Summary. A procedure for the regeneration of complete plantlets of Tylophora indica from cultured leaf callus via somatic embryogenesis is described.
In Vitro Cell. Dev. Biol.ÐPlant 37:576±580, September±October 2001 q 2001 Society for In Vitro Biology 1054-5476/01 $10.0010.00
DOI:10.1079/IVP2001211
PLANT REGENERATION THROUGH SOMATIC EMBRYOGENESIS AND RAPD ANALYSIS OF REGENERATED PLANTS IN TYLOPHORA INDICA (BURM. F. MERRILL.) M. JAYANTHI* and P. K. MANDAL²
M. S. Swaminathan Research Foundation, III Cross Road, Taramani, Chennai 600 113, India (Received 31 August 2000; accepted 23 March 2001; editor M. Madkour)
Summary A procedure for the regeneration of complete plantlets of Tylophora indica from cultured leaf callus via somatic embryogenesis is described. Callus induction from leaf explants was on Murashige and Skoog (MS) medium with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D; 0.0±3 mg l21; 0.0±13.56 mM) and kinetin (Kn; 0.01 mg l21; 0.05 mM). The best response for callus induction was obtained on MS medium containing 2 mg l21 (9.04 mM) 2,4-D and 0.01 mg l21 (0.05 mM) Kn. After two subcultures on the same medium the embryogenic callus was transferred to MS medium with different concentrations of the cytokinin, 6-benzyladenine (0.5±3 mg l21; 2.22±13.32 mM) and 2-isopentenyladenine (2ip; 0.53 mg l21; 2.46±14.76 mM) along with 0.01 mg l21 (0.05 mM) indole-3-butyric acid (IBA) for somatic embryo development and maturation. MS medium with 2 mg l21 (9.84 mM) 2ip produced the maximum number of mature somatic embryos. The mature embryos were bipolar and on transfer to MS basal medium produced complete plantlets. After hardening the regenerants were planted in the Gudalur forests of Western Ghats. Total DNA was extracted from 14 regenerants and the mother plant. Random amplified polymorphic DNA (RAPD) analysis was carried out using 20 arbitrary oligonucleotides. The amplification products were monomorphic among all the plants revealing the genetic homogeneity and true-to-type nature of the regenerants. Key words: medicinal plant; callus induction; somatic embryos; plant regeneration; DNA isolation; genetic homogeneity. regeneration via somatic embryogenesis is considered to be more efficient than shoot multiplication (Roberts et al., 1995) and opens up the prospects of applying recombinant DNA technology (Hassig et al., 1987). In addition, in medicinal plants, it has been reported that productivity in terms of quantity and quality of secondary metabolites was enhanced in plants derived from somatic embryogenesis (Gastaldo et al., 1994). The only report on somatic embryo formation in T. indica was that of Rao et al. (1970), who reported somatic embryo formation from stem callus, but the details of the protocol were very limited. Yet another concern in clonal propagation and in vitro conservation is retaining the genetic integrity of micropropagated plants (Rani et al., 2000). Hence testing the genetic homogeneity of regenerated plants is very important. This paper reports for the first time plant regeneration from leaf callus via somatic embryogenesis and the use of random amplified polymorphic DNA (RAPD) technique to assess the genetic homogeneity of the regenerated plants of T. indica.
