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A grobacterium-mediated genetic transformation in hot chilli (Capsicum annuum L. var. Pusa jwala)
Plant Science 131 (1998) 77 – 83
Agrobacterium-mediated genetic transformation in hot chilli (Capsicum annuum L. var. Pusa jwala) M. Manoharan, C.S. Sree Vidya, G. Lakshmi Sita * Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012, India Received 1 April 1997; received in revised form 14 October 1997; accepted 14 October 1997
Abstract A protocol for regeneration and genetic transformation has been established for chilli (Capsicum annuum L. var. Pusa jwala). High frequency regeneration of shoot buds from cotyledonary leaves was achieved with Murashige and Skoog’s (MS) medium supplemented with 0.5 mg/l thidiazuron. Elongation of shoot buds and subsequent rooting was obtained on half-strength MS medium with 0.5 mg/l IAA. The Agrobacterium tumefaciens strain EHA 105 carrying a binary vector plasmid pBI 121 has been used for transformation. The cotyledonary explants from in vitro grown shoots were cocultivated for 2 days. Shoot buds were produced on the regeneration medium containing kanamycin (50 mg/l) and cefotaxime (400 mg/l). The shoot buds were elongated and rooted in the presence of kanamycin (25 mg/l). The transgenic nature of the regenerated plants were confirmed by the histochemical staining of GUS, polymerase chain reaction (PCR) and Southern hybridization analyses of nptII gene. © 1998 Elsevier Science Ireland Ltd. Keywords: Regeneration; Thidiazuron (TDZ); Genetic transformation; b-Glucuronidase (GUS); Neomycin phosphotransferase (NPT II); Chilli (Capsicum annuum L).
1. Introduction Recent advances in genetic engineering of plants have evoked great interest in developing modern technology for crop improvement. In many countries efforts have been made at cloning genes for use potential in agriculture and intro-
* Corresponding author. E-mail: [email protected]
ducing them gradually into plants of important crop species. Consequently, many transgenic plants have been successfully produced which showed remarkable results such as resistance to chemicals, pests and disease [1]. Chilli is a spice cum vegetable of commercial importance. In India, the production of chilli has declined from 779 thousand tonnes during 1992– 93 to 730 thousand tonnes during 1993–94, the overall percentage in decrease is − 6.25. Conse-
0168-9452/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S 0 1 6 8 - 9 4 5 2 ( 9 7 ) 0 0 2 3 1 - 8
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M. Manoharan et al. / Plant Science 131 (1998) 77–83
quently, the export of chilli fell during the 1994 – 95 season [2]. There are many factors which contributed to this decline. The most important among them are the diseases caused by viruses, bacteria, fungi and insects. Spraying of fungicides and pesticides can control the diseases to some extent, however, effective resistance against several destructive pathogens is still not possible. Efforts are being made to produce disease resistant plants through genetic engineering. Genetic transformation through Agrobacterium tumefaciens is now a routine procedure for introducing foreign genes into many plant species including several vegetable crop plants such as tomato, brinjal, Brassica etc. [3 – 5]. The two most important pre-requisites for the success of the method are the availability of a plant regeneration system from the explants and suitable method for transformation. Although chilli belongs to the Solanaceae family, whose members are easily amenable to tissue culture and transformation practices, it is highly recalcitrant. Several reports of plant regeneration either through organogenesis [6–16] or embryogenesis [17,18] are evident. However, these reports are genotype-specific and consequently, the regeneration protocol as well as viable transformation has to be established for each commercial cultivar for exploiting the potential of genetic engineering. Recently transformation has been reported [19,20] in sweet pepper with viral coat protein gene and herbicide resistant gene. In this study, we show the successful establishment of plant regeneration and genetic transformation with marker genes (GUS and NPT II) from the cotyledonary leaves of chilli var. Pusa jwala.
2. Materials and methods
2.1. Establishment of aseptic plants Seeds of Capsicum annuum L. cv. Pusa jwala were surface disinfected with the fungicide, Bavistin, for 1 h and rinsed repeatedly with running tap water. The seeds were sterilized for 2 min in 0.1% HgCl2 and then rinsed in several changes
of sterile distilled water. The seeds were germinated in a magenta box containing solidified MS basal medium [21].
2.2. Culture condition and media The cotyledonary leaves from 3 week old seedlings were used as explants and inoculated on the MS medium with 2% sucrose and supplemented with cytokinins (BAP, TDZ) and auxin (IAA) either singly or in combination. The medium was adjusted to pH 5.7 with 1 N HCl or 1 N NaOH phyta gel (0.2%, Sigma) or agar agar (0.8%, BDH) was used to solidify the medium. The shoot buds were transferred to shoot elongation and rooting (SER) medium containing halfstrength MS medium with 0.5 mg/l IAA for further elongation and rooting.
