Appl Biochem Biotechnol DOI 10.1007/s12010-014-1226-2
Transformation of Blackgram (Vigna mungo (L.) Hepper) by Barley Chitinase and Ribosome-Inactivating Protein Genes Towards Improving Resistance to Corynespora Leaf Spot Fungal Disease Rajan Chopra & Raman Saini
Received: 5 May 2014 / Accepted: 8 September 2014 # Springer Science+Business Media New York 2014
Abstract Blackgram (Vigna mungo (L.) Hepper), an important grain legume crop, is sensitive to many fungal pathogens including Corynespora cassiicola, the causal agent of corynespora leaf spot disease. In the present study, plasmid pGJ42 harboring neomycin phosphotransferase (nptII) a selectable marker gene, the barley antifungal genes chitinase (AAA56786) and ribosome-inactivating protein (RIP; AAA32951) were used for the transformation, to develop fungal resistance for the first time in blackgram. The presence and integration of transgene into the blackgram genome was confirmed by PCR and Southern analysis with an overall transformation frequency of 10.2 %. Kanamycin selection and PCR analysis of T0 progeny revealed the inheritance of transgene in Mendelian fashion (3:1). Transgenic plants (T1), evaluated for fungal resistance by in vitro antifungal assay, arrested the growth of C. cassiicola up to 25–40 % over the wild-type plants. In fungal bio-assay screening, the transgenic plants (T1) sprayed with C. cassiicola spores showed a delay in onset of disease along with their lesser extent in terms of average number of diseased leaves and reduced number and size of lesions. The percent disease protection among different transformed lines varies in the range of 27–47 % compare to control (untransformed) plants. These results demonstrate potentiality of chitinase and RIP from a heterologous source in developing fungal disease protection in blackgram and can be helpful in increasing the production of blackgram. Keywords Agrobacterium tumefaciens . Blackgram . Chitinase . Fungal resistance . Ribosomeinactivating protein . Transgenic plants Abbreviations BAP 6-Benzylaminopurine IBA Indole-3-butyric acid nptII Neomycin phosphotransferase RIP Ribosome-inactivating protein RM Rooting media SR Shoot regeneration medium R. Chopra : R. Saini (*) Department of Biotechnology, Kurukshetra University, Kurukshetra 136119, India e-mail: [email protected]
Appl Biochem Biotechnol
Introduction Blackgram (Vigna mungo (L.) Hepper) is an important source of vegetarian dietary protein with relatively low antinutritional factors to the large population of world particularly in developing countries. India is its main producer by contributing about 70 % of the world’s total blackgram production [1], but there is no substantial increase in its production during the last five decades due to its prominent susceptibility to several biotic and abiotic factors. Among biotic stresses, a high incidence of viral diseases, fungal pathogens, and insects contribute altogether more than 70 % of the yield loss. The estimated yield loss has been reported up to 50 % by the fungal diseases [2]. The most destructive and widespread fungal diseases affecting the productivity of blackgram are the cercospora and corynespora leaf spots and powdery mildew caused by Cercospora canescens, Corynespora cassiicola, and Erysiphe polygoni, respectively [3]. Chemical control measures for fungal diseases are expensive and ineffective. Moreover, the increasing use of chemically synthesized fungicides is adversely affecting the seed and fodder qualities of this crop. Further, indiscriminate use of fungicides pollutes the environment and thereby posing a threat to the ecosystem [4]; it also results in evolution of resistant biotypes of fungal pathogens, making management of fungal diseases altogether more difficult. Improvement in fungal resistance by classical breeding is limited due to the lack of sufficient and satisfactory levels of genetic variability within cultivated blackgram germplasm. Sources of resistance have been identified in wild and closely related Vigna species that are sexually incompatible to cultivated species. Further, conventional breeding approaches are expensive and time consuming, and often in addition to the desired characters, some of the undesirable characters may get transferred. Hence, it becomes imperative to look for effective alternative methods not only to protect crops but also to ensure a safe and clean environment. Genetic engineering technique has made it possible to transfer genes from sources which are otherwise difficult to introduce through conventional breeding. The simplest means of genetic engineering for resistance to fungal diseases entails the constitutive expression of one or more antifungal protein genes in transgenic plants. Among the antifungal proteins, pathogenesis-related (PR) proteins such as hydrolytic enzymes (chitinases and glucanases) and ribosome-inactivating proteins (RIP) either singly or in different combination are well known to provide resistance to fungal infection in various plants [5–9]. The constitutive expression of a single antifungal gene has been shown to provide resistance against fungal pathogens in several crops such as peanut, rice, Brassica juncea, tomato, finger millet, etc. [10–16]. However, combined expression of these antifungal genes showed significantly higher level of resistance than that of expression of either gene alone in several crop plants such as tobacco, rice, wheat, soybean, carrot, etc. [17–22]. Thus, adoption of combined gene expression-mediated protection against fungal pathogens in economically important crops like blackgram can become an important aspect of crop improvement programs. Blackgram is notoriously recalcitrant to in vitro regeneration and genetic transformation [23, 24]. There are only few reports on genetic transformation of blackgram using Agrobacterium tumefaciens containing mostly selectable marker and reporter genes [25–29]. However, no published reports are available to date on transfer of any antifungal gene for resistance to fungal diseases in this crop. In this direction, the present study was envisaged for the first time in blackgram to prove the efficacy of barley antifungal protein class II chitinase (AAA56786) and RIP (AAA32951) in combination for enhanced protection against C. cassiicola, an important fungal pathogen of blackgram, using transgenic approach.