Introduction Tylophora indica Burm. f. Merrill. (Asclepiadaceae) is a perennial branching climber found in several parts of India. This indigenous medicinal plant has multifarious uses. The roots and leaves of this plant have long been used for the treatment of asthma, bronchitis, whooping cough, dysentery, rheumatic gouty pains and hydrophobia (Anonymous, 1976). The roots are known to possess bacteriostatic properties (Bhutani et al., 1985) and therefore it was suggested to be a good natural preservative of food. Moreover, the root of the plant contains a potential anti-tumor alkaloid tylophorinidine (Mulchandani et al., 1971), besides other alkaloids such as tylophorine and tylophorenine found in the other parts of the plant. In addition to its therapeutic value, the plant is reported to yield a fine silky and strong fiber, which may be useful in the manufacture of extra-fine fabrics (Anonymous, 1976). Wild populations of this medicinal plant are declining fast due to over-exploitation and lack of organized cultivation. Realizing this, Sharma and Chandel (1992) established a protocol for axillary bud induction and shoot multiplication from T. indica. However, plant
Materials and Methods Plant material and explant preparation. Leaves were collected from a single mature plant of T. indica, which was obtained from the forests of Western Ghats and maintained in the glass house at M. S. Swaminathan Research Foundation, India. The leaves were washed with 1% Teepol detergent solution and then washed thoroughly in running tap water. Surface sterilization was performed by immersion in 0.1% aqueous mercuric chloride for 5 min followed by five rinses in sterile double-distilled water.
*Author to whom correspondence should be addressed (present address): Plant Biotechnology Division, Tropical Botanical Garden and Research Institute, Palode, Thiruvananthapuram 695 562, India. Email jayman21@ rediffmail.com ²Present address: National Research Center for Oil Palm, Palode, Thiruvananthapuram 695 562, India.
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SOMATIC EMBRYOGENESIS AND STABILITY ANALYSIS IN T. INDICA The leaves were then cut into 0.5 cm2 segments and cultured on callus induction medium. Culture media and growth conditions. The medium used in all experiments consisted of MS (Murashige and Skoog, 1962) basal salts supplemented with 3% sucrose (Himedia, Mumbai, India) and 0.2% phytagel (Sigma, St. Louis, MO, USA). The pH of the medium was adjusted to 5.8 before the addition of phytagel. Culture tubes
150 25 mm were filled with 15 ml of the medium and plugged with non-absorbent cotton plugs before autoclaving. The media was autoclaved at 1218C for 15 min. For callus induction, MS medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) concentrations of 0.5, 1.0, 2.0, and 3.0 mg l21 (2.26, 4.52, 9.05, and 13.56 mM) and a fixed concentration of kinetin (Kn) at 0.01 mg l21 (0.05 mM) was used. Cultures were incubated in the dark at 25 ^ 18C for 4 wk. The best callus induction medium, as identified in the preliminary experiment, was MS basal salts medium supplemented with 2 mg l21 2.4-D (9.05 mM) and 0.01 mg l21 Kn (0.05 mM). The callus induced on the above media was subcultured every 2 wk onto the same media. After two subcultures, 0.5 g of the embryogenic callus was transferred to MS medium supplemented with different cytokinins, either 6-benzyladenine (BA) or 2-isopentenyladenine (2ip), at concentrations of 0.5, 1.0, 1.5, 2.0, and 3.0 mg l21 in combination with indole-3-butyric acid (IBA) at 0.01 mg l21 (0.05 mM). These cultures were transferred to a culture room with light provided by fluorescent lamps (50 mmol m22 s21) under a 10-h photoperiod. After 4 wk of culture, the percentage of callus that initiated somatic embryos and the number of embryos were calculated. Each experiment was carried out with a minimum of 25 replicates and repeated three times. Statistical analysis of the data was conducted using the Duncan's multiple range test. Completely developed embryos were transferred to basal MS medium for regeneration and kept under the same light conditions under a 10-h photoperiod. After a sufficient growth period the plantlets were removed from the culture tubes, the culture medium sticking to the roots was washed off, and the plantlets were planted in plastic pots
