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LIST OF PUBLICATIONS

Kumara Swamy M, Balasubramanya S, Anuradha M (2010). In vitro

multiplication of Pogostemon cablin Benth. through direct

organogenesis. Afr J Biotechnol 9(14):2069–2075.

Kumara Swamy M, Balasubramanya S, Anuradha M (2009).

Germplasm conservation of Patchouli (Pogostemon cablin Benth.).

Int J Biodvers Conserv 1(8):224–230.

Kumara Swamy M, Sudipta KM, Balasubramanya S, AnuradhaM

(2010). Effect of different carbon sources on in vitro

morphogenetic response of Patchouli (Pogostemon cablin benth.).

J Phytol 2(8):11–17.

Kumara Swamy M, Anuradha M. Analysis of genetic variability in

Patchouli cultivars (Pogostemon cablin Benth.) using RAPD

Markers. Research in Biotechnology 2(6): 64– 71.

African Journal of Biotechnology Vol. 9(14), pp. 2069-2075, 5 April, 2010 Available online at http://www.academicjournals.org/AJB ISSN 1684–5315 © 2010 Academic Journals Full Length Research Paper

In vitro multiplication of Pogostemon cablin Benth. through direct regeneration

M. Kumara Swamy1,2, S. Balasubramanya2 and M. Anuradha2*

1Padmashree Institute of Management and Sciences, Kommagatta campus, Kengeri, Bangalore- 560060.

2Rishi Foundation, #234, 10th C main, 1st Block, Jayanagar, Bangalore- 560011.

Accepted 14 January, 2010

An efficient method was developed to initiate multiple shoots from nodal explants of Pogostemon cablin Benth. MS medium supplemented with 0.5 mg/L BA initiated a mean of 45.66 shoots/nodal explant. Within 4 weeks of initiation, regenerated multiple shoots attained a height of 3.6 cm. Subsequent transfer of these in vitro derived nodal segments onto MS medium supplemented with BA and KN ( 0.5 mg/L), induced mean of 62.45 shoots. Higher concentration of either BA or KN more than 0.5 mg/L resulted in callus proliferation and showed hyperhydric shoots with morphological abnormalities. Rooting was readily achieved upon transfer of shoots on half strength MS medium supplemented with 100mg/L activated charcoal. Rooted shoots, following acclimatization in green house, were successfully transferred to soil with 91% survival. Also shoots regenerated in vitro were directly transplanted to soil and acclimatized. Tissue cultured plants were analyzed for oil content by employing gas chromatography and found that the patterns were similar to mother plants. Key words: Growth regulators, regeneration, activated charcoal, patchouli, organogenesis, callus, gas chromatography.

INTRODUCTION Patchouli (Pogostemon cablin Benth.), belonging to the family Lamiaceae is an aromatic plant, native to tropical Asia and is widely grown in India, Malaysia, Philippines, Indonesia and Singapore. The patchouli oil, obtained by steam distillation of shade-dried leaves is commercially used in perfumes and cosmetics (Hasegawa et al., 1992; Maheswari et al., 1993). It also possesses anti insecticidal activities, anti-fungal and bacteriostatic properties (Kukreja et al., 1990; Yang, 1996; Pattnaik et al., 1996). In aromatherapy, it is used to calm nerves, relieve depression and stress (Bowel et al., 2002). Fibrinolytic and anti thrombotic (Sumi, 2003; Eunkyung et al., 2002) activity of this essential oil is also been reported.

The plant never flowers in India and hence vegetative propagation through stem cuttings is in practice. However, the feasibility of mass production of patchouli through *Corresponding author. Email: swamy.bio@gmail.com. Abbreviations: BA, Benzyl-6-adenine; KN, kinetin; MS, Murashige and Skoog; NAA, α-naphthalene acetic acid; IAA, 3-indol acetic acid; GC, gas chromatography.

conventional methods has been limited due to recurrence of mosaic virus (Sastri and VasanthKumar, 1981), root knot nematodes and insect pests. Apart from this, propa-gation through vegetative cuttings is slow and insufficient for large-scale cultivation. Natural variations occurring in this plant may result in yield fluctuations. The rapidness of tissue culture techniques can be advantageous for the continuous provision of plantlet stock for field cultivation (Reddy et al., 2001) and may further compliment breeding programmes.

Several authors have envisaged feasibility of mass propagation of high yielding and disease/ pathogen resistant patchouli through tissue culture. Patchouli plants regeneration from stem tip, leaf and nodal callus (Misra, 1996; Padmanabhan et al., 1981), plant regeneration from protoplasts encapsulated in alginate beads (Kageyama et al., 1995), mass production of virus free plants by in vitro culture and somatic embryogenesis (Kukreja et al., 1990; Rajan et al., 1997) have been reported. Hembrom et al. (2006) has reported the produc-tion of true to type plants of Pogostemon heyneanus through dedifferentiated axillary buds. However, there are limited efforts to study direct organogenesis, which supports

2070 Afr. J. Biotechnol.

Table 1. Effect of different cytokinins on shoot proliferation from nodal segments of patchouli grown on MS medium after 30 days of first subculture.

Medium + concentration of cytokinins (mg/L)

Mean number of shoots/explants

± SD

Mean length of the shoots

(cm) ± SD

Mean fresh weight of the shoots (g) ±

SD

Callus formation

MS (CONTROL) 13.62 ± 1.05 1.23 ± 1.52 1.06 ± 1.39 - MS + 0.25 mg/L BA 36.66 ± 1.81 1.73 ± 1.71 1.42 ± 2.02 - MS + 0.5 mg/L BA 45.66 ± 0.71 3.60 ± 2.08 2.94 ± 2.01 - MS + 1.0 mg/L BA 20.00 ± 1.53 1.65 ± 1.72 1.97 ± 1.84 + MS + 2.0 mg/L BA 00.00 0.00 0.00 + MS + 0.25 mg/L KN 23.33 ± 0.35 1.93 ± 1.90 1.08 ± 1.98 - MS + 0.5 mg/L KN 34.56 ± 0.38 2.43 ± 2.10 2.87 ± 1.23 - MS + 1.0 mg/L KN 21.53 ± 0.98 2.23 ± 1.21 2.44 ± 1.31 - MS + 2.0 mg/L KN 00.00 0.00 0.00 + F- value * * * CD 8.46 0.04 0.04

* Significant at 5% level. +: Callus induction. -: No callus. cultivation by providing true to type plants in large numbers. Hence the present study is aimed to establish suitable protocol for rapid regeneration of patchouli by direct organogenesis using nodal explants. Also the essential oil from mother plant and in vitro grown plant was analyzed by using gas chromatography to check the quality of the oil obtained. This method can mitigate the problem of non-availability of planting materials to meet the global demand. MATERIALS AND METHODS Nodal segments were procured from elite mother plants, maintained at Rishi Herbal Garden, Bangalore, India. All expanded leaves and petioles were removed and the explants were cut into 1 - 2 cm length. The explants were washed 3 - 4 times in the tap water and treated with liquid soap, teepol for 15 min followed by thorough washing under tap water. These were then surface sterilized with 0.5% HgCl2 for 10 min. Rinsing was done five times with sterile distilled water to remove traces of HgCl2 completely. Under aseptic conditions, explants were inoculated on MS (Murashige and Skoog, 1962) medium, containing 2% (w/v) sucrose, supplemented with different concentrations and combinations of BA (0.25, 0.5, 1.0 and 2.0 mg/L) and KN (0.25, 0.5 and 1.0, 2.0 mg/L) for shoot proliferation and multiplication. The pH of the medium was adjusted to 5.8 prior to the addition of 0.8% agar and autoclaved at 121°C, 15 lb pressure for 15 min. All the cultures were incubated at 25 ± 2°C under a 16 h light and 8 h dark regimes with a light intensity of 3000 lux provided by cool-white fluorescent tubes. Weekly observations were recorded. In vitro derived shoots from both the explants were excised after 30 days and sub cultured on to fresh medium with the same concentrations of growth regulators unless otherwise mentioned.

For rooting, 5 - 6 cm long regenerated shoots bearing at least 4 - 5 internodes were excised and cultured on freshly prepared rooting medium containing half strength or full strength MS medium supplemented with different concentrations of activated charcoal (100 and 200 mg/L), IAA and NAA (0.5 and 1.0 mg/L). Rooted plantlets were transferred to sterile soilrite in net pots. For direct acclimatization, the in vitro derived shoots with thick stems were cut

off and directly transferred to sterile soilrite in net pots. Plantlets were hardened for 4 weeks in a moisture saturated chamber with 80% relative humidity. Hardened plants were transferred to pots containing soil: manure: sand (1:1:2) under shade condition. The experiments were set up in completely randomized design with different treatments replicated thrice. 20 cultures were raised for each treatment. Data recorded after 30 days of culture were subjected to Fisher’s method of analysis of variance. Wherever, the ‘F’ test was significant for comparison of treatment means, C. D value was worked out at probability level of 5%.

Fresh leaves of P. cablin after 4 months were hydrodistilled by a Clevenger-type apparatus. The essential oil was collected and stored at 4°C until being analyzed for its chemical constituents by Gas chromatography (GC).

GC analysis of the oil was performed on Varian 3400 (Varian, Les Ulis, France) with an FID and an electronic integrator. The instrument was fitted with a 30 m × 0.25 mm non-polar CP-Sil-5-CB-MS column, film thickness 0.25 mm. Oven temperature was programmed from 50 - 220°C at 5°C /min, held at 120°C for 10 min. Injector and detector temperatures were 250 and 280°C. Carrier gas was helium at 16 psi. 1 ml of oil dissolved in acetone was introduced into the gas Chromatograph with a split mode ratio of 1:100. The constituents of the oil were identified by running the reference sample under similar condition. RESULTS AND DISCUSSION The nodal explants underwent direct organogenesis when cultured on MS using various concentrations of BA and KN (0.25, 0.5 and 1.0 mg/L) separately or in combinations. Comparatively, BA showed the strongest effect than KN in terms of shoot induction. It also increased mean shoot length and shoot weight (Table 1). According to George et al. (2008), BA is most effective in enhancing shoot multiplication and triggering shoot elongation. The use of MS medium supplemented with 0.5 mg/L BA was best suitable for bud break (Figure 1a) and resulted maximum number of shoots/explant (45.66 ± 0.71); higher shoot length (3.60 ± 2.08 cm) and fresh

Swamy et al. 2071

Figure 1. In vitro regeneration of Pogostemon cablin Benth. from nodal explants. a. Induction of shoots in 4 weeks of culture on MS + 0.5 mg/l BA. b. Multiple shoot regeneration from nodal segments on MS + 0.5 mg/l BA and 0.5 mg/l KN. c. Rooting of shoots on MS (½) + Activated charcoal (100 mg/l). d. In vitro raised plantlets transplanted to soil rite in net pots. e. Directly acclimatized plantlet in the soil. f. In vitro regenerated plant in the field.

weight of the shoot (2.94 ± 2.01 g) without callus induction. The result is in accordance with the findings of Bharati (2002) but contradictory to the findings of Kukreja et al. (1990) and Hembrom et al. (2006), who reported the higher requirement of cytokinin (2 mg/L BA) for maximum multiple shoot regeneration in patchouli. In our study, higher concentrations of cytokinins resulted in callus formation. However, BA at 0.5 mg/L when used for subsequent sub cultures resulted in callus, indicating the elevation of endogenous hormonal levels. Hence though

initiation was made on medium supplemented with 0.5 mg/L BA, multiplication is better evidenced on 0.25 mg/L BA (Figure 2). As the concentration of cytokinins was increased beyond 0.5 mg/L, it resulted in decrease in number of shoot buds coupled with callus proliferation.

