Production of marker-free transgenic rice expressing tissue-specific Bt gene

Rice blast is one of the most serious diseases of rice and a major threat to rice production. Breeding disease-resistant rice is one of the most economical, safe, and effective measures for the control of rice blast. As a complement to traditional crop breeding, the transgenic method can avoid the time-consuming process of crosses and multi-generation selection. In this study, maize (Zea mays) Activator (Ac)/Dissociation (Ds) transposon vectors carrying green fluorescent protein (GFP) and red fluorescent protein (mCherry) genetic markers were used for generating marker-free transgenic rice. Double fluorescent protein-aided counterselection against the presence of T-DNA was performed together with polymerase chain reaction (PCR)-based positive selection for the gene of interest (GOI) to screen marker-free progeny. We cloned an RNAi expression cassette of the rice Pi21 gene that negatively regulates resistance to rice blast as a GOI into the Ds element in the Ac/Ds vector and obtained marker-free T1 rice plants from 13 independent transgenic lines. Marker-free and Ds/GOI-homozygous rice lines were verified by PCR and Southern hybridization analysis to be completely free of transgenic markers and T-DNA sequences. qRT-PCR analysis and rice blast disease inoculation confirmed that the marker-free transgenic rice lines exhibited decreased Pi21 expression levels and increased resistance to rice blast. TAIL-PCR results showed that the Ds (Pi21-RNAi) transgenes in two rice lines were reintegrated in intergenic regions in the rice genome. The Ac/Ds vector with dual fluorescent protein markers offers more reliable screening of marker-free transgenic progeny and can be utilized in the transgenic breeding of rice disease resistance and other agronomic traits.

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Inheritance of NmDef02 synthetic defensin gene and hpt selectable marker in four successive generations of transgenic rice (Oryza sativa, cv. J-104)

International Journal of Current Research in Biosciences and Plant Biology ◽

10.20546/ijcrbp.2021.809.005 ◽

2021 ◽

Vol 8 (9) ◽

pp. 34-41

Author(s):

Maylin Pérez-Bernal ◽

Magali Delgado ◽

Daymí Abreu ◽

Onel Valdivia ◽

Raúl Armas

Keyword(s):

Transgenic Rice ◽

Selectable Marker ◽

Marker Gene ◽

Rice Cultivar ◽

Future Research ◽

Maturation Stage ◽

Defensin Gene ◽

Regenerated Plants ◽

Marker Free ◽

Successive Generations

The evaluation of the inheritance and stability of the transgenes is essential for the application of transgenic plants in agriculture. In this work, we studied the inheritance of two transgenes in T1, T2, T3 and T4 rice progenies. Transgenic rice plants (cv. J-104) was obtained by biolistic method using a synthetic defensin gene (NmDef02) and hpt as selectable marker gene, co-transformed in a 4:1 proportion, respectively. Regenerated plants were acclimated under natural conditions. The study started from the primary transformants that fulfilled the agronomic characters reported by the experts for the J-104 rice cultivar in the maturation stage. The integration and relative expression of NmDef02 in T1 plants was verified by Southern blot and qRT-PCR, respectively. The inheritance of transgenes over four generations was analyzed by PCR. The following transgene combinations were identified: NmDef02(+)hpt(+), NmDef02(+)hpt(-) and NmDef02(-)hpt(-). The most advantageous combination was NmDef02(+)hpt(-), which corresponded to the marker-free plants harboring the gene of interest. The inheritance of NmDef02 was confirmed in T1 and T2 progenies, but in some T3 and T4 lines the loss of this gene was verified. This inheritance analysis provided information concerning the transgenic population, but the mechanisms that destabilize the inheritance of the gene of interest will be the goal of future research.

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