Efficient genetic transformation of Jatropha curcas L. by means of vacuum infiltration combined with filter-paper wicks

Abstract

Jatropha curcas L. (Jatropha) is a promising plant for the production of biodiesel in arid regions. To improve its productivity, it is important to understand the molecular functions of key genes in Jatropha, and to seek ways to modify important agronomic traits of Jatropha via molecular breeding. However, Jatropha is notorious for its recalcitrance to transformation. Thus, an improved protocol for efficient and reproducible Agrobacterium tumefaciens-mediated transformation is essential for molecular biology research and breeding in this species. Because resistance of Jatropha to A. tumefaciens infection is one of the key factors limiting transformation efficiency, we attempted to improve infection efficiency by comparing various co-culture conditions with the use of a β-glucuronidase (GUS) assay. The LBA4404 strain of A. tumefaciens was more efficient than the EHA105 strain for transformation of Jatropha. Vacuum infiltration of an A. tumefaciens suspension with Jatropha explants, combined with co-cultivation on filter-paper wicks moistened with coculture medium instead of on solid medium, significantly improved transformation efficiency. In these conditions, GUSpositive sectors were observed in 93.0±23.6% of infected explants. Moreover, a variant of the yellow fluorescent protein gene, Venus, was used as a visible marker, which proved effective for discrimination of candidate transgenic shoots from escape shoots. Transgene integration was confirmed by means of polymerase chain reaction and Southern hybridization analyses. Transgenic shoots were regenerated from 23% of explants from the vacuum-filter-paper treatment, which is sufficient for practical use.

Adenine sulfate and glutamine enhanced shoots multiplication in Jatropha curcas L. a potent biofuel plant

“…The results showed that BA and IBA affected the shoot with 0.5 mgL -1 TDZ, and the combination of 1.5 mgL -1 BA and 0.05 mgL -1 IBA was the most effective in regenerating shoots from calli (3.82 ± 0.18 shoots/explant) (Table 1 and Figure 1c, d). This finding was similar to previous results reported by Chiangmai et al (2015), Nanasato et al (2015) and Fufa et al (2019). The study was divergent from previous study in our lab by Khemkladngoen et al (2011) which showed that the combination of 3 mgL -1 BA and 0.1 mgL -1 IBA produced the highest regeneration frequency from calli due to genotype difference.…”

Differential physiological responses and tolerance to potentially toxic elements in biodiesel tree Jatropha curcas

“…Seeds of Jatropha ( Jatropha curcas L.) variety IP-3P obtained from The Plantation Research and Development Center (Pakuwon), Ministry of Agriculture, Indonesia were used in this study 45 . Jatropha seeds were planted in a calcined granular diatomaceous earth (Isolite CG No.…”

Agrobacterium-mediated transformation of Jatropha curcas leaf explants with a fungal chitinase gene

“…To date, plant regeneration protocols have been developed using various types of explants (Wei et al, 2004;Jha et al, 2007;Deore and Johnson, 2008;Sharma et al, 2011;Toppo et al, 2012;Franco et al, 2014). Based on these regeneration systems, stable transformation procedures have been developed employing both Agrobacterium tumefaciens (Li et al, 2008;Kumar et al, 2011;Pan et al, 2010;Misra et al, 2012;Fu et al, 2015;Nanasato et al, 2015) or particle bombardment (Joshi et al, 2011;Purkayastha et al, 2010).…”

“…Li et al (2008) first reported J. curcas genetic transformation via A. tumefaciens infection of cotyledon disc using the phosphinothricin herbicide as selective agent, however, the transformation efficiency was low (13%). Recently, Nanasato et al (2015) improved the rate of cotyledon transformation efficiency to 23.3% using vacuum infiltration for the step of bacterial inoculation and co-culture on filter-paper wicks instead of agar for the step of latency before shoot regeneration. Fu et al (2015) further increased the rate of transformation efficiency to 56% by optimizing kanamycin concentration in the selective medium and the duration of the selection process.…”