The decrease in shoot production at higher concentration of BA may be due to the inhibition of organogenesis and induction of callus proliferation. Patchouli is a very sensitive plant and it expresses its morphogenetic potentiality even at very low concentrations of cytokinins.

2072 Afr. J. Biotechnol.

Table 2. Effect of combination of cytokinins on elongation of shoots regenerated from primary node cultures of patchouli grown on MS medium after 30 days.

Cytokinins (mg/L) Mean Shoot length

(cm) ± SD

Number of multiple

shoots ± SD

Fresh weight of the shoots

(g) ± SD

Callus formation BA KN

0.0 0.0 0.21 ± 1.3 13.14 ± 0.4 1.10 ± 1.9 - 0.25 0.25 1.95 ± 1.8 62.66 ±0.7 2.46 ± 1.7 - 0.5 0.25 4.65 ± 1.4 61.33 ± 1.0 4.67 ± 1.6 - 1.0 0.25 1.66 ±0.9 22.66 ± 1.1 1.86 ±1.7 + 0.25 0.5 3.31 ± 1.1 41.66 ± 0.8 3.06 ± 1.2 - 0.5 0.5 5.20 ± 1.7 62.45 ± 0.6 5.07 ± 0.9 - 1.0 0.5 2.05 ± 1.7 21.37 ± 0.4 2.35 ± 1.4 + 0.25 1.0 2.32 ± 0.8 23.00 ± 1.2 2.96 ± 1.4 - 0.5 1.0 2.67 ± 1.4 42.10 ± 1.5 4.14 ± 1.3 - 1.0 1.0 1.92 ± 1.2 21.33 ± 0.8 2.35 ± 1.1 + F- value * * * CD 0.03 2.93 0.03

* Significant at 5% level. +: Callus induction. -: No callus. Shoot proliferation was satisfactory on MS medium supplemented with 0.25 mg/L BA and 0.5 mg/L KN separately. However, kinetin has no significant effect on multiple shoot regeneration but played a role in increasing the length and strength of shoots. The combination treatment (0.5 mg/L BA along with 0.5 mg/L KN) was found to exhibit highest frequency of shoot multiplication (62.45 ± 0.6%). The highest mean shoot length (5.20 ± 1.7 cm) and mean fresh weight of the shoot (5.07 ± 0.9 g) was also evidenced in the same treatment (Table 2). The efficacy of BA over KN, when used singly and in combi-nations has been demonstrated for the axillary bud proliferation in many medicinal plants of Lamiaceae like Mentha spicata and Lavendula viridis (Hirata and Kukreja, 1990; Dias and Nickell, 2002). Superior effect of the combination of BA and KN may be due to the synergy of cytokinins as reported in Rollinia mucosa and Solanum surrattense (Figueiredo, 2001; Pawar, 2002). The above result clearly indicates that combination of BA and KN is a better choice for patchouli as it significantly exhibited better morphogenetic response in terms of multiple shoot regeneration, length of the shoots and biomass (Figure 1b).

The effect of the strength of MS basal media, MS media with activated charcoal, IAA and NAA at different concentrations on rhizogenesis was studied (Table 3). Among the treatments tried, ½ strength MS medium is enough to get better rooting. This is in conformity with the results obtained by Bharati (2002) in patchouli. However, we could able to induce high frequency of rooting (93.33 ± 0.9%) when shoots implanted on ½ strength MS media with activated charcoal with 100 mg/l activated charcoal. Mean number of roots/shoot (15.23 ± 0.6) and root length (6.23 ± 1.8 cm) was found to be superior among all other treatments (Figure 1c). This is the first report of its kind in

patchouli. Activated charcoal is an anti-oxidant and known to induce rhizogenesis in Decalepis hamiltonii (Obul et al., 2001) and Annona cherimoya (Padilla and Encina, 2004). This is because it provides darkness in the medium, which is essential for rooting. The result obtained by using half strength MS medium and activated charcoal is superior to the results obtained by using auxins. Both IAA and NAA were shown to induce rooting with varying degrees, however not suitable for patchouli as both the auxins invariably triggered callus proliferation. The results of Misra (1996) support the usage of auxins for rhizogenesis in patchouli which is in contrary to the present observation. This suggests that although the addition of auxins is beneficial for rooting, their use is not essential in patchouli. The similar report is published in Ulmus species (Conde et al., 2008). After 4 weeks, 89% of in vitro derived plants were directly acclimatized (Figure 1e) suggesting that the formation of in vitro roots prior to acclimatization is not needed and this can reduce time and cost. The similar observations are reported by Conde et al. (2008) and Cheng and Shi (1995).

In vitro raised plantlets were transplanted to the soil in net pots (Figure 1d). During the early hardening phase, maintenance of 80% relative humidity in the chamber showed 91% plantlet survival. After 4 weeks of hardening, the plantlets were transferred to pots filled with sand: soil: manure (2:1:1) under shade (Figure 1e). Gradual transfer of the established plants to the sunlight was ideal for tissue culture derived patchouli plants in the field (Figure 1f) rather than a direct transfer to sunlight, which caused wilting of plants and charring of leaves. Similar observations are recorded by Misra (1996).

All the regenerated plants grown for 4 months were similar in leaf morphology, plant height and number of branches per plant. The essential oils were extracted

Swamy et al. 2073

Table 3. Effect of various concentrations IAA, NAA and activated charcoal on rooting of proliferated shoots of patchouli.

Medium (strength) + Auxin (mg/L)

Root induction (%) ± SD

Mean number of roots/shoot (cm) ± SD

Mean root length (cm) ±SD

MS (½) 91.01 ± 1.0 13.00 ± 1.2 5.40 ± 1.9 MS (½) + IAA (0.5) 71.31 ± 1.0 12.10 ± 0.8 5.13 ±1.8 MS (½) + IAA (1.0) 64.33 ± 0.9 13.66 ± 0.5 5.16 ± 1.4 MS (½) + NAA (0.5) 65.66 ± 1.6 14.06 ± 0.3 5.43 ± 0.9 MS (½) + NAA (1.0) 61.00 ± 2.1 13.78 ± 1.3 5.66 ± 1.5 MS (½) + Activated charcoal (100) 93.33 ± 0.9 15.23± 0.6 6.23 ± 1.8 MS (½) + Activated charcoal (200) 92.66 ± 1.0 15.00 ± 0.7 6.10 ± 1.5 MS 82.12 ± 0.8 11.20 ± 0.8 5.10 ± 1.4 MS + IAA (0.5) 51.00 ± 1.0 11.66 ± 1.1 5.14 ± 1.3 MS + IAA (1.0) 67.66 ±1.7 12.12 ±1.4 5.23 ± 1.8 MS + NAA (0.5) 63.33 ± 0.9 12.00 ± 1.6 5.45 ± 0.9 MS + NAA (1.0) 55.33 ± 1.2 12.01 ± 0.8 5.50 ± 1.5 MS + Activated charcoal (100) 90.66 ± 1.2 13.21 ± 0.6 5.76 ±0.9 MS + Activated charcoal (200) 85.00 ± 1.0 13.00 ± 0.5 5.80 ± 0.8 F-value * * * CD 1.00 1.13 0.12

* Significant at 5% level.

Figure 2. Gas chromatographic profiles of essential oils extracted from (a) the leaves of mother plant and (b) in vitro derived regenerants.

2074 Afr. J. Biotechnol.

Figure 2. Continued. from the leaves of in vitro grown plants and mother plant. The oil yield was found to be 0.30% (v/w) of fresh weight. The essential oils were analyzed by using GC and their patterns were compared. Patchouli alcohol at 30.31% was found to be the predominant component in the oil. The uniform pattern of essential oils in the GC profile of the regenerants and the mother plant (Figures 2a and b) suggests the feasibility of the protocol.

In conclusion, the above protocol describes an efficient protocol for rapid multiplication of patchouli by direct regeneration, which is preferred for generating true-to-type plants over callus regeneration. The present protocol can ensure a stable supply of this commercial crop irrespective of seasonal variations and thus meet the global demand for its essential oil. REFERENCES Bharati N (2002). Biotechnology in commercial production of patchouli in

North Eastern Region. In: Patchouli; National Workshop on Commercialization of patchouli in North Eastern region held at Guwahati, Assam. NEDFC and NHB, pp. 46 -51.

Bowel EJ, Griffiths DM, Quirk L, Brownriggs A, Croot K (2002). Effect of essential oils and touch on resistance to nursing care procedures and

other dementia-related behaviours in a residential care facility. Int. J. Aromather. 12: 22-29.

Cheng ZM, Shi NQ (1995). Micropropagation of mature Siberian elm in two steps. Plant Cell Tissue Organ Cult. 41(2): 197-199.

Conde P, Sousa A, Costa A, Santos C (2008). A protocol for Ulmus minor Mill. Micropropagation and acclimatization. Plant Cell Tissue Organ. Cult. 92(1): 113-119.

Dias MC, Nickell GL (2002). Rapid multiplication of Lavandula viridis L through in vitro axillary shoots proliferation. Plant Cell Tissue Organ Cult. 68: 99-102.

Eunkyung P, Kyung YH, Hyun KD (2002). Antithrombotic activity of Sunghyangjunggisan. Nat. Prod. Sci. 8(2): 71-75.

Figueiredo SFL (2001). Micro propagation of Rollinia mucosa (Jacq.) Baill. In Vitro Cell. Dev. Biol. Plant. 37: 4 71-475.