Appl Biochem Biotechnol
Materials and Methods Plant Material, Bacterial Strain, and Vector Seeds of a commercially grown cultivar, PS-1 of V. mungo, were obtained from the Pulse Research Laboratory, Division of Genetics, Indian Agricultural Research Institute, New Delhi. The disarmed A. tumefaciens strain EHA105 harboring binary vector pGJ-42 was used for the transformation studies. The plasmid contains two antifungal genes, barley chitinase (AAA56786) and RIP (AAA32951)—both driven by the cauliflower mosaic virus (CaMV) 35S promoter and a selectable marker gene, neomycin phosphotransferase (nptII) driven by nos promoter (Fig. 1). The A. tumefaciens was grown in liquid YEM (yeast extract, 1.0 g l−1; mannitol, 10 g l−1; NaCl, 0.1 g l−1; MgSO4·7H2O, 0.2 g l−1; K2HPO4, 0.5 g l−1; pH 7.2) medium containing 50 mg l−1 kanamycin and 20 mg l−1 rifampicin overnight at 28 °C on a shaker at 200 rpm. Bacteria were pelleted at 4000 rpm for 10 min and re-suspended at a density of 108 cells ml−1 in liquid shoot regeneration (SR) medium containing 0.5 μM 6benzylaminopurine (BAP), acetosyringone (50 μM), and pH 5.2. Transformation and Plant Regeneration The stable transformation of blackgram was carried out using the cotyledonary node explants as described by Saini and Jaiwal [27] along with short vacuum infiltration during inoculation with the Agrobacterium suspension. The cotyledonary nodes without cotyledon explants of blackgram cv. PS-1 were excised from 16-h-old water-soaked seedlings and precultured for 3 days on shoot regeneration (SR) medium containing Murashige and Skoog (MS) salts [30], B5 vitamins [31], 3 % sucrose, and BAP (0.5 μM). Precultured explants were gently stabbed at the nodal region 4–5 times using a sterile fine hypodermic needle and inoculated with A. tumefaciens suspension. The explants were then subjected to vacuum of 660 mm of Hg for 5 min. After physical treatment, the Petri dish was left for 30 min on a rotary shaker at 25 °C and 70 rpm; thereafter, explants were blot dried on a sterilized filter paper (Whatman, www.whatman.com) and co-cultivated with A. tumefaciens on co-cultivation medium (semisolid SR medium having pH 5.2) for 3 days under 16-h photoperiod of cool-white fluorescent light (80 μ mol m−2 s−1) at 25±2 °C. After co-cultivation, the explants were washed 3–4 times with liquid SR medium and cultured on semisolid SR medium containing 75 mg l−1 kanamycin and 500 mg l−1 cefotaxime for shoot regeneration. The explants were transferred onto fresh medium containing the same levels of antibiotics every 2 weeks for a total of 4–6 weeks, until the shoots attained a height of 2–3 cm. Green shoots were transferred to rooting medium (RM) containing half-strength MS salts, full-strength MS vitamins, indole-3-butyric acid (IBA) (2. 5 μM), 3 % sucrose, and 10 mg l−1 kanamycin. The putative transformed plants were established in soil and grown to maturity to collect T0 seeds. HindIII
RB
p nos
npt II
nos t
p 35S
RIP
35 St
p 35 S
LB CHI
35 St
4kb Fig. 1 Schematic representation of the T-DNA region of binary vector pGJ42 contains a selectable marker gene neomycin phosphotransferase (nptII) and barley-derived chitinase (CHI) and ribosome-inactivating protein (RIP) antifungal genes
Appl Biochem Biotechnol
DNA Extraction and Molecular Analysis of Putative Transformants Total genomic DNA was extracted from fresh leaves of putative transformants (T0) and nontransformed (control) plants by the cetyltrimethyl ammonium bromide (CTAB) method [32]. Putative transformants were screened by the polymerase chain reaction (PCR) for the presence of the nptII gene. The 540-bp coding region of nptII was amplified using 20-bp oligonucleotide primers (5′ CCACCATGATATTCGGCAAC-3′ and 5′-GTGGAGAGGCTATTCGGCTA -3′). The amplification reaction was carried out using a thermal cycler (Bioer, www.bioer.com. cn) under the following conditions: one cycle of 94 °C for 1 min; 38 cycles of 94 °C for 1 min (denaturation), 58 °C for 1 min (annealing), 72 °C for 2 min (extension); and a final extension at 72 °C for 7 min (one cycle). The PCR was performed using approximately 100 ng of