy root cultures. Acknowledgements The authors are thankful to the Department of Biotechnology, Government of India for the financial support and the Regional Sophistication Instrumentation Facility, CDRI, Lucknow for GC-MS of the oils. Thanks are also due to S. Sharma and S.A. Hasan for their help in manuscript preparation and A.P. Dhiman for photography.
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Plant Cell Tiss Organ Cult (2007) 90:215–223 Rodrigues MMA, Stafford A, Cresswell R, Ariascastro C (1991) Bioreactors for growth of plant roots. Enz Microb Technol 13:697–702 Saxena G, Banerjee S, Rahman L, Mallavarapu GR, Sharma S, Sushil Kumar (2000) An efficient in vitro procedure for micropropagation and generation of somaclones of rosescented Pelargonium. Plant Sci 155:133–140 Shoyama Y, Zhu X, Nakai R, Shiraishi S, Kohda H (1997) Micropropagation of Panax notoginseng by somatic embryogenesis and RAPD analysis of regenerated plantlets. Plant Cell Rep 16:450–453 Teisseire P (1987) Industrial quality control of essential oils by capillary GC. In: Sandri P, Beichi C (ed) Capillary gas chromatography in essential oil analysis. Huethig, Heidelberg, pp 215–258 Tempe J, Casse-Deebart F (1989) Plant gene vectors and genetic transformation: Agrobacterium Ri plasmids. In: Cell culture and somatic cell genetics of plants. Molecular
223 Biology of Plant Nuclear genes, vol. 6. Academic Press, pp 26–49 Tepfer D (1984) Transformation of several species of higher plants by Agrobacterium rhizogenes: sexual transmission of the transformed genotype and phenotype. Cell 37: 659–967 Yamazaki M, Kobayashi M, Saito K (1997) Transformation of Perilla frutescens var, crispa using Agrobacterium –Ri binary vector system. Plant Biotechnol 14:169–173 Zehra M, Banerjee S, Naqvi AA, Kumar S (1998) Variation in the growth and a production capability of the hairy roots of Hyoscyamus albus, H. muticus and their somatic hybrid. Plant Sci 136:93–99 Zehra M, Banerjee S, Sharma S, Kumar S (1999) Influence of Agrobacterluin rhizogenes strains on biomass and alkaloid productivity in hairy root of Hyoscyamus muticus and H. albus. Planta Med 65:60–63
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ORIGINAL PAPER
Rose-scented geranium (Pelargonium sp.) generated by Agrobacterium rhizogenes mediated Ri-insertion for improved essential oil quality Gauri Saxena Æ Suchitra Banerjee Æ Laiq-ur-Rahman Æ Praveen Chandra Verma Æ G. R. Mallavarapu Æ Sushil Kumar
Received: 20 February 2007 / Accepted: 12 June 2007 / Published online: 7 July 2007 Springer Science+Business Media B.V. 2007
Abstract Transgenic plants of rose-scented geranium (Pelargonium graveolens cv. Hemanti) have been produced from Agrobacterium rhizogenes (strains A4 and LBA9402) mediated hairy root cultures. Amongst the explants tested, leaves were most responsive followed by the petioles and internodal segments, respectively. The A4 strain performed better for all the three explants both in terms of frequency of response and time requirement for hairy root induction. Transgenic shoots could be obtained by spontaneous regeneration without intervening callus phase amongst 16% and 12% root lines of A4 and LBA 9402 origin, G. Saxena (&) Department of Botany, Lucknow University, Lucknow 226001, India e-mail: [email protected] S. Banerjee Central Institute of Medicinal and Aromatic Plants, P.O. CIMAP, Lucknow 226015, India Laiq-ur-Rahman CIMAP Field Station, Puraura, India P. C. Verma National Botanical Research Institute, Rana Pratap Marg, Lucknow 226001, India G. R. Mallavarapu CIMAP Field Station, Bangalore, India S. Kumar National Centre for Plant Genome Research, JNU Campus, P.O. Box 10531, New Delhi 110067, India
