Background

Genetic transformation and expression of recombinant proteins in plant cells are major factors for plant genetic engineering. This is also a powerful tool for discovering novel genes and exploring genetically controlled traits. Agrobacterium tumefaciens-mediated transformation has been known for its unique ability to transfer a DNA segment from a specialized plasmid into a host plant cell (Gelvin, 2010). This feature allows efficient insertion of stable, unrearranged, single-copy DNA into plant genomes, which may lead to more stable expression than multiple gene copies or scrambled inserts (Iglesias et al., 1997). Since the initial reports in the early 1980s using Agrobacterium to generate transgenic plants, efforts were made to improve the tool in plant transformation. In most instances, major improvements involved alterations in plant tissue culture transformation and regeneration conditions rather than manipulation of host or bacterial genes. The first effective method for Agrobacterium-mediated transformation of japonica rice is now a common protocol in many laboratories (Chan et al., 1993; Hiei et al., 1994; Park et al., 1996). Many functional analyses, for example, PCR, GUS assay, and southern blot analysis, have confirmed the integration of foreign genes into transgenic rice plants obtained by Agrobacterium-mediated transformation (Dai et al., 2001); moreover, Mendelian inheritance of the transgenes was also reported (Pawlowski and Somers, 1996; Hiei et al., 1997).

The transfer of T-DNA and its integration into the rice genome is influenced by numerous factors, such as genotype, explants, Agrobacterium strains, plasmid vectors, addition of vir-gene inducing synthetic phenolic compounds, selection marker, and various conditions of tissue culture and regeneration. In tissue culture systems, selection of actively growing regenerable calli is a principal factor for efficient plant transformation. Likewise, optimization of culture conditions for co-cultivation of rice calli with Agrobacterium (Ozawa and Takaiwa, 2010) and suppression of Agrobacterium overgrowth may be applied to improve the transformation efficiency. Scientists have attempted to improve the plasmid constructions and rice transformation by Agrobacterium-mediated transformation to investigate the function of seed storage protein gene factors as well as cloning genomic candidate regions of more than 10 kb, which may have low restriction enzymes sites required for cloning. In particular, previous limitations of efficient promoter expression were overcome (Gupta et al., 2001; Furtado et al., 2008), especially for endosperm-specific expression (Zhou et al., 2013).

Several transformation methods have been established by many laboratories using mature japonica and indica seeds. Taichung 65 is a japonica rice variety, selected from a cross between the two Japanese varieties, Shinriki and Kameji, in 1923 (Iso, 1957). Taichung 65 is considered an important cultivar for studying rice genetics and breeding. Several point mutations were induced to the genetic stock of Taichung 65 using the methylnitrosourea (MNU) mutagen and have been maintained at the institute of plant genetic resources of Kyushu university. Taichung 65 has been extensively used as a model cultivar for rice biology, breeding research, and genomic studies initiated in many laboratories. However, transformation efficiency is still low in most indica and many japonica varieties due to the difficult regeneration; consequently, the procedure to obtain transgenic plants takes an average of 5 months (Nishimura et al., 2006). Thus, additional improvements in the transformation potentiality are still possible.

Here, we describe a protocol for Agrobacterium-mediated transformation in rice using mature embryos. This protocol is based on the method described by Toki (Toki, 1997), with several modifications; in particular, meropenem is used for plant regeneration (Ogawa and Mii, 2007). Meropenem exhibits the highest antibacterial activity and also achieves a high shoot formation rate in rice and tomato (Ogawa and Mii, 2007). Using this protocol, we succeeded to produce hundreds of independent transgenic lines and transformed several novel genes over the past decade, including Endosperm Storage Protein (ESP1), encoding a eukaryotic chain release factor1 (eRF1) (Elakhdar et al., 2019); Esp2, encoding the protein disulfate isomerase 1-1 (PDI 1-1) (Satoh-Cruz et al., 2010); Glup3, encoding a vacuole processing enzyme (VPE) (Kumamaru et al., 2010); Glup4, encoding the small GTPase Rab5a (Fukuda et al., 2011); and Glup6, encoding the guanine nucleotide exchange factor (GEF) (Fukuda et al., 2013).

In summary, this protocol is a step-by-step approach to obtain stably transformed plants from mature rice embryos by optimizing several stages of Agrobacterium transformation and standard regeneration medium components, including culture conditions, timing, and growth hormones. In this method, we use a 13.8 kb construct carrying the hygromycin phosphotransferase gene (hpt), a firefly luciferase reporter gene (LUC), spectinomycin (Sp), an attR1 site, the chloramphenicol resistance gene (Cm^R), the ccdB gene, the attR2 site, and the ZmUbi1 promoter (Figure 1 and Figure 2).

Figure 1. Flow chart for Agrobacterium-mediated transformation and regeneration of rice calli using mature embryogenic seed (cv; Taichung 65). (a) Seed embryos. (b) Calli pieces with granular structure, yellowish-white. (c) Growth of transformed Agrobacterium harboring the pSMAH638OX/Ubilp binary vector. (d) Co-cultivation of infected calli. (e) De-colonization of Agrobacterium tumefaciens. (f) Shoot regeneration. (g) Rooting in regenerated shoots. (h) Hardening of rooted plants. (i) Transplanted hardened plants.

Figure 2. Schematic showing embryo calli infection by Agrobacterium. (a) Growth of Agrobacterium-mediated harboring pSMAH638OX/Ubilp vector on LB medium. (b) Mix a small portion of the positive colonies. (c) Collect healthy calli for infection. (d) Calli infection. (e) Remove excess Agrobacterium suspension.