purified genomic DNA and Taq polymerase (Bangalore Genei, www.bangaloregenei.com). To ensure that reagents were not contaminated, DNA from non-transformed (control) plants was included in the experiments. The amplified products were separated by electrophoresis on a 1 % agarose gel and visualized with ethidium bromide [33]. Southern hybridization analysis was carried out to confirm the stable integration of nptII gene. DNA samples (~20 μg) from transformed (PCR-positive T0 plants) and untransformed (control) plants were digested with HindIII and separated on a 0.9 % agarose gel, blotted on positively charged nylon membrane (Whatman) by capillary blotting, and fixed by UV crosslinking. The membrane was then hybridized using the non-radioactive DIG-labeled probe having the coding regions of nptII (710 bp), and immunological detection of the hybridized probe was carried out following the supplier’s instructions (Roche, www.roche-appliedscience.com). Analysis of Transgene Inheritance To determine the stability and inheritance of transgenes, T0 seeds of Southern-positive transformed plants were germinated on MS medium containing 2500 mg l−1 kanamycin (this concentration was chosen based on kanamycin toxicity tests from our preliminary experiments) for 2 weeks. The green healthy seedlings were considered as resistant whereas non-germinated/brown/bleached and weak seedlings as sensitive. The kanamycin-resistant plants (T1) of each transgenic line were transferred to soil and further analyzed by PCR as above for T0 plants to check the presence of transgene in the progeny plants.
Evaluation of Transgenic Plants for Fungal Resistance In Vitro Antifungal Assay Water-soluble protein extracted from transgenic leaves obtained randomly from kanamycinresistant PCR-positive plants (T1) of each transformed line was assayed for their inhibitory effect on hyphal growth of C. cassiicola. The autoclaved transgenic leaf extract and protein extraction buffer (phosphate buffer, pH 7.0) served as a control. Wells on antibiotic assay agar plate were filled with 40 μl of spore suspension. After incubating overnight at 30 °C, each well was differently treated with 40 μl each of transgenic leaf extract, autoclaved transgenic leaf extract, and protein extraction buffer. The plates were incubated at 25 °C, and the radial growth of C. cassiicola was recorded up to 3 days. The percent inhibition of hyphal growth was calculated based on the following formula:
Appl Biochem Biotechnol
Percent inhibition ¼ 2
3
7 6 7 6 7 6 6diameter of fungal colony in untransformed plantðmmÞ−diameter of fungal colony in transgenic plantðmmÞ 7 100 7 6 5 4 . diameter of fungal colony in controlðuntransformed plantÞðmmÞ
Bio-assay of Transgenic Plants (T1) Progeny plants (T1) of each of the independent transgenic lines were assayed against C. cassiicola. Artificial epiphytotic conditions were created by maintaining the temperature at 26–28 °C and humidity at greater than 80 % by covering with a polypropylene bag for 8 h before inoculation. The plants were inoculated with spore suspension of C. cassiicola by atomizing. Untransformed plants were used as a control. The mean data by counting number of diseased leaves on each plant of control and transformed lines as well as lesions/patches per 2 cm2 leaf area (average of three leaves per plant taken randomly from one plant of each line) and individual lesion/patch diameter (in mm) were recorded from the tenth to 15th day of inoculation to calculate percent disease protection and to check the severity of infection. Among the other parameters of disease severity, shot holing and defoliation which are a marked symptom in advanced stages of infection were also taken to compare the control and transgenic plants. The percentage of disease protection was calculated as number of diseased leaves in untransformed plant minus number of diseased leaves in transgenic divided by number of diseased leaves in untransformed plant, multiplied by 100. Similarly, disease protection was also calculated from the data of average number of lesions per leaf in control and transformed plants.