George EF, Hall MA, Klerk GJD (2008). Plant propagation by Tissue culture: Volume 1. The background. Third Edition, Springer Publisher: Dordrecht; London.

Hasegawa Y, Tajima K, Toi N, Sugimura Y (1992). An additional constituent occurring in the oil from a patchouli cultivar. Flav. Fragr. J. 7: 333-335.

Hembrom, Emanuel M, Martin KP, Patchathundikandi, Suneesh K, Madasser Y, Joseph (2006). Rapid in vitro production of true to type plants of Pogostemon heyneanus through dedifferentiated axillary buds. In vitro. Cell. Dev. Biol. Plant. 42(3): 283-286.

Hirata T, Kukreja AK (1990). Volatile monoterpenoids constituents of the plantlets of Mentha spicata produced by shoot tip culture. Phytochemistry, 29: 955-959.

Kageyama Y, Honda Y, Sugimura Y (1995). Plant regeneration from patchouli protoplasts encapsulated in alginate beads. Plant Cell Tissue

Organ Cult. 41(1): 65-70. Kukreja AK, Mathur AK, Zaim M (1990). Mass production of virus free

patchouli plants [Pogostemon cablin (Blanco) Benth.] by in vitro culture. Trop. Agrc. 67: 101-104.

Maheswari ML, Vasantha Kumar T, Neelam Sharma, Chandel KPS (1993). Patchouli- An Indian perspective. Indian Perf. 37: 9-11.

Misra M (1996). Regeneration of Patchouli (Pogostemon cablin Benth.) plants from leaf and node callus and evaluation after growth in the field. Plant Cell Rep. 15: 991-994.

Murashige T, Skoog F (1962). A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol. Plant. 15: 473-497.

Obul RB, Giridhar P, Ravishankar GA (2001). In vitro rooting of Decalepis hamiltonii. Wight and Arn, an endangered shrub, by auxins and root promoting agents. Curr. sci. 81(11): 26-29.

Padilla MG, Encina CL (2004). Micro propagation of adult Cherimoya (Annona cherimoya). In vitro Cell. Dev. Biol. Plant. 40(2): 210-214.

Padmanabhan C, Sukumar S, Sreerangaswamy SR (1981). Patchouli plants differentiated in vitro from stem tip and callus cultures. Curr. Sci. 50: 195-197.

Pattnaik S, Subramanyam VR, Kole C (1996). Antibacterial and antifungal activity of ten essential oils in vitro. Microbios. 86: 237-246.

Pawar PK (2002). A technique for rapid propagation of Solanum surrattense Burm. F. Indian. J. Biotechnol. 1: 201-204.

Swamy et al. 2075 Rajan GB, Shakila A, Rajasekaran LR (1997). Mass propagation of

Pogostemon patchouli through somatic embryogenesis. South Indian Hort. 45: 45-49.

Reddy PS, Rodrigues R, Rajasekharan R (2001). Shoot organogenesis and mass propagation of Coleus forskohlii from leaf derived callus. Plant Cell Tissue Organ Cult. 66: 183-188

Sastri KS, Vasantha Kumar T (1981). Yellow mosaic of patchouli (Pogostemon patchouli) in India. Curr. Sci. 50: 767-768.

Sumi H (2003). Fibrinolysis-accelerating activity of the essential oils and Shochu aroma. Aroma Res. 4(3): 264-267.

Yang D (1996). Michel, Mandin andriamboavonjy, Poitry, Chaumont, Mellet C Antifungal and antibacterial properties in vitro of three patchouli oils from different origins. Acta Botanica. Gallica. 143(1): 29-35.

International Journal of Biodiversity and Conservation Vol. 1(8) pp. 224-230 December, 2009 Available online http://www.academicjournals.org/ijbc ©2009 Academic Journals Full Length Research Paper

Germplasm conservation of patchouli (Pogostemon cablin Benth.) by encapsulation of in vitro derived

nodal segments

M. Kumara Swamy1, S. Balasubramanya2 and M. Anuradha2

1Padmashree Institute of Management and Sciences, Kommagatta Campus, Kengeri, Bangalore- 560060, India. 2Rishi Foundation, #234, 10th C main, 1st Block, Jayanagar, Bangalore- 560011, India.

Accepted 22 October, 2009

Encapsulation of in vitro derived nodal segments of patchouli (Pogostemon cablin Benth.) was done successfully by employing sodium alginate gel. Among various concentrations of sodium alginate tried to optimize the strength of the bead, 4% sodium alginate produced firm beads and showed the highest percentage of shoot emergence (73.3%). The best storage temperature was found to be 25°C. The encapsulated beads retained regeneration potentiality up to 6 months and later gradually declined. Browning and loss of regeneration was more after 9 months. Various growth regulating factors (6- benzyl adenine, kinetin, coconut water and tomato juice) at different concentrations were tested for their conversion frequency of encapsulated buds. Murashige and Skoog media supplemented with 2.22 µM/l 6- benzyl adenine showed the highest conversion percentage (91.1%) followed by, 10% coconut water (85.4%). Plants retrieved from the encapsulated buds were rooted on half strength Murashige and Skoog basal medium and acclimatized successfully in the soil. This technology can be adopted for ex situ germplasm conservation of elite plants of patchouli. Key words: Conversion frequency, germplasm conservation, growth regulators, encapsulated buds, patchouli.

INTRODUCTION Patchouli (Pogostemon cablin Benth.), belonging to Lamiaceae, yields an aromatic oil and is commercially used in perfumes and cosmetics (Hasegawa et al., 1992; Maheswari et al., 1993). Patchouli is propagated by rooting the vegetative cuttings of stem, and there are reports of in vitro propagation and mass production of virus-free patchouli plants (Kukreja et al., 1990). The possible methods of conservation of improved lines of patchouli are maintaining the stock plants in the field or as in vitro cultures. The conservation of elite germplasm in the field is prone to diseases, pests and other environ-mental hazards. Alternatively maintenance of in vitro cultures for conservation is associated with somaclonal variations, and not cost effective because of monthly sub *Corresponding author. E-mail: swamy.bio@gmail.com. Abbreviations: BA; 6- benzyl adenine, KN; kinetin, MS; Murashige and Skoog, NAA; α-naphthalene acetic acid, IAA; 3-indol acetic acid.

cultures and labour intensive processes. In this regard, synthetic seed technology offers an excellent scope for conservation of rare hybrids, elite genotypes and genetically engineered patchouli plants. During the last four decades, synthetic seed technology has gained considerable importance in plant biotechnology as a potential, viable and valuable system for ex situ con-servation of commercially important plants (Kavyashree et al., 2004). Encapsulation and storage of the buds at freezing temperatures offers a long-term storage capability, maximal stability of phenotypic and genotypic characteristics, minimum space and maintenance. Low production costs, ease of storage and transport are the additional advantages (Ghosh and Sen, 1994).

Studies on the in vitro germplasm conservation by encapsulation of somatic embryos is reported in many plant species, including cereals, vegetables, fruits, ornamentals and medicinal plants (Bapat and Rao, 1988; Ghosh and Sen, 1994; Fowke et al., 1994; Onay et al., 1996; Castillo et al., 1998). In recent years encapsulation of in vitro derived shoot tips or axillary buds has become a suitable alternative in place of somatic embryos (Bapat

et al., 1987; Mathur et al., 1989; Sharma et al., 1994; Maruyama et al., 1997; Sarkar and Naik, 1997; Adriani et al., 2000). This technology is quite promising as developing somatic embryo system is difficult for many plant species. Kageyama et al., (1995) has reported encapsulation and regeneration of patchouli protoplasts, however encapsulation of in vitro derived nodal segments is being reported for the first time in the present study, which is a simpler and easier technique.

The present report describes the encapsulation of nodal segments of micropropagated patchouli in calcium alginate hydrogen. The evaluation of in vitro response of encapsulated micropropagules to various concentrations of growth regulators and also the effect of temperature and storage period on conversion rate is also reported in this study Murashige and Skoog, 1962. MATERIALS AND METHODS Healthy patchouli plants were selected from the herbal garden of Rishi Herbal Technologies, pvt. Ltd., Bangalore. Nodal segments and shoot tips (2 - 3 cm) of these selected plants were surface sterilized with 0.1% (w/v) HgCl2 for 10 min and washed thoroughly with sterile distilled water. Later, the explants were implanted on MS medium supplemented with 2.22 µM/l BA. The pH of the medium was adjusted to 5.7 prior to autoclaving at 121°C for 20 min. Cultures were maintained at a temperature of 25 ± 2°C under 16 h light (2000 lux) / 8 h dark photoperiod and sub cultured every 4 weeks. Multiple shoots regenerated on this medium were used for further studies. Nodal segments measuring about 5 mm were explanted carefully and used for immobilization experiments.

The in vitro derived explants isolated were immersed for a few seconds in 2 - 6% sodium alginate solution prepared in full strength MS basal medium with 2% sucrose (w/v). Later, the micro-propagules in alginate medium were picked up by tweezers and dropped into a sterile solution of 100mM calcium chloride. The drops, each containing a single micropropagule, were placed in this solution for half an hour to allow polymerization. Calcium alginate beads containing the micropropagule were retrieved from the solution and rinsed twice with autoclaved distilled water to remove the traces of calcium chloride. The beads were then transferred to sterile filter paper in Petri dishes. Blot dried beads were stored in a petri dishes sealed with para film for 1 - 6 months at refrigerator (4°C), incubator (20°C) and plant tissue culture room(25°C).

