J Plant Biotechnol 2022; 49(1): 61-73
Published online March 31, 2022
https://doi.org/10.5010/JPB.2022.49.1.061
© The Korean Society of Plant Biotechnology
Correspondence to : e-mail: peddaboina@kakatiya.ac.in
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
In this study, we developed a reliable and efficient Agrobacterium-mediated genetic transformation system by applying sonication and vacuum infiltration to six chickpea cultivars (ICCV2, ICCV10, ICCV92944, ICCV37, JAKI9218, and JG11) using embryo axis explants. Wounded explants were precultured for 3 days in shoot induction medium (SIM) before sonication and vacuum infiltration with an Agrobacterium suspension and co-cultivated for 3 days in co-cultivation medium containing 100 μM/l of acetosyringone and 200 mg/l of L-cysteine. Responsive explants with putatively transformed shoots were selected using a gradual increase in kanamycin from 25 mg/l to 100 mg/l in selection medium to eliminate escapes. Results showed optimal transformation efficiency at a bacterial density of 1.0, an optical density at 600 nm wavelength (OD600), and an infection duration of 30 min. The presence and stable integration of the β-glucuronidase (gusA) gene into the chickpea genome were confirmed using GUS histochemical assay and polymerase chain reaction. A high transformation efficiency was achieved among the different factors tested using embryo axis explants of cv. JAKI 9218. Of the six chickpea cultivars tested, JAKI9218 showed the highest transformation efficiency of 8.6%, followed by JG11 (7.2%), ICCV92944 (6.8%), ICCV37 (5.4%), ICCV2 (4.8%), and ICCV10 (4.6%). These findings showed that the Agrobacterium-mediated genetic transformation system will help transfer novel candidate genes into chickpea.
Keywords Agrobacterium, chickpea, embryo axis, sonication, transformation efficiency, vacuum infiltration
Chickpea is one of the most significant dietary sources of protein for the vegetarian population of India (Gaur et al. 2014; Jacob et al. 2016). Chickpea is considered the third most crucial edible grain legume, with about 17.2 million metric tonnes production worldwide in 2018 (FAO 2020). It is cultivated in more than fifty countries, covering all parts of the globe (Muehlbauer and Sarker 2017). India is the prominent producer of chickpea, with around 66% of global production with 11.38 million tonnes per annum (FAO 2020). Chickpea production is severely impeded by several biotic and abiotic stresses, notably fungal diseases like fusarium wilt (caused by
The development of biotic (fungal and insect pest) stress-resistant chickpea varieties using conventional breeding methods is not feasible due to constricted genetic variability in its germplasm (Gatti et al. 2016) and the existence of strong sexual incompatibility for genetic hybridization in chickpea (van Rheenen et al. 1993). Therefore, introducing biotic stress-resistant candidate genes into chickpea through genetic engineering could be a good choice for developing transgenic plants. A proficient and repeatable plant regeneration protocol amenable to gene transfer must produce transgenic chickpea plants (Leonetti et al. 2018; Pandey et al. 2021; Yadav et al. 2017). In general, chickpea is considered one of the highly recalcitrant species of legumes to manipulate through plant regeneration and genetic engineering (Atif et al. 2013). This problem could also be because of the unavailability of regenerable target tissue for efficient delivery and integration of transgene from
Due to genotype specificity in chickpea,
Therefore, the present study was undertaken to explore the conditions influencing
Chickpea genotypes JAKI9218, JG11, ICCC37, ICCV10, and ICCV92944 (JG14) (desi) and ICCV2 (Swetha) (Kabuli) seeds were procured from Germplasm Resource Unit, International Crops Research Institute for Semi-Arid Tropics (ICRISAT), Hyderabad, Telangana State, India. Uniform and healthy seeds of the JAKI9218 genotype were chosen for explant preparation. The seeds were sterilized with 70% (v/v) ethanol for 2 min, followed by 0.1% mercuric chloride (HgCl2) for 10 min. The sterilized seeds were rinsed with sterile distilled water 5 to 6 times. The sterile seeds were soaked overnight in water, and the seeds were blotted dry. Then the seed coat was removed and germinated on medium amended with 1.0 mg l-1 BAP (15 per plate) at 24 ± 2°C in dark condition for one day. The embryo axis explants were prepared by removing radicle (root apex), shoot (plumule), and cotyledons. These embryo axes were used as explants for transformation experiments (Pathak and Hamzah 2008; Sadhu et al. 2020).
Decapitate embryo axis explants were inoculated on MSB5 medium augmented with 6-Benzyl amino purine (BAP) (2.0 mg l-1) and Indole-3-butyric acid (IBA) (0.05 mg l-1) designated as shoot induction medium (SIM) (Sadhu et al. 2020). Shoot induction medium (SIM) added with different kanamycin concentrations (25, 50, 75, and 100 mg l-1) and 300 mg l-1 cefotaxime designated as selection medium. Responsive explants with a bunch shoots were inoculated onto shoot elongation medium (SEM) amended with BAP (1.0 mg l-1), Indole-3-acetic acid (IAA) (0.05 mg l-1), Gibberellic acid (GA3) (1.0 mg l-1), 50 mg l-1 kanamycin and 100 mg l-1 cefotaxime. Putative transgenic shoots (~3.0 cm length) were transferred into the root induction medium (RIM) fortified with 2.0 mg l-1 IBA, 50 mg l-1 kanamycin, and 100 mg l-1 cefotaxime. The pH of the media was adjusted to 5.6 using 0.1N NaOH /0.1N HCl, then gelled with 0.8% agar before autoclaving at 121°C for 20 min. All cultures were maintained at 24 ± 2°C with a 16/8 h photoperiod using cool fluorescent lights (50 μMol m2 S-1).
