In vitro shoot grafting on rootstock: An effective tool for <i>Agrobacterium</i>-mediated transformation of pigeonpea (<i>Cajanus cajan</i> (L.) Millsp.)

Abstract

Lack of reproducible in vitro transformation method in pigeonpea limits the application of biotechnological breeding approaches for its genetic improvement. The present study describes a transformation method using novel in vitro shoot grafting technique for two cultivars ICPL87 and ICPL87119. Modified Murashige and Skoog (MS) medium with 1 mg l −1 6-benzylaminopurine and 0.2 mg l −1 α-naphthaleneacetic acid induced an average of 25 shoots from decapitated embryonic axis explants after six weeks of culture. These shoots were further elongated in a modified MS medium containing 0.5 mg l −1 6-benzylaminopurine along with 0.5 mg l −1 gibberellic acid for another four weeks. Grafting of pigeonpea shoots to seedling rootstock allowed 95% recovery of shoots. The whole regeneration process, starting from explant preparation to complete plant development, took 12-13 weeks. Further, the explants were infected with Agrobacterium tumefaciens harboring a binary vector pBI121. Transient and constitutive β-glucuronidase expressions were obtained in putative transgenic shoots selected at 100 mg l −1 kanamycin. The selected shoots were grafted on non-transgenic root stock to establish putative transformants. T 0 and T 1 transformants were confirmed through polymerase chain reaction for presence of neomycin phosphotransferase gene. An overall 9% of transformation efficiency was recorded in both cultivars.

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

“…Regenerated pigeonpea shoots showed enhanced elongation on MS medium containing 0.2 mg/L GA3 in our experiments. Likewise, other researchers have communicated results wherein, the additions of GA3 alone or in combination with BAP lead to efficient shoot regeneration as well as elongation [55,56,57].…”

In vitro grafting procedure for facilitating growth of separated shoots in castor

“…It may be because of the good activity of meristem region in the grafting junction, which is important to combine rootstocks with scions (Pina & Errea, 2005). However, a sterile Teflon ring is often used to support the combine zone of scion inserted into rootstock with the conventional method (Ghosh, Purohit, Ganguly, Chaudhuri, & Chakraborti, 2014). Therefore, our method might be more convenient and efficient than the traditional method.…”

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

“…The use of acetosyringone was not reported in the majority of the previous pigeon pea genetic transformation studies. 13,14,21,37,38 Another study reported the use of 150 µM acetosyringone to obtain an average transformation efficiency of 9% using embryonic axis as explants. 39 The results of the present study indicated that the use of 100 µM acetosyringone is ideal for high transformation efficiency.…”