After storage, the encapsulated buds were cultured in regeneration medium and incubated in a culture room maintained at 25 ± 2°C. Various regeneration media tested were MS medium supplemented with different concentrations of BA (1.11, 2.22 and 4.44 µM/l) or Kinetin (KN) (1.16, 2.32 and 4.65 µM/l). The percentage of shoot emergence from the encapsulated beads was calculated after 3 - 4 weeks of inoculation. To further improve the shoot multiplication and morphology of the plantlet, MS media fortified with 5 - 15% coconut water and tomato juice (5 - 15 g of tomato is added to 100 ml of distilled water and grinded in a mixer to get juice). Similar experiments were also carried out by using filter paper bridges in MS liquid medium in culture tubes to check the regeneration efficiency. For rooting, developed shoots were transferred to half strength and full strength MS basal medium with or without α-naphthalene acetic acid (NAA) at 2.68 and 5.37 µM/l or 3-indol acetic acid (IAA) at 2.85 and 5.71 µM/l. The rooted shoots were planted in net pots containing sterile Soilrite and hardened for 4 weeks in a moisture saturated glass chamber with 80% relative humidity. Hardened plantlets were transferred to pots containing

Swamy et al. 225 garden soil: manure: sand (1:1:2) under shade conditions. For each treatment 20 replicates were used and the experiments were repeated thrice. The data were subjected to Fisher’s method of analysis of variance. RESULTS AND DISCUSSION Establishment of micropropagated plants The surface sterilized explants inoculated on to MS medium fortified with 2.22 µM/l BA resulted in multiple shoot regeneration after 25 days. The development of complete multiple shoots were observed after 3 - 4 weeks of incubation. The nodal segments from these micropropagated plants were used for encapsulation. Effect of different concentrations of sodium alginate on encapsulation Sodium alginate is a copolymer composed of D-mannuronic acid and L-glucuronic acid units and has been extensively studied because of its biocompatibility, biodegradability and its capability to form hydro gels in the presence of divalent cations. The rigid structure and large pore size of these gels, which are insoluble in water, make them useful for the encapsulation of live cells of plants. Polymer concentration, degree of viscosity of the alginate used, CaCl2 concentration, and curing time are important parameters determining the permeability, resistance and hardness of the resulting beads and the subsequent success of the encapsulation method (Block, 2003). In the present study, the polymerizing ability of sodium alginate at different concentrations (2 - 6%) varied markedly when used to encapsulate the buds (Table 1). Very firm, clear, isodiametric beads of uniform size and shape, was achieved using 4% sodium alginate solution and 100 mM calcium chloride (Figure 1a). Concentrations of sodium alginate lower than 4% were not suitable as the beads were too soft to handle, while at higher concentrations (5 and 6%), they were too viscous, harder and hindered the emergence of shoot in patchouli. The influence of optimum concentration (4%) of sodium alginate on bead quality and shoot emergence is in agreement with the earlier reports (Mathur et al., 1989; Ghosh and Sen, 1994; Castillo et al., 1998; Jaydip Mandal et al., 2000). The conversion frequency was highest (73.3%) when 4% sodium alginate was used and decreased with increase in sodium alginate concentration to 16.7% at 6%. Different stages of sprouting and regeneration from encapsulated buds are depicted in Figure 1b. Effect of storage period and temperature on shoot emergence from encapsulated buds Conversion frequency of shoots is directly dependent on storage period and temperature (Table 2). It was observed

226 Int. J. Biodvers. Conserv.

Table 1. Effect of sodium alginate concentration on quality of beads and shoot emergence.

Concentration of sodium alginate (%) Quality of beads Conversion frequency (%) 2 Too soft 50 ± 2.51 3 Soft 60 ± 2.56 4 Firm 73.3 ± 2.53 5 Hard 30.0 ± 2.65 6 Hard 16.7 ± 2.62

Data shown are means of ± standard error from 20 beads for each of three replicates per treatments.

Table 2. Effect of storage period and temperature on shoot emergence from encapsulated buds.

Storage period (Months) Temperature (°C) Conversion frequency (%) 4 53.3 ± 2.86

20 66.0 ± 2.89 2 25 82.3 ± 2.62

4

39.3 ± 2.87

20 46.7 ± 2.68

4 25 63.1 ± 2.68

4

36.7 ± 2.69

20 43.3 ± 3.67

6 25 52.3 ± 3.68

Data shown are means of ± standard error from 20 beads for each of three replicates per treatments. observed that 2 months old beads resulted in higher emergence of plants (53.3-73.3%) when compared to the germination response of 4 and 6 months old beads (33.3 - 50.0% and 36.7 - 43.3% respectively). The decline in the germination percentage among the synthetic seeds of shoots stored for a period of 2 - 6 months may be due to inhibited respiration of plant tissues by alginate leading to loss of viability (Redenbaugh et al., 1987). The best storage temperature is 25°C during 2, 4 and 6 months periods. The morphology and growth of regenerated shoots is not affected at 25°C. However, at 4 and 20°C the emerged shoots exhibited slow growth with necrotic and vitrification symptoms. After 6 months of storage, the percent frequency of conversion was reduced, along with death and decay of the encapsulated buds. This might be due to the cracks and dehydration of the bead. Effect of various growth regulating factors on bud sprouting and regeneration of encapsulated buds Different growth regulating factors were supplemented to MS media to increase the sprouting and multiplication rate of encapsulated buds (Table 3 and 4). There was a significant difference in percent frequency and nature of response due to different growth regulator supplemented media. The regeneration potentiality of a tissue/organ

culture can vary according to type and concentration of growth regulator, the type and age of explant and the species from which it is derived (George, 1993). The percentage of encapsulated buds exhibiting multiple shoots emergence was highest (91.1%) on MS medium supplemented with 2.22 µM/l BA followed by µM/l KN (86.3%). MS basal medium (control) exhibited only single shoot formation. The use of filter paper bridges on the liquid medium was more suitable for regeneration (Figure 1c). This is a useful approach to avoid abnormality and hyperhydricity of the recovered shoots.

Among the natural growth regulating factors supple-mented to MS medium, 10% coconut water resulted in good response (Figure 1d). Maximum percentage of multiple shoot emergences with an average shoot length of 2 cm was observed on media supplemented with 10% coconut water and this response is superior to the other treatments (Table 4). Being complex mixtures and inexpensive nature, natural additives provide a good scope for substituting the expensive growth regulators. Coconut water stimulates cell division in other cultured tissues and its use as a supplement is adopted in many laboratories (Morel, 1950; Nickell, 1950; Henderson et al. 1952; Archibald, 1954). Similarly Straus (1960) has shown that tomato juice function by supplying a form of organic nitrogen to in vitro cultured explants. Optimizing cost effective protocol by using natural extracts for the

Swamy et al. 227

Table 3. Effect of plant growth regulators on shoot emergence from encapsulated buds. MS + Plant growth Regulator

Concentration (µM/l)

% of beads Exhibiting multiple shoots

% of beads exhibiting shoot length more than 2 cm

0.25 78.3 ± 2.68 86.0 ± 1.14 0.50 91.1 ± 2.66 93.6 ± 1.16

BA

1.00 70.0 ± 2.45 58.3 ± 1.15 0.25 71.7 ± 2.66 34.3 ± 1.19 0.50 86.3 ± 2.66 38.4 ± 1.16

KN

1.00 70.2 ± 2.71 29.0 ± 1.86 Control - 0.00 0.00 F-value - 19.62* 255.31* SEm ± - 2.318 2.54 CD at 5% Level - 5.169 5.96

* Significant at 5% Level. Data shown are from 20 beads for each of three replicates per treatments.

Table 4. Effect of natural extracts on multiplication of shoots emerged from encapsulated buds.

MS + natural extracts Concentration ( % )

% of beads exhibiting multiple shoots

% of beads exhibiting shoot length more than 2 cm

5 76.6 ± 2.67 80.0 ± 1.14 10 85.4 ± 2.68 91.6 ± 1.16

Coconut water

15 66.6 ± 2.58 48.3 ± 1.15 5 13.3 ± 2.66 28.3 ± 1.19

10 28.3 ± 2.58 38.3 ± 1.16 Tomato juice 15 20.0 ± 2.86 21.0 ± 1.86 Control - 0.00 0.00 F-value - 117.55* 255.31* SEm ± - 1.10 2.54 CD at 5% Level - 2.46 5.96

* Significant at 5% Level. Data shown are from 20 beads for each of three replicates per treatments.

Figure 1A. Encapsulated buds of patchouli in calcium alginate.

228 Int. J. Biodvers. Conserv.

Figure 1B. Different stages of sprouting and multiplication of encapsulated buds.

Figure 1c. Multiplication of encapsulated buds in MS liquid medium supported on filter paper bridges.

Figure 1d. Multiple shoots regeneration of encapsulated buds on MS + 10% coconut water.

Swamy et al. 229

Figure 1E. Rooted plantlets of patchouli regenerated from encapsulated buds.

Figure 1f. Acclimatization of regenerated plantlets from encapsulated buds.

using natural extracts for the recovery of plants from encapsulated propagules of patchouli perhaps is a first report made in the present study. Rooting and acclimatization of plantlets regenerated from encapsulated buds Individual shoots were rooted on half strength MS medium without hormones to produce whole plants (Figure 1e). The use of NAA and IAA in the media in-duced rooting with varying degrees with callus formation at the base of the shoot. This suggests that although the addition of auxins is beneficial for rooting, their use is not essential and half strength MS medium is enough to get

vigorous rooting. This is in conformity with the results obtained by Bharati (2002). Rooted shoots were successfully hardened off in net pots containing sterile soilrite (Figure 1f). After 4 weeks of hardening, these plantlets were acclimatized well and transferred to green house and planted in the field. Maintenance of high humidity during the early hardening phase in glass cham-ber was found to be essential for good plantlet survival (90 - 93%). The above results coincides with the findings of Misra (1996) who has reported that the maintenance of 80% relative humidity increase the plant survival rate. To date there are no reports on encapsulation of nodal segments in patchouli. The use of this method can avoid minimum of six subcultures and variations due to pro-longed cultures on a growth regulator containing medium.

230 Int. J. Biodvers. Conserv. Conclusions The present study shows the feasibility of using nodal segments for encapsulation and germplasm conservation of rare hybrids, elite genotype and genetically engineered patchouli plants. Germplasm conservation through tissue culture requires about 5 - 6 subcultures in fresh medium and this can cause variations in the plant morphology. Hence, this encapsulation technology can be adapted to reduce the risk of sub-culturing, the handling cost and risk of contamination during sub culturing. However, further investigations are needed to extend the duration of storage before making the technique applicable to patchouli germplasm storage. REFERENCES Adriani M, Piccioni E , Standardi (2000). Effects of different treatments

on the conversion of Hayward kiwifruit synthetic seeds to whole plants following encapsulation of in vitro derived buds. NZ. J. Crop. Hort. Sci., 28: 59-67.

Archibald JF (1954). Culture in vitro of cambial tissues of Cacao. Nature, 173: 351-353.

Bapat VA, Mhatre M , Rao PS (1987). Propagation of Morus indica L. (mulberry) by encapsulated shoot buds. Plant Cell. Rep., 6: 393-395.

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Bharati N (2002). Biotechnology in commercial production of patchouli in NE Region.In: Patchouli. National Workshop on Commercialization of patchouli in NE region dated 9-11 April, 2002 at Guwahati, Assam organized by NEDFC and NHB, pp: 46 -51.

Block W (2003). Water status and thermal analysis of alginate beads used in cryopreservation of plant germplasm. Cryobiology. 47: 59-72.

Castillo B, Smith MAL and Yadava UL (1998). Plant regeneration from encapsulated somatic embryos of Carica papaya L. Plant Cell. Rep., 17: 171-176.