Results Regeneration of Transformants From 350 explants inoculated with A. tumefaciens in different experiments, a total of 49 shoots were produced on kanamycin containing selection medium (Fig. 2a–c) (Table 1). Out of these, 20 shoots (40.8 %) formed roots in the presence of kanamycin at second round of selection (Fig. 2d). These plantlets were subsequently transferred to soil, where 11 plants survived, grew to maturity, and produced seeds (Fig. 2e). The time required to generate the putatively transformed plantlets from initiation of culture was only 6 weeks. Molecular Analysis of Transformants PCR analysis showed amplification of a 0.54-kb band corresponding to the coding region of nptII gene, indicating the presence of transgene in five (45.45 %) out of total 11 putative transformed plants established in soil (Fig. 2f) (Table 1). Southern analysis was performed to confirm the integration and copy number of the transgene in the genome of the putative transgenic plants. All PCR-positive putative transgenic plants checked with Southern analysis were found positive and showed integration of transgenes as the detected band sizes are higher than 4.0 kb (size of the entire 35S-RIP/35S-
Appl Biochem Biotechnol
a
b
c
M PC1 2 3 4 5 6 7 8 9 1 kb 0.5
d kb
M +C C 1
f
e
M P C1 2 3 4 5 6 7 8 9
2 3 4 5
1 kb
23 9.4 6.5 4.3 2
0.5
g
h
Fig. 2 In vitro regeneration of transformants from cotyledonary node explants of Vigna mungo cv. PS-1 and molecular analysis of primary transformants and their T1 progeny plants. a Multiple shoot regeneration from nontransformed cotyledonary node explants cultured on shoot regeneration medium (SR) containing (MS) salts, (B5) vitamins, and BAP (0.5 μM). b Non-transformed cotyledonary node explants cultured on SR medium supplemented with kanamycin (75 mg l−1). c Cotyledonary nodes co-cultivated with Agrobacterium cultured on SR medium+kanamycin (75 mg l−1)+cefotaxime (500 mg l−1). d Putative transformed shoot rooted on RM containing half-strength MS salts, full-strength MS vitamins, IBA (2.5 μM), kanamycin (10 mg l−1), and cefotaxime (500 mg l−1). e Putative transgenic plants (T0) in the pots. f PCR analysis of control and primary transformed plants using the nptII primers. Lane M marker DNA, lane P plasmid DNA, lane C DNA from untransformed control, lane 1–9 putative transformants. g Southern analysis of HindIII-digested genomic DNA of putative transformants and non-transformed (control) plants using the nptII DIG-labeled probe. Lane M marker DNA, lane +C PCR amplicon product of nptII gene (710 bp), lane C DNA from untransformed (control), lane 1–5 putative transformants. h PCR analysis of T1 plants using the nptII primers. Lane M marker DNA, lane P plasmid DNA, lane C DNA from untransformed control, lane 1–9 DNA from T1 progeny of five different primary transformants
CHI region of pGJ42 cassette cuts with HindIII). The different hybridization band patterns showed that these transgenic plants were derived from independent transformation events with four plants having a single copy and one having two copies of the T-DNA in their genome (Fig. 2g). DNA isolated from non-transformed plants did not hybridize with the nptII probe. An overall transformation frequency of 10.2 % (five Southern-positive plants out of 49 shoots regenerated from explants inoculated with Agrobacterium) was achieved (Table 1).
Appl Biochem Biotechnol Table 1 Summary of transformation of cotyledonary node explants of V. mungo cv. PS-1 co-cultured with A. tumefaciens strain EHA105 carrying binary vector pGJ42 Total explants inoculated in Agrobacterium
Total shoots regenerated on selection mediuma
Total shoots rooted on selection mediumb
Plants established in soil
% plants positive by PCR
Plants positive by Southern hybridization
350
49
20
11/20
45.45 (5/11)
5
SR medium containing MS salts, B5 vitamins, 3 % sucrose, BAP (0.5 μM), 75 mg l−1 kanamycin and 500 mg l−1 cefotaxime a
RM medium containing half-strength MS salts, full-strength MS vitamins, IBA (2.5 μM), 3 % sucrose, 10 mg l−1 kanamycin and 500 mg l−1 cefotaxime
b
Analysis of Transgene Inheritance The stability and inheritance of transgenes to the next generation of Southern-positive transformed plants were checked by germination of seeds under kanamycin selection followed by PCR analysis for the presence of transgene in the progeny plants. Out of total 48 seeds (T0) collected from five different transformed lines, 35 seedlings were found kanamycin tolerant and rest 13 either not germinates or found sensitive to selective concentration of kanamycin. All the kanamycin-tolerant plants were checked by PCR analysis; out of 35 plants, 32 were found positive for nptII gene while three plants negative, one each in line number 1, 3, and 4 (Table 2) (Fig. 2h). The segregation analysis in all of the five lines showed that the observed ratio is in accordance to the expected (3:1) Mendelian pattern of inheritance with no significant difference at P
Transformation of Blackgram (Vigna mungo (L.) Hepper) by Barley Chitinase and Ribosome-Inactivating Protein Genes Towards Improving Resistance to Corynespora Leaf Spot Fungal Disease Rajan Chopra & Raman Saini
Received: 5 May 2014 / Accepted: 8 September 2014 # Springer Science+Business Media New York 2014