respectively, or they were induced in 29% and 22% hairy root lines of A4 and LBA9402 origin, respectively, with different hormonal supplementation. These transgenic plants showed 30% survival as against 90% of their control under the confined environment of glasshouse. The transgenic plants were of similar morphotype having increased branching, higher number of leaves with increased dentations, short and round stature, highly branched root system and absence of leaf wrinkling. These transgenic plants showed opine positive results even after 5 months of their transfer to the glasshouse. The essential oil compositions of 81% of these transgenics were qualitatively similar to that of the wild type parent. However, two transgenic plants (LZ-3 and 14TG) showed increase in concentrations of geraniol and geranyl esters signifying improved oil quality with respect to the citronellol:geraniol ratio. These two oils having better olfactory value represent an improvement over that of the wild type parent from the commercial point of view. Keywords Geranium Pelargonium Agrobacterium rhizogenes Essential oil Abbreviations MS Murashige and Skoog’s medium IBA Indole butyric acid BAP 6-Benzylaminopurine NAA Naphthaleneacetic acid GC Gas chromatography GC/MS Gas chromatography mass spectroscopy
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Introduction Geranium oils extracted from Pelargonium species (scented geraniums) have been used in the perfumery, cosmetic and pharmaceutical industries and also in food industries (Narayana et al. 1986). Due to limited genetic resources, area of cultivation is generally limited to the hilly regions of Southern India. As a result, looking at the high demand of geranium oil, India imports about 100 tons of geranium oil annually (Khanuja et al. 2005). Extension of the area of cultivation of this crop and/or improvement in product yield are feasible alternatives which can solve the above-mentioned constraints for which widening of the genetic base is absolutely essential. But conventional breeding in the case of geranium is very much hampered due to its high degree of male and female sterility (Kubba and Tilney-Basset 1981). Agrobacterium mediated transformation is one of the most efficient techniques to introduce variability due to stable integration and appropriate expression of the limited copy numbers of the transgenes (Finnegan and Elroy 1994; Kumpatla et al. 1998). The concept of using Ri-T-DNA as a vector for regenerating transgenic plants from hairy root cultures has already been in use in a number of plant species due to the fact that roots do not produce chimeras or somaclonal variants (Ooms et al. 1985; Tepfer 1984; Ottaviani et al. 1990; Yamazaki et al. 1997). Genetic transformation systems through Agrobacterium species have been reported for ornamental scented geranium species, in zonal and regal cultivars (Robichon et al. 1995; Boase et al. 1996, 1998), with successful regeneration of whole plants with A. rhizogenes mediated hairy root cultures of scented geraniums (Pellgrineschi et al. 1994; Pellgrineschi and Mariani 1996). Agrobacterium tumefaciens mediated transformation and regeneration of transgenic plants through somatic embryogenesis has also been reported in the case of Pelargonium sp. cultivar Frensham (Krishnaraj et al. 1997), Zonal (Pelargonium X hortorum) and scented geranium (P. capitata) (Hassanein et al. 2005). In continuation of our effort for the genetic improvement of rose-scented geraniums (Saxena et al. 2000), protocol has been established for both spontaneous and induced regeneration of
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whole plants from A. rhizogenes mediated hairy root cultures of the cultivated variety of P. graveolens cv. Hemanti. We felt necessary to evaluate the morphological features and oil characters of as many transformants as possible since the different inserts are independent and each individual transformation event represents a cellular clone (Tempe and Casse-Deebart 1989). The present paper reports successful regeneration studies on transgenic variants of Pelargonium sp. obtained through A. rhizogenes mediated hairy root cultures.
Materials and methods Plant material Leaf, petiole and internodal segments excised from the shoots of in vitro grown plants of Pelargonium graveolens cv. Hemanti (Saxena et al. 2000) were used as explants for the present study.
Bacterial strains Two strains of A. rhizogenes namely A4 and LBA 9402 (Courtesy Dr. David Tepfer, France) were used in the present study. A day-old bacterial suspension grown in liquid YMB medium (Hooykass et al. 1977) at 28 ± 2C on rotary shaker at a speed of 100 rpm was used for experimentation.
Establishment of non-transformed root The non-transformed roots were obtained from the in vitro grown, P. graveolens cv. Hemanti plantlets maintained on a modified Murashige and Skoog’s (MS) half strength medium supplemented with 1 mg/l IBA (Saxena et al. 2000).
