Hitesh V. Jasani ¹, Nimita G. Umretiya ², Manthan N. Kapuria ³, Darshan T. Dharajiya ¹, Tarun J. Khatrani ¹, Nalin K. Pagi ¹, Karen P. Pachchigar ¹, L. D. Parmar ¹

Affiliations
1 Department of Plant Molecular Biology and Biotechnology, C. P. College of Agriculture, Sardarkrushinagar Dantiwada Agricultural University, Sardarkrushinagar-385506, Gujarat, IN
2 Department of Biotechnology, Junagadh Agricultural University, Junagadh-362 001, Gujarat, IN
3 Shree P. M. Patel Institute of Integrated M. Sc. in Biotechnology, Anand 388001, Gujarat, IN

Abstract

Objectives: A protocol was developed for plant regeneration using cotyledonary node of high-yielding indigenous pigeonpea [Cajanus cajan (L.) Millsp.] genotype GT 101. Methods: Induction of multiple shoots directly was completed from cotyledonary node as an explants on Murashige and Skoog’s (MS) medium supplemented with various concentrations and combination of 6-benzylaminopurine (BAP), kinetin and α-naphthalene acetic acid (NAA). Elongation of multiplied shoots was performed on MS medium supplemented with different combination of BAP and GA3. These well elongated plantlets were further transferred on MS medium supplemented with various concentrations of indole-3-butyric acid (IBA) for ischolar_main induction. Regenerated plants were transferred to cocopeat:soil:vermiculite (2:1:2) for acclimation. Findings: The frequency of multiplication and number of multiple shoots from cotyledonary node explant were influenced on various types and concentrations of cytokinin. For multiple shoots induction, 3.0 mg/L BAP with 0.5 mg/L NAA was superior as compared to other combinations. The elongation of multiplied shoots was carried out on MS medium supplemented with 0.5 mg/L BAP and 0.5 mg/L GA3. The developed shoots were advanced to ischolar_maining on the medium supplemented with 0.5 mg/L IBA. They were subsequently grown in pots with 80% survival rate and these plants produced viable seeds. Improvement: The protocol for the production of in vitro multiple shoots with high frequency and their successive conversion to whole plants agreements potential for use in the improvement of protocol for development of transgenic in pigeonpea.

Keywords

Cajanus cajan (L.), Cotyledonary Node, GT101, In vitro regeneration, Pigeonpea.

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Darshan T. Dharajiya ¹, Satish M. Khadia ², Nalin K. Pagi ², Tarun J. Khatrani ¹, Hitesh V. Jasani ¹, Akshay D. Khunt ¹, Y. Ravindrababu ³

Affiliations
1 Department of Plant Molecular Biology and Biotechnology, C. P. College of Agriculture, Sardarkrushinagar Dantiwada Agricultural University, Sardarkrushinagar – 385506, Gujarat, IN
2 Department of Genetics and Plant Breeding, CPCA, SDAU, Sardarkrushinagar – 385506, Gujarat, IN
3 Pulses Research Station, SDAU, Sardarkrushinagar – 385506, Gujarat, IN

Abstract

Objectives: Mungbean is one among the foremost necessary pulse crops. It is necessary to extract good quality genomic DNA for molecular work in plant science. Hence, present study intended to establish a robust and cost effectively genomic DNA extraction method for mungbean. Methods/Statistical Analysis: The prevailing CTAB method for genomic DNA extraction from plant was altered by avoiding usage of liquid nitrogen or lyophilization. Genomic DNA of plant was extracted from leaves of seven mungbean genotypes by grinding in pre-heated CTAB extraction buffer. Increased concentration of CTAB, PVP, β-mercaptoethanol, use of RNase treatment and repetition of purification step with C:I after P:C:I treatment will suppress the interference of the contaminants. The effectiveness of DNA extraction protocol for PCR amplification was evaluated using SSR and RAPD markers. Findings: The quality of DNA was assured by the presence of intact bands of genomic DNA separated on 0.8% agarose gel. The yield of genomic DNA ranged from 871.6 to 1143.2 ng/μL with an average of 979.14 ng/μL. When measured spectrophotometrically, the A₂₆₀/A₂₈₀ ratio was in the range of 1.78 to 1.86, with an average of 1.82, indicating very low levels of contamination such as polysaccharides, phenolics and protein. The results of PCR amplification using SSR and RAPD markers indicated that the DNA extracted by this modified protocol was of decent quality and appropriate for various molecular studies. This protocol is suitable for underdeveloped countries, where resources for high throughput DNA isolation are not or less available. Application/Improvement: An effective, simple and cost-effective protocol for genomic DNA extraction with good yield as well as high purity was developed for mungbean which can be applied to other pulse crops also.

Keywords

Cetyl Trimethyl Ammonium Bromide (CTAB), Genomic DNA Extraction, Mungbean [Vigna radiata (L.) Wilczek], PCR Amplification.

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