Abstract Blackgram (Vigna mungo (L.) Hepper), an important grain legume crop, is sensitive to many fungal pathogens including Corynespora cassiicola, the causal agent of corynespora leaf spot disease. In the present study, plasmid pGJ42 harboring neomycin phosphotransferase (nptII) a selectable marker gene, the barley antifungal genes chitinase (AAA56786) and ribosome-inactivating protein (RIP; AAA32951) were used for the transformation, to develop fungal resistance for the first time in blackgram. The presence and integration of transgene into the blackgram genome was confirmed by PCR and Southern analysis with an overall transformation frequency of 10.2 %. Kanamycin selection and PCR analysis of T0 progeny revealed the inheritance of transgene in Mendelian fashion (3:1). Transgenic plants (T1), evaluated for fungal resistance by in vitro antifungal assay, arrested the growth of C. cassiicola up to 25–40 % over the wild-type plants. In fungal bio-assay screening, the transgenic plants (T1) sprayed with C. cassiicola spores showed a delay in onset of disease along with their lesser extent in terms of average number of diseased leaves and reduced number and size of lesions. The percent disease protection among different transformed lines varies in the range of 27–47 % compare to control (untransformed) plants. These results demonstrate potentiality of chitinase and RIP from a heterologous source in developing fungal disease protection in blackgram and can be helpful in increasing the production of blackgram. Keywords Agrobacterium tumefaciens . Blackgram . Chitinase . Fungal resistance . Ribosomeinactivating protein . Transgenic plants Abbreviations BAP 6-Benzylaminopurine IBA Indole-3-butyric acid nptII Neomycin phosphotransferase RIP Ribosome-inactivating protein RM Rooting media SR Shoot regeneration medium R. Chopra : R. Saini (*) Department of Biotechnology, Kurukshetra University, Kurukshetra 136119, India e-mail: [email protected]
Appl Biochem Biotechnol
Introduction Blackgram (Vigna mungo (L.) Hepper) is an important source of vegetarian dietary protein with relatively low antinutritional factors to the large population of world particularly in developing countries. India is its main producer by contributing about 70 % of the world’s total blackgram production [1], but there is no substantial increase in its production during the last five decades due to its prominent susceptibility to several biotic and abiotic factors. Among biotic stresses, a high incidence of viral diseases, fungal pathogens, and insects contribute altogether more than 70 % of the yield loss. The estimated yield loss has been reported up to 50 % by the fungal diseases [2]. The most destructive and widespread fungal diseases affecting the productivity of blackgram are the cercospora and corynespora leaf spots and powdery mildew caused by Cercospora canescens, Corynespora cassiicola, and Erysiphe polygoni, respectively [3]. Chemical control measures for fungal diseases are expensive and ineffective. Moreover, the increasing use of chemically synthesized fungicides is adversely affecting the seed and fodder qualities of this crop. Further, indiscriminate use of fungicides pollutes the environment and thereby posing a threat to the ecosystem [4]; it also results in evolution of resistant biotypes of fungal pathogens, making management of fungal diseases altogether more difficult. Improvement in fungal resistance by classical breeding is limited due to the lack of sufficient and satisfactory levels of genetic variability within cultivated blackgram germplasm. Sources of resistance have been identified in wild and closely related Vigna species that are sexually incompatible to cultivated species. Further, conventional breeding approaches are expensive and time consuming, and often in addition to the desired characters, some of the undesirable characters may get transferred. Hence, it becomes imperative to look for effective alternative methods not only to protect crops but also to ensure a safe and clean environment. Genetic engineering technique has made it possible to transfer genes from sources which are otherwise difficult to introduce through conventional breeding. The simplest means of genetic engineering for resistance to fungal diseases entails the constitutive expression of one or more antifungal protein genes in transgenic plants. Among the antifungal proteins, pathogenesis-related (PR) proteins such as hydrolytic enzymes (chitinases and glucanases) and ribosome-inactivating proteins (RIP) either singly or in different combination are well known to provide resistance to fungal infection in various plants [5–9]. The constitutive expression of a single antifungal gene has been shown to provide resistance against fungal pathogens in several crops such as peanut, rice, Brassica juncea, tomato, finger millet, etc. [10–16]. However, combined expression of these antifungal genes showed significantly higher level of resistance than that of expression of either gene alone in several crop plants such as tobacco, rice, wheat, soybean, carrot, etc. [17–22]. Thus, adoption of combined gene expression-mediated protection against fungal pathogens in economically important crops like blackgram can become an important aspect of crop improvement programs. Blackgram is notoriously recalcitrant to in vitro regeneration and genetic transformation [23, 24]. There are only few reports on genetic transformation of blackgram using Agrobacterium tumefaciens containing mostly selectable marker and reporter genes [25–29]. However, no published reports are available to date on transfer of any antifungal gene for resistance to fungal diseases in this crop. In this direction, the present study was envisaged for the first time in blackgram to prove the efficacy of barley antifungal protein class II chitinase (AAA56786) and RIP (AAA32951) in combination for enhanced protection against C. cassiicola, an important fungal pathogen of blackgram, using transgenic approach.