An initial study was conducted to check the influence of kanamycin (selection agent) on decapitated embryo axis explants were cultured in SIM along with different concentrations (0, 25, 50, 75, 100, and 150 mg l-1) of kanamycin. The explants were subcultured three times into a fresh SIM medium with respective concentrations of kanamycin (at ten days intervals) after ten days of subculture. Kanamycin (50 mg/ml) stocks were prepared; filter-sterilized (0.22 μm) and stored as aliquots at -20°C. The aliquots were added to the warm autoclaved medium to obtain the needed concentration of kanamycin. A control without kanamycin was also maintained for the explants.
The embryo axis explants were subjected to infection in batches of 50 explants/30 ml of
For standardization of transformation efficiency, hundred embryo axis explants were infected with
After 3 days of cocultivation, embryo axis explants were thoroughly washed with the sterile liquid washing medium (LWM) comprising MS salts and B5 vitamins, sucrose (30%), MES buffer (3 mM), along with cefotaxime (200 mg l-1) twice to eliminate the excess of
The elongated putative transgenic shoots (> 3 cm) were separated from a bunch of multiple shoots then inoculated onto RIM amended with cefotaxime (100 mg l-1) and kanamycin (50 mg l-1) incubated 4 weeks. The complete plantlets were obtained after rooting of putative transgenic shoots. The complete plantlets were carefully removed from culture tubes and washed with sterile distilled to eliminate the traces of agar sticking to roots. The complete plantlets were shifted to plastic cups filled with an equal ratio (1 : 1 : 1) of sterile sand, soil, and vermiculite. The plantlets were enclosed with polythene bags with the slightest puncture, then placed culture for two to three weeks and wetted once in two days. After three weeks, plantlets were shifted to pots and grown in the greenhouse.
Histochemical GUS assay was carried out to verify in embryo axis explants after each treatment and T0 putative plantlets using the substrate X-GlucA (5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid) (Jefferson 1987). The plant material was washed with methanol for two h to remove chlorophyll pigments. The explants with positive transformants showed blue staining, and the explant with staining was recorded as the GUS positive. The non-transformed explants (control), cultured under similar conditions, served as control.
Genomic DNA from leaflets of the putative T0 transformants regenerated from the embryo axis explants of chickpea was isolated (Doyle and Doyle 1990). Wild-type (untransformed control) plant genomic DNA and plasmid pCAMBIA2301 (positive control) were also used for amplification. The polymerase chain reaction (PCR) was performed with specific primer sets to
The data were scored for each treatment, and each experiment was repeated three times. Each replicates with 100 embryo axis explants to optimize different factors on transformation efficiency. The data were scored for each treatment to evaluate various factors using 30 randomly selected embryo axis explants for
Explant type is also one of the highly imperative factors affecting plant genetic transformation. The
The identification and selection of transformants and non-transformants generally depend on the growth difference of transformed against non-transformed tissues in the presence of a selection agent. Several selectable marker genes encode resistance to an antibiotic which has been used in genetic transformation experiments. Legumes’ genetic improvement programmes were deterred due to the lack of an efficient transformation protocol and reliable selection strategy (Nyaboga et al. 2014). Kanamycin resistance is one the most commonly used markers in legumes, including chickpea (Chakraborty et al. 2016; Das et al. 2017; Srivastava et al. 2017). For the selection of transformed shoots, different kanamycin concentrations ranging from 50-150 mg l-1 were used (Anbazhagan et al. 2015; Chakraborty et al. 2016; Das et al. 2017; Srivastava et al. 2017). Based on these studies, to decide the most favourable dose of kanamycin on the survival and regeneration response of the embryo axes were evaluated in chickpea. The survival and regeneration response of the explants was tested on different concentrations of kanamycin (25, 50, 75, and 100 mg l-1), and the explant browning was recorded periodically (7, 14, and 21 days after culture initiation). The increase in kanamycin concentration severely decreased the survival and regeneration response of the embryo axis explants. Our study has used four to five selection cycles with diverse selection regimes of kanamycin (25-100 mg l-1). The different selection regimes were helpful in the efficient recovery of putative transformants and successful prevention escapes during selection. The step-wise selection pressure permits transformed explants to express the antibiotic-resistance gene efficiently and induce cell division, thus recovering the regeneration of explants to produce plants (Bull et al. 2009). The low antibiotic concentration at early stages promotes transformed cell recovery, and a gradual improvement of antibiotic concentration efficiently eliminates non-transformed cells in the selection medium (Anbazhagan et al. 2015; Burgos and Alburquerque 2003). The final selection of putatively transformed shoots on a higher concentration of antibiotics eliminates chimeras (escapes) and generates positive plants. Finally, a low to stringent selection (25-100 mg l-1) regime of kanamycin was adopted for all the transformation experiments. The kanamycin was one of the most efficiently used selectable markers for generating transgenic plants of a variety of legumes like
