Development of a rapid and highly efficient
            <i>Agrobacterium</i>
            -mediated transformation system for pigeon pea [
            <i>Cajanus cajan</i>
            (L.) Millsp]

Karmakar, Subhasis; Molla, Kutubuddin A.; Gayen, Dipak; Karmakar, Aritra; Das, Kaushik; Sarkar, Sailendra Nath; Datta, Karabi; Datta, Swapan K.

doi:10.1080/21645698.2019.1625653

Cited by 18 publications

(10 citation statements)

References 38 publications

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“…F. hindsii is a model species for citrus studies and has complete genome sequencing and assembly [ 18 ]. However, due to the limitation of genotype dependence, the transformation efficiency of F. hindsii is much lower than that of other crops, which increases the workload and further hinders research on citrus genetics and functional genomics [ 35 , 36 ]. In this study, we confirmed that the CRISPR/Cas9 system with pK7WG2D as the binary vector can efficiently edit the genome of F. hindsii , while the pMDC32 vector was not suitable for F. hindsii ( Figs 2 and 3 ).…”

Section: Discussionmentioning

confidence: 99%

An efficient CRISPR/Cas9 system for simultaneous editing two target sites in Fortunella hindsii

Zhang

et al. 2022

Horticulture Research

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The CRISPR/Cas9 system is a revolutionary genome editing technique, that has been widely used in numerous plants. For plants (e.g. citrus) with very low transformation efficiency, how to optimize gene editing efficiency and induce large fragment deletion has been the focus of research. Here, we reported that CRISPR/Cas9 induces efficient deletion of 16–673 bp fragments in the genome of Fortunella hindsii (F. hindsii). The ability of two binary vectors, pK7WG2D and pMDC32, to introduce specific mutations into the genome of F. hindsii was evaluated. Double sgRNAs were designed to achieve precise editing of two sites of a gene and deletion of fragments between two sites. The construction of vectors based on Golden Gate assembly and Gateway recombination cloning is simple and efficient. pK7WG2D is more suitable for F. hindsii genome editing than the pMDC32 vector. The editing efficiency using the pK7WG2D vector reached 66.7%. The allele mutation frequency was 7.14%–100%. The plants with 100% allele mutations accounted for 39.4% (13 100% allele mutation plants/33 mutants). The proportion of mutant plants with fragment deletion induced by this editing system was as high as 52.6% (10 fragment deletion mutants/19 FhNZZ mutants). Altogether, these data suggest that our CRISPR/Cas9 platform is capable of targeted genome editing in citrus and has broad application in research on the citrus functional genome and citrus molecular breeding.

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Section: Discussionmentioning

confidence: 99%

An efficient CRISPR/Cas9 system for simultaneous editing two target sites in Fortunella hindsii

Zhang

et al. 2022

Horticulture Research

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“…Karmakar et al reported the use of a non-tissue culture-based method to obtain the transformation efficiency of 83% from embryonic axis-attached cotyledons [60]. Low transformation frequencies from 13.7 % to 32% have been shown in T1 generation using embryogenic calli obtained from different explants via agrobacterium-mediated transformation [61,62,63].…”

Section: Discussionmentioning

confidence: 99%

An improved protocol for efficient regeneration, transformation and gene-editing using CRISPR/Cas9 in pigeonpea (Cajanus cajan(L.) Millsp.)

