In Vitro Cell. Dev. Biol.—Plant 42:283–286, May –June 2006 q 2006 Society for In Vitro Biology 1054-5476/06 $18.00+0.00
DOI: 10.1079/IVP2006757
RAPID IN VITRO PRODUCTION OF TRUE-TO-TYPE PLANTS OF POGOSTEMON HEYNEANUS THROUGH DEDIFFERENTIATED AXILLARY BUDS MANOJ EMANUEL HEMBROM, K. P. MARTIN, SUNEESH KUMAR PATCHATHUNDIKANDI,
AND
JOSEPH MADASSERY*
Department of Biotechnology, University of Calicut, Kerala 673 635, India (Received 25 January 2005; accepted 9 February 2006; editor P. Saxena)
Summary Rapid propagation of Pogostemon heyneanus Benth. (Lamiaceae) was accomplished through culture of node explants on Murashige and Skoog (MS) medium containing N 6-benzyladenine (BA). Random amplified polymorphic DNA (RAPD) and gas chromatographic (GC) analysis of in vitro-derived progenies were used to determine the true-to-type nature of in vitroderived plantlets. At the optimum level of BA (2.22 mM), the axillary buds underwent a degree of dedifferentiation to become small globular green masses from which a mean of 17.1 shoots were developed within 40 d. Retaining the culture without subculture enhanced the number of shoots (.30 shoots). Increased callus proliferation was observed at higher concentrations of BA in concomitance with a reduction in number of shoots. However, prolonged culture without subculture (more than 60 d) initiated 25 – 30 shoot buds from the callus. Culture of node segments excised from in vitro shoots on fresh medium with optimal BA (2.22 mM) exhibited a similar response, but with an increase of shoots (mean of 26.3 shoots per node) within 40 d. Subculture of shoot clumps on half-strength MS basal medium resulted in elongation (more than 4 cm) of most of the shoots along with the development of new shoots. Shoots developed were rooted most successfully on half-strength MS medium with 4.9 mM indole-3-butyric acid (IBA). Plantlets derived from the best rooting medium established in small cups exhibited 95% survival. Plantlets successfully established in field conditions exhibited morphological characteristics identical to the source plant. The RAPD profile of the in vitro-derived plants and source plant, using 10 random primers, was similar. The gas chromatogram of the extracted oils from in vitro-derived plants and the source plant showed similar patterns. Key words: clonal propagation; gas chromatography; micropropagation; patchouli oil; Pogostemon heyneanus. valuable medicinal species. In addition to in vitro propagation, the clonal nature of the progenies using random amplified polymorphic DNA (RAPD) assay and gas chromatographic (GC) analysis of the oils extracted from source and in vitro-derived plants of P. heyneanus were also studied in the present investigation.
Introduction Pogostemon heyneanus Benth., a native of the Indo-Malaysian region, is a highly aromatic herb widely distributed and frequently cultivated in India. Essential oils of the members of the Lamiaceae are valuable commercially as additives for cosmetics and pharmaceuticals. The plant is an important source of pogostemonine, patchouli resinoid, and trimethylamine, and the oil extracted from the leaves contains patchoulene, patchouli alcohol, and eugenol as the major components (Anonymous, 1976). Patchouli oil (. US$100 kg21) is one of the most important raw materials in perfumes, soaps, and cosmetics. The oil also possesses antibacterial activity against Escherichia coli, Staphylococcus aureus, Streptococcus pyogenes, Bacterium coli, Bacterium typhosum, and Mycobacterium tuberculosis (Anonymous, 1976). The oil is also useful as an ingredient in insect-repellent preparations, for moths in particular, and the leaves are applied to repel leeches (Anonymous, 1976). Propagation of P. heyneanus via vegetative means is insufficient to meet the requirement of disease-free raw materials. Low or no seed setting curtails propagation and improvement of P. heyneanus. To date, no in vitro studies have been reported on this