2.3. Establishment of plants into soil The plantlets were transferred to liquid medium consisting of half strength MS salts for hardening of roots for a week. Rooted plants measuring about 7–10 cm were transferred to paper cups filled with autoclaved soilrite and covered with plastic bags. Pots were kept in the culture room for 1 week before being transferred to soil.
2.4. Bacterial strain and 6ector The disarmed hypervirulent A. tumefaciens strain EHA 105 carrying the binary plasmid pBI 121 (Fig. 1) was used as a vector system. The T-DNA contains the neomycin phosphotransferase II (NPT II) gene, driven by the nopaline synthase (NOS) promoter and terminator sequences, which provides resistance to kanamycin and was used as selectable marker, and the b-glucuronidase (GUS) gene, driven by the cauliflower mosaic virus (CaMV) 35S promoter, was used as reporter gene. Bacterial strain was grown overnight in a Luria broth (LB) medium with appropriate antibiotics and collected in the log phase, when the absorbance at 550 nm was between 0.4 and 0.8.
M. Manoharan et al. / Plant Science 131 (1998) 77–83
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Fig. 1. Plasmid pBI 121 binary vector map 0.7 kb PCR fragment of nptII gene (approximately indicated as — ) was used as probe for Southern hybridization.
2.5. Transformation and regeneration
2.7. Polymerase chain reaction (PCR)
The cotyledonary leaves were precultured in the regeneration medium (MS+ 0.5 mg/l TDZ) for 2 days. Then, the explants were infected for 2 – 5 min with Agrobacterium suspension, blotted dry with Whatman No. 1 filter paper and returned to the regeneration medium for co-cultivation. Following the 48 h co-cultivation, the explants were washed with MS liquid medium. Subsequently, the explants were placed on selection medium, which is the regeneration medium, as described above, complemented with 50 mg/l kanamycin and 400 mg/l cefotaxime. The explants were transferred to fresh selection medium after 15 days of culture. The explants with putative transgenic shoot buds were transferred to SER medium with 25 mg/l kanamycin and 200 mg/l cefotaxime. After hardening The transgenic plants were transferred to soil.
For PCR analysis, DNA was isolated from leaves according to Edwards et al. [23]. Two primers of the NPT II gene were used: 5%-GAG GCT ATT CGG CTA TGA CTG-3% and 5%ATC GGG AGG GGC GAT ACC GTA-3%. Expected size of the fragment was 700 bp. PCR amplification was carried out in 50 ml containing 1 ml of the DNA solution, 200 mM dNTPs, 2.5 mM MgCl2, 50 mM KCl, 10 mM Tris–HCl pH 9.0, 0.1% (v/v) Triton X-100, 0.25 mM of each primer and two units of Taq DNA polymerase. DNA was subjected to 35 cycles of 1 min at 92, 55 and 72°C. Amplified DNA fragments were electrophoresed on 0.8% agarose ethidium bromide gel and observed under ultraviolet.
2.6. Histochemical GUS assay
Genomic DNA was extracted by the method of Dellaporta et al. [24]. About 10 mg of DNA from transformed and non transformed plants and 1 mg DNA of plasmid pBI 121 were digested with HindIII separated by electrophoresis through a 0.8% agarose gel and transferred onto Hybond–Nylon membrane. Hybridizations were performed according to Sambrook et al. [25]. A 0.7 kb PCR fragment of the nptII gene was used as a probe. Probe was labelled with 32P dCTP (Amersham) using a random-primed labelling kit (Amersham).
The histochemical GUS assay was conducted essentially as described by Jefferson et al. [22]. Leaf segments were immersed in an X-Gluc solution consisting of 2 mM X-Gluc, 100 mM Tris – HCl, pH 7.0, 50 mM NaCl, 2 mM potassium ferricyanide and 0.1% (v/v) Triton X-100. Tissues were stained overnight in dark at 37°C. The tissues were cleared through ethanol series to remove chlorophyll. Assayed tissues were observed under a microscope and photographed.
2.8. Southern hybridization analysis
M. Manoharan et al. / Plant Science 131 (1998) 77–83
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Table 1 Effect of growth regulators on shoot bud formation from the cotyledonary leaves of Capsicum annuum L. var. Pusa jwala Hormone concentration (mg/l) BAP TDZ IAA
No. of explants cultured
No. of explants responded
Regeneration frequency (%) (mean 9S.D.)