For optimum delivery of T-DNA from
The influence of the preculture period on transformation efficiency was determined by culturing the explants for different time durations. The explants were precultured before
The influence of different bacterial cell densities was evaluated on transformation efficiency with the embryo axis explants of chickpea. The histochemical
The embryo axes were infected in
The embryo axis explants were co-cultivated for 1, 2, 3, 4, and 5 days after infection with
Table 1 Influence of co-cultivation duration and acetosyringone and cysteine concentrations on transformation efficiency in embryo axis explants of chickpea cv. JAKI92181
Co-cultivation duration (days) | Acetosyringone concentration (µM/l) | Cysteine concentration (mg/l) | Explants infected, | |
---|---|---|---|---|
0 | - | - | 100 | 36.6 ± 1.47op |
1 | - | - | 100 | 38.2 ± 1.58op |
2 | - | - | 100 | 42.6 ± 1.63lm |
3 | - | - | 100 | 46.4 ± 1.74kl |
4 | - | - | 100 | 38.8 ± 1.68no |
5 | - | - | 100 | 36.4 ± 1.83op |
3 | 0 | - | 100 | 46.4 ± 1.36kl |
3 | 50 | - | 100 | 48.2 ± 1.65jk |
3 | 100 | - | 100 | 66.2 ± 1.82cd |
3 | 150 | - | 100 | 58.4 ± 1.68fg |
3 | 200 | - | 100 | 56.2 ± 1.72gh |
3 | 250 | - | 100 | 54.6 ± 1.66hi |
3 | 100 | 0 | 100 | 66.2 ± 1.81cd |
3 | 100 | 50 | 100 | 68.4 ± 1.65cd |
3 | 100 | 100 | 100 | 70.6 ± 1.48cb |
3 | 100 | 200 | 100 | 76.5 ± 1.60a |
3 | 100 | 300 | 100 | 62.2 ± 1.78ef |
3 | 100 | 400 | 100 | 60.8 ± 1.64ef |
1The control group consisted of wounded explants precultured for 3 days at a bacterial cell density 1.0 at OD6005 and an infection duration of 30 min. Transformation efficiency = (GUS explants/total explants infected) × 100. We used 100 embryo axis explants per treatment, and each treatment was repeated thrice.
2gus: β-glucuronidase.
3SE: standard error.
4Means ± SEs of values of three independent experiments. Values with the same letter within columns are not significantly different according to the DMRT6 at a 5% level.
5OD600: optical density at 600 nm wavelength.
6DMRT: Duncan’s multiple range test.
Acetosyringone (AS), one of the plant phenolic compounds produced during plant cell wounding, stimulates bacterial attachment and triggers the transcription of virulence genes that control the processing and transfer of T-DNA from
In the present investigation, we have noticed that the embryo axis explants exhibited enzymatic browning and tissue necrosis at the wounded sites following cocultivation, which were likely to affect the efficient transformation. L-cysteine is being considered one of the most useful anti-necrotic agents. Therefore, to reduce
In chickpea, explants with meristematic cells are the primary target for
Table 2 Influence of sonication and vacuum infiltration on transformation efficiency in embryo axis explants of chickpea cv. JAKI92181
Sonication duration (s) | Vacuum infiltration duration (min) | Explants infected, | |
---|---|---|---|
0 | - | 100 | 76.5 ± 1.38ef |
30 | - | 100 | 78.6 ± 1.62de |
60 | - | 100 | 81.4 ± 1.64cd |
90 | - | 100 | 73.2 ± 1.68gh |
120 | - | 100 | 70.4 ± 1.82hi |
150 | - | 100 | 73.2 ± 1.56gh |
90 | 0 | 100 | 81.4 ± 1.62cd |
90 | 5 | 100 | 82.6 ± 1.86bc |
90 | 10 | 100 | 82.8 ± 1.46bc |
90 | 15 | 100 | 84.8 ± 1.58ab |
90 | 20 | 100 | 78.8 ± 1.66de |
90 | 25 | 100 | 74.6 ± 1.62fg |
1The control group consisted of wounded explants precultured for 3 days at a bacterial cell density of 1.0 at OD6005 and an infection duration of 30 min, with 100 µM/l of acetosyringone and 200 mg/l of L-cysteine. Transformation efficiency = (GUS explants/total explants infected) × 100. We used 100 embryo axis explants per treatment, and each treatment was repeated thrice.
2
3SE: standard error.
4Means ± SEs of values of three independent experiments. Values with the same letter within columns are not significantly different according to the DMRT6 at a 5% level.
5OD600: optical density at 600 nm wavelength.
6DMRT: Duncan’s multiple range test.
The sonication treatment increased the
To further evaluate the transformation efficiency in chickpea, the
An efficient selection approach is highly significant for developing a reproducible plant transformation protocol. The co-cultivated explants were washed to remove the excess
In the present investigation, repeated transfer of the embryo axis explants to a medium containing kanamycin facilitated uniformly GUS expressing shoots. The non-transformed kanamycin-sensitive shoots of the embryo axis explants were completely bleached and showed retarded growth. The observation suggested that incremental kanamycin (25 to 100 mg l-1) resulted in the efficient selection of transformed cells from non-transformed cells (Fig. 1b). The kanamycin induced the rapid death of non-transformed cells (sensitive cells) throughout the cycles of the selection period. The selection period improved the growth and development of healthy shoots resistant to kanamycin and avoided the problem of chimerism. The comparable selection regime in the presence of kanamycin was reported in chickpea as an earlier study (Anbazhagan et al. 2015; Srivastava et al. 2017). The complete plants were obtained after rooting the putatively transformed shoots on a rooting medium augmented with IBA 2.0 mg l-1, 100 mg l-1 cefotaxime, and 50 mg l-1 kanamycin (Fig. 1d). The putatively transformed plantlets were successfully acclimatized under greenhouse conditions (Fig. 1e). The regenerated plants established under greenhouse for morphologically similar and flowered normally.
