Stable transformation of rice ( Oryza sativa L.) via microprojectile bombardment of highly regenerative, green tissues derived from mature seed

Abstract

A highly efficient and reproducible transformation system for rice ( Oryza sativa L. cv. Taipei 309) was developed using microprojectile bombardment of highly regenerative, green tissues. These tissues were induced from mature seeds on NB-based medium containing 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BAP) and high concentrations of cupric sulfate under dim light conditions; germinating shoots and roots were completely removed. Highly regenerative, green tissues were proliferated on the same medium and used as transformation targets. From 431 explants bombarded with transgenes [i.e. a hygromycin phosphotransferase ( hpt) gene plus one of a wheat thioredoxin h ( wtrxh), a barley NADP-thioredoxin reductase ( bntr), a maize Mutator transposable element ( mudrB) or beta-glucuronidase ( uidA; gus) gene], 28 independent transgenic events were obtained after an 8- to 12-week selection period, giving a 6.5% transformation frequency. Of the 28 independent events, 17 (61%) were regenerable. Co-transformation of the second introduced transgene was detected in 81% of the transgenic lines tested. Stable integration and expression of the foreign genes in T(0) plants and T(1) progeny were confirmed by DNA hybridization, western blot analyses and germination tests.

A highly efficient plant regeneration system through multiple shoot differentiation from commercial cultivars of barley (Hordeum vulgare L.) using meristematic shoot segments excised from germinated mature embryos

“…The differentiation of multiple shoot clumps from shoot tips was first described for maize (Zhong et al 1992), and the suitability of these cultures for transformation was demonstrated by Zhong et al (1996). In recent years protocols for establishing shoot meristematic cultures have been successfully developed and used for gene transfer in oat Cho et al 2003), barley (Zhang et al 1998(Zhang et al , 1999, pennisetum (Devi et al 2000;Goldman et al 2003) and rice (Cho et al 2004).…”

“…Therefore, a highly efficient regeneration system with either no callus phase or only a very minimum one is needed for the genetic transformation of present-day barley cultivars. Plant regeneration and the subsequent generation of transgenic plants have recently been obtained in different cereals using more easily available explants such as mature embryos (Torbert et al 1998), mature seeds (Cho et al 2004), leaf-base segments (Gless et al 1998;Chugh and Khurana 2003) or in vitro shoot meristematic cultures derived from germinated seedlings Zhang et al 1999;Goldman et al 2003).…”

Genotypic differences in callus induction and plant regeneration from mature embryos of barley (Hordeum vulgare L.)

“…In cereals, green plantlets can be successfully developed from a variety of explants, including embryos (Chauhan et al, 2007), mature embryos (Cho et al, 2004), leaves (Chugh and Khurana, 2003) or seedling segments (Becher et al, 1992), apical meristems (Goldman et al, 2003), coleoptiles (Sahrawat and Chand, 2004), and microspores (Jähne et al, 1994). For barley, morphogenic calli can be induced more frequently from immature embryos (Caligari et al, 1987;Bregitzer et al, 1998).…”

Morphogenic Regulators Baby boom and Wuschel Improve Monocot Transformation

“…Among reports of successful transformation of callus, the explants from which the callus was first derived include (1) immature tassels, immature ears, or anthers in maize (Cheng et al, 2004); (2) leaf bases from maize (Sidorov et al, 2006;Ahmadabadi et al, 2007); and (3) scutellum from mature seeds in rice (Chen et al, 1998;Dai et al, 2001). Alternatively, explants used to initiate proliferating meristem cultures for subsequent transformation have included maize apical or nodal meristems (Zhong et al, 1996;Zhang et al, 2002) or mature seeds in species such as rice (Cho et al, 2004), oat (Avena sativa; Cho et al, 1999), orchardgrass (Dactylis glomerata; Cho et al, 2000a), Kentucky bluegrass (Poa pratensis; Ha et al, 2000), and fescue (Festuca sp; Cho et al, 2000b). Regardless of the culture type, all of these reports have relied on the manipulation of exogenous hormones in the culture media to produce either embryogenic callus or multiple meristems for use as the transformation target.…”