2.0 5.0 10.0 20.0
33 32 37 35
15.0 21.0 27.0 24.0
45.4 90.8 65.6 91.3 78.9 91.2 68.5 91.6
33 33 33 32
7.0 13.0 30.0 27.0
21.2 92.1 39.3 92.1 90.9 92.4 84.3 92.6
32 33 33 30
23.0 21.0 19.0 17.0
71.8 92.1 63.6 91.2 57.5 91.6 56.5 9 1.8
1.0 1.0 1.0 1.0 0.1 0.2 0.5 1.0
2.0 5.0 10.0 20.0
0.5 0.5 0.5 0.5
1.0 1.0 1.0 1.0
Observations were made after 1 month of culture. All experiments were repeated thrice
3. Results and discussion
3.1. Regeneration The composition of the media used in the regeneration of shoot buds and their frequency of induction from the cotyledonary explants are shown in Table 1. On the media containing BAP and IAA, the maximum regeneration frequency of 72.9% was obtained with 10 mg/l BAP and 1 mg/l IAA. Our results are consistent with those in previous reports in which BAP and IAA were used successfully to regenerate shoot buds [6 – 14,16]. However, the highest frequency of 90.9% of shoot buds regeneration was obtained in TDZ medium (0.5 mg/l). On the media containing various concentrations of TDZ (0.1 – 1.0 mg/l), callus formation was found to be less frequent and the buds induced on the cotyledons were green and vigorous. Our observations show that on media with BAP and IAA, callus formation interferes with shoot buds formation and that could be the reason for less frequency induction of shoot buds. Similar regeneration was reported in pepper plants by organogenesis from explants treated with TDZ [15]. TDZ has a high efficiency in stimulating cytokinin-dependent shoot regenera-
tion from a wide variety of plants [26,27]. On the medium containing TDZ, BAP and IAA, the frequency of shoot buds formation decreased with the increase in BAP concentration due to callus growth. Although efficient shoot buds induction was observed in the present study and those of others [6–16], elongation of shoot buds into long shoots has been a consistent problem. The shoot buds which formed at the cut end of the distal part of the cotyledonary leaves after 30–40 days of culture (Fig. 2A), were transferred to shoot elongation and rooting medium (SER). The SER contain half-strength MS supplemented with 0.5 mg/l IAA. After 90 days of culture, both shoot elongation and rooting were observed simultaneously (Fig. 2B,C). The transfer of shoot buds to the medium containing GA (1 mg/l) and Kinetin (1 mg/l) leads to excessive callus growth and did not support shoot bud elongation and rooting (data not shown). By transferring the shoot buds to SER, a total of 30 normal plants were obtained. Normally one, and occasionally two, elongated shoots were produced from the total of ten to 60 shoot buds per explant (observed under stereo microscope). Only vigorous grown shoot buds after transfer to SER produced elongated
M. Manoharan et al. / Plant Science 131 (1998) 77–83
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of TDZ, BAP and IAA. Liu et al. [28] failed to induce rooting whereas Ebida et al. [13] rooted the rossette buds and transferred to soil where the shoots are elongated. In our study, both elongation and rooting were obtained before transferring to soil. More than 95% of the elongated shoots were rooted. The plants were transferred to soil after hardening (Fig. 2E) and started producing flowers and normal sized and shaped fruits (Fig. 2F). Interestingly, in vitro flowering was also observed (Fig. 2D). However, there was no fruit set.
3.2. Transformation
Fig. 2. Regeneration of plants from the cotyledonary leaves of chilli (Capsicum annuum L. var. Pusa jwala). A: Initiation of shoot buds; B: Elongation of shoos; C: Rooted plants; D: In vitro flowering; E: Hardening of the in vitro grown plants; and F: Plants in the soil.
shoots. More number of vigorous grown shoot buds were obtained in TDZ medium than the medium containing BAP and IAA or combination
For transformation, around 200 cotyledonary leaves were co-cultivated with Agrobacterium for 48 h. The shoot bud induction was observed after 15 days of inoculation on the selection medium. The explants with shoot buds (about 5%) were subcultured in the fresh selection medium. After 40–45 days culture, the putatively transformed shoot buds were transferred to the SER medium containing 25 mg/l kanamycin and 200 mg/l cefotaxime. Of the ten explants with shoot buds, only four elongated shoots with roots were obtained (Fig. 3A). These putative transgenics were subsequently transferred to soil (Fig. 3B).The trans-
Fig. 3. Genetically transformed chilli (Capsicum annuum L. var. Pusa jwala) plants. A: Kanamycin resistant transgenic plants; B: Transgenic plants in the soil; C: Untransformed leaf tissue assayed for GUS activity (control); and D: Transgenic leaf tissue showing characteristic blue colour of the GUS gene.
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Fig. 4. PCR analysis of kanamycin resistant transgenic plants showing the presence of an expected 0.7 kb DNA fragment of nptII gene. Lane 1: l HindIII marker; Lane 2: Positive control (pBI 121); Lane 3: negative control (untransformed); Lanes 4–7: Transgenic plants; and Lane 8: fX 174 molecular weight marker digested with HaeIII.