Materials and Methods Young shoot tip segments (3 –4 cm length) of P. heyneanus were collected from a 5-yr-old plant grown (a vegetative clone) in Calicut University Botanical Garden. Shoot segments were washed under running tap water followed by immersion in 2% (v/v) Extran (a neutral liquid detergent; Merck India Ltd., Mumbai) for 2 min. After repeated washing in double-distilled water, the shoot tip segments were surface sterilized using 0.1% (w/v) mercuric chloride solution for 12–14 min with continuous stirring. The sterilized segments, after a thorough wash in sterile double-distilled water, were cut to lengths of 1.0 – 1.5 cm each with one node, and cultured on MS (Murashige and Skoog, 1962) medium supplemented with 3% (w/v) sucrose. The media were gelled with 0.8% (w/v) agar (Merck India Ltd.). For node culture, MS medium was supplemented with N 6-benzyladenine (BA), kinetin, indole-3-butyric acid (IBA), and a-naphthaleneacetic acid (NAA) at different concentrations (Table 1). Half-strength MS solid media modified with 0.54–5.37 mM NAA, 0.57–5.71 mM indole-3-acetic acid (IAA), and 0.49– 4.90 mM IBA were tested for in vitro rooting. The pH of all media was adjusted to 5.8 before the addition of agar. Test tubes (25 £ 200 mm) and 100 ml conical flasks closed by cotton plugs were used as culture vessels. Twenty and 40 ml media were dispensed, respectively, to test tubes and conical flasks. All media were sterilized in an autoclave at a pressure of 1.06 kg cm22 (temperature
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HEMBROM ET AL. TABLE 1
SHOOT INDUCTION FROM NODE EXPLANTS OF POGOSTEMON HEYNEANUS ON MS MEDIUM WITH DIFFERENT GROWTH REGULATORS Growth regulators (mM) BA 00 1.11 2.22 3.33 4.44 6.66 8.87 13.31 Kinetin 2.32 4.65 6.97 BA þ kinetin 2.22 þ 0.46 2.22 þ 2.32 4.44 þ 0.46 4.44 þ 2.32 BA þ IBA 2.22 þ 0.49 2.22 þ 2.46 4.44 þ 0.49 4.44 þ 2.46 BA þ NAA 2.22 þ 0.54 2.22 þ 2.69 4.44 þ 0.54 4.44 þ 2.69
Explants inducing shoots (%)
Shoots per node (mean)
20 55 80 70 60 65 80 70
1.8 g 5.2 f 17.1 a 14.6 b 12.5 c 7.9 e 9.7 d 5.3 f
60 75 60
9.3 d 7.7 e 4.9 f
70 75 70 70
12.3 c 9.3 d 9.9 e 7.3 e
80 70 75 75
14.8 b 11.9 c 12.3 c 11.7 c
80 75 80 75
8.1 de 4.5 f 7.6 e 5.1 f
Values are mean of 20 replicates. Mean values followed by different letters are significantly different at 5% level. Culture duration was 40 d. 1218C) for 20 min. All cultures were incubated at 25 ^ 28C with 16 h dark/8 h photoperiod under white fluorescent tubes (25 mmol m22 s21). The experiments were conducted in a complete randomized block design. Twenty replicates were raised for each plant growth regulator treatment, and all treatments were repeated once. Healthy plantlets (.4 cm height) planted in small cups containing soil and sand (1:1) were acclimatized in a greenhouse and later transferred to field conditions. The survival rate of the plantlets derived from the best rooting medium was recorded 30 d after transplantation. The mean values of different treatments were compared using Duncan’s (1955) multiple range test. DNA isolation and RAPD assay. As the plants were regenerated from dedifferentiated axillary buds, the clonal nature of the field-established plantlets were tested using the RAPD profile. Leaves of five randomly selected in vitro-derived plants as well as the source (control) plant were selected for the RAPD assay. The leaves of in vitro-derived plants as well as of the control plant were separately washed in sterile double-distilled water followed by blotting with tissue paper, and 4 g of each was frozen for 1 h at 2708C, keeping in separate sterilized mortars. After 1 h, the leaves were homogenized for the isolation of genomic DNA using separate pestles. Plant genomic DNA was extracted according to the method of Dellaporta et al. (1983) with 3% b-mercaptoethanol and with 3% polyvinylpyrrolidone. The purity was examined by agarose (0.8%) gel electrophoresis, and the concentration of DNA was determined by ultraviolet–visible spectrophotometer (UV-1601, Shimadzu, Kyoto, Japan). RAPD profiling was performed using the following 10 random primers (Integrated DNA Technologies Inc, Coralville, IA, USA): S-01, 50 -GTTTCGCTCC-30 ; S-04, 50 -GGACTGGAGT-30 ; S-20, 50 -GGACCCTTAC-30 ; S-24, 50 -AATCGGGCTG-30 ; S-39, 50 -CAAACGTCGG-30 ; S-58, 50 -AGGGCGTAAG-30 ; S-304, 50 -CCGCTACCGA-30 ; S-322, 50 -CCTACGGGGA-30 ; S-1387, 50 -CTACGCTCAC-30 ; S-1401, 50 -GGAAACCCCT-30 .