Establishment of hairy root cultures Internode, leaf and petiole explants were wounded with sterile needles dipped in bacterial suspension. The inoculated explants were placed on a hormone-free MS
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medium with 3% sucrose. After 24-48 h of inoculation, the explants were transferred to the hormone free MS medium containing 1 mg/ml cephalexine. The hairy roots which emerged from the infected sites were maintained at standard culture conditions of 22 ± 2C temperature and 55–60% relative humidity in liquid half strength hormone free liquid MS medium with 3% sucrose on a rotary shaker at 80 rpm. These hairy root lines were repeatedly subcultured after every 2 weeks. The concentration of antibiotic was gradually reduced and finally withdrawn after six sub-cultures. Regeneration of transgenic plants from hairy root cultures Some of the hairy root lines were regenerated on the same subculture medium while the others had to be transferred to 1/2 MS medium with 1.5% sucrose and various hormonal combinations of BAP (0.25– 0.75 mg/l) and NAA (0.05 and 0.1 mg/l) for promoting regeneration (Table 1). Rooting of the transgenics and acclimatization of transgenic plants In vitro regenerated transgenic shoots of size 1.5– 2.0 cm with 2–3 expanded leaves were excised and transferred to semi-solid hormone free half strength MS medium. The rooted transgenic plantlets were transplanted to pots containing sand and soil in equal ratio and were maintained within the confined Table 1 Shoot regeneration response of Pelargonium graveolens cv. Hemanti hairy roots on semi-solid and liquid MS media containing various combinations of BAP and NAA Phytohormones supplements (mg/l)
1/2 MS + 1.5% sucrose
NAA
BAP
Semi-solid
Liquid
0.05
0.25
-NR
-NR
0.05
0.50
-NR
*OR
0.05
0.75
-NR
-NR
0.1
0.25
**OR
**OR
0.1
0.50
***OR
***OR
0.1
0.75
-NR
-NR
* No. of shoots = 1–2 ** No. of shoots = 3–4 *** No. of shoots = 6–8 NR = No response OR = Organogenesis
environment of the glasshouse. The morphological parameters of transformed as well as non-transformed plants were noted after 3 months of their growth. The flowering behaviour and floral morphology was also recorded in comparison to the control plants. The plants were maintained through cuttings and tested for their morphological and biochemical behaviour for three successive years. Detection of opines About 200 mg of fresh hairy roots and non-transformed roots and also 200 mg of leaves each from the transformed and non-transformed plants were separately homogenized with distilled water, centrifuged at 15,000 rpm for 5 min. The supernatant was used for detection of opines according to the protocol established by Morgan et al. (1987). Isolation of essential oils and GC analysis The aerial parts (25 gm, leaf:stem ratio = 1:1) were hydrodistilled for 3 h using mini-Clevenger apparatus for extraction of the essential oils. GC analysis of the oil samples of the transformed plants and the control was carried out on Perkin–Elmer gas chromatograph 8500 using BP-I capillary column (dimethyl poly siloxane) (25 m · 0.5 mm id · 0.25 mm film thickness) with nitrogen as carrier gas at a flow rate of 40 ml/min and 10 psi inlet pressure. Temperature was programmed to increase from 60 to 220C at 5C/min. Compounds were identified by their kovats retention indices (relative to C8–C20 alkanes) (Jenmings and Shibamoto 1980; Adams 1995) peak enrichment on co-infection with standards, and comparison of mass spectral fragments of the peaks in GC/MS with literature values (Jenmings and Shibamoto 1980; Adams 1995). The compounds and their relative concentrations in the oils were identified by electronic integration in GC. RAPD analysis of the transgenic and control plants The DNA from 11 transgenic plants and their control parental cultivar Hemanti were isolated from approximately 1 g fresh leaf tissue from glasshouse grown plants following the protocol given by Khanuja et al. (1998). The PCR amplification reactions and the
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RAPD analysis was done according to the protocol by Shoyama et al. (1997). Two transgenic plants (2TG and 14TG) were analyzed in replicates in order to demonstrate genetic uniformity within the clone.