Appl Biochem Biotechnol
Materials and Methods Plant Material, Bacterial Strain, and Vector Seeds of a commercially grown cultivar, PS-1 of V. mungo, were obtained from the Pulse Research Laboratory, Division of Genetics, Indian Agricultural Research Institute, New Delhi. The disarmed A. tumefaciens strain EHA105 harboring binary vector pGJ-42 was used for the transformation studies. The plasmid contains two antifungal genes, barley chitinase (AAA56786) and RIP (AAA32951)—both driven by the cauliflower mosaic virus (CaMV) 35S promoter and a selectable marker gene, neomycin phosphotransferase (nptII) driven by nos promoter (Fig. 1). The A. tumefaciens was grown in liquid YEM (yeast extract, 1.0 g l−1; mannitol, 10 g l−1; NaCl, 0.1 g l−1; MgSO4·7H2O, 0.2 g l−1; K2HPO4, 0.5 g l−1; pH 7.2) medium containing 50 mg l−1 kanamycin and 20 mg l−1 rifampicin overnight at 28 °C on a shaker at 200 rpm. Bacteria were pelleted at 4000 rpm for 10 min and re-suspended at a density of 108 cells ml−1 in liquid shoot regeneration (SR) medium containing 0.5 μM 6benzylaminopurine (BAP), acetosyringone (50 μM), and pH 5.2. Transformation and Plant Regeneration The stable transformation of blackgram was carried out using the cotyledonary node explants as described by Saini and Jaiwal [27] along with short vacuum infiltration during inoculation with the Agrobacterium suspension. The cotyledonary nodes without cotyledon explants of blackgram cv. PS-1 were excised from 16-h-old water-soaked seedlings and precultured for 3 days on shoot regeneration (SR) medium containing Murashige and Skoog (MS) salts [30], B5 vitamins [31], 3 % sucrose, and BAP (0.5 μM). Precultured explants were gently stabbed at the nodal region 4–5 times using a sterile fine hypodermic needle and inoculated with A. tumefaciens suspension. The explants were then subjected to vacuum of 660 mm of Hg for 5 min. After physical treatment, the Petri dish was left for 30 min on a rotary shaker at 25 °C and 70 rpm; thereafter, explants were blot dried on a sterilized filter paper (Whatman, www.whatman.com) and co-cultivated with A. tumefaciens on co-cultivation medium (semisolid SR medium having pH 5.2) for 3 days under 16-h photoperiod of cool-white fluorescent light (80 μ mol m−2 s−1) at 25±2 °C. After co-cultivation, the explants were washed 3–4 times with liquid SR medium and cultured on semisolid SR medium containing 75 mg l−1 kanamycin and 500 mg l−1 cefotaxime for shoot regeneration. The explants were transferred onto fresh medium containing the same levels of antibiotics every 2 weeks for a total of 4–6 weeks, until the shoots attained a height of 2–3 cm. Green shoots were transferred to rooting medium (RM) containing half-strength MS salts, full-strength MS vitamins, indole-3-butyric acid (IBA) (2. 5 μM), 3 % sucrose, and 10 mg l−1 kanamycin. The putative transformed plants were established in soil and grown to maturity to collect T0 seeds. HindIII
RB
p nos
npt II
nos t
p 35S
RIP
35 St
p 35 S
LB CHI
35 St
4kb Fig. 1 Schematic representation of the T-DNA region of binary vector pGJ42 contains a selectable marker gene neomycin phosphotransferase (nptII) and barley-derived chitinase (CHI) and ribosome-inactivating protein (RIP) antifungal genes
Appl Biochem Biotechnol
DNA Extraction and Molecular Analysis of Putative Transformants Total genomic DNA was extracted from fresh leaves of putative transformants (T0) and nontransformed (control) plants by the cetyltrimethyl ammonium bromide (CTAB) method [32]. Putative transformants were screened by the polymerase chain reaction (PCR) for the presence of the nptII gene. The 540-bp coding region of nptII was amplified using 20-bp oligonucleotide primers (5′ CCACCATGATATTCGGCAAC-3′ and 5′-GTGGAGAGGCTATTCGGCTA -3′). The amplification reaction was carried out using a thermal cycler (Bioer, www.bioer.com. cn) under the following conditions: one cycle of 94 °C for 1 min; 38 cycles of 94 °C for 1 min (denaturation), 58 °C for 1 min (annealing), 72 °C for 2 min (extension); and a final extension at 72 °C for 7 min (one cycle). The PCR was performed using approximately 100 ng of