The visual or selectable markers are required for detection due to the relatively small number of cells in which integration of the foreign DNA occurs during the development of gene transfer protocols in plant species (Xing et al. 2000). After three successive subcultures, the explants with shoots were examined for
The PCR analysis confirmed the existence of the
The transformation efficiency strongly depends upon genotypes, and transformation efficiency may be different in various genotypes. Similarly, different transformation efficiencies have been reported with multiple explants from different chickpea genotypes (Tripathi et al. 2013). Hence, the
Table 3 Influence of different cultivars of chickpea on
Chickpea genotype | Explants infected, | Explants that responded (after 3 subcultures), mean ± SE2,3 | Transformation efficiency (%)5 | |
---|---|---|---|---|
JAKI9218 | 100 | 12.8 ± 0.76a | 8.6 ± 0.84a | 8.6 |
JG11 | 100 | 10.4 ± 0.68b | 7.2 ± 0.78b | 7.2 |
ICCC37 | 100 | 9.6 ± 0.86d | 5.4 ± 0.92d | 5.4 |
ICCV10 | 100 | 7.3 ± 0.82e | 4.6 ± 0.83e | 4.6 |
ICCV92944 (JG14) | 100 | 10.2 ± 0.92bc | 6.8 ± 0.76bc | 6.8 |
ICCV2 (Swetha) | 100 | 6.3 ± 0.88f | 3.8 ± 0.78ef | 4.8 |
1We used 100 embryo axis explants per treatment, and each treatment was repeated thrice.
2SE: standard error.
3Means ± SEs of values of three independent experiments. Values with the same letter within columns are not significantly different according to the DMRT5 at a 5% level.
4
5Transformation efficiency = (GUS explants/total explants infected) × 100.
6DMRT: Duncan’s multiple range test.
In the present investigation, we have standardized several factors for efficient
The authors are grateful to the Department of Atomic Energy (DAE), Board of Research in Nuclear Sciences (BRNS), Government of India for financial assistance in the form of a project (DAE-BRNS No.2013/35/36/BRNS/1254, Dt: 30.07.2013).
J Plant Biotechnol 2022; 49(1): 61-73
Published online March 31, 2022 https://doi.org/10.5010/JPB.2022.49.1.061
Copyright © The Korean Society of Plant Biotechnology.
Suman Kalyan Sadhu ・Phanikanth Jogam ・Kranthikumar Gande ・Raghu Banoth ・Suprasanna Penna ・ Venkataiah Peddaboina
Department of Microbiology, Kakatiya University, Vidyaranyapuri, Warangal 506009, Telangana, India
Department of Biotechnology, Kakatiya University, Vidyaranyapuri, Warangal 506009, Telangana, India
Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre (BARC), Mumbai 400085, Maharashtra, India
Correspondence to:e-mail: peddaboina@kakatiya.ac.in
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
In this study, we developed a reliable and efficient Agrobacterium-mediated genetic transformation system by applying sonication and vacuum infiltration to six chickpea cultivars (ICCV2, ICCV10, ICCV92944, ICCV37, JAKI9218, and JG11) using embryo axis explants. Wounded explants were precultured for 3 days in shoot induction medium (SIM) before sonication and vacuum infiltration with an Agrobacterium suspension and co-cultivated for 3 days in co-cultivation medium containing 100 μM/l of acetosyringone and 200 mg/l of L-cysteine. Responsive explants with putatively transformed shoots were selected using a gradual increase in kanamycin from 25 mg/l to 100 mg/l in selection medium to eliminate escapes. Results showed optimal transformation efficiency at a bacterial density of 1.0, an optical density at 600 nm wavelength (OD600), and an infection duration of 30 min. The presence and stable integration of the β-glucuronidase (gusA) gene into the chickpea genome were confirmed using GUS histochemical assay and polymerase chain reaction. A high transformation efficiency was achieved among the different factors tested using embryo axis explants of cv. JAKI 9218. Of the six chickpea cultivars tested, JAKI9218 showed the highest transformation efficiency of 8.6%, followed by JG11 (7.2%), ICCV92944 (6.8%), ICCV37 (5.4%), ICCV2 (4.8%), and ICCV10 (4.6%). These findings showed that the Agrobacterium-mediated genetic transformation system will help transfer novel candidate genes into chickpea.
