Epicotyl sections as targets for plant regeneration and transient transformation of common bean using Agrobacterium tumefaciens

Abstract

Common bean is recalcitrant to genetic transformation, due to limited regeneration capacity and low DNA transfer rates. The effect of different parameters on T-DNA transfer from Agrobacterium tumefaciens, was studied by measuring transient expression of the β-glucuronidase gene in Phaseolus vulgaris L. cv. CIAP7247F. Epicotyl containing seedling explants were inoculated with Agrobacterium EHA101 and C58C1Rif R (pMP90) strains harboring the binary vector pTJK136 with GusA gene on the T-DNA. Parameters studied were temperature and light regime during co-cultivation, explant injury, and the acetosyringone concentration for vir gene induction. The co-cultivation temperature and photoperiod had a significant effect on Agrobacterium DNA transfer. In addition, explant injury and supplementation of the cocultivation medium with acetosyringone increased the GUS activity. Optimal T-DNA transfer was obtained under the following conditions: co-cultivation at 25°C in darkness, injuring the explants with carborundum, and supplementation of the co-cultivation medium with 200 μM acetosyringone. This T-DNA delivery system was combined with a direct organogenesis protocol using epicotyl explants and fertile regenerants were recovered from tissue transformed with Agrobacterium. However, no transmission of transgenes to progeny could be observed, suggesting that the obtained plants were chimeras.

Agrobacterium tumefaciens-mediated transformation of common bean (Phaseolus vulgaris) var. Brunca

“…Bean EAs co-cultured for three days under 0.5 OD600nm and incubated at 25 ºC showed β-glucuronidase activity. This suggests a transient transformation of 45 %, which is similar to that recently reported by Collado et al (2016), (46 %) using the C58C1RifR strain and a higher co-culture time (five days). This transformation efficiency increased (80 %) with the EHA101 strain under the same conditions Morphologic traits and frequency of regenerants of beans co-cultured with LBA4404-ElectroMAX® pBindrd29A and selected with 50 mg/L kanamycin after more than 120 days ( Collado et al, 2016).…”

“…Three-days co-cultures favored the insertion of the marker gene (GUS or nptII), which agrees with the results reported by (Amugune et al, 2011). On the other hand, previous studies indicate long-term co-cultures of five days (Collado et al, 2016), six days (Collado et al, 2015), and eight days (Espinosa-Huerta et al, 2013;Mukeshimana et al, 2013) increase the temporary expression of the transgene (Zambre et al, 2003;Mukeshimana et al, 2013) especially in embryonic axes (Mukeshimana et al, 2013). The presence of BAP in the co-culture and regeneration media favored transformation and survival of the explants.…”

“…According to previous results (data not shown), the bacteria (LBA4404-ElectroMAX®) were used at an OD600nm of 0.5. Previous reports used similar conditions (Mukeshimana et al, 2013;Collado et al, 2015Collado et al, , 2016.…”

“…Regeneration of plants from transformed explants is difficult in genetic transformation protocols, and for recalcitrant species like common bean is one of the unsolved aspects (Liu, Park, Kanno, & Kameya, 2005;Amugune et al, 2011;Collado et al, 2015). Recently the green nodular compact calli were used as explants to obtain transformed plants, based on the indirect organogenesis regeneration pathway (Collado et al, 2015(Collado et al, , 2016).…”

A simple and efficient Agrobacterium-mediated in planta transformation protocol for horse gram (Macrotyloma uniflorum Lam. Verdc.)

“…There are several conventional breeding methods adopted to develop hybrid varieties with disease and pest resistance. Significant success was achieved in the transformation of forage and pasture legumes via Agrobacterium-mediated transformation, which is the most popular and efficient method of plant genetic engineering [16][17][18][19]. In this study, the "in planta" transformation method was studied, where Agrobacterium is used to infect the seedlings.…”