genic nature of these plants were confirmed by histochemical localization of GUS gene and PCR and Southern analyses of nptII gene. Histochemical assay of the leaves for GUS activity, after staining with X-Gluc demonstrated the presence of blue coloured cells typical of the gusA gene (Fig. 3D). Leaf tissues from control plants did not show GUS activity (Fig. 3C). PCR was used to demonstrate the presence of T-DNA in the transgenic plants. Two specific primers derived from nptII gene sequences were used to detect a 0.7 kb fragment. The amplified DNA samples were electrophoresed on 0.8% agarose gel. As shown in Fig. 4, the 0.7 kb fragment co-running with the amplified product from pBI 121 could be detected from the transgenic plants but not from nontransformed plant. Southern hybridization was also carried out in order to further confirm the T-DNA integration. A 0.7 kb PCR fragment of the nptII gene was used as a probe. We obtained hybridization signal in the undigested positive control at 13 kb.(Two bands corresponding to two forms of plasmid). However, undigested DNA from transgenic plants gave signal at above
23 kb size indicating the integration of nptII gene in to the genome of transgenic plants. To confirm further, DNA from transgenic plants and plasmid DNA was digested with HindIII, which cuts once in the plasmid. Hybridization results clearly show variation in pattern among transgenic plants, indicating independent transformation events, and differed from the 13 kb band corresponding to the plasmid DNA disgested with HindIII. No hybridization signals were observed in genomic DNA isolated from non-transformed plants (Fig. 5.) Thus, genomic DNA blot hybridization data confirmed the introduction of nptII gene in the genome of transgenic chilli plants. Although it is desirable to have segregation data of the transgenic plants it has not been done since only marker genes were used. The results presented above demonstrate the potential of genetic transformation for the introduction of useful traits such as disease resistance in chilli.
Fig. 5. Southern blot analysis of total DNA isolated from transgenic plants. DNAs were digested with HindIII probed with 0.7 kb PCR fragment of the nptII gene. Lane 1: Undigested pBI 121 binary vector; Lane 2: pBI 121 digested with HindIII; Lanes 3 – 6: Undigested transgenic DNA; Lane 7: Undigested untransformed DNA; Lane 8: Untransformed DNA digested with HindIII; Lanes 9 – 12: Transgenic DNA digested with HindIII
M. Manoharan et al. / Plant Science 131 (1998) 77–83
Acknowledgements The corresponding author is grateful to Council of Scientific and Industrial Research (CSIR) for sanctioning the project entitled ‘In vitro development of transgenic plants in tomato and Capsicum’.
References [1] D.M. Shah, C.M.T. Rommens, R.N. Beachy, Resistance to diseases and insects in transgenicplants: progress and applications to agriculture, Trends Biotechnol. 13 (1995) 362 – 368. [2] K.V. Peter, Spices research and development: an updated overview, Plant Chron. 90 (1995) 471–477. [3] S. McCormick, J. Niedermeyer, J. Fry, A. Barnason, R. Horsch, R. Fraley, Leaf disc transformation of cultivated tomato (L. esculentum) using Agrobacterium tumefaciens, Plant Cell Rep. 5 (1986) 81–84. [4] M. Fari, I. Nagy, M. Csanyi, J. Mityko, A. Andrasfalvy, Agrobacterium mediated genetic transformation and plant regeneration via organogenesis and somatic embryogenesis from cotyledon leaves in egg plant (Solanum melongena L. cv. kecskemet; lila), Plant Cell Rep. 15 (1995) 82 – 86. [5] T.D. Metz, R. Dixit, E.D. Earle, Agrobacterium tumefaciens-mediated transformation of brocoli (Brassica oleracea var. italica) and cabbage (B. oleracea var. capitata), Plant Cell Rep. 15 (1995) 287–292. [6] A.L. Gunay, P.S. Rao, In vitro plant regeneration from hypocotyl and cotyledon explants of red pepper (Capsicum), Plant Sci. Lett. 11 (1978) 365–372. [7] M. Fari, M. Czako, Relationship between position and morphogenetic response of pepper hypocotyl explants cultured in vitro, Sci. Hort. 15 (1981) 207–213. [8] G.C. Phillips, J.F. Hubstenberger, Organogenesis in pepper tissue cultures, Plant Cell Tissue Organ Cult. 4 (1985) 261 – 269. [9] P. Sripichitt, E. Nawata, S. Shigenaga, In vitro shoot forming capacity of cotyledon explants in red pepper (Capsicum annuum L. cv. yatsufusa), Jpn. J. Breed. 37 (1987) 133 – 142. [10] S. Agrawal, N. Chandra, S.L. Kothari, Plant regeneration in tissue cultures of pepper (Capsicum annuum L. cv. mathania), Plant Cell Tissue Organ Cult. 16 (1989) 47– 55. [11] R. Arroyo, R. Revilla, In vitro plant regeneration from cotyledon and hypocotyl segments in two bell pepper cultivars, Plant Cell Rep. 10 (1991) 414–416. [12] L.L. Valera-Montero, N. Ochoa-Alejo, A novel approach for chili pepper (Capsicum annuum L.) plant regeneration: shoot induction in rooted hypocotyls, Plant Sci. 84 (1992) 215 – 219.