Keywords: Agrobacterium, chickpea, embryo axis, sonication, transformation efficiency, vacuum infiltration
Chickpea is one of the most significant dietary sources of protein for the vegetarian population of India (Gaur et al. 2014; Jacob et al. 2016). Chickpea is considered the third most crucial edible grain legume, with about 17.2 million metric tonnes production worldwide in 2018 (FAO 2020). It is cultivated in more than fifty countries, covering all parts of the globe (Muehlbauer and Sarker 2017). India is the prominent producer of chickpea, with around 66% of global production with 11.38 million tonnes per annum (FAO 2020). Chickpea production is severely impeded by several biotic and abiotic stresses, notably fungal diseases like fusarium wilt (caused by
The development of biotic (fungal and insect pest) stress-resistant chickpea varieties using conventional breeding methods is not feasible due to constricted genetic variability in its germplasm (Gatti et al. 2016) and the existence of strong sexual incompatibility for genetic hybridization in chickpea (van Rheenen et al. 1993). Therefore, introducing biotic stress-resistant candidate genes into chickpea through genetic engineering could be a good choice for developing transgenic plants. A proficient and repeatable plant regeneration protocol amenable to gene transfer must produce transgenic chickpea plants (Leonetti et al. 2018; Pandey et al. 2021; Yadav et al. 2017). In general, chickpea is considered one of the highly recalcitrant species of legumes to manipulate through plant regeneration and genetic engineering (Atif et al. 2013). This problem could also be because of the unavailability of regenerable target tissue for efficient delivery and integration of transgene from
Due to genotype specificity in chickpea,
Therefore, the present study was undertaken to explore the conditions influencing
Chickpea genotypes JAKI9218, JG11, ICCC37, ICCV10, and ICCV92944 (JG14) (desi) and ICCV2 (Swetha) (Kabuli) seeds were procured from Germplasm Resource Unit, International Crops Research Institute for Semi-Arid Tropics (ICRISAT), Hyderabad, Telangana State, India. Uniform and healthy seeds of the JAKI9218 genotype were chosen for explant preparation. The seeds were sterilized with 70% (v/v) ethanol for 2 min, followed by 0.1% mercuric chloride (HgCl2) for 10 min. The sterilized seeds were rinsed with sterile distilled water 5 to 6 times. The sterile seeds were soaked overnight in water, and the seeds were blotted dry. Then the seed coat was removed and germinated on medium amended with 1.0 mg l-1 BAP (15 per plate) at 24 ± 2°C in dark condition for one day. The embryo axis explants were prepared by removing radicle (root apex), shoot (plumule), and cotyledons. These embryo axes were used as explants for transformation experiments (Pathak and Hamzah 2008; Sadhu et al. 2020).
Decapitate embryo axis explants were inoculated on MSB5 medium augmented with 6-Benzyl amino purine (BAP) (2.0 mg l-1) and Indole-3-butyric acid (IBA) (0.05 mg l-1) designated as shoot induction medium (SIM) (Sadhu et al. 2020). Shoot induction medium (SIM) added with different kanamycin concentrations (25, 50, 75, and 100 mg l-1) and 300 mg l-1 cefotaxime designated as selection medium. Responsive explants with a bunch shoots were inoculated onto shoot elongation medium (SEM) amended with BAP (1.0 mg l-1), Indole-3-acetic acid (IAA) (0.05 mg l-1), Gibberellic acid (GA3) (1.0 mg l-1), 50 mg l-1 kanamycin and 100 mg l-1 cefotaxime. Putative transgenic shoots (~3.0 cm length) were transferred into the root induction medium (RIM) fortified with 2.0 mg l-1 IBA, 50 mg l-1 kanamycin, and 100 mg l-1 cefotaxime. The pH of the media was adjusted to 5.6 using 0.1N NaOH /0.1N HCl, then gelled with 0.8% agar before autoclaving at 121°C for 20 min. All cultures were maintained at 24 ± 2°C with a 16/8 h photoperiod using cool fluorescent lights (50 μMol m2 S-1).
An initial study was conducted to check the influence of kanamycin (selection agent) on decapitated embryo axis explants were cultured in SIM along with different concentrations (0, 25, 50, 75, 100, and 150 mg l-1) of kanamycin. The explants were subcultured three times into a fresh SIM medium with respective concentrations of kanamycin (at ten days intervals) after ten days of subculture. Kanamycin (50 mg/ml) stocks were prepared; filter-sterilized (0.22 μm) and stored as aliquots at -20°C. The aliquots were added to the warm autoclaved medium to obtain the needed concentration of kanamycin. A control without kanamycin was also maintained for the explants.
The embryo axis explants were subjected to infection in batches of 50 explants/30 ml of
For standardization of transformation efficiency, hundred embryo axis explants were infected with
After 3 days of cocultivation, embryo axis explants were thoroughly washed with the sterile liquid washing medium (LWM) comprising MS salts and B5 vitamins, sucrose (30%), MES buffer (3 mM), along with cefotaxime (200 mg l-1) twice to eliminate the excess of
The elongated putative transgenic shoots (> 3 cm) were separated from a bunch of multiple shoots then inoculated onto RIM amended with cefotaxime (100 mg l-1) and kanamycin (50 mg l-1) incubated 4 weeks. The complete plantlets were obtained after rooting of putative transgenic shoots. The complete plantlets were carefully removed from culture tubes and washed with sterile distilled to eliminate the traces of agar sticking to roots. The complete plantlets were shifted to plastic cups filled with an equal ratio (1 : 1 : 1) of sterile sand, soil, and vermiculite. The plantlets were enclosed with polythene bags with the slightest puncture, then placed culture for two to three weeks and wetted once in two days. After three weeks, plantlets were shifted to pots and grown in the greenhouse.
Histochemical GUS assay was carried out to verify in embryo axis explants after each treatment and T0 putative plantlets using the substrate X-GlucA (5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid) (Jefferson 1987). The plant material was washed with methanol for two h to remove chlorophyll pigments. The explants with positive transformants showed blue staining, and the explant with staining was recorded as the GUS positive. The non-transformed explants (control), cultured under similar conditions, served as control.