Fowke LC, Attree SM and Pomeroy MK (1994). Production of vigorous desiccation tolerant white spruce (Piceca glauca [Moench.] Voss.)

synthetic seeds in a bioreactor. Plant Cell Rep., 13: 601-606. George EF (1993). Plant growth regulation. In: Plant propagation by

tissue culture. Part-1, pp: 420-450. Ghosh B , Sen S (1994). Plant regeneration from alginate encapsulated

somatic embryos of Asparagus cooperi Barker. Plant Cell. Rep., 13: 381-385.

Hasegawa Y, Tajima K, Toi N , Sugimura Y (1992). An additional constituent occurring in the oil from a patchouli cultivar. Flavour Frag. J., 7: 333-335.

Henderson JHM, Durell ME , Bonner J (1952). The cultures of normal

sunflower callus. Am. J. Bot., 39: 467-472. Jaydip M, Sitakanta P, Pradeep K and Chand (2000). Alginate en-

capsulation of axillary buds of Ocimum americanum L. (Hoary basil), O. basilicum L. (Sweet basil), O. gratissimum L. (Shrubby basil) and O. sanctum L. (Sacred basil) In vitro. Cell Dev. Biol., 36: 287-292.

Kageyama Y, Yasuki Honda, Yukio Sugimura (1995). Plant regeneration from patchouli protoplasts encapsulated in alginate beads. Plant Tiss. Organ Cult., 41: 65-70.

Kavyashree R, Gayathri MC, Revanasiddaiah HM (2004). Regeneration of encapsulated apical buds of mulberry variety- S54 (Morus indica L.). Serologia. 44(1): 83-89.

Kukreja AK, Mathur AK, Zaim M (1990). Mass production of virus free patchouli plants (Pogostemon cablin (Blanco) Benth.) by in vitro culture. Trop. Agric., 67: 101-104.

Maheshwari, ML, Vasantha Kumar T, Neelam Sharma and Chandel KPS (1993). Patchouli- An Indian perspective. Indian Perfumer, 37: 9-11.

Mathur J, Ahuja PS, Lai N , Mathur AK (1989). Propagation of Valeriana wallichii D.C. using encapsulated apical and axial shoot buds. Plant Sci., 60: 111-116.

Meena M (1996). Regeneration of patchouli (Pogostemon cablin Benth.) plants from leaf and node callus, and evaluation after growth in the field. Plant Cell Rep., 15: 991-994.

Morel G (1950) Sur la culture des tissues de deux monocotyledons. Comp. Rendus. De L’Acad. Des. Sci., 230: 1099-1105.

Murarshige T , Skoog F (1962). A revised medium for rapid growth and bioassays with tobacco cell cultures. Physiol. Plant, 15: 473-497. Maruyama E, Kinoshita I, Ishii K, Ohba K and Saito A (1997). Germplasm conservation of the tropical forest trees, Cedrele odorata L., Guazauma Crinata Mart. and Jacaranda mimosaefolia D. Don., by shoot tip encapsulation in calcium alginate and storage at 12-25°C. Plant Cell Rep., 16: 393-396.

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Onay A, Jeffree CE , Yeoman MM (1996). Plant regeneration from encapsulated embryoids and embryogenic mass of pistachio (Pistachia vera L.). Plant Cell Rep., 15: 723-726.

Redenbaugh K, Slade P, Viss PR , Fugii JA (1987). Encapsulation of a somatic embryos in synthetic seed coats. Hort. Sci., 22: 803-809.

Sarkar D , Naik PS (1997). Synseeds in potato: an investigation using nutrient encapsulated in vitro nodal segments. Sci. Hortic. 73: 179-184.

Sharma D , Naik PS (1998). Synseeds in potato: an investigation using nutrient encapsulated in vitro nodal segments. Sci. Hort., 73: 179-184

Sharma TR, Singh BM and Chauhan RS (1994). Production of disease free encapsulated buds of Zingiber officinale Rosc. Plant Cell Rep., 13: 300-302.

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Journal of Phytology 2010, 2(8): 11–17 ISSN: 2075-6240 Tissue Culture Available Online: www.journal-phytology.com

REGULAR ARTICLE

EFFECT OF DIFFERENT CARBON SOURCES ON IN VITRO MORPHOGENETIC RESPONSE OF PATCHOULI

(POGOSTEMON CABLIN BENTH.) M. Kumara Swamy1,2*, K. M. Sudipta1, S. Balasubramanya2 and M. Anuradha1,2

1Padmashree Institute of Management and Sciences, Kommagatta, Kengeri, Bangalore- 560060 2Rishi Foundation, #234, 10th C main, 1st Block, Jayanagar, Bangalore- 560011

SUMMARY

The effect of various carbon sources, sucrose, glucose, fructose, table sugar and sugarcane juice was investigated on in vitro growth and physiology of Pogostemon cablin Benth. The entire morphogenetic pattern was influenced by nature and concentration of carbon source used. The maximum shoot length (4.87±0.41cm) and higher number of multiple shoots (61.43±0.l9) was observed on MS media fortified with 20% sugarcane juice. The maximum fresh weight of shoots was recorded on MS medium containing 2% sucrose (4.89±0.19g). Sugarcane juice at 20% resulted in maximum chlorophyll content (0.81±2.0mg/g tissue). The protein content was maximum on media supplemented with 20% sugarcane juice (18.8±0.24mg/ml) followed by 2% sucrose (18.5±0.25mg/ml). The least content was observed on media supplemented with 3% fructose (12.2±0.32mg/ml). Maximum accumulation of carbohydrate content was observed on MS supplemented with 3% sucrose (18.2±0.30mg/ml) and the least carbohydrate content (11.6±0.53mg/ml) was observed on MS media with 1% glucose. This is the first report on the use of sugarcane juice in tissue culture studies of patchouli.

Key words: Patchouli, Carbon sources, Sugarcane juice, Multiple shoots, Physiology M Kumara swamy et al. Effect of Different Carbon Sources on In Vitro Morphogenetic Response of Patchouli (Pogostemon cablin Benth.). J Phytol 2/8 (2010) 11-17. *Corresponding Author, Email: swamy.bio@gmail.com

1. Introduction Patchouli (Pogostemon cablin Benth.),

belonging to the family Lamiaceae is an aromatic plant. The oil, obtained by steam distillation of shade dried leaves is commercially used in perfumes and cosmetics because of its strong fixative property. It possesses anti insecticidal activities, anti-fungal and bacteriostatic properties [1, 2]. In aromatherapy, it is used to calm nerves, relieve depression and stress. In Chinese medicine decoction from the leaves are used with other drugs to treat nausea, vomiting, diarrhea, cold and headaches [3]. Feasibility of mass propagation of high yielding and disease/pathogen resistant patchouli through tissue culture has been envisaged by several authors [1, 4, 5, 6 and 7].

The growth and multiplication of shoots in vitro are affected by many factors, one of which is the type of exogenous carbon source added to the medium [8]. The carbon sources serve as energy and osmotic agents to support the growth

of plant tissues [9]. There have been various opinions on the beneficial effects of various carbon sources (sucrose, fructose, glucose, table sugar etc,.) on the growth of plants in vitro.

Sucrose (2-5%) is the most popular carbohydrate used in tissue culture [10]. In general most of the tissue culture studies are performed using sucrose as the sole carbon source due to its efficient uptake across the plasma membrane. Glucose also has been reported to have various effects on the in vitro growth of plants. Cunha and Fernandes-Ferreira (1999) on Linum usitatissium showed that medium supplemented with monosaccharides (glucose or fructose) at concentrations of 4% gave consistently highly embryonic culture with higher somatic embryo frequencies and higher growth rate compared with medium supplemented with either sucrose or maltose [11]. The use of fructose is considered as an excellent source of carbohydrate for embryo culture [12].

M Kumara swamy et al./J Phytol 2/8 (2010) 11-17

Kaufman et al. (1962) and Dickinson (1966) used fructose as a good source for the culture of stem segments and pollen [13, 14]. However, use of fructose in the medium results in hyper-hydricity which leads to low chlorophyll contents and abnormal nitrogen and sugar metabolism [15]. Table sugar is used as an alternative low cost medium component for in vitro micropropagation of potato [16]. It has been reported that in many plant species, addition of plant extracts/ juices of coconut, tomato, banana, orange, apple and yeast to the culture medium enhanced the growth of tissues [17, 18, 19 and 20].

It is evident that various carbon sources affect the growth of in vitro cultured plants differently. Therefore the present work was undertaken to study the effects of sucrose, fructose, glucose, table sugar and sugarcane juice on the growth of patchouli in vitro. Since the use of analytical grade sucrose contributes to the higher costs of media (34% of the production cost) [16], an attempt is made to reduce the cost of the medium by using low cost supplements like locally available table sugar and sugarcane juice. 2. Materials and Methods

Healthy patchouli plants grown in herbal garden of Rishi Herbal Technologies Pvt. Ltd., Bangalore were selected. Nodal segments and shoot tips of these selected plants were surface sterilized with 0.1% (w/v) HgCl2 for 10 min and washed thoroughly with sterile distilled water. Later, the explants were implanted on Murashige and Skoog (MS) [21] medium supplemented with 0.5 mg/L 6-benzylaminopurine (BA). The pH of the medium was adjusted to 5.7 prior to autoclaving at 121ºC for 20 min. Cultures were maintained at a temperature of 25±2ºC under 16 h light/ 8 h dark photoperiod and sub cultured every 4 weeks. Multiple shoots regenerated on this medium were used for further studies. Established cultures were subjected to sub culturing in same media. Uniform proliferated shoots (4-5 cm in length) resulted from direct organogenesis were transferred to MS basal

medium supplemented with 0.5 mg/L BA and kinetin (KN). The media was further supplemented with different carbon sources viz., sucrose, glucose, fructose, commercially available table sugar (1, 2 and 3%) and sugarcane juice (10, 20 and 30%).