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[13] A.I.A. Ebida, C.Y. Hu, In vitro morphogenetic responses and plant regeneration from pepper (Capsicum annuum L. cv. Early california wonder) seedling explants, Plant Cell Rep. 13 (1993) 107 – 110. [14] T. Christopher, M.V. Rajam, In vitro clonal propagation of Capsicum spp., Plant Cell Tissue Organ Cult. 38 (1994) 25 – 27. [15] A. Szasz, G. Nervo, M. Fari, Screening for in vitro shoot forming capacity of seedling explants in bell pepper (Capsicum annuum L.) genotypes and efficient plant regeneration using thidiazuron, Plant Cell Rep. 14 (1995) 666 – 669. [16] C.L. Hyde, G.C. Phillips, Silver nitrate promotes shoot development and plant regeneration of chile pepper (Capsicum annuum L.) via organogenesis, In Vitro Cell Dev. Biol-Plant 32 (1996) 72 – 80. [17] I. Harini, G. Lakshmi Sita, Direct somatic embryogenesis and plant regeneration from immature embryos of chilli (Capsicum annuum L.), Plant Sci. 89 (1993) 107 – 112. [18] M.L. Binzel, N. Sankhla, S. Joshi, D. Sankhla, Induction of direct somatic embryogenesis and plant regeneration in pepper (Capsicum annuum L.), Plant Cell Rep. 15 (1996) 536 – 540. [19] K. Zhu, Y.F. Zhang Wen-Jun, Z.L. Chen Ou-Yang, Transgenic sweet pepper plants from Agrobacterium mediated transformation, Plant Cell Rep. 16 (1996) 71 – 75. [20] A. Tsafataris, The development of herbicide tolerant transgenic crops, Field Crop Res. 45 (1996) 115 – 124. [21] T. Murashige, F.A. Skoog, A revised medium for rapid growth and bioassays with tobacco tissue cultures, Physiol. Plant 15 (1962) 473 – 497. [22] R.A. Jefferson, T.A. Kavanagh, M.W. Bevan, GUS fusions: b-glucronidase as a sensitive and versatile gene fusion marker in higher plants, EMBO J. 6 (1987) 3901 – 3907. [23] K. Edwards, C. Johnstone, C. Thompson, A simple and rapid method for the preparation of plant genomic DNA for PCR analysis, Nucleic Acid Res. 19 (1991) 1349. [24] S.L. Dellaporta, J. Wood, J.B. Hicks, A plant DNA minipreparation: version II, Plant Mol. Biol. Rep. 4 (1983) 19 – 21. [25] J. Sambrook, E.F. Fritsch, T. Maniatis (Eds.), Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. [26] A.K. Malik, P.K. Saxena, Regeneration in Phaseolus 6ulgaris L.: High frequency induction of direct shoot formation in intact seedlings by N 6-benzylaminopurine and thidiazuron, Planta 186 (1992) 384 – 389. [27] C.A. Huettemann, J.E. Preece, Thidiazuron: a potent cytokinin for woody plant tissue culture, Plant Tissue Organ Cult. 33 (1993) 105 – 119. [28] W. Liu, W.A. Parrott, D.F. Hildebrand, G.B. Collins, E.G. Williams, Agrobacterium-induced gall formation in bell pepper (Capsicum annuum L.) and formation of shoot-like structures expressing introduced genes, Plant Cell Rep. 9 (1990) 360 – 364.
Agrobacterium-mediated genetic transformation in hot chilli (Capsicum annuum L. var. Pusa jwala) M. Manoharan, C.S. Sree Vidya, G. Lakshmi Sita * Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012, India Received 1 April 1997; received in revised form 14 October 1997; accepted 14 October 1997
Abstract A protocol for regeneration and genetic transformation has been established for chilli (Capsicum annuum L. var. Pusa jwala). High frequency regeneration of shoot buds from cotyledonary leaves was achieved with Murashige and Skoog’s (MS) medium supplemented with 0.5 mg/l thidiazuron. Elongation of shoot buds and subsequent rooting was obtained on half-strength MS medium with 0.5 mg/l IAA. The Agrobacterium tumefaciens strain EHA 105 carrying a binary vector plasmid pBI 121 has been used for transformation. The cotyledonary explants from in vitro grown shoots were cocultivated for 2 days. Shoot buds were produced on the regeneration medium containing kanamycin (50 mg/l) and cefotaxime (400 mg/l). The shoot buds were elongated and rooted in the presence of kanamycin (25 mg/l). The transgenic nature of the regenerated plants were confirmed by the histochemical staining of GUS, polymerase chain reaction (PCR) and Southern hybridization analyses of nptII gene. © 1998 Elsevier Science Ireland Ltd. Keywords: Regeneration; Thidiazuron (TDZ); Genetic transformation; b-Glucuronidase (GUS); Neomycin phosphotransferase (NPT II); Chilli (Capsicum annuum L).