5 5 cm containing sterile sand and kept in a controlled growth chamber (NK Systems, Japan) (temperature of 28 ^ 18C and 70% relative humidity under a 10-h photoperiod). After 2±3 wk, these plantlets were transferred to bigger pots containing sand, soil, and cow dung (1:1:1) and kept in the mist chamber. The well-established plants were then transported and planted at Gudalur forests of Western Ghats. DNA isolation. Total genomic DNA was isolated from the leaf tissue of the mother plant and regenerated plants using the CTAB method (Saghai-Maroof et al., 1984) with minor modifications. Fourteen plants were selected randomly from a batch of 126 somatic embryo regenerants. Two grams of leaf tissue were ground in liquid nitrogen and suspended in three volumes of CTAB extraction buffer (2% cetyltrimethylammonium bromide (CTAB), 100 mM Tris±HCl (pH 8.0), 20 mM EDTA, and 1% b-mercaptoethanol). The suspension was incubated at 608C for 25 min, extracted with an equal volume of chloroform: isoamyl alcohol (24:1) and centrifuged at 5000 g for 10 min. The DNA was precipitated from the aqueous phase with a two-third volume of isopropanol at 2208C for 1 h. The pellet, recovered by centrifugation at 10 000 g for 10 min, was dissolved in TE buffer (10 mM Tris±HCl, 1 mM EDTA, pH 8.0) and treated with RNAse at 378C for 1 h. The DNA was purified by phenol/ chloroform extraction and ethanol precipitation in the presence of 0.3 M sodium acetate (pH 5.2). The pellet was dissolved in TE buffer and the DNA concentration was estimated in 1% agarose gel. RAPD analysis. RAPD analysis of genomic DNA was carried out using 20 decamer random oligonucleotide primers (OPB 1±20) obtained from Operon Tech. (California, USA). The polymerase chain reaction (PCR) was carried out in a volume of 25 ml containing 20 ng of genomic DNA, 2.5 ml of 10 assay buffer, 2 ml of 5 mM MgCl2, 0.5 ml of 5 mM dNTPs (Pharmacia), 15 ng of primer and 1 unit of Taq DNA Polymerase (Bangalore Genei). The reaction mixture was overlaid with 25 ml of mineral oil and amplification was carried out in a DNA thermal cycler (Perkin-Elmer 480) programmed for 45 cycles. Each cycle comprised 1 min at 948C (first cycle was for 3 min at 948C), 1 min at 378C and 2 min at 728C. An additional cycle of 15 min at 728C was used for primer extension. The amplified samples were electrophoresed in 1.5% agarose gels in 0:5 TBE (Tris±borate±EDTA) buffer. Amplification with each primer was repeated twice to confirm reproducibility of the results.
Results and Discussion Callus initiation, embryogenesis and regeneration. Callus initiation was observed on the cut edges of the leaf explants 10 d after
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culture initiation. The induction of callus was mainly influenced by the auxin concentration. Although callus was produced in MS medium supplemented with different concentrations of 2,4-D (0.5± 3 mg l21), maximum callus induction was observed in MS supplemented with 2 mg l21 2,4-D (Fig. 1a). Growth of the callus on MS medium with 2,4-D (2 mg l21) and Kn (0.01 mg l21) was slow at the beginning, but by the end of the second subculture onto the same medium, it produced numerous highly organized structures. The capacity of the auxin 2,4-D to induce embryogenic callus has been reported previously in T. indica and in several other crops (Rao et al., 1970; Chang, 1991; Joseph et al., 2000). When the cultures of T. indica were exposed to light and transferred to MS medium containing cytokinins, such as BA or 2ip at different concentrations ranging from 0.5 to 3 mg l21, the cultures grew rapidly and produced clusters of somatic embryos on their surfaces (Fig. 1b). The critical step in somatic embryogenesis is the ability to form embryos that will develop into complete plantlets. The importance of some compounds, such as abscissic acid and charcoal, for maturation has been reported in some plants (Mathews et al., 1993). However, in our study, somatic embryogenesis was evident by the addition of either BA or 2ip concentrations ranging from 0.5 to 2 mg l21 along with a fixed concentration of IBA (0.01 mg l21). An increase in the concentration of BA up to 1 mg l21 or 2ip up to 2 mg l21 improved the embryogenesis frequency. Callus cultured on MS medium containing 1 mg l21 BA produced 75% embryogenesis while MS medium supplemented with 2 mg l21 2ip revealed the highest percentage of embryogenesis (85%). The average number of somatic embryos per 500 mg callus produced on the different media is presented in Table 1. MS medium supplemented with 1 mg l21 BA and 0.01 mg l21 IBA produced a maximum of 18.20 embryos per 500 mg callus, while the addition of 3 mg l21 BA reduced this