purified genomic DNA and Taq polymerase (Bangalore Genei, www.bangaloregenei.com). To ensure that reagents were not contaminated, DNA from non-transformed (control) plants was included in the experiments. The amplified products were separated by electrophoresis on a 1 % agarose gel and visualized with ethidium bromide [33]. Southern hybridization analysis was carried out to confirm the stable integration of nptII gene. DNA samples (~20 μg) from transformed (PCR-positive T0 plants) and untransformed (control) plants were digested with HindIII and separated on a 0.9 % agarose gel, blotted on positively charged nylon membrane (Whatman) by capillary blotting, and fixed by UV crosslinking. The membrane was then hybridized using the non-radioactive DIG-labeled probe having the coding regions of nptII (710 bp), and immunological detection of the hybridized probe was carried out following the supplier’s instructions (Roche, www.roche-appliedscience.com). Analysis of Transgene Inheritance To determine the stability and inheritance of transgenes, T0 seeds of Southern-positive transformed plants were germinated on MS medium containing 2500 mg l−1 kanamycin (this concentration was chosen based on kanamycin toxicity tests from our preliminary experiments) for 2 weeks. The green healthy seedlings were considered as resistant whereas non-germinated/brown/bleached and weak seedlings as sensitive. The kanamycin-resistant plants (T1) of each transgenic line were transferred to soil and further analyzed by PCR as above for T0 plants to check the presence of transgene in the progeny plants.
Evaluation of Transgenic Plants for Fungal Resistance In Vitro Antifungal Assay Water-soluble protein extracted from transgenic leaves obtained randomly from kanamycinresistant PCR-positive plants (T1) of each transformed line was assayed for their inhibitory effect on hyphal growth of C. cassiicola. The autoclaved transgenic leaf extract and protein extraction buffer (phosphate buffer, pH 7.0) served as a control. Wells on antibiotic assay agar plate were filled with 40 μl of spore suspension. After incubating overnight at 30 °C, each well was differently treated with 40 μl each of transgenic leaf extract, autoclaved transgenic leaf extract, and protein extraction buffer. The plates were incubated at 25 °C, and the radial growth of C. cassiicola was recorded up to 3 days. The percent inhibition of hyphal growth was calculated based on the following formula:
Appl Biochem Biotechnol
Percent inhibition ¼ 2
3
7 6 7 6 7 6 6diameter of fungal colony in untransformed plantðmmÞ−diameter of fungal colony in transgenic plantðmmÞ 7 100 7 6 5 4 . diameter of fungal colony in controlðuntransformed plantÞðmmÞ
Bio-assay of Transgenic Plants (T1) Progeny plants (T1) of each of the independent transgenic lines were assayed against C. cassiicola. Artificial epiphytotic conditions were created by maintaining the temperature at 26–28 °C and humidity at greater than 80 % by covering with a polypropylene bag for 8 h before inoculation. The plants were inoculated with spore suspension of C. cassiicola by atomizing. Untransformed plants were used as a control. The mean data by counting number of diseased leaves on each plant of control and transformed lines as well as lesions/patches per 2 cm2 leaf area (average of three leaves per plant taken randomly from one plant of each line) and individual lesion/patch diameter (in mm) were recorded from the tenth to 15th day of inoculation to calculate percent disease protection and to check the severity of infection. Among the other parameters of disease severity, shot holing and defoliation which are a marked symptom in advanced stages of infection were also taken to compare the control and transgenic plants. The percentage of disease protection was calculated as number of diseased leaves in untransformed plant minus number of diseased leaves in transgenic divided by number of diseased leaves in untransformed plant, multiplied by 100. Similarly, disease protection was also calculated from the data of average number of lesions per leaf in control and transformed plants.