Genomic DNA from leaflets of the putative T0 transformants regenerated from the embryo axis explants of chickpea was isolated (Doyle and Doyle 1990). Wild-type (untransformed control) plant genomic DNA and plasmid pCAMBIA2301 (positive control) were also used for amplification. The polymerase chain reaction (PCR) was performed with specific primer sets to
The data were scored for each treatment, and each experiment was repeated three times. Each replicates with 100 embryo axis explants to optimize different factors on transformation efficiency. The data were scored for each treatment to evaluate various factors using 30 randomly selected embryo axis explants for
Explant type is also one of the highly imperative factors affecting plant genetic transformation. The
The identification and selection of transformants and non-transformants generally depend on the growth difference of transformed against non-transformed tissues in the presence of a selection agent. Several selectable marker genes encode resistance to an antibiotic which has been used in genetic transformation experiments. Legumes’ genetic improvement programmes were deterred due to the lack of an efficient transformation protocol and reliable selection strategy (Nyaboga et al. 2014). Kanamycin resistance is one the most commonly used markers in legumes, including chickpea (Chakraborty et al. 2016; Das et al. 2017; Srivastava et al. 2017). For the selection of transformed shoots, different kanamycin concentrations ranging from 50-150 mg l-1 were used (Anbazhagan et al. 2015; Chakraborty et al. 2016; Das et al. 2017; Srivastava et al. 2017). Based on these studies, to decide the most favourable dose of kanamycin on the survival and regeneration response of the embryo axes were evaluated in chickpea. The survival and regeneration response of the explants was tested on different concentrations of kanamycin (25, 50, 75, and 100 mg l-1), and the explant browning was recorded periodically (7, 14, and 21 days after culture initiation). The increase in kanamycin concentration severely decreased the survival and regeneration response of the embryo axis explants. Our study has used four to five selection cycles with diverse selection regimes of kanamycin (25-100 mg l-1). The different selection regimes were helpful in the efficient recovery of putative transformants and successful prevention escapes during selection. The step-wise selection pressure permits transformed explants to express the antibiotic-resistance gene efficiently and induce cell division, thus recovering the regeneration of explants to produce plants (Bull et al. 2009). The low antibiotic concentration at early stages promotes transformed cell recovery, and a gradual improvement of antibiotic concentration efficiently eliminates non-transformed cells in the selection medium (Anbazhagan et al. 2015; Burgos and Alburquerque 2003). The final selection of putatively transformed shoots on a higher concentration of antibiotics eliminates chimeras (escapes) and generates positive plants. Finally, a low to stringent selection (25-100 mg l-1) regime of kanamycin was adopted for all the transformation experiments. The kanamycin was one of the most efficiently used selectable markers for generating transgenic plants of a variety of legumes like
For optimum delivery of T-DNA from
The influence of the preculture period on transformation efficiency was determined by culturing the explants for different time durations. The explants were precultured before
The influence of different bacterial cell densities was evaluated on transformation efficiency with the embryo axis explants of chickpea. The histochemical
The embryo axes were infected in
The embryo axis explants were co-cultivated for 1, 2, 3, 4, and 5 days after infection with
Table 1 . Influence of co-cultivation duration and acetosyringone and cysteine concentrations on transformation efficiency in embryo axis explants of chickpea cv. JAKI92181.
Co-cultivation duration (days) | Acetosyringone concentration (µM/l) | Cysteine concentration (mg/l) | Explants infected, | |
---|---|---|---|---|
0 | - | - | 100 | 36.6 ± 1.47op |
1 | - | - | 100 | 38.2 ± 1.58op |
2 | - | - | 100 | 42.6 ± 1.63lm |
3 | - | - | 100 | 46.4 ± 1.74kl |
4 | - | - | 100 | 38.8 ± 1.68no |
5 | - | - | 100 | 36.4 ± 1.83op |
3 | 0 | - | 100 | 46.4 ± 1.36kl |
3 | 50 | - | 100 | 48.2 ± 1.65jk |
3 | 100 | - | 100 | 66.2 ± 1.82cd |
3 | 150 | - | 100 | 58.4 ± 1.68fg |
3 | 200 | - | 100 | 56.2 ± 1.72gh |
3 | 250 | - | 100 | 54.6 ± 1.66hi |
3 | 100 | 0 | 100 | 66.2 ± 1.81cd |
3 | 100 | 50 | 100 | 68.4 ± 1.65cd |
3 | 100 | 100 | 100 | 70.6 ± 1.48cb |
3 | 100 | 200 | 100 | 76.5 ± 1.60a |
3 | 100 | 300 | 100 | 62.2 ± 1.78ef |
3 | 100 | 400 | 100 | 60.8 ± 1.64ef |
1The control group consisted of wounded explants precultured for 3 days at a bacterial cell density 1.0 at OD6005 and an infection duration of 30 min. Transformation efficiency = (GUS explants/total explants infected) × 100. We used 100 embryo axis explants per treatment, and each treatment was repeated thrice..
2gus: β-glucuronidase..
3SE: standard error..
4Means ± SEs of values of three independent experiments. Values with the same letter within columns are not significantly different according to the DMRT6 at a 5% level..
5OD600: optical density at 600 nm wavelength..
6DMRT: Duncan’s multiple range test..