Results and discussion A transformation frequency from leaf explants was the highest, followed by the petioles and internodal segments (Table 2). However, young leaf petioles did not produce hairy roots in any of the Pelargonium species tested (Pellegrineschi and Mariani 1996). Although transformation frequencies was high with both the bacterial strains used in this study, as has also been reported in lemon-scented geranium (Pellegrineschi et al. 1994). Amongst the two bacterial strains used, A4 proved to be effective than LBA 9402, both in terms of frequency of response and time requirement for hairy-root induction (Table 2). Similar observations had also been reported earlier for diverse plant species (Porter 1991; Rodrigues et al. 1991; Banerjee et al. 1994; Giri 1997; Zehra 1998). In view of the fact that each transformation event is distinct from the other due to different integration sites and copy numbers of Ri T-DNA (Tepfer 1984), 90 independent hairy-root lines of A4 origin and 60 of LBA 9402 origin were selected for regeneration studies (Fig. 1A). Amongst these, 14 regenerants by A4 and seven root lines by LBA 9402 origin showed spontaneous regeneration without the intervening callus phase within 8–10 weeks of subculturing in hormone-free liquid and semi-solid MS medium (Fig. 1B and C; Table 1). Spontaneous regeneration of plants from hairy root cultures has already been reported 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 of the hairy root lines of A4 and LBA 9402 origin,
Table 2 Susceptibility of different explants of Pelargonium graveolens cv. Hemanti to A4 and LBA 9402 strains of Agrobacterium rhizogenes
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Bacterial strains
respectively (Table 3), within 6–8 weeks of culture with different hormonal combinations. Only two of the five regeneration media tested with varying concentrations of NAA and BAP responded both in their semi-solid and liquid state. A maximum of 6–8 shoots per root explant was produced after 7 weeks of culture containing 0.5 mg/l BAP and 0.1 mg/l NAA (Table 1). These concentrations were lower than those as reported in regal Pelargonium (Pelargonium · domesticum Dubonnet) (Boase et al. 1998). Various combinations of different hormones including NAA, BAP, zeatin as well as supplementary vitamin source and PVP-10 have been used for inducing direct and indirect regeneration of transgenic 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 to different hormonal combinations, reduction in the concentrations of sucrose as well as that of other nutrients to half appears to be an essential prerequisite of the present study in order to optimize the shoot regeneration efficiency from hairy root cultures which has not been considered earlier (Boase et al. 1998). All the transgenic plantlets of the present study, irrespective of their spontaneous or hormone-mediated origin, exhibited spontaneous rhizogenesis upon transfer to hormone-free half strength MS medium within 2 weeks of transfer (Fig. 1E). A total of 11 transgenic plants out of the total 36 tested were successfully transferred to the glasshouse. The leaves of all transformed plants were darker in colour, more dentated and leaf wrinkling was absent in all of them as has also been reported earlier (Pellegrineschi et al. 1994). The transformants also had highly branched root systems, shorter internodes, increased number of leaves and branches and short and round stature of
Transformation frequency Internode
Petiole
Leaf
A4
50 ± 3.2
71.3 ± 3.3
100 ± 0.0
LBA 9402
20 ± 1.9
50 ± 2.6
82 ± 4.1
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Fig. 1 Generation of Agrobacterium rhizogenes mediated transgenic plants in Pelargonium graveolens Indian cultivar Hemanti; A; Hairy root culture maintained for 2 weeks in half strength, hormone free liquid MS medium; B–C: regeneration of plantlets from hairy root culture in semi-solid and liquid culture media, respectively; D–E: in vitro raised rooted control and transformed plants; F: Control (left) and transformed (right) plants after 10 weeks of growth in pots exhibiting increased branching and higher number of leaves in the latter (· 0.10); G–H: flower of control and transformed (LZ-3) plant exhibiting altered morphology (· 3.5)
Table 3 Frequency of regeneration from hairy roots of Pelargonium graveolens cv. Hemanti induced with A4 and LBA 9402 strains of Agrobacterium rhizogenes
Genetic resource
Bacterial strain used for hairy root induction
Total number of root lines
A4
LBA 9402
90
60
Total number of root lines that exhibited regeneration 40
20
Number of lines showing spontaneous regeneration
14
07
Number of lines where regeneration was induced
26
13
the 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 reported
in the case of other Pelargonium species (Pellegrineschi et al. 1994; Pellegrineschi and Mariani 1996). All the transgenics showed opine positive results even after 5 months of transfer to the pot.
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220 Fig. 2 Comparative account of various morphological characters of transgenic and control plants of Pelargonium graveolens cv. Hemanti
Plant Cell Tiss Organ Cult (2007) 90:215–223 80 Contr ol Plants
70
Transgenic Plants
60 50 40 30 20 10
) (c m en gt h Ro ot l
Ro ot s/ pl an t
Le av es /b ra nc h
No de s/ br an ch
of b No .