Results Regeneration of Transformants From 350 explants inoculated with A. tumefaciens in different experiments, a total of 49 shoots were produced on kanamycin containing selection medium (Fig. 2a–c) (Table 1). Out of these, 20 shoots (40.8 %) formed roots in the presence of kanamycin at second round of selection (Fig. 2d). These plantlets were subsequently transferred to soil, where 11 plants survived, grew to maturity, and produced seeds (Fig. 2e). The time required to generate the putatively transformed plantlets from initiation of culture was only 6 weeks. Molecular Analysis of Transformants PCR analysis showed amplification of a 0.54-kb band corresponding to the coding region of nptII gene, indicating the presence of transgene in five (45.45 %) out of total 11 putative transformed plants established in soil (Fig. 2f) (Table 1). Southern analysis was performed to confirm the integration and copy number of the transgene in the genome of the putative transgenic plants. All PCR-positive putative transgenic plants checked with Southern analysis were found positive and showed integration of transgenes as the detected band sizes are higher than 4.0 kb (size of the entire 35S-RIP/35S-
Appl Biochem Biotechnol
a
b
c
M PC1 2 3 4 5 6 7 8 9 1 kb 0.5
d kb
M +C C 1
f
e
M P C1 2 3 4 5 6 7 8 9
2 3 4 5
1 kb
23 9.4 6.5 4.3 2
0.5
g
h
Fig. 2 In vitro regeneration of transformants from cotyledonary node explants of Vigna mungo cv. PS-1 and molecular analysis of primary transformants and their T1 progeny plants. a Multiple shoot regeneration from nontransformed cotyledonary node explants cultured on shoot regeneration medium (SR) containing (MS) salts, (B5) vitamins, and BAP (0.5 μM). b Non-transformed cotyledonary node explants cultured on SR medium supplemented with kanamycin (75 mg l−1). c Cotyledonary nodes co-cultivated with Agrobacterium cultured on SR medium+kanamycin (75 mg l−1)+cefotaxime (500 mg l−1). d Putative transformed shoot rooted on RM containing half-strength MS salts, full-strength MS vitamins, IBA (2.5 μM), kanamycin (10 mg l−1), and cefotaxime (500 mg l−1). e Putative transgenic plants (T0) in the pots. f PCR analysis of control and primary transformed plants using the nptII primers. Lane M marker DNA, lane P plasmid DNA, lane C DNA from untransformed control, lane 1–9 putative transformants. g Southern analysis of HindIII-digested genomic DNA of putative transformants and non-transformed (control) plants using the nptII DIG-labeled probe. Lane M marker DNA, lane +C PCR amplicon product of nptII gene (710 bp), lane C DNA from untransformed (control), lane 1–5 putative transformants. h PCR analysis of T1 plants using the nptII primers. Lane M marker DNA, lane P plasmid DNA, lane C DNA from untransformed control, lane 1–9 DNA from T1 progeny of five different primary transformants
CHI region of pGJ42 cassette cuts with HindIII). The different hybridization band patterns showed that these transgenic plants were derived from independent transformation events with four plants having a single copy and one having two copies of the T-DNA in their genome (Fig. 2g). DNA isolated from non-transformed plants did not hybridize with the nptII probe. An overall transformation frequency of 10.2 % (five Southern-positive plants out of 49 shoots regenerated from explants inoculated with Agrobacterium) was achieved (Table 1).
Appl Biochem Biotechnol Table 1 Summary of transformation of cotyledonary node explants of V. mungo cv. PS-1 co-cultured with A. tumefaciens strain EHA105 carrying binary vector pGJ42 Total explants inoculated in Agrobacterium
Total shoots regenerated on selection mediuma
Total shoots rooted on selection mediumb
Plants established in soil
% plants positive by PCR
Plants positive by Southern hybridization
350
49
20
11/20
45.45 (5/11)
5
SR medium containing MS salts, B5 vitamins, 3 % sucrose, BAP (0.5 μM), 75 mg l−1 kanamycin and 500 mg l−1 cefotaxime a
RM medium containing half-strength MS salts, full-strength MS vitamins, IBA (2.5 μM), 3 % sucrose, 10 mg l−1 kanamycin and 500 mg l−1 cefotaxime
b
Analysis of Transgene Inheritance The stability and inheritance of transgenes to the next generation of Southern-positive transformed plants were checked by germination of seeds under kanamycin selection followed by PCR analysis for the presence of transgene in the progeny plants. Out of total 48 seeds (T0) collected from five different transformed lines, 35 seedlings were found kanamycin tolerant and rest 13 either not germinates or found sensitive to selective concentration of kanamycin. All the kanamycin-tolerant plants were checked by PCR analysis; out of 35 plants, 32 were found positive for nptII gene while three plants negative, one each in line number 1, 3, and 4 (Table 2) (Fig. 2h). The segregation analysis in all of the five lines showed that the observed ratio is in accordance to the expected (3:1) Mendelian pattern of inheritance with no significant difference at P