Data was taken on the following parameters; fresh shoot weight (g), number of shoots, shoot length (cm), number of roots and root length (cm). The total chlorophyll content of the regenerated plantlets was measured by following the method explained by Yadava [22] and was expressed in mg/g tissue. The total protein content of the regenerated plantlets was measured using standard Lowry’s method [23] and was expressed in mg/ml. The total sugar content of the regenerated plantlets was measured using Anthrone method explained by Sadasivam and Manickam [24] and was expressed in mg/ml. The experiments were set up in completely randomized design with different treatments replicated thrice. Data recorded after 30 days of culture were subjected to Fisher’s method of analysis of variance. 3. Results

The growth, multiplication rate and other physiological parameters were affected by type and concentration of carbon source used. Number of shoots regenerated per explant

Sugar cane juice at 20% level is not only effective in elongating the shoots, but also regenerated higher number of multiple shoots (61.43±0.19). The next best rate of multiplication was recorded on MS medium supplemented with 2% sucrose (60.4±0.26) followed by 10% sugarcane juice (56.1±0.22). The use of table sugar at 2% showed 55.7±0.36 shoots while at 1% level resulted in 50.57±0.49 shoots per explant. However, at higher concentrations of carbon sources in the medium resulted in more of callus proliferation than shoot differentiation (Table 1). There was no significant difference in multiplication of shoots when glucose and fructose were used in the medium.

M Kumara swamy et al./J Phytol 2/8 (2010) 11-17

Table 1: Effect of carbon sources on in vitro shoot proliferation and growth of Pogostemon cablin Benth. after 30 days in MS medium supplemented with 0.5mg/L BA and 0.5mg/L KN.

Carbon sources Number of multiple

shoots ± SD a

Shoot Length (cm) ± SD a

Fresh weight of the shoots (g) ± SD a

Callus formation

Sucrose 1% 45.00±0.78 3.57±0.15 3.10±0.17 – 2% 60.40±0.26 4.57±0.22 4.89±0.19 – 3% 30.13±0.67 2.43±0.34 3.30±0.07 +

Glucose 1% 32.50±0.95 3.27±0.44 2.73±0.10 – 2% 36.50±0.43 3.63±0.04 2.80±0.38 – 3% 36.00±0.38 3.20±0.08 3.73±0.10 +

Fructose 1% 32.10±0.94 2.87±0.39 2.67±0.14 – 2% 32.63±0.13 3.30±0.10 3.40±0.13 + 3% 35.53±0.53 2.77±0.29 3.43±0.11 +

Table sugar 1% 50.57±0.49 3.47±0.45 4.53±0.31 – 2% 55.77±0.36 4.20±0.10 4.81±0.12 + 3% 33.80±0.60 2.20±0.46 3.47±0.16 +

Sugarcane juice

10% 56.10±0.22 4.03±0.37 4.60±0.35 –

20% 61.43±0.19 4.87±0.41 4.87±0.56 – 30% 49.13±0.62 3.37±0.07 4.80±0.19 +

Control _ _ _ F- value 384* 5.66 * 9.08*

CI at 95% *Significant at 5% level +: Callus induction, –: No callus a Data indicate mean ± standard deviation. Ten replicates were used per treatments and experiment was repeated trice.

Mean length of the shoots

Shoots induced on MS media supplemented with 20% sugarcane juice resulted in maximum shoot length (4.87±0.41cm) when compared to other carbon sources (Table -1). The second best carbon source which exhibited positive influence is sucrose at 2 % level (4.57±0.22cm). The plants grown on fructose and glucose showed reduced shoot length. Fresh weight of the shoots

The maximum average fresh weight of shoots was recorded on MS medium containing 2% sucrose (4.89±0.19g) followed by 20% sugarcane juice (4.87±0.56g). Glucose and fructose at 3% level exhibited 3.73±0.10g and 3.43±0.11g respectively. While table sugar at 2% level showed 4.81±0.12g. There was a steady increase in the fresh weight of shoot as the

concentration of glucose and fructose was increased in the medium from 1% -3%. Higher concentrations of carbon sources resulted in callus formation (Table 1). Chlorophyll content

Plants cultured on media supplemented with sugarcane juice at 20% level had the highest photosynthetic activity, and the plants cultured on fructose and glucose the lowest (Table 2). The leaves of the plants cultured on sugarcane juice (20%) had the highest chlorophyll content (0.81±2.0mg/g tissue) followed by those of the plants cultured on 2% sucrose (0.63±2.0mg/g tissue). The leaves of the plants cultured on glucose and fructose had the lowest chlorophyll content.

M Kumara swamy et al./J Phytol 2/8 (2010) 11-17

Table 2: Effect of carbon sources on the physiology of in vitro grown plants of Pogostemon cablin Benth. after 30 days in MS medium supplemented with 0.5mg/L BA and 0.5mg/L KN.

Carbon sources Chlorophyll

content (mg/g) ±SD a

Total protein content (mg/ml) ± SD a

Carbohydrate content (mg/ml) ± SD a

Sucrose 1% 0.42±1.4 12.8±0.21 15.2±0.11 2% 0.63±2.0 18.5±0.25 17.4±0.34 3% 0.33±1.5 12.4±0.23 18.2±0.30

Glucose 1% 0.33±2.1 13.6±0.26 11.6±0.53 2% 0.31±1.8 12.9±0.10 12.3±0.36 3% 0.22±2.1 12.3±0.15 13.0±0.36

Fructose 1% 0.33±2.5 12.2±0.20 12.9±0.20 2% 0.32±1.5 13.2±0.25 12.9±0.26 3% 0.26±2.0 12.2±0.32 13.5±0.20

Table sugar 1% 0.46±2.8 14.5±0.35 14.5±0.26 2% 0.52±2.5 15.6±0.15 14.7±0.10 3% 0.43±2.1 12.3±0.25 15.3±0.15

Sugarcane juice 10% 0.73±1.5 16.1±0.23 16.2±0.12 20% 0.81±2.0 18.8±0.24 17.5±0.25 30% 0.79±2.5 15.3±0.21 17.9±0.13

Control _ _ _ F- value 256* 245.4 * 265.6*

CI at 95%. *Significant at 5% level a Data indicate mean ± standard deviation. Ten replicates were used per treatments and experiment was repeated trice. Total protein content

The protein content of the plants differed significantly with the type and concentration of the carbon sources used in the treatments (Table 2). The maximum protein content was observed on media supplemented with 20% sugarcane juice (18.8±0.24mg/ml) followed by 2% sucrose (18.5±0.25mg/ml). The least content was observed on 3% fructose containing media (12.2±0.20mg/ml). Table sugar also showed a better result which can be comparable with the use of sucrose in the medium.

Total carbohydrate content The highest carbohydrate content was

observed on the medium supplemented with 3% carbon sources. Sucrose at 3% produced highest protein content of 18.2±0.30mg/ml. This was followed by 30% sugarcane juice (17.9±0.13mg/ml). The lower content was observed when glucose and fructose were used in the medium. From the table 2, it is evident that irrespective of the carbon sources used, increase in the concentration of the carbon sources resulted in increasing the total protein content.

4. Discussion Different types and levels of carbon sources

were tried to study their effect on in vitro growth of patchouli. Previous report by ILL- Whan and Korban [8] indicated that the type of carbon source used in the culture medium affects the growth of in vitro plants in various ways. In our study also, growth of patchouli is strongly influenced by different carbon sources supplemented in the media. Even though carbohydrates are of prime importance for cell growth, maintenance and differentiation in vitro, the fundamental aspects of carbon utilization and metabolism in cell and tissue cultures have yet to be fully understood [25, 26].

Normally analytical grade sucrose is used for tissue culture studies. In plant tissue culture, sucrose serves as a carbohydrate supply to provide energy for cell. In order to reduce the cost of the culture medium, commercially available table sugar and sugarcane juice at different levels were studied. Many authors have reported that various sources of carbon such as glucose, fructose, mannitol and sorbitol play an important role in tissue culture of asparagus [27],

M Kumara swamy et al./J Phytol 2/8 (2010) 11-17

cucumber [28]. For the first time sugar cane extract is supplemented to MS medium, as a source of carbon on which the growth and multiplication of shoots is vigorous. The use of sugarcane juice at 20% showed better response towards multiple shoot formation (61), shoot elongation (4.8cm), increased chlorophyll content (0.8mg/g tissue) and total protein content (18.1mg/ml). This might be due to the fact that sugarcane juice is one of the best sources of energy. Also it contains 15% of natural sugar and is a good source of riboflavin, calcium, magnesium and potassium. These additional factors would have possibly affected overall response of patchouli multiplication in vitro. Similar results were obtained in other studies related to addition of plant extracts/juice of coconut, tomato, potato, onion, banana, orange, apple, pineapple and yeast to the culture medium [17, 18, 19, 20 and 29].

Sucrose has been reported to be the best source of carbon and energy [10]. However in the present study, the use of sugarcane juice has shown better results than the use of sucrose. The results of commercial table sugars and sucrose in the media have shown comparable results. This suggests that sucrose can be replaced by table sugar for patchouli tissue culture. Many laboratories have reported the use of table sugar in plant propagation medium [30, 31]. Zapata [32] has successfully reduced the cost of banana tissue culture by 90% by replacing the tissue culture sucrose grade with a commercial sugar. The use of sugarcane juice can further reduce the cost of the media since commercial sugars are processed from sugarcane. It is therefore recommended that sugarcane juice can be considered as low cost substitute for patchouli micropropagation.

The plants cultured on glucose and fructose had poor growth compared to other carbon sources. Similar results are reported in Pinus sylvestris by ILL- Whan and Korban [8]. Bouza et al. [15] reported that the addition of fructose to the medium results in hyperhydricity which leads to low cellulose and chlorophyll contents, less ethylene production and abnormal nitrogen and sugar metabolism.

The decrease in shoot multiplication at higher concentration of carbon sources may be due to the inhibition of organogenesis and induction of callus proliferation. The differences

in shoot length, multiple shoots, fresh weight, total protein and carbohydrate content could be due to the differences in their photosynthetic activities (chlorophyll content). The substantial increase in the total carbohydrate content at higher concentration of carbon sources could be attributed to sugar accumulation.

5. Conclusion It can be concluded that various carbon

sources used in our experiment affected the growth of patchouli plants. Furthermore, 20% sugarcane juice and 2% table sugar can be used totally as a replacement of 2% sucrose which was the carbon source used in most of the plant tissue culture including patchouli. Since sucrose is expensive, the present investigation suggests a new source of carbon in the form of sugarcane juice and table sugar which are not expensive and available easily. However, further research is required to explore the possible growth promoting factors in sugarcane juice.

Acknowledgement The authors are grateful to the Director, Rishi

Foundation and the Chairman, Padmashree Group of Institutions, Bangalore for their encouragement and providing special permission to use the research facilities to undertake this programme. References 1. Kukreja A.K., A.K. Mathur, M. Zaim.

1990. Mass production of virus free patchouli plants (Pogostemon cablin (Blanco) Benth.) by in vitro culture. Trop. Agri., 67: 101-104.

2. Yang D., Michel, Mandin, Andriamboavonjy, Poitry, Chaumont, Mellet Clerc. 1996. Antifungal and antibacterial properties in vitro of three patchouli oils from different origins. Acta Botanica Gallica., 143(1): 29-35.