Pl
an th
ei gh t(
cm
)
ra nc he s
0
Morphological characters
One of the 11 transgenic plants, LZ-3, produced flowers in 3 months with two inflorescences, each bearing four flowers could be seen during the entire cultivation period and the flower size was smaller with reduced petals and abnormality in their shape (Fig. 1G and H). The small flower size in the transgenic plant in comparison to control was also reported in tobacco (Tepfer 1984). A hundred percent pollen sterility could be recorded through acetocarmine staining technique. The cuttings were used for maintaining and further multiplication of transgenic plants for three successive years. All the 11 transgenic plants along with their control were subjected to quantitative and qualitative analysis of their essential oils after 5 months of transfer to the pots. It was observed that all the plants showed stable behaviour in this duration. The oil contents of all the transgenics were comparatively lower than the wild type (Table 4) in contrast to an earlier report (Pellegrineschi et al. 1994). Taking into consideration the number of leaves and branches, an overall increase in the oil yield can be predicted from the transgenics since higher herb yield normally has a stronger correlation with the oil yield per plant (Saxena et al. 2000). The analysis showed that the oils of nine transgenic plants (A6, 2TG, 5TG, 6TG, 10TG, 12TG, 13TG, 15TG and 20TG) had composition similar to that of the control Hemanti type (Table 4) which represents Chinese geranium oil in having higher amounts of citronellol and lower amounts of geraniol (Saxena et al. 2000). The
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concentrations of citronellol and geraniol as well as their esters were found to be at par in these transgenics with those of the wild type oil. Among these nine transgenics, the oil of A6 was unique in having very low concentration of 10-epi-c-eudesmol (0.2%) which signifies better odour value (Teisseire 1987). As compared to the oils of the other eight transgenics and the control, the oil of 6TG was found to contain lower concentration of isomenthone. Unaltered oil profiles of these transgenics might represent the fact that the insertion sites of Ri TDNA in these plants do not interfere with the secondary metabolites biosynthetic pathway as is mostly noted in majority of medicinal plants (Jung and Tepfer 1987; Banerjee et al. 1998; Zehra et al. 1999). The RAPD analysis of these transgenics also revealed similarity at genetic level with that of the wild type in terms of most of the primers used. However, the primer OPT 1 revealed distinct differences between the wild type and A6 in terms of the polymorphic bands (Fig. 3) which might account for the distinctiveness of A6 in terms of its unique oil profile in having very low concentrations of 10-epi-ceudesmol. On the other hand, the difference in polymorphic bands between the 15TG and control with regard to the same primer could not be corelated with any biochemical or morphological parameters in consideration, however changes in other physiological characters cannot be ruled out. Besides the above nine transgenics, the transgenics LZ-3 and 14TG were found to differ in their oil
Fig. 3 PCR profiles of DNA isolated from 11 transgenic plants and their parent Pelargonium graveolens cv. Hemanti amplified with primer OPT1 50 GGGCCACTCA30 and resolved on 0.8% agarose gel. Samples in gel lanes (from right to left) are as follows: Lane 1: marker k DNA digested with HindIII ; Lane 2: wild type Pelargonium graveolens cv. Hemanti; Lanes 3,4 and 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-6
1.80 ± 0.12 2.60 ± 0.09 1.90 ± 0.04 1.50 ± 0.03 2.20 ± 0.09 1.50 ± 0.06 Geranyl esters
tr, trace
14.9 ± 0.88
1.80 ± 0.03
9.00 ± 0.19
14.8 ± 0.73
221
2.30 ± 0.12
1.10 ± 0.05
2.00 ± 0.16
10.3 ± 0.27
12.9 ± 0.65 22.7 ± 0.78 23.2 ± 0.81 22.8 ± 0.72 20.4 ± 0.43 23.8 ± 0.49 Citronellyl esters
19.7 ± 0.41
19.6 ± 0.96
17.0 ± 0.69
21.6 ± 0.62
17.5 ± 1.16
0.30 ± 0.016
6.60 ± 0.15 3.00 ± 0.12 4.40 ± 0.14 3.00 ± 0.21 3.80 ± 0.15 0.20 ± 0.01 10-epi-Y-Eudesmol