1. Introduction Recent advances in genetic engineering of plants have evoked great interest in developing modern technology for crop improvement. In many countries efforts have been made at cloning genes for use potential in agriculture and intro-
* Corresponding author. E-mail: [email protected]
ducing them gradually into plants of important crop species. Consequently, many transgenic plants have been successfully produced which showed remarkable results such as resistance to chemicals, pests and disease [1]. Chilli is a spice cum vegetable of commercial importance. In India, the production of chilli has declined from 779 thousand tonnes during 1992– 93 to 730 thousand tonnes during 1993–94, the overall percentage in decrease is − 6.25. Conse-
0168-9452/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S 0 1 6 8 - 9 4 5 2 ( 9 7 ) 0 0 2 3 1 - 8
78
M. Manoharan et al. / Plant Science 131 (1998) 77–83
quently, the export of chilli fell during the 1994 – 95 season [2]. There are many factors which contributed to this decline. The most important among them are the diseases caused by viruses, bacteria, fungi and insects. Spraying of fungicides and pesticides can control the diseases to some extent, however, effective resistance against several destructive pathogens is still not possible. Efforts are being made to produce disease resistant plants through genetic engineering. Genetic transformation through Agrobacterium tumefaciens is now a routine procedure for introducing foreign genes into many plant species including several vegetable crop plants such as tomato, brinjal, Brassica etc. [3 – 5]. The two most important pre-requisites for the success of the method are the availability of a plant regeneration system from the explants and suitable method for transformation. Although chilli belongs to the Solanaceae family, whose members are easily amenable to tissue culture and transformation practices, it is highly recalcitrant. Several reports of plant regeneration either through organogenesis [6–16] or embryogenesis [17,18] are evident. However, these reports are genotype-specific and consequently, the regeneration protocol as well as viable transformation has to be established for each commercial cultivar for exploiting the potential of genetic engineering. Recently transformation has been reported [19,20] in sweet pepper with viral coat protein gene and herbicide resistant gene. In this study, we show the successful establishment of plant regeneration and genetic transformation with marker genes (GUS and NPT II) from the cotyledonary leaves of chilli var. Pusa jwala.
2. Materials and methods
2.1. Establishment of aseptic plants Seeds of Capsicum annuum L. cv. Pusa jwala were surface disinfected with the fungicide, Bavistin, for 1 h and rinsed repeatedly with running tap water. The seeds were sterilized for 2 min in 0.1% HgCl2 and then rinsed in several changes
of sterile distilled water. The seeds were germinated in a magenta box containing solidified MS basal medium [21].
2.2. Culture condition and media The cotyledonary leaves from 3 week old seedlings were used as explants and inoculated on the MS medium with 2% sucrose and supplemented with cytokinins (BAP, TDZ) and auxin (IAA) either singly or in combination. The medium was adjusted to pH 5.7 with 1 N HCl or 1 N NaOH phyta gel (0.2%, Sigma) or agar agar (0.8%, BDH) was used to solidify the medium. The shoot buds were transferred to shoot elongation and rooting (SER) medium containing halfstrength MS medium with 0.5 mg/l IAA for further elongation and rooting.
2.3. Establishment of plants into soil The plantlets were transferred to liquid medium consisting of half strength MS salts for hardening of roots for a week. Rooted plants measuring about 7–10 cm were transferred to paper cups filled with autoclaved soilrite and covered with plastic bags. Pots were kept in the culture room for 1 week before being transferred to soil.
2.4. Bacterial strain and 6ector The disarmed hypervirulent A. tumefaciens strain EHA 105 carrying the binary plasmid pBI 121 (Fig. 1) was used as a vector system. The T-DNA contains the neomycin phosphotransferase II (NPT II) gene, driven by the nopaline synthase (NOS) promoter and terminator sequences, which provides resistance to kanamycin and was used as selectable marker, and the b-glucuronidase (GUS) gene, driven by the cauliflower mosaic virus (CaMV) 35S promoter, was used as reporter gene. Bacterial strain was grown overnight in a Luria broth (LB) medium with appropriate antibiotics and collected in the log phase, when the absorbance at 550 nm was between 0.4 and 0.8.
M. Manoharan et al. / Plant Science 131 (1998) 77–83
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Fig. 1. Plasmid pBI 121 binary vector map 0.7 kb PCR fragment of nptII gene (approximately indicated as — ) was used as probe for Southern hybridization.
2.5. Transformation and regeneration
2.7. Polymerase chain reaction (PCR)
The cotyledonary leaves were precultured in the regeneration medium (MS+ 0.5 mg/l TDZ) for 2 days. Then, the explants were infected for 2 – 5 min with Agrobacterium suspension, blotted dry with Whatman No. 1 filter paper and returned to the regeneration medium for co-cultivation. Following the 48 h co-cultivation, the explants were washed with MS liquid medium. Subsequently, the explants were placed on selection medium, which is the regeneration medium, as described above, complemented with 50 mg/l kanamycin and 400 mg/l cefotaxime. The explants were transferred to fresh selection medium after 15 days of culture. The explants with putative transgenic shoot buds were transferred to SER medium with 25 mg/l kanamycin and 200 mg/l cefotaxime. After hardening The transgenic plants were transferred to soil.
