Development of transgenic pigeonpea using high throughput plumular meristem transformation method

Ganguly, Shreeparna; Ghosh, Gourab; Purohit, Arnab; Chaudhuri, Rituparna Kundu; Chakraborti, Dipankar

doi:10.1007/s11240-018-1444-3

Cited by 16 publications

(6 citation statements)

References 25 publications

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“…Interestingly out of 465 short plants 6 plants were found as PCR positive. Similarly Ganguly et al (2018) [5] used 100 ppm kanamycin concentration to check segregation pattern of T1 seeds and observed 12 Cry1Ac and 15 Cry2Aa T1 events exhibit segregation of 3:1 ratio (KanR : KanS). Singh et al (2018) [10] used 70 ppm kanamycin concentration for quartz sand-based screening of a large number of T1 generation seedlings, resulted in the identification of 59 cry2Aa putative transformants.…”

Section: Resultsmentioning

confidence: 99%

Optimization of kanamycin concentration for screening of primary transformants in pigeonpea

Baburao¹,

Bhat²

2020

J Pharmacogn Phytochem

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Pigeonpea is an important pulse crop which is widely cultivated throughout the tropical and sub-tropical regions. It is the major source of protein for the people with vegetarian diet. Major constraint in pigeonpea is the attack by pod borer. Conventional plant breeding techniques have limitations to develop resistance to the pest due to non-availability of resistance in the available germplasm. Therefore genetic engineering tools can be employed to transfer foreign genes into pigeonpea to impart pod borer resistance. Development of a reliable regeneration system coupled with efficient transformation has been a major limitation in developing transgenic pigeonpea with useful genes. In planta is an alternative approach to obtain transformed plants in short duration without involving tissue culture regeneration. However major limitations with this approach is screen more number of seeds to identify putative transformants which is time consuming and expensive. Therefore the present investigation was carried out to optimize the concentration of kanamycin for quick identification of putative transformants carrying cry1Ac and npt II using sand based assay. Seeds of Asha and BSMR736 were soaked in different concentrations of kanamycin (10-100, 200, 300, 400 and 500 ppm) for 5hrs and later sown in sand based medium. There was no effect of kanamycin on seedling growth up to 60 ppm and decrease in survival percentage in both the genotypes beyond this concentration. There was significant differences in growth of the seedlings in terms of shoot and root length at 100 ppm and beyond. Therefore this concentration was used to screen seeds of 32 primary transformants carrying cry1Ac and npt II and subjected to gene specific PCR analysis to confirm the usability of simple screening on kanamycin based sand medium for the identification of true transgenic plants. However some discrepancies between the growth inhibition and PCR results were observed.

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Section: Resultsmentioning

confidence: 99%

Optimization of kanamycin concentration for screening of primary transformants in pigeonpea

Baburao¹,

Bhat²

2020

J Pharmacogn Phytochem

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“…Much lower transformation efficiency was reported earlier. 8,9,12,17,35,36 In this study, we used four different confirmation methods to identify positive transgenic plants; PCR amplification of inserted genes (Fig. 3(c-f)), Southern hybridization (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…The transformation efficiency observed in our study (83%) was the highest among the pigeon pea genetic transformation studies done to date. 17,35,36 The use of tobacco leaf extract in Agrobacterium suspension to introduce vir gene was reported in an in-planta pigeon pea transformation method. 18 An earlier report showed that the use of wounded plant tissues, present in an active metabolic state, secrete vir-inducing signaling molecules into the surrounding culture medium.…”

Section: Discussionmentioning

confidence: 99%

“…Agrobacterium-mediated genetic transformation and ultimately plant regeneration of pigeon pea has been demonstrated via embryonic axis or cotyledonary node, 6,[12][13][14][15] embryonic axis with single cotyledon, 16 plumular and intercotyledonary meristem of the seedling axis. 17,18 However, the efficiency of transformation of these protocols is quite low, and they are also time-consuming. The success of Agrobacteriummediated transformation depends on lot of factors such as wound-induced signal transduction in the meristematic tissue of explants, media components, delivery of T-DNA, and its integration into the plant genome, precise selection of transformed cells, and characterization of transgenic plants.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2019

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An efficient genetic transformation system is a prerequisite for studying gene functions, molecular breeding program, and introducing new traits. Agrobacterium tumefaciens-mediated genetic transformation is a widely preferred and accepted method for many plants, including pigeon pea. However, the efficiency of transformation of pigeon pea using the existing protocols is low and time-consuming. In the present study, we developed a rapid and highly efficient transformation system of pigeon pea, using embryonic axis-attached cotyledons as explants. We systematically investigated the influence of varying optical densities of Agrobacterium suspension, duration of incubation, and co-cultivation on the transformation efficiency. In our system, a transformation efficiency of approximately 83% was achieved using Agrobacterium cells at an optical density (OD 600 ) of 0.25, infection time of 15 min, and co-culturing with explants for 72 h in the light with 100µM acetosyringone. The entire procedure, starting from seed to establishment of transformed plants in soil, was achieved in 35-40 days. This is a rapid and highly efficient protocol for

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“…Amongst the different protocols using direct de novo shoot organogenesis, the plumular meristem strategy was proposed as a time-efficient direct regenerationbased transformation approach with high transformation rates for chickpea [22,146] and pigeon pea [147,148]. In this system, three-day-old seedlings are decapitated at the shoot apex and pricked in the apical portion and cotyledonary nodes [22,147] (Fig. 6b).…”

Section: Plumular Meristem Strategymentioning

confidence: 99%

A comprehensive review of in planta stable transformation strategies

Bélanger,

Copley,

Hoyos-Villegas

et al. 2024

Plant Methods

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Plant transformation remains a major bottleneck to the improvement of plant science, both on fundamental and practical levels. The recalcitrant nature of most commercial and minor crops to genetic transformation slows scientific progress for a large range of crops that are essential for food security on a global scale. Over the years, novel stable transformation strategies loosely grouped under the term “in planta” have been proposed and validated in a large number of model (e.g. Arabidopsis and rice), major (e.g. wheat and soybean) and minor (e.g. chickpea and lablab bean) species. The in planta approach is revolutionary as it is considered genotype-independent, technically simple (i.e. devoid of or with minimal tissue culture steps), affordable, and easy to implement in a broad range of experimental settings. In this article, we reviewed and categorized over 300 research articles, patents, theses, and videos demonstrating the applicability of different in planta transformation strategies in 105 different genera across 139 plant species. To support this review process, we propose a classification system for the in planta techniques based on five categories and a new nomenclature for more than 30 different in planta techniques. In complement to this, we clarified some grey areas regarding the in planta conceptual framework and provided insights regarding the past, current, and future scientific impacts of these techniques. To support the diffusion of this concept across the community, this review article will serve as an introductory point for an online compendium about in planta transformation strategies that will be available to all scientists. By expanding our knowledge about in planta transformation, we can find innovative approaches to unlock the full potential of plants, support the growth of scientific knowledge, and stimulate an equitable development of plant research in all countries and institutions.

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Development of transgenic pigeonpea using high throughput plumular meristem transformation method

Cited by 16 publications

References 25 publications

Optimization of kanamycin concentration for screening of primary transformants in pigeonpea

Optimization of kanamycin concentration for screening of primary transformants in pigeonpea

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

A comprehensive review of in planta stable transformation strategies

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