5.00 ± 0.07
4.20 ± 0.05
4.20 ± 0.09
4.20 ± 0.08
3.00 ± 0.14
2.00 ± 0.05
0.30 ± 0.02 1.80 ± 0.06 1.40 ± 0.11 1.80 ± 0.06 2.70 ± 0.12 2.20 ± 0.09 Guaia-6,9- diene
1.40 ± 0.07
2.50 ± 0.10
2.00 ± 0.01
2.30 ± 0.08
1.70 ± 0.54
7.90 ± 0.74
1.30 ± 0.06 1.60 ± 0.08 2.40 ± 0.10 2.30 ± 0.12 2.50 ± 0.21 2.00 ± 0.09 2.10 ± 0.09 6- Caryophyllene
2.20 ± 0.05
2.10 ± 0.06
1.70 ± 0.03
2.30 ± 0.16
9.60 ± 0.40
33.3 ± 0.88 35.7 ± 0.30
0.50 ± 0.08
2.00 ± 0.10
46.9 ± 0.74
0.40 ± 0.12 1.40 ± 0.06 0.80 ± 0.14
48.3 ± 0.78 54.4 ± 0.69
1.10 ± 0.11 0.70 ± 0.004 1.20 ± 0.06 0.50 ± 0.08 Geraniol
45.6 ± 0.27 47.0 ± 0.62 47.0 ± 0.65 46.1 ± 0.45 Citronellol
1.90 ± 0.09
52.4 ± 0.32
46.3 ± 0.66
52.7 ± 0.55
tr
1.80 ± 0.12 7.90 ± 0.33 9.50 ± 0.36 10.3 ± 0.49 8.20 ± 0.11 9.50 ± 0.68 9.60 ± 0.40 10.9 ± 0.13 Isomenthone
4.0 ± 0.28
8.00 ± 0.31
9.30 ± 0.37
9.40 ± 0.51
1.60 ± 0.04
0.90 ± 0.010 1.60 ± 0.04
2.30 ± 0.02 2.40 ± 0.07 2.60 ± 0.16 1.80 ± 0.07 0.70 ± 0.015 1.60 ± 0.09 1.60 ± 0.06 1.90 ± 0.07 Rose oxides (cis + trans) 2.50 ± 0.09 2.60 ± 0.14
0.10 ± 0.017 0.11 ± 0.010 0.11 ± 0.010
0.60 ± 0.016 0.40 ± 0.020 0.30 ± 0.005 0.90 ± 0.01
0.09 ± 0.005 0.11 ± 0.005 0.05 ± 0.001 0.10 ± 0.04
Linalool
0.10 ± 0.06 0.06 ± 0.001 0.10 ± 0.01 0.15 ± 0.01 0.11 ± 0.01
0.50 ± 0.04 1.00 ± 0.02
Oil content (%)
0.50 ± 0.02 0.30 ± 0.004 0.60 ± 0.008 0.50 ± 0.01
14TG LZ-3 2OTG 15TG I3TG 12TG 10TG 6TG 5TG 2TG A-6 Control Compound
Table 4 Oil content and percent concentration (±SD) of the terpenoid constituents in essential oils of transgenic plants and the wild type Pelargonium graveolens cv. Hemanti
Plant Cell Tiss Organ Cult (2007) 90:215–223
profiles from that of the control oil (Table 4). These two oils had comparatively lower concentration of citronellol (33.3% and 35.7%) and higher concentration of geraniol (6.6% and 10.3%) than the control oil where citronellol was 47% and geraniol was 2.1%. Since the citronellol:geraniol ratio in geranium oils determines their odour value (Lawrence 1984) these two oils represent better quality oils resembling Bipuli type (Rao et al. 1999). The oils of these two transgenic plants also possess relatively higher amounts of linalool, geranyl esters and 10-epi-ceudesmol as compared to that of the control. On the other hand, the concentrations of 6, 9-guaiadiene and the esters of citronellol were found to be low in these two oils as compared to the oil of the control. Further, the concentrations of isomenthone and rose oxides were quite low (1.8% and trace, respectively) in one of these two oils (14TG) as compared to the oil of control (9.6% and 2.5%, respectively). At the genetic level these two transgenics resembled each other with regard to OPT 1 primer while distinct differences could be noted between them from the others along with the wild type control with regards to same primer (Fig. 3) which further substantiates their totally altered oil profile in terms of the major terpenoid constituents. On the basis of overall analysis, it can be stated that since the increase in geraniol and geranyl esters concentration signifies improved olfactory value (Rao et al. 1999), the oils of these two transgenics definitely represent an improvement over the control from the commercial point of
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view. Reduction in citronellol concentration and increase in geraniol concentration had earlier been noted in three transgenic plants of lemon-geranium of hairy root origin (Pellegrineschi et al. 1994). The present study once again highlights the success of hairy root transformation strategy for the creation of new fragrances through the easiest and quickest method of establishment of transgenic plants from hairy root cultures. Acknowledgements The authors are thankful to the Department of Biotechnology, Government of India for the financial support and the Regional Sophistication Instrumentation Facility, CDRI, Lucknow for GC-MS of the oils. Thanks are also due to S. Sharma and S.A. Hasan for their help in manuscript preparation and A.P. Dhiman for photography.
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