Abstract
Genetic engineering can add new capabilities or traits and direct method using biolistic particle delivery holds key for rapid, routine and efficient transformation of chickpea. Regeneration efficiencies of five different explants derived from BAP pre-treated breeders’ chickpea seeds (cv. DCP 92-2) raised in phytohormones combinations (BAP and KIN for shoot primordia induction; GA3 for shoot elongation and NAA for rooting) were compared. Best response was obtained using the embryonic axis (EAX) explants with 86.69% regeneration efficiency followed by epicotyl (EPI) explants (78.69%). Direct genetic transformation were demonstrated in two responding explants by bombarding with pre-treated tungsten, coated with plant expression cassette (harboring Bt and nptII gene) from a distance of 4 cm with 1100 psi helium pressure. Transgenic chickpea lines with multiple and single copy integrations were obtained with transformation frequency of 0.72% for EPI explants and 1.21% for EAX explants, significantly higher than Agrobacterium tumefaciens mediated transformation of same genotype (0.076%). Southern blot based analyses of seven single copy transgenic chickpea lines exhibited presence and transmission to subsequent generations (T1 and T2). Presence of Bt protein were detected in the leaves of transgenic chickpea lines at pre-flowering (6.63–11.95 ng/mg TSP) and post flowering stages (4.85–8.93 ng/mg TSP). Genetic fidelity analysis using genome wide SSR markers of ten independent transgenic lines indicated true to type with original genotype. Taken together, this study describes a protocol that can be adapted for direct genetic transformation of chickpea with high efficiency.
Key message
Routine protocol for direct transformation of grain legume, chickpea.
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Data availability
All data generated or analyzed during this study are included in this published article. Transgenic chickpea materials reported in the study are available in Seed Repository, ICAR-Indian Institute of Pulses Research, Kanpur, INDIA (https://iipr.icar.gov.in/).
Code availability
Not Applicable.
Abbreviations
- DT:
-
Direct transformation
- Bt :
-
Bacillus thuringiensis gene
- nptII :
-
Neomycin phosphotransferase II
- BAP:
-
6-Benzyl amino purine
- KIN:
-
Kinetin
- TDZ:
-
Thidiazuron
- 2iP:
-
2-Isopentenyladenine
- GA3 :
-
Gibberellic acid
- IAA:
-
Indole-3-acetic acid
- IBA:
-
Indole-3-butyric acid
- NAA:
-
1-Naphthalene acetic acid
- KNO3 :
-
Potassium nitrate
- SSR:
-
Simple sequence repeat
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Acknowledgements
The authors are thankful to ICAR-National Institute of Plant Biotechnology, New Delhi, for providing the plant expression vector harboring the Bt gene; Institutional Biosafety Committee (IBSC), IIPR, Kanpur for approving the activities for development of experimental transgenic chickpea lines in Plant Tissue Culture Facility and Transgenic Containment Facility (PBSL1), as per extant Indian Biosafety Regulations (Rules, 1989 and Regulations and Guidelines for Recombinant DNA Research and Bio-Containment, 2017). Thanks are due to Scientific Technical Staffs: Mr. Ajeet Pratap Singh, Mr. Malkhan Singh and Mr. Ravi Ranjan Singh, Division of Plant Biotechnology, ICAR-Indian Institute of Pulses Research, Kanpur, INDIA, for overall help in maintaining the experiments in Containment Facility, chemicals and seed inventory maintenance.
Funding
Research was partially supported by Network Project on Transgenics in Crops (NPTC), National Agricultural Science Fund (NASF) and Indian Council of Agricultural Research-Indian Institute of Pulses Research, Kanpur, India.
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AD, PGP, MR, OPV, NPS: Conception and designing of the experiments; PS, JA, AD, ST, MR: Standardization of in vitro regeneration parameters for particle bombardment of chickpea, JA, AS, AD, ST, NPS: Particle bombardment of chickpea with Bt gene, molecular analyses of transgenic lines, interpreted and compiled the results; NNT, PGP, AD, ST: Conducted genetic fidelity and interpreted the data related to the experiments. All authors compiled the text presented in the manuscript, proof-read and approved the final manuscript.
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The authors declare that there are no competing interests. ICAR-Indian Institute of Pulses Research, Kanpur is the sole owner and holds all rights pertaining to transgenic chickpea materials developed in the study.
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Communicated by Sergio J. Ochatt.
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11240_2022_2230_MOESM1_ESM.ppt
Supplementary Figure 1: Five different explants derived from mature chickpea seeds (DCP 92-3). Supplementary Figure 2: Map of plant expression vector harboring insecticidal Bt gene and selectable marker nptII. Supplementary Figure 3. Regeneration and establishment of EAX (a-d) and EPI (e-h) explant (a) Shoot primordial in SIM 3, EAX explants inset (b) Elongated multiple shoots on SEM 3 (c) Regenerated rooting system on RM 3 (d) Establishment of in-vitro regenerated plantlet (e) Shoot primordial in SIM 3, EPI explants inset (f) Elongated multiple shoots on SEM 3 (g) Regenerated rooting system on RM 3 (h) Establishment of in-vitro regenerated plantlet. Supplementary Figure 4: Original blot of genomic Southern blotting after single digestion (HindIII). Supplementary Figure 5: Original blot of genomic Southern blotting after double digestion (HindIII and EcoRI). Supplementary Figure 6: Original western blot. Supplementary file1 (PPT 2854 KB)
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Singh, P., Shukla, A., Tiwari, N.N. et al. Routine and efficient in vitro regeneration system amenable to biolistic particle delivery in chickpea (Cicer arietinum L.). Plant Cell Tiss Organ Cult 148, 699–711 (2022). https://doi.org/10.1007/s11240-022-02230-7
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DOI: https://doi.org/10.1007/s11240-022-02230-7