For PCR analysis, DNA was isolated from leaves according to Edwards et al. [23]. Two primers of the NPT II gene were used: 5%-GAG GCT ATT CGG CTA TGA CTG-3% and 5%ATC GGG AGG GGC GAT ACC GTA-3%. Expected size of the fragment was 700 bp. PCR amplification was carried out in 50 ml containing 1 ml of the DNA solution, 200 mM dNTPs, 2.5 mM MgCl2, 50 mM KCl, 10 mM Tris–HCl pH 9.0, 0.1% (v/v) Triton X-100, 0.25 mM of each primer and two units of Taq DNA polymerase. DNA was subjected to 35 cycles of 1 min at 92, 55 and 72°C. Amplified DNA fragments were electrophoresed on 0.8% agarose ethidium bromide gel and observed under ultraviolet.
2.6. Histochemical GUS assay
Genomic DNA was extracted by the method of Dellaporta et al. [24]. About 10 mg of DNA from transformed and non transformed plants and 1 mg DNA of plasmid pBI 121 were digested with HindIII separated by electrophoresis through a 0.8% agarose gel and transferred onto Hybond–Nylon membrane. Hybridizations were performed according to Sambrook et al. [25]. A 0.7 kb PCR fragment of the nptII gene was used as a probe. Probe was labelled with 32P dCTP (Amersham) using a random-primed labelling kit (Amersham).
The histochemical GUS assay was conducted essentially as described by Jefferson et al. [22]. Leaf segments were immersed in an X-Gluc solution consisting of 2 mM X-Gluc, 100 mM Tris – HCl, pH 7.0, 50 mM NaCl, 2 mM potassium ferricyanide and 0.1% (v/v) Triton X-100. Tissues were stained overnight in dark at 37°C. The tissues were cleared through ethanol series to remove chlorophyll. Assayed tissues were observed under a microscope and photographed.
2.8. Southern hybridization analysis
M. Manoharan et al. / Plant Science 131 (1998) 77–83
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Table 1 Effect of growth regulators on shoot bud formation from the cotyledonary leaves of Capsicum annuum L. var. Pusa jwala Hormone concentration (mg/l) BAP TDZ IAA
No. of explants cultured
No. of explants responded
Regeneration frequency (%) (mean 9S.D.)
2.0 5.0 10.0 20.0
33 32 37 35
15.0 21.0 27.0 24.0
45.4 90.8 65.6 91.3 78.9 91.2 68.5 91.6
33 33 33 32
7.0 13.0 30.0 27.0
21.2 92.1 39.3 92.1 90.9 92.4 84.3 92.6
32 33 33 30
23.0 21.0 19.0 17.0
71.8 92.1 63.6 91.2 57.5 91.6 56.5 9 1.8
1.0 1.0 1.0 1.0 0.1 0.2 0.5 1.0
2.0 5.0 10.0 20.0
0.5 0.5 0.5 0.5
1.0 1.0 1.0 1.0
Observations were made after 1 month of culture. All experiments were repeated thrice
3. Results and discussion
3.1. Regeneration The composition of the media used in the regeneration of shoot buds and their frequency of induction from the cotyledonary explants are shown in Table 1. On the media containing BAP and IAA, the maximum regeneration frequency of 72.9% was obtained with 10 mg/l BAP and 1 mg/l IAA. Our results are consistent with those in previous reports in which BAP and IAA were used successfully to regenerate shoot buds [6 – 14,16]. However, the highest frequency of 90.9% of shoot buds regeneration was obtained in TDZ medium (0.5 mg/l). On the media containing various concentrations of TDZ (0.1 – 1.0 mg/l), callus formation was found to be less frequent and the buds induced on the cotyledons were green and vigorous. Our observations show that on media with BAP and IAA, callus formation interferes with shoot buds formation and that could be the reason for less frequency induction of shoot buds. Similar regeneration was reported in pepper plants by organogenesis from explants treated with TDZ [15]. TDZ has a high efficiency in stimulating cytokinin-dependent shoot regenera-
tion from a wide variety of plants [26,27]. On the medium containing TDZ, BAP and IAA, the frequency of shoot buds formation decreased with the increase in BAP concentration due to callus growth. Although efficient shoot buds induction was observed in the present study and those of others [6–16], elongation of shoot buds into long shoots has been a consistent problem. The shoot buds which formed at the cut end of the distal part of the cotyledonary leaves after 30–40 days of culture (Fig. 2A), were transferred to shoot elongation and rooting medium (SER). The SER contain half-strength MS supplemented with 0.5 mg/l IAA. After 90 days of culture, both shoot elongation and rooting were observed simultaneously (Fig. 2B,C). The transfer of shoot buds to the medium containing GA (1 mg/l) and Kinetin (1 mg/l) leads to excessive callus growth and did not support shoot bud elongation and rooting (data not shown). By transferring the shoot buds to SER, a total of 30 normal plants were obtained. Normally one, and occasionally two, elongated shoots were produced from the total of ten to 60 shoot buds per explant (observed under stereo microscope). Only vigorous grown shoot buds after transfer to SER produced elongated
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of TDZ, BAP and IAA. Liu et al. [28] failed to induce rooting whereas Ebida et al. [13] rooted the rossette buds and transferred to soil where the shoots are elongated. In our study, both elongation and rooting were obtained before transferring to soil. More than 95% of the elongated shoots were rooted. The plants were transferred to soil after hardening (Fig. 2E) and started producing flowers and normal sized and shaped fruits (Fig. 2F). Interestingly, in vitro flowering was also observed (Fig. 2D). However, there was no fruit set.