number approximately three-fold (6.33 embryos per 500 mg callus). On the other hand, MS medium supplemented with 2 mg l21 2ip produced maximum numbers of embryos (25.00). Statistical analysis showed that the numbers of somatic embryos produced in MS medium supplemented with 2ip at 1, 2, and 3 mg l21 were significantly higher than all other treatments; whereas, the numbers of somatic embryos produced on MS medium supplemented with 1 mg l21 BA were significantly higher than those found on MS supplemented with 0.5, 2.0, and 3.0 mg l21 BA. Therefore it could be concluded that the addition of cytokinin 2ip to the MS medium enhanced embryogenesis significantly when compared to BA. It has been reported that cytokinins have an important role in promoting embryogenesis by stimulating divisions in the proembryogenic cells (Fujimura and Komamine, 1980). After 4 wk in culture, the somatic embryos developed into mature embryos with distinct shoot and root poles (Fig. 1c). The formation of distinct root and shoot poles may be attributed to the presence of cytokinins, since it has been reported that cytokinins such as BA stimulate shoot and tap root formation (Chang, 1991). Maturation of somatic embryos was asynchronous and some of the somatic embryos produced multiple shoots (Fig. 1d). This may be due to the early development of embryo axillary buds. On transfer to MS basal medium these somatic embryos formed complete plantlets with well-developed shoot and root systems (Fig. 1e). Regenerated plants grew well under both growth chamber and mist chamber conditions (Fig. 1f ). Survival rate during the hardening was always more than 80%. After a month, the well-established
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Fig. 1. Plant regeneration via somatic embryogenesis in Tylophora indica. a, Callus induction from cut edges of leaf after 4 wk of culture. b, Clusters of somatic embryos produced after two subcultures. c, Mature somatic embryos with root and shoot pole. d, Somatic embryos developed with multiple shoots. e, Complete plantlet formation in MS basal medium. f, Hardened plants transferred to mist chamber. g, Well-established plants grown at Gudalur forests, Western Ghats.
plants were transported to Gudalur forests of Western Ghats region and planted (Fig. 1g). RAPD analysis. Usually, in medicinal plants genetic homogeneity is tested through analysis of secondary metabolites. This is time consuming and requires large amounts of plant material (Shoyama et al., 1997). Moreover, in plants regenerated via somatic embryogenesis the quality of somatic embryos rests on the production of true-to-type plants. Molecular tools are more reliable than phenotypic observations for evaluating variations (Leroy et al., 2000). Recently the RAPD technique has been reported to be a powerful tool to analyze variation among in vitro-regenerated plants (Isabel et al., 1993; Rani et al., 1995; Shoyama et al., 1997; Goto et al., 1998). In this study, DNA from two or three large leaves (2 g)
was adequate for the RAPD analysis. Out of the 20 arbitrary OPB primers tested, 18 produced amplification products. OPB 3 and 7 did not produce any amplification. In order to confirm genetic integrity, the DNA of 14 regenerated plants was compared to the DNA of the mother plant. The number of bands produced by each primer ranged from 2 to 13. All the 18 primers produced monomorphic bands confirming the genetic homogeneity of the regenerated plants. Fig. 2a, b shows representative amplification patterns obtained with primers OPB 8 and 11, respectively. Although many authors have reported that dedifferentiation of plant tissues leads to genetic modifications (Taylor et al., 1995; Hashmi et al., 1997; Rani et al., 2000), several reports confirm the genetic integrity of somatic embryo-regenerated plants. For
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SOMATIC EMBRYOGENESIS AND STABILITY ANALYSIS IN T. INDICA TABLE 1 EFFECT OF GROWTH REGULATORS ON EMBRYOGENESIS AND THE NUMBER OF SOMATIC EMBRYOS FORMED IN T. INDICA No. 1 2 3 4 5 6 7 8
Growth regulator concentration BA (mg l21) 0.5 1.0 2.0 3.0 2ip (mg l21) 0.5 1.0 2.0 3.0
IBA (mg l21) 0.01 0.01 0.01 0.01 IBA (mg l21) 0.01 0.01 0.01 0.01
Percentage of embryogenesis
Mean number of embryos
62 75 72 61
13.63 18.20 11.70 6.33
e d fg h
78 82 85 75
12.87 23.00 25.00 19.90
ef b a c
Values are the mean of 25 replicates repeated three times. Any two treatments having a common letter are not significantly different (Duncan's multiple range test).