Verma¹,

Bharti²,

kaul³

et al. 2022

Preprint

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Background: Pigeonpea (Cajanus cajan (L.) Millsp.; 2n = 2× = 22) is a drought-tolerant perennial grain legume commonly cultivated in India's rain-fed and dry land zones, and it is a remarkable natural source of minerals and protein worldwide. Despite decades of research, the constraints linked to tissue culture continues to hinder the genetic improvement of the crop for instances; the explants inability to produce an embryogenic calli, direct shoot formation, and limited regeneration potential, as well as a lack of an effective transformation system. Thereby, efficient calli production, in-vitro recovery, and a systematic technique to co-delivery of Cas9 and sgRNA into the plant remain pre-requisites for efficient genome alteration. Results: To establish a CRISPR/Cas9 based genome editing platform in pigeonpea, a CRISPR/Cas9 mediated binary vector for coherent gene modification (for example, knockout, Homology Donor Repair) was designed. Calli production was achieved at a rate of over 70% by combining with a standardized robust genetic transformation method of pigeonpea using embryonic axis and cotyledonary node explants. The addition of tailored growth regulators and silver nitrate to shoot induction media boosted in-vitro regeneration by up to 89 %. Finally, as a hallmark, for biolistic-mediated transformation, an improved regeneration strategy was used, resulting in the successful incorporation of the transforming binary vector into the embryogenic calli with a transformation efficiency of 46%. PCR and Southern blot analyses were used to validate the results in the end.Conclusion: The standardized shoot regeneration method and successful transformation method of Cas9 in this research, made pioneer steps for facilitating the procedures for the improvement of this crop, which is as yet considered as recalcitrant. Furthering the protocol uncovers significant contributions in both effectiveness and convenience over existing pigeonpea transformation techniques, thereby enabling genetic research on other legumes/dicot crops achievable, as well.

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“…It was used as a selection marker for the production of recombinant proteins in the pea transient expression by agroinfection (Fan et al 2011). Its successful usage for Arobacterium-mediated transformation of pigeon peas has been described by Karmakar et al (2019). A summary of the selection and reporter genes used for the pea transformation in different studies is presented in Table 2.…”

Section: Selective and Reporter Genes Used For Pea Transformationmentioning

confidence: 99%

Pea transformation: History, current status and challenges

Ludvíková¹,

Griga²

2022

Czech J. Genet. Plant Breed.

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This review recapitulates the history, important milestones, the current status, and the perspectives of the pea (Pisum sativum L.) transformation as a tool for pea crop breeding. It summarises the developments of the pea transformation from the first methodological experiments to achieving the complete transformation and regeneration of genetically modified (GM) plants, transformation with the first genes of interest (GOI), to recent techniques of targeted genome editing. We show how recent biotechnological methods and genetic engineering may contribute to pea breeding in order to speed up the breeding process and for the creation of new pea cultivars. The focus is laid on genetic engineering which represents an excellent technology to enhance the pea gene pool with genes of interest which are not naturally present in the pea genome. Different methods of pea transformation are mentioned, as well as various GOI that have been used for pea transformation to date, all aimed at improving transgenic pea traits. Tolerance to herbicides or resistance to viruses, fungal pathogens, and insect pests belong, among others, to the pea traits that have already been modulated by methods of genetic engineering. The production of phytopharmaceuticals is also an important chapter in the use of genetically modified peas. We compare different methods of introducing transgenes to peas and also the usage of different selective and reporter genes. The transformation of other major legumes (soybeans, beans) is marginally mentioned. The effect of genetically modified (GM) peas on animal health (feeding tests, allergenicity) is summarised, the potential risks and benefits of pea modification are evaluated and also the prime expectations of GM peas and the real current state of this technology are compared. Unfortunately, this technology or, more precisely, the products created by this technology are under strict (albeit not scientifically-based) legislative control in the European Union.

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Development of a rapid and highly efficient Agrobacterium -mediated transformation system for pigeon pea [ Cajanus cajan (L.) Millsp]

Cited by 18 publications

References 38 publications

An efficient CRISPR/Cas9 system for simultaneous editing two target sites in Fortunella hindsii

An efficient CRISPR/Cas9 system for simultaneous editing two target sites in Fortunella hindsii

An improved protocol for efficient regeneration, transformation and gene-editing using CRISPR/Cas9 in pigeonpea (Cajanus cajan(L.) Millsp.)

Pea transformation: History, current status and challenges

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