Polymerase chain reaction (PCR) was performed in a volume of 25 ml using PCR buffer containing 3 mM MgCl2, 250 mM each of dNTPs, 0.2 nM primer, 200 ng genomic DNA, and 2 units of Taq polymerase (Biogene, CA, USA). DNA amplification was performed in a programmable Ericomp Inc. (Powersysteme, San Diego, USA) thermal cycler. The PCR program consisted of an initial denaturation for 1 min at 948C, then 40 cycles of 1 min at 948C, 1 min at 368C, and 2 min at 728C. For each primer, tubes containing all the reaction components except the DNA template served as a (negative) control to check for contamination. The amplification products were stored at 48C until analyzed. Amplification products were analyzed by electrophoresis in 1.5% agarose gels using Tris–acetic acid–EDTA buffer, stained with ethidium bromide, and documented using AlphaImager gel documentation analysis system (Alpha Innotech, San Leandro, CA, USA). A DNA ladder of 500 bp was used as a marker (Genei, Bangalore, India). RAPD assays of each in vitro-derived plant were performed twice to confirm the results. GC analysis. The essential oils extracted from 500 mg leaf tissue of the source (control) plant and in vitro-derived (shoots from MS with 2.22 mM BA rooted on half-strength MS with 0.49 mM IBA) randomly selected plants (seven) were analyzed by GC. The chopped leaves of the source and in vitroderived plants were first treated with 2 ml of methanol and incubated overnight, and then 2 ml of 2 mM potassium chloride were added. The mixture recovered was then mixed with an equal volume of n-heptane. The top layer containing the essential oil, carefully recovered using a micropipette, was transferred to clean Eppendorf tubes and stored at 48C. The GC analyses were performed using a Perkin-Elmer Autosystem equipped with a PE Nelson 1022 GC plus integrator (Boston, USA). The column and the oven program used were, respectively, OV-1 and 70–2108C at 88C min21. The packed column used was stainless steel, and the flame ionization detector (FID) temperature was 3008C. The carrier gas nitrogen flow rate was 30 ml min21 and the injection port temperature was 2008C. GC analyses of each in vitro-derived plant were performed twice.
Results and Discussion Node explants cultured on MS basal medium facilitated initiation of axillary buds. All concentrations of BA tested induced shoots after dedifferentiation of axillary buds (Table 1). Culture on MS medium containing 2.22 mM BA was optimal for induction of shoots with minimal callus. On the optimal medium, axillary buds dedifferentiated to a small globular green mass of callus from which a mean of 17.1 shoots was developed within 40 d (Table 1 and Fig. 1A, B). Retaining the culture without subculture enhanced the number of shoots to more than 30 shoots within 60 d. Increase of BA showed a concomitant increase of callus, and callusing was high on medium containing 13.3 mM BA. However, retention of the culture on the same medium without subculture induced shoot buds (25– 30) after 60 d. With increasing amount of BA, the shoots exhibited a stunted nature. Kinetin was inferior to BA, but in comparison to BA-supplemented medium, the shoots on medium with optimal kinetin (2.32 mM) concentration grew to more than 3 cm in height. Kinetin-enriched medium induced callus from the proximal cut end of the node explants, and the amount of callus depended on the concentration. Addition of different levels of kinetin, NAA, and IBA singly to media with optimal concentration of BA (2.22 mM) was inferior to medium supplemented with BA alone (Table 1). Medium with BA and auxin showed an increased amount of callus by the dedifferentiation of axillary buds; however, this increased amount of callus depended on the type and concentration of auxin. NAA, even at 0.54 mM, resulted in the formation of higher amounts of callus (data not shown). The formation of shoots from dedifferentiated axillary buds on medium with BA and auxin was delayed compared to that on medium enriched with optimal BA alone. Plant propagation in vitro, using axillary buds, has been shown to be a simple and reliable strategy for the rapid production of elite clones of
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FIG . 1. In vitro propagation of P. heyneanus. A, Formation of green callus mass by the dedifferentiation of axillary buds and shoot initiation on MS medium with 2.22 mM BA (25 d) (bar ¼ 3 mm). B, The above after 40 d (bar ¼ 3 mm). C, Development of a large number of shoots from the shoot clumps transferred onto fresh medium with 2.22 mM BA (60 d) (bar ¼ 3 mm). D, Established plant after 60 d (bar ¼ 5 mm).
