Factors influencing Agrobacterium mediated genetic transformation of kenaf

Abstract

As a first step in the development of a successful Agrobacterium tumefaciens mediated transformation method for kenaf, factors influencing the successful T-DNA integration and expression (as measured by the GUS expression) were investigated. Transformation was carried out using two kenaf cultivars and Agrobacterium strain EHA 105 carrying different vectors, plasmid pIG 121-Hm or pEC:gus. Pre-culturing the explants for 2 days in benzyl adenine containing medium, and wounding the explant before inoculation were found to enhance the transient GUS expression. Increasing the duration of pre-culture and co-culture period enhanced the transient GUS expression up to a threshold level. Increased transient GUS expression did not correlate with an increase in stable expression. Gene integration was confirmed by PCR analysis.Kenaf (Hibiscus cannabinus L.), an economically important industrial fiber crop, belongs to family Malvaceae. Success in in vitro manipulations of kenaf has been achieved very slowly. Banks et al. (1993) first reported the expression of trans genes in kenaf callus. However, shoot regeneration from kenaf callus has not been successful though direct shoot regeneration without a callus phase has been achieved (Srivatanakul et al., 2000;Herath et al., 2004). Srivatanakul et al. (2001) reported on the effect of Agrobacterium strain, temperature, and acetosyringone on T-DNA integration into kenaf shoot apices; no stable transformants were obtained. Hence, we further investigated the influence of some factors such as duration and cytokinin concentration during pre-culture and co-culture, and wounding the explant on successful transient and stable gene expression of kenaf.Since several factors had to be tested two independent experiments were carried out to determine the effect of the BA concentration during pre-culture and co-culture periods and to determine the effect of the duration of pre-culture and co-culture periods. Effect of wounding the explant was determined in a preliminary experiment and was carried out in all experiments. In experiment one, treatment factors were BA concentration during pre-culture (0, 4.4, and 8.8 lM) and coculture (8.8, 13.2, and 22 lM). Each of the 9 treatment combinations was replicated three times with at least 50 explants for each replicate. Number of plants inoculated differed for each treatment since some plants were damaged during wounding. In experiment 2, treatment factors were duration of pre-culture (0, 1, 2 and 4 days), and duration of coculture (2 and 3 days). All 8-treatment combinations were replicated three times with at least 60 explants for each replicate. This experiment was duplicated using the two vectors (pIG 121 (Hm) and pEC: GUS). Kenaf cultivar Tainung 2 (provided by the Oji Paper Company Ltd. Japan) was used for all experiments. Seed germination and shoot regeneration was carried out according to the previous published method (Herath et al., 2004) unless otherwise stated. After 7-10 days in the germination medium, the shoot tip of the seedling

Agrobacterium tumefaciens-mediated transformation of axillary bud callus of Hibiscus rosa-sinensis L. ‘Ruby’ and regeneration of transgenic plants

“…Procedures to produce transformed plants are usually empirically determined as the type of explant, and the ratio and type of PGR needed for successful transformation and regeneration, can vary considerably depending upon plant genotype. This has clearly been illustrated for kenaf (Herath et al 2005), Prunus avium (Feeney et al 2007), chickpea (Tripathi et al 2013) and Miscanthus (Hwang et al 2014). In Actinidia eriantha for example, BAP in combination with zeatin was critical for successful plant regeneration, including those of transformed plants (Wang et al 2006).…”

Agrobacterium-mediated genetic transformation of plants: The role of host

“…In barley, dicamba in the callus induction and maintenance media was generally promoting transient expression and subsequent stable transformation (Trifonova et al 2001). In kenaf, pre-culturing the explants for 2 d in benzyladenine (BA) containing medium, was found to enhance the transient GUS expression (Herath et al 2005).…”

Efficient callus induction and a temperature condition for flowering and seed setting in kenaf <i>Hibiscus cannabinus</i>

“…In addition to the agrobacterium infection method that is still incomplete for the production of the kenaf T 2 generation (Banks et al 1993;Herath et al 2005;Kojima et al 2004), a new gene transformation method with functional peptide has recently been developed in plants (Chuah and Numata 2018;Lakshmanan et al 2013;Miyamoto et al 2018). In combination with the transformation methods, our complete regeneration method would produce stable genetically transformed kenaf.…”