3. Bowles E.J., D.M. Griffiths, L. Quirk, A. Brownriggs, K. Croot. 2002. Effect of essential oils and touch on resistance to nursing care procedures and other dementia-related behaviors in a residential care facility. International Journal of Aromatherapy., 12: 22- 29.

4. Kageyama Y., Y. Honda, Y. Sugimura. 1995. Plant regeneration from patchouli protoplasts encapsulated in alginate

M Kumara swamy et al./J Phytol 2/8 (2010) 11-17

beads. Plant Cell Tiss. Organ Cult., 41(1): 65-70.

5. Misra M. 1996. Regeneration of Patchouli (Pogostemon cablin Benth.) plants from leaf and node callus, and evaluation after growth in the field. Plant Cell Rep., 15: 991-994.

6. Kumara swamy M., S. Balasubramanya, M. Anuradha. 2009. Germplasm conservation of patchouli (Pogostemon cablin Benth.). Int. J. Biodvers. Conserv., 1(8): 224- 230.

7. Kumara swamy M., S. Balasubramanya, M. Anuradha. 2010. In vitro multiplication of patchouli through direct organogenesis. Afr. J. Biotechnol., 9(14):2069- 2075.

8. ILL- Wan S., and S.S. Korban. 1998. Effects of media, carbon sources and cytokinins on shoot organogenesis in the Christmas tree, Scot pine (Pinus sylvestris). J. Hort. Sci. Biotech., 73: 822- 827.

9. Lipavska H., and H. Konradova. 2004. Somatic embryogenesis in conifers: The role of carbohydrate metabolism. In Vitro Cell. Dev. Biol.-Plant., 40: 23-30.

10. Bridgen M.P. 1994. A review of plant embryo culture. Hort. Science., 29:1243-1245.

11. Cunha A., and Fernandes-Ferreira. 1999. Influence of medium parameters on somatic embryogenesis from hypocotyls explants and flx (Linum usitatissium L.). J. Plant Physiol., 155:591-597.

12. Mauney J.R. 1961. The culture in vitro of immature cotton embryos. Bot. Gaz., 122: 205- 209.

13. Kaufman P.B., J. M. Katz, M. E. Yoder. 1962. Growth responses of Avena stem segments to various sugars. Nature., 196: 1332- 1333.

14. Dickinson D. B. 1996. Relation between external sugars and respiration of germinating lilly pollen. Proc. Am. Soc. Hort., 88: 651- 656.

15. Bouza L., M. Jaques, Y. Maziere, Y. Arnaud. 1992. In vitro propagation of Prunus tenella Batsch. cv. ‘Firehill’: Control of vitrification increase of the multiplication rate and growth by chilling. Scientia Hort., 52: 143-155.

16. Demo P. 2008. Table sugar as an alternative low cost medium component for in vitro micropropagation of potato (Solanum tuberosum L.). Afr. J. Biotechnol., 7: 2578-2584.

17. He S.L., K. DeZheng, Y.S. Qiu, Z. QiXiang. 2003. Effect of carbon sources and organic compounds on the multiplication of Oncidium aloha var. Iwanaga protocorm like body. Journal of Henon Agricultural University., 37: 154-157.

18. Hong E.Y., Y.S. Jong, K. IkHwan, Y. Tae, I. CheolHee, K. TaeSu, P. Kee Yoeup. 2003. Growth, flowering and variation of somaclones as affected by subcultures and natural materials supplemented to media in Phalaenopsis. Korean Journal of Horticultural Science & technology., 21: 362-368.

19. Amo-Marco J.B., and I. Picazo. 1994. In vitro culture of albedo tissue from fruits of Citrus sinensis cv. Washington Navel: effect of fruit age and orange juice. Journal of Horticultural Science., 69: 929-935.

20. Siddique A. B., and L. Paswan. 1998. Effect of growth regulators and organic supplements on differentiation of cymbidium longifolium protocorm in vitro. Journal of Hill Research., 11: 234-236.

21. Murashige T., and F. Skoog. 1962. A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol. Plant., 15: 473-497.

22. Yadava U.L. 1986. A rapid and non destructive method to determine chlorophyll in intact leaves. Hort. Science., 21:1 449-1450.

23. Lowry O.H., N.J. Rosebrough, A.L. Farr, R.J. Randal. 1951. Protein measurement with folin-phenol reagent. J. Biol. Chem. 193: 265-275.

24. Sadasivam S., A. Manickam. 1992. In; Biochemical Methods for Agricultural Sciences. Wiley Eastern Limited, New Delhi, pp. 11-12.

25. Romano A., C. Norohna, M.A. Martins-Loucao. 1995. Role of carbohydrates in micropropagation of cork oak. Plant Cell Tiss. Org. Cult., 40(2): 159-167.

26. Vu J.C.V., R.P. Niedz, G. Yelenosky. 1995. Activities of sucrose metabolism

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enzymes in glycerol-grown suspension cultures of sweet orange (Citrus sinensis L. Osbeck). Env. Exp. Bot., 35(4): 455-463.

27. Mamiya K., and Y. Sakamoto. 2000. Effects of sugar concentration and strenght of basal medium on conversion of somatic embryos in Asparagus officinalis L. Scientia Horticulturae., 84: 15-26.

28. Lou H., and S. Sako. 1995. Role of high sugar con-centration in inducing somatic embryogenesis from cucumber cotyledons. Scientia Horticulturae., 64: 11-20.

29. Puchooa D., and R. Ramburn, 2004. A study on the use of carrot juice in the tissue culture of Daucus carota. Afr. J. Biotechnol., 3(4): 248-252.

30. Ganapati T.R., J.S. Mohan, P. Suprasanna, V.A. Bapat, P.S. Rao. 1995. A

low cost strategy for in vitro propagation of Banana. Curr. Sci., 68:646- 665.

31. Kaur R., H. Gowtham, D.R. Sharma. 2005. A low cost strategy for micropropagation of strawberry (Fragaria × Ananassa Duch.) Cv. Chandler. Acta Hort., (ISHS) 696: 129- 133.

32. Zapata A. 2001. Cost reduction in tissue culture of banana. (Special leaflet), Int. Atom Energy Labs. Agric. and Biotech. Lab. Austria.

33. Obul Reddy B., P. Giridhar, G.A. Ravishankar. 2001. In vitro rooting of Decalepis hamiltonii. Wight and Arn, an endangered shrub, by auxins and root promoting agents. Curr. sci., 81(11): 26-29.

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Research in Biotechnology, 2(6): 64-71, 2011 ISSN: 2229-791X

www.researchinbiotechnology.com

Research Article

Analysis of genetic variability in patchouli cultivars (Pogostemon cablin Benth.) by using RAPD Markers

Kumara Swamy M1, 2*, and Anuradha M2, 3

1Department of Biotechnology, Acharya Nagarjuna University, Nagarjunanagar, Guntur, India 2Rishi Foundation, #234, 10th C main, 1st Block, Jayanagar, Bangalore- 560011, India

3Padmashree Institute of Management and Sciences, Kommagatta, Kengeri, Bangalore- 560060 *Corresponding Author Email: swamy.bio@gmail.com

The genetic relationships among patchouli cultivars were determined by using

Random Amplified Polymorphic DNA (RAPD) technology. Among 45 decamer random primers used for PCR reactions, 10 primers showed reproducible results. Out of 98 amplification products recorded, 16.7 per cent were monomorphic and 83.3 per cent were polymorphic. The highest dissimilarity (7.35) was detected between KSM 4 and 5 and the least 3.61 between KSM 2 and 3. Dendrogram constructed by cluster analysis of RAPD markers using Unweighted Pair Group Method of Arithmetic Averages (UPGMA) produced two major clusters ‘A’ and ‘B’. Cluster ‘A’ consisted of five cultivars that segregated into two sub clusters ‘A1’ and ‘A2’. Overall, RAPD analysis revealed the existence of considerable genetic variations in patchouli cultivars. This information regarding genetic variability at the molecular level could be used to identify and develop genetically unique germplasm that complements existing cultivars.

Key words: Pogostemon cablin, RAPD marker, genetic variability, cluster analysis

Patchouli (Pogostemon cablin Benth.)

belonging to the family Lamiaceae, is an aromatic herb cultivated on a commercial scale in India, Indonesia, Malaysia, China and Singapore. The commercial oil from patchouli is extensively used in perfumes and cosmetics (Hasegawa et al., 1992, Maheswari et al., 1993). The oil is widely used in the manufacture of soaps, scents, body lotions and detergents. It is been used to treat dysentery, diarrhea, colds without fevers, vomiting and nausea. The essential oil may be used to treat acne, dry skin, fungal infections, dermatitis, dandruff and eczema (Kalra et al., 2006). The fresh leaves can help in healing burns. In aromatherapy, it is used

to calm nerves, control appetite, relieves depression, stress and lack of sexual interest (Bowel et al., 2002). It also possesses insecticidal, antibacterial and antifungal properties (Kukreja et al., 1990, Yang, 1996, Pattnaik et al., 1996). Fibrinolytic and anti thrombotic activity of this essential oil is also been reported (Sumi, 2003, Eunkyung et al., 2002).

Indian demand for patchouli oil is around 220 tonnes valued at 33 crores while global demand is to the tune of 1600 tonnes of oil per annum with a value of 240 crores (Vijaya Kumar, 2004). India is importing annually about 20 tonnes of pure patchouli oil and 100 tonnes of formulated oil which is

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certainly a very huge quantity. Patchouli oil’s growing demand can be understood as it can neither be replaced inorganically nor synthesized because of its complex molecular structure (Farooqi et al., 2001). Hence the only alternative is to cultivate the plant extensively.

Worldwide research activities are aiming at improving the plant quality. The increasing number of varieties and the importance of their choice make it necessary to strengthen user’s guarantee concerning purity and identity. Hence, unambiguous characterization of varieties and conservation of elite germplasm is of prime importance. At present there are only few commercially grown varieties in patchouli (http://nhb. gov.in/bulletin_files/aromatic/patchouli/ pat012.pdf). Many people have named the same lines according to their company or other local names. Hence, accurate identification of plant materials is essential for effective germplasm characterization.

Despite the commercial importance of the crop, genetic data on patchouli in India are scarce. The identification of different varieties/cultivars/germplasm based on morphological traits implies culture inspection at different stages and is not reliable because many traits are governed by complex genetic interactions. Molecular markers based on DNA sequences offer means of identification with much greater reliability than the morphological traits.