Fig. 2. RAPD analysis of mother plant and regenerated plants of Tylophora indica. M-l-HindIII marker. Lane 1, mother plant; lanes 2±15 regenerants. a, Amplification products obtained with OPB 8. b, Amplification products obtained with OPB 11.
example, uniformity among regenerated plants produced from somatic embryogenesis has been reported in Pennisetum purpureum, Panicum maximum and Lolium multiflora (Dale et al., 1981; Haydu and Vasil, 1981; Hanna et al., 1989). Isozyme analysis of 60 plants derived from somatic embryogenesis from a hybrid between Carica papaya and Carica cauliflora showed a high degree of uniformity (Moore and Litz, 1984). Studies in some medicinal plant species like Foeniculum vulgare (Miura et al., 1987) and Bupleurum falcatum (Hiraoka et al., 1986) have shown that the coefficient of variation between plants obtained via embryogenesis was lower when compared with plants obtained from seeds. In Picea mariana RAPD analysis of somatic embryogenesis-derived populations revealed nil variation (Isabel et al., 1993). Similarly, Shoyama et al. (1997) reported nil variation among regenerants through somatic embryogenesis in Panax notoginseng by using RAPD analysis. Very recently, the use of inter-simple sequence repeats (ISSR) for assessing the genetic integrity of somatic embryogenesisregenerated plants have been reported in Brassica oleracea var. botrytis (Leroy et al., 2000). They have reported absolute uniformity among regenerants derived via somatic embryogenesis. These studies and the present study confirm the superiority of somatic embryogenesis over other methods of plant regeneration for the production of true-to-type plants. This lack of variation may be due to the fact that developmental constraints required by embryos exert selection against variant cells. Leroy et al. (2000) also stated that the lack of variation in somatic embryo regenerants is due to the
stringent internal genetic controls throughout embryo formation causing selection pressure against abnormal types. The present investigation describes a regeneration procedure for T. indica with several advantages: (1) the initial explants are mature leaves which are available irrespective of the season; (2) more than 80% of the explants showed callus induction on MS medium supplemented with 2,4-D (2 mg l21) and Kn (0.01 mg l21); (3) since formation of distinct bipolar embryos was observed, there was no need of a separate root induction medium; (4) more than 80% of the regenerated plants survived in the growth chamber; (5) RAPD analysis proved that regenerated plants were true-to-type, and hence this protocol can be used for the clonal propagation and in vitro conservation of this important medicinal plant. Using this optimized protocol it would be possible to produce an average of 50 plantlets with as little as 1 g of callus within a period of 5 mo. In addition, this procedure of regenerating uniform plants as described here may lead to the use of biotechnological tools for the improvement of T. indica. Acknowledgments M. Jayanthi wishes to thank the Council of Scientific and Industrial Research, India, for providing a Senior Research Fellowship. Prof. M. S. Swaminathan is gratefully acknowledged for providing the facilities and guidance. Dr. N. Anil Kumar is acknowledged for collecting the plant material.
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