Improved protocols for transformation of indica rice mediated by Agrobacterium tumefaciens

Abstract

A highly efficient gene transfer method mediated by Agrobacterium tumefaciens was developed for Group I indica rice, which had been quite recalcitrant in tissue culture and transformation. Freshly isolated immature embryos from plants grown in a greenhouse were inoculated with A. tumefaciens LBA4404 that harbored super-binary vector pTOK233 or pSB134, which had a hygromycin-resistance gene and a GUS gene in the T-DNA. The efficiency of gene transfer varied with the kinds of gelling agents and the basic compositions of co-cultivation media. The highest activity of GUS after co-cultivation was observed when NB medium solidified with agarose was used. For the subsequent cultures, two types of media (modified NB and CC) were chosen to recover hygromycin-resistant cells efficiently. The transformation protocol thus developed worked very well in all of the varieties tested in this study, and the transformation frequency (number of independent hygromycin-resistant and GUS-positive plants per embryo) reached more than 30% in IR8, IR24, IR26, IR36, IR54, IR64, IR72, Xin Qing Ai 1, Nan Jin 11, and Suewon 258. Most of the transformants (T 0 ) were normal in morphology and fertile. Stable integration, expression and inheritance of transgenes were demonstrated by molecular and genetic analysis of transformants in the T 0 and T 1 generations. For the recovery of multiple independent transgenic events from a single immature embryo, procedures were developed to section the embryo into as many as 30 pieces after non-selective cultures following co-cultivation. Transformants were then obtained from the pieces cultured on the selective media, and, in the highest case, more than seven independent transgenic plants per original embryo (transformation frequency of 738%) were produced. Thus, the efficiency of transformation was remarkably improved.

NAL1 allele from a rice landrace greatly increases yield in modern indica cultivars

“…The fragment was ligated into the binary vector pCAMBIA1300int-prUbi1-tNOS (provided by Emmanuel Guiderdoni, Centre de Coopération Internationale en Recherche Agronomique pour le Développement, Montpellier, France) between the maize ubiquitin promoter and the nopaline synthase terminator to generate the overexpression vector. Using Agrobacterium-mediated transformation, we introduced the vector into IR64 (42). The regenerated plants were evaluated for transgene copy numbers by Southern blot analysis.…”

“…The amplified fragment was ligated into the binary vector pCAMBIA0380 (Cambia) upstream of the GUS reporter gene. This vector was introduced into IR64 by Agrobacterium-mediated transformation (42). Organs of the regenerated plants were sampled to analyze GUS activity as described (44).…”

Overexpression of OsNAC6 transcription factor from Indonesia rice cultivar enhances drought and salt tolerance

“…Subsequently, they were sub-cultured onto the NBM primary selection medium containing hygromycin (30 mg 1 -1 ) and cefotaxime (250 mg l -1 ) for primary selection and maintained for 3 weeks. Hygromycin-resistant callus lines developed from the rice scutella were isolated and cultured onto NBM primary selection medium for another 10 days and ones clearly hygromycin resistant were transferred onto NBPR medium consisted of N6 major salts, B5 minor salts and vitamins, vitamin assay casamino acids (0.5 g l -1 ), L-proline (0.5 g l ), pH 5.8, supplemented with hygromycin (30 mg l -1 ) and cefotaxime (250 mg l -1 ) as described by Hiei and Komari (2006).…”

Protocol for Artificial MicroRNA Mediated Over-Expression of miR820 in Indica Rice

“…Rice transformation is a challenging task as it is comparatively less amenable to genetic manipulation, especially the indica cultivars [3]. Several methods have been reported for indica rice transformation, but are still unpredictable in terms of transformation efficiency and regenerative capacity of the explants [4]- [7]. This is mainly due to their dependence on the genotype of the plant, age of the callus, type of gene introduced into the genome etc.…”