RAPD remains one of the most extensively used molecular techniques due to its simplicity, low cost and high speed. Thus, RAPD markers have been successfully used in many crops in providing a convenient and rapid assessment of genetic diversity among different genotypes (Williams et al., 1990, Rafalski and Tingey, 1993, Ragot and Hoisington, 1993). RAPD markers have been already successfully used on other medicinal and aromatic crops (Kasaian et al., 2011, Salim Khan et al., 2010, Verma et al., 2009, Bharmauria et al., 2009, Padmalatha and Prasad, 2007). The present study therefore, was undertaken to study genetic diversity in patchouli cultivars. This might serve as a valuable tool for effective screening of genetic resources for future research and to improve and sustain genetic diversity of different patchouli varieties/cultivars. This would help in the identification and differentiation of various cultivars being cultivated.

Materials and methods Plant material

The details of 6 patchouli cultivars selected for RAPD analysis are given in the table 1. All the cultivars were maintained in the herbal garden of Rishi Foundation, Bangalore, India. The fresh leaf sample collected were wiped with 70% alcohol and dried at 400 C for 24 hours. The dried leaves are then sealed in plastic bags and used later for DNA isolation.

Table 1. Details of patchouli cultivars used in the present study

S. No Cultivar code Cultivar details

1 KSM 1 Johor variety: Collected from the Aromatic section, Department of

Horticulture, Gandhi Krishi Vignana Kendra (GKVK), Bangalore, India

2 KSM 2 Singapore variety: Collected from the Aromatic section, Department of Horticulture, Gandhi Krishi Vignana Kendra (GKVK), Bangalore, India

3 KSM 3 Simshreshta variety: Collected from the Aromatic section, Department of Horticulture, Gandhi Krishi Vignana Kendra (GKVK), Bangalore, India

4 KSM 4 Collected from Rishi Herbal Technologies Pvt Ltd, Bangalore, India

5 KSM 5 Collected from Rishi Herbal Technologies Pvt Ltd, Bangalore, India

6 KSM 6 Collected from Rishi Herbal Technologies Pvt Ltd, Bangalore, India

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DNA extraction protocol The method used here is a

modification of the original Cetyl trimethyl ammonium bromide (CTAB) method outlined by Doyle and Doyle (1987). 500 mg of leaf powder is transferred to a sterile centrifuge tube containing 15ml of extraction buffer (50mM Tris-HCl pH 8.0, 10mM EDTA pH 8.0, 0.7MNaCl, 0.4M LiCl, 1% w/v CTAB, 1% w/v PVP and 0.25% beta mercaptoethanol) preheated at 650 C. The contents were then incubated in a water bath at 650 C for 30 minutes with intermittent shaking. After incubation the contents are brought to room temperature and 10ml of chloroform: isoamylalcohol (24:1) was added to the tube. The mixture is agitated thoroughly and centrifuged at 6000 rpm for 5 minutes. The aqueous phase is transferred to a new tube and centrifuged for 5minutes at 6000 rpm in order to pellet possible debris. The supernatant is then transferred to a new tube and an equivalent volume of isopropanol is added to the aqueous solution. The tube is swirled gently to precipitate DNA. The tube is then centrifuged for 5minutes at 6000rpm and the supernatant is withdrawn. The DNA pellet is washed with 70% ethanol and is air dried for 10minutes. DNA pellet is resuspended in 500µl of Tris-EDTA buffer (10mM tris-HCl and 1mM EDTA, pH 8.0). Concentration and quality of genomic DNA was verified by using gel electrophoresis and spectrophotometer.

Primer selection

Out of 140 primers (Operon Technologies, Alameda, CA, US) screened, 42 primers produced at least one band. During preliminary screening, 22 out of 42 primers yielding more than 6 bands were selected. Finally, 10 (out of 22) primers producing strong, intense and unambiguous bands were selected for fingerprinting patchouli cultivars. The selected primer details are shown in the table 2.

Table 2: Details of random primers used in RAPD analysis

S. No Primer code Sequence (5’ to 3’) 1 OPA02 5’ TGCCGAGCTG 3’ 2 OPA11 5’ CAATCGCCGT 3’ 3 OPC04 5’ CCGCATCTAC 3’ 4 OPC07 5’ GTCCCGACGA 3’ 5 OPD03 5’ GTCGCCGTCA 3’ 6 OPF19 5’ CCTCTAGACC 3’ 7 OPF08 5’ GGGATATCGG 3’ 8 OPG05 5’ CTGAGACGGA 3’ 9 OPG12 5’ CAGCTCACGA 3’ 10 OPG17 5’ ACGACCGACA 3’

DNA amplification Amplification was achieved by

following the protocol outlined by Williams et al., (1990) with slight modifications. Polymerase reactions were carried out in a final volume of 25 µl reaction mixture containing 25 ng of template DNA, 10 p mol random decamer primer, 0.2 mM dNTPs, 2 U Taq polymerase (Genei, Bangalore, India) and 10 x PCR buffer (10 mM tris pH 8.0, 50 mM KCl, 1.5 mM MgCl2 and 0.1% gelatin, pH 9.0). Amplification was achieved in a Eppendorf Thermocycler programmed for initial denaturation at 950 C for 4 minutes, followed by 45 cycles; each cycle consisting of denaturation at 940 C for 1 minute, primer annealing at 350 C for 2 minutes, primer extention at 720 C for 2 minutes and a final extention of 10 minutes at 720 C. The PCR reactions were repeated 3 times, using the same conditions to check the repeatability of amplification products.

RAPD data analysis

Amplification of DNA fragments by random primers was scored “1” for the presence of the band and “0” for absence of the band and the data was converted into a matrix of binary data, where the presence of the band corresponded to value 1 and the absence to value 0. The genetic similarity was calculated using Squared Euclidean Distance

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matrix based on RAPD markers amplified with the ten primers. The matrix was subjected to UPGMA (Unweighted Pair wise Methods with Arithmetic averages) cluster analysis to generate a dendrogram. All analyses were worked out using the software STATISTICA, version 3.0 (STATISTICA for Windows, Stat Soft Inc, Tulsa. OK, USA, 1996).

Results and Discussion

A total of 98 RAPD markers of size ranging from 80bp to 3kbp that were consistent, unambiguous and repeatable were produced from the selected 10 primers. These markers were used for fingerprinting and to estimate genetic diversity among the cultivars of patchouli. The number of markers or bands scored for each primer varied from 7 to 10 with an average of 9.6 bands per primer (Fig 1a-b). Out of 98 amplification products recorded, 16.7 per cent were monomorphic and 83.3 per cent were polymorphic bands (Table 3). The bands amplified were of

uniform intensity and did not vary significantly with respect to the initial concentration of DNA. Such unique bands can be converted into genotype specific RAPD markers, which may be used for the identification of genotypes. This pool of primers yielded reasonable number of amplification products for all the cultivars examined. Hence, the result of the preliminary RAPD method is capable of revealing nuclear DNA variation in patchouli cultivars. The high number polymorphic markers detected in this study could be result of high diversity among the material used. The utility of RAPD markers in estimating genetic variability has been demonstrated in several studies on medicinal and aromatic plants (Kasaian et al., 2011, Salim Khan et al., 2010, Verma et al., 2009, Ganjewala, 2008, Bharmauria et al., 2009, Padmalatha and Prasad, 2007, Sangwan et al., 1999, Lal et al., 1997).

Table 3. Total number of amplified fragments, number of monomorphic and polymorphic bands

generated by PCR using 10 selected primers

Primers No of monomorphic bands

No of polymorphic bands

Polymorphism (%)

Total no of bands

1 0 12 100 12

2 0 11 100 11

3 4 6 60 10

4 0 10 100 10

5 2 7 77.7 9

6 0 10 100 10

7 4 4 50 8

8 1 6 85.7 7

9 2 7 77.7 9

10 3 7 70 10

Total 16 80 83.3 96

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Fig 1a: RAPD banding pattern of patchouli cultivars generated by random primer OPC07.

Lanes 1-6 contain the amplification profile obtained using the cultivars (KSM 1, KSM 2, KSM 3,

KSM 4, KSM 5, KSM 6). Lane M1 contains 100 bp DNA ladder and M2 contains 500bp DNA

ladder.

Fig 1b: RAPD banding pattern of patchouli cultivars generated by random primer OPG12.

Lanes 1-6 contain the amplification profile obtained using the cultivars (KSM 1, KSM 2, KSM 3,

KSM 4, KSM 5, KSM 6). Lane M1 contains 100 bp DNA ladder and M2 contains 500bp DNA

ladder.

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chemical composition of the essential oil viz., patchouli alcohol content. The sub cluster ‘A1’ was divided into two minor clusters ‘A1a’ and ‘A1b’ at 5.75 linkage distance. The minor cluster ‘A1a’ consisted of one cultivar i.e., KSM 1. The minor cluster ‘A1b’ consisted of 3 cultivars segregated into two groups at 5.3 linkage distance. Group I of ‘A1b’ consisted of one cultivar, KSM 6. The cultivars, KSM 2 and 3 were closely linked at 3.61 linkage distance. Closeness of these cultivars may be due to the fact that they have originated from a single parent or a narrow genetic base. The major cluster ‘B’ consist only one cultivar (KSM 4) and was linked to cluster ‘A’ at 6.5 linkage distance. It is clear from the dendrogram that KSM 4 was very distinct from the rest of the cultivars, which might be due to its superior qualities with respect to morphology, yield and patchouli alcohol content. This cultivar might be an improved variety which is commercially available for mass cultivation.

This information regarding genetic

variability at the molecular level could be used to identify and develop genetically unique germplasm that complements existing cultivars. Pan et al., (2006) have studied the genetic polymorphism and intra specific genetic differentiation of five plant populations of Pogostemon cablin in China. Four original plants of Xihuangcao (P. cablin) have been differentiated from each other by random amplified polymorphic DNA polymorphism (Chen et al., 2001). To the best of our knowledge in India, a little information is available for patchouli at the genetic level. This is the first report on the use of molecular markers for fingerprinting and evaluating genetic relationship of patchouli cultivars in India. Thus, RAPD method allowed us to access genetic diversity between patchouli cultivars. This assessment is fundamental because genetic diversity in the future could be exploited through molecular approaches or plant breeding techniques to improve

patchouli cultivars for disease resistance or to increase essential oil yield. Acknowledgement

The authors are grateful to the Director, Rishi Foundation and the Chairman, Padmashree Group of Institutions, Bangalore for their encouragement and providing special permission to use the research facilities to undertake this programme.

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http://nhb.gov.in/bulletin_files/aromatic/patchouli/pat012.pdf