Acetosyringone (AS), one of the plant phenolic compounds produced during plant cell wounding, stimulates bacterial attachment and triggers the transcription of virulence genes that control the processing and transfer of T-DNA from
In the present investigation, we have noticed that the embryo axis explants exhibited enzymatic browning and tissue necrosis at the wounded sites following cocultivation, which were likely to affect the efficient transformation. L-cysteine is being considered one of the most useful anti-necrotic agents. Therefore, to reduce
In chickpea, explants with meristematic cells are the primary target for
Table 2 . Influence of sonication and vacuum infiltration on transformation efficiency in embryo axis explants of chickpea cv. JAKI92181.
Sonication duration (s) | Vacuum infiltration duration (min) | Explants infected, | |
---|---|---|---|
0 | - | 100 | 76.5 ± 1.38ef |
30 | - | 100 | 78.6 ± 1.62de |
60 | - | 100 | 81.4 ± 1.64cd |
90 | - | 100 | 73.2 ± 1.68gh |
120 | - | 100 | 70.4 ± 1.82hi |
150 | - | 100 | 73.2 ± 1.56gh |
90 | 0 | 100 | 81.4 ± 1.62cd |
90 | 5 | 100 | 82.6 ± 1.86bc |
90 | 10 | 100 | 82.8 ± 1.46bc |
90 | 15 | 100 | 84.8 ± 1.58ab |
90 | 20 | 100 | 78.8 ± 1.66de |
90 | 25 | 100 | 74.6 ± 1.62fg |
1The control group consisted of wounded explants precultured for 3 days at a bacterial cell density of 1.0 at OD6005 and an infection duration of 30 min, with 100 µM/l of acetosyringone and 200 mg/l of L-cysteine. Transformation efficiency = (GUS explants/total explants infected) × 100. We used 100 embryo axis explants per treatment, and each treatment was repeated thrice..
2
3SE: standard error..
4Means ± SEs of values of three independent experiments. Values with the same letter within columns are not significantly different according to the DMRT6 at a 5% level..
5OD600: optical density at 600 nm wavelength..
6DMRT: Duncan’s multiple range test..
The sonication treatment increased the
To further evaluate the transformation efficiency in chickpea, the
An efficient selection approach is highly significant for developing a reproducible plant transformation protocol. The co-cultivated explants were washed to remove the excess
In the present investigation, repeated transfer of the embryo axis explants to a medium containing kanamycin facilitated uniformly GUS expressing shoots. The non-transformed kanamycin-sensitive shoots of the embryo axis explants were completely bleached and showed retarded growth. The observation suggested that incremental kanamycin (25 to 100 mg l-1) resulted in the efficient selection of transformed cells from non-transformed cells (Fig. 1b). The kanamycin induced the rapid death of non-transformed cells (sensitive cells) throughout the cycles of the selection period. The selection period improved the growth and development of healthy shoots resistant to kanamycin and avoided the problem of chimerism. The comparable selection regime in the presence of kanamycin was reported in chickpea as an earlier study (Anbazhagan et al. 2015; Srivastava et al. 2017). The complete plants were obtained after rooting the putatively transformed shoots on a rooting medium augmented with IBA 2.0 mg l-1, 100 mg l-1 cefotaxime, and 50 mg l-1 kanamycin (Fig. 1d). The putatively transformed plantlets were successfully acclimatized under greenhouse conditions (Fig. 1e). The regenerated plants established under greenhouse for morphologically similar and flowered normally.
The visual or selectable markers are required for detection due to the relatively small number of cells in which integration of the foreign DNA occurs during the development of gene transfer protocols in plant species (Xing et al. 2000). After three successive subcultures, the explants with shoots were examined for
The PCR analysis confirmed the existence of the
The transformation efficiency strongly depends upon genotypes, and transformation efficiency may be different in various genotypes. Similarly, different transformation efficiencies have been reported with multiple explants from different chickpea genotypes (Tripathi et al. 2013). Hence, the
Table 3 . Influence of different cultivars of chickpea on
Chickpea genotype | Explants infected, | Explants that responded (after 3 subcultures), mean ± SE2,3 | Transformation efficiency (%)5 | |
---|---|---|---|---|
JAKI9218 | 100 | 12.8 ± 0.76a | 8.6 ± 0.84a | 8.6 |
JG11 | 100 | 10.4 ± 0.68b | 7.2 ± 0.78b | 7.2 |
ICCC37 | 100 | 9.6 ± 0.86d | 5.4 ± 0.92d | 5.4 |
ICCV10 | 100 | 7.3 ± 0.82e | 4.6 ± 0.83e | 4.6 |
ICCV92944 (JG14) | 100 | 10.2 ± 0.92bc | 6.8 ± 0.76bc | 6.8 |
ICCV2 (Swetha) | 100 | 6.3 ± 0.88f | 3.8 ± 0.78ef | 4.8 |
1We used 100 embryo axis explants per treatment, and each treatment was repeated thrice..
2SE: standard error..
3Means ± SEs of values of three independent experiments. Values with the same letter within columns are not significantly different according to the DMRT5 at a 5% level..
4
5Transformation efficiency = (GUS explants/total explants infected) × 100..
6DMRT: Duncan’s multiple range test..
In the present investigation, we have standardized several factors for efficient
The authors are grateful to the Department of Atomic Energy (DAE), Board of Research in Nuclear Sciences (BRNS), Government of India for financial assistance in the form of a project (DAE-BRNS No.2013/35/36/BRNS/1254, Dt: 30.07.2013).