3.2. Transformation
Fig. 2. Regeneration of plants from the cotyledonary leaves of chilli (Capsicum annuum L. var. Pusa jwala). A: Initiation of shoot buds; B: Elongation of shoos; C: Rooted plants; D: In vitro flowering; E: Hardening of the in vitro grown plants; and F: Plants in the soil.
shoots. More number of vigorous grown shoot buds were obtained in TDZ medium than the medium containing BAP and IAA or combination
For transformation, around 200 cotyledonary leaves were co-cultivated with Agrobacterium for 48 h. The shoot bud induction was observed after 15 days of inoculation on the selection medium. The explants with shoot buds (about 5%) were subcultured in the fresh selection medium. After 40–45 days culture, the putatively transformed shoot buds were transferred to the SER medium containing 25 mg/l kanamycin and 200 mg/l cefotaxime. Of the ten explants with shoot buds, only four elongated shoots with roots were obtained (Fig. 3A). These putative transgenics were subsequently transferred to soil (Fig. 3B).The trans-
Fig. 3. Genetically transformed chilli (Capsicum annuum L. var. Pusa jwala) plants. A: Kanamycin resistant transgenic plants; B: Transgenic plants in the soil; C: Untransformed leaf tissue assayed for GUS activity (control); and D: Transgenic leaf tissue showing characteristic blue colour of the GUS gene.
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Fig. 4. PCR analysis of kanamycin resistant transgenic plants showing the presence of an expected 0.7 kb DNA fragment of nptII gene. Lane 1: l HindIII marker; Lane 2: Positive control (pBI 121); Lane 3: negative control (untransformed); Lanes 4–7: Transgenic plants; and Lane 8: fX 174 molecular weight marker digested with HaeIII.
genic nature of these plants were confirmed by histochemical localization of GUS gene and PCR and Southern analyses of nptII gene. Histochemical assay of the leaves for GUS activity, after staining with X-Gluc demonstrated the presence of blue coloured cells typical of the gusA gene (Fig. 3D). Leaf tissues from control plants did not show GUS activity (Fig. 3C). PCR was used to demonstrate the presence of T-DNA in the transgenic plants. Two specific primers derived from nptII gene sequences were used to detect a 0.7 kb fragment. The amplified DNA samples were electrophoresed on 0.8% agarose gel. As shown in Fig. 4, the 0.7 kb fragment co-running with the amplified product from pBI 121 could be detected from the transgenic plants but not from nontransformed plant. Southern hybridization was also carried out in order to further confirm the T-DNA integration. A 0.7 kb PCR fragment of the nptII gene was used as a probe. We obtained hybridization signal in the undigested positive control at 13 kb.(Two bands corresponding to two forms of plasmid). However, undigested DNA from transgenic plants gave signal at above
23 kb size indicating the integration of nptII gene in to the genome of transgenic plants. To confirm further, DNA from transgenic plants and plasmid DNA was digested with HindIII, which cuts once in the plasmid. Hybridization results clearly show variation in pattern among transgenic plants, indicating independent transformation events, and differed from the 13 kb band corresponding to the plasmid DNA disgested with HindIII. No hybridization signals were observed in genomic DNA isolated from non-transformed plants (Fig. 5.) Thus, genomic DNA blot hybridization data confirmed the introduction of nptII gene in the genome of transgenic chilli plants. Although it is desirable to have segregation data of the transgenic plants it has not been done since only marker genes were used. The results presented above demonstrate the potential of genetic transformation for the introduction of useful traits such as disease resistance in chilli.
Fig. 5. Southern blot analysis of total DNA isolated from transgenic plants. DNAs were digested with HindIII probed with 0.7 kb PCR fragment of the nptII gene. Lane 1: Undigested pBI 121 binary vector; Lane 2: pBI 121 digested with HindIII; Lanes 3 – 6: Undigested transgenic DNA; Lane 7: Undigested untransformed DNA; Lane 8: Untransformed DNA digested with HindIII; Lanes 9 – 12: Transgenic DNA digested with HindIII
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Acknowledgements The corresponding author is grateful to Council of Scientific and Industrial Research (CSIR) for sanctioning the project entitled ‘In vitro development of transgenic plants in tomato and Capsicum’.
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