desired plants (Elangomathavan et al., 2003; Lai-Keng and Leng, 2004). However, in the present study, the shoots were developed after a degree of dedifferentiation of axillary buds. The potential of BA, especially compared to kinetin, has been documented to be effective for propagation using axillary buds among the Lamiacean medicinal plants, viz. Orthosiphon spiralis (Elangomathavan et al., 2003), Orthosiphon stamineus (Lai-Keng and Leng, 2004), and Mentha piperita (Sunandakumari et al., 2004). As in the present study, a reduced propagation rate using axillary buds by the synergy of BA and an auxin compared to medium supplemented with BA alone has been reported in O. stamineus (Lai-Keng and Leng, 2004) and M. piperita (Sunandakumari et al., 2004). The stunted nature of shoots developed on medium corresponding to the increased concentrations of BA, as in the present study, has been reported in Orthosiphon (Lai-Keng and Leng, 2004) and Eupatorium (Martin, 2004).
Transfer of shoot clumps onto fresh MS medium with 2.22 mM BA induced numerous shoots (Fig. 1C). The shoots developed were short and slender. Nevertheless, as the culture period progressed, shoots showed elongation (Fig. 1C). Subculture of shoot clumps to half-strength MS basal medium facilitated elongation (more than 4 cm) of most of the shoots along with the development of new shoots. The shoots were healthy with 2 – 5 nodes. Some of the shoots induced roots from the base. As pointed out in O. spiralis (Elangomathavan et al., 2003), the lack of shoot elongation in subsequent cultures is due to the super-optimal effect of BA, which is also associated with the suppression of shoot elongation and the tendency for callus formation at high concentration in the primary culture of the present study. The reversion of the suppressive effect of BA by growth regulator-free medium strengthens the above conclusion.
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FIG . 2. The RAPD profile of the in vitro-derived and source plants (5-yr-old vegetative clone) (FG, field-grown; IN, in vitro; M, marker).
Half-strength MS medium containing IBA was superior to IAA and NAA for root induction in vitro. Half-strength MS medium with 0.49 mM IBA induced a mean of 10.7 roots per shoot, and was the best for root induction. The influence of IBA in in vitro rooting has been documented among Lamiacean medicinal plants, viz. O. spiralis (LaiKeng and Leng, 2004) and M. piperita (Sunandakumari et al., 2004). Healthy plantlets (more than 4 cm height) transferred to small pots containing soil and sand (1:1) kept in a greenhouse revived within 15 d and grew well (Fig. 1D). Ninety-five percent of the plantlets survived in the greenhouse, and established plantlets were morphologically identical to the source plant. The genomic DNA isolated from the source plant as well as from five randomly selected in vitro-derived progenies had low protein contamination. Spectrophotometric analysis (A 260/A 280) of DNA in all cases was above 1.8. Agarose gel electrophoresis of the genomic DNA isolated in all cases showed a single 21 kbp band after ethidium bromide staining, which confirmed the purity of the isolated genomic DNA. The RAPD profile using random primers obtained through amplification of genomic DNA of the randomly selected in vitro-derived progenies and that of the source plant was similar in all respects (Fig. 2). This indicates the true-to-type nature of the progenies. RAPD has been proven to be a suitable molecular technique to analyze the clonal fidelity of descendents of a single individual as well as to detect the variation that is induced or occurs during in vitro culture (Shu et al., 2003; Prakash et al., 2004). The occurrence of genetic defects arising from variation in the regenerants seriously affects the true-to-type nature of the plants, which in turn hampers the production of clonal propagation of plants with desired traits and also the production of a specific secondary metabolite of commerce. Thus, the production of genetically uniform and stable plants is a necessity for commercial purposes (Shu et al., 2003; Prakash et al., 2004). Considering P. heyneanus as a valuable source of essential oil (mainly patchouli oil), the maintenance of the genetic stability of the progenies without any alternation in structure is highly important in terms of the production of essential oils. The GC profile of the oil extracted from leaves of field-grown and in vitro-derived plants was similar with major peaks at retention times of 14.687 and 14.570 min, respectively. As the plant is a leading source of patchouli oil, the major peak in the chromatogram
may be of the patchouli oil. Similar patterns of GC profiles between protoplast-derived plants and the source plant have been demonstrated in Pogostemon cablin, the main source of patchouli oil (Kageyama et al., 1995). Efficient plant regeneration, the indication of the true-to-type nature of the progenies by RAPD, and uniform pattern of essential oils in the GC profile of the source and progeny plants exemplify the feasibility of the protocol. The present study emphasizes that shoot differentiation after slight dedifferentiation of axillary buds does not necessarily impede the clonal fidelity of the in vitro-derived progenies. The protocol enables large-scale clonal propagation of patchouli plants and thus meets the demand for raw materials for oil extraction.
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