Table 1 . Influence of co-cultivation duration and acetosyringone and cysteine concentrations on transformation efficiency in embryo axis explants of chickpea cv. JAKI92181.
Co-cultivation duration (days) | Acetosyringone concentration (µM/l) | Cysteine concentration (mg/l) | Explants infected, | |
---|---|---|---|---|
0 | - | - | 100 | 36.6 ± 1.47op |
1 | - | - | 100 | 38.2 ± 1.58op |
2 | - | - | 100 | 42.6 ± 1.63lm |
3 | - | - | 100 | 46.4 ± 1.74kl |
4 | - | - | 100 | 38.8 ± 1.68no |
5 | - | - | 100 | 36.4 ± 1.83op |
3 | 0 | - | 100 | 46.4 ± 1.36kl |
3 | 50 | - | 100 | 48.2 ± 1.65jk |
3 | 100 | - | 100 | 66.2 ± 1.82cd |
3 | 150 | - | 100 | 58.4 ± 1.68fg |
3 | 200 | - | 100 | 56.2 ± 1.72gh |
3 | 250 | - | 100 | 54.6 ± 1.66hi |
3 | 100 | 0 | 100 | 66.2 ± 1.81cd |
3 | 100 | 50 | 100 | 68.4 ± 1.65cd |
3 | 100 | 100 | 100 | 70.6 ± 1.48cb |
3 | 100 | 200 | 100 | 76.5 ± 1.60a |
3 | 100 | 300 | 100 | 62.2 ± 1.78ef |
3 | 100 | 400 | 100 | 60.8 ± 1.64ef |
1The control group consisted of wounded explants precultured for 3 days at a bacterial cell density 1.0 at OD6005 and an infection duration of 30 min. Transformation efficiency = (GUS explants/total explants infected) × 100. We used 100 embryo axis explants per treatment, and each treatment was repeated thrice..
2gus: β-glucuronidase..
3SE: standard error..
4Means ± SEs of values of three independent experiments. Values with the same letter within columns are not significantly different according to the DMRT6 at a 5% level..
5OD600: optical density at 600 nm wavelength..
6DMRT: Duncan’s multiple range test..
Table 2 . Influence of sonication and vacuum infiltration on transformation efficiency in embryo axis explants of chickpea cv. JAKI92181.
Sonication duration (s) | Vacuum infiltration duration (min) | Explants infected, | |
---|---|---|---|
0 | - | 100 | 76.5 ± 1.38ef |
30 | - | 100 | 78.6 ± 1.62de |
60 | - | 100 | 81.4 ± 1.64cd |
90 | - | 100 | 73.2 ± 1.68gh |
120 | - | 100 | 70.4 ± 1.82hi |
150 | - | 100 | 73.2 ± 1.56gh |
90 | 0 | 100 | 81.4 ± 1.62cd |
90 | 5 | 100 | 82.6 ± 1.86bc |
90 | 10 | 100 | 82.8 ± 1.46bc |
90 | 15 | 100 | 84.8 ± 1.58ab |
90 | 20 | 100 | 78.8 ± 1.66de |
90 | 25 | 100 | 74.6 ± 1.62fg |
1The control group consisted of wounded explants precultured for 3 days at a bacterial cell density of 1.0 at OD6005 and an infection duration of 30 min, with 100 µM/l of acetosyringone and 200 mg/l of L-cysteine. Transformation efficiency = (GUS explants/total explants infected) × 100. We used 100 embryo axis explants per treatment, and each treatment was repeated thrice..
2
3SE: standard error..
4Means ± SEs of values of three independent experiments. Values with the same letter within columns are not significantly different according to the DMRT6 at a 5% level..
5OD600: optical density at 600 nm wavelength..
6DMRT: Duncan’s multiple range test..
Table 3 . Influence of different cultivars of chickpea on
Chickpea genotype | Explants infected, | Explants that responded (after 3 subcultures), mean ± SE2,3 | Transformation efficiency (%)5 | |
---|---|---|---|---|
JAKI9218 | 100 | 12.8 ± 0.76a | 8.6 ± 0.84a | 8.6 |
JG11 | 100 | 10.4 ± 0.68b | 7.2 ± 0.78b | 7.2 |
ICCC37 | 100 | 9.6 ± 0.86d | 5.4 ± 0.92d | 5.4 |
ICCV10 | 100 | 7.3 ± 0.82e | 4.6 ± 0.83e | 4.6 |
ICCV92944 (JG14) | 100 | 10.2 ± 0.92bc | 6.8 ± 0.76bc | 6.8 |
ICCV2 (Swetha) | 100 | 6.3 ± 0.88f | 3.8 ± 0.78ef | 4.8 |
1We used 100 embryo axis explants per treatment, and each treatment was repeated thrice..
2SE: standard error..
3Means ± SEs of values of three independent experiments. Values with the same letter within columns are not significantly different according to the DMRT5 at a 5% level..
4
5Transformation efficiency = (GUS explants/total explants infected) × 100..
6DMRT: Duncan’s multiple range test..
Hyun A Jang, Setyo Dwi Utomo, Suk Yoon Kwon, Sun-Hwa Ha, Ye Xing-guo, and Pil Son Choi
J Plant Biotechnol 2016; 43(3): 341-346
Journal of
Plant Biotechnology