The Competence of Maize Shoot Meristems for Integrative Transformation and Inherited Expression of Transgenes

Abstract

We have developed a nove1 and reproducible system for recovery of fertile transgenic maize (Zea mays L.) plants. The transformation was performed using microprojectile bombardment of cultured shoot apices of maize with a plasmid carrying two linked genes, the Strepfomyces bygroscopicus phosphinothricin acetyltransferase gene (bar) and the potato proteinase inhibitor II gene, either alone or in combination with another plasmid containing the 5' region of the rice actin 1 gene fused to the Escbericbia coli p-glucuronidase gene (gus). Bombarded shoot apices were subsequently multiplied and selected under 3 to 5 mg/L glufosinate ammonium. Co-transformation frequency was 100% (146/146) for linked genes and 80% (41/51) for unlinked genes. Co-expression frequency of the bar and gus genes was 5 7 % (29/51). The co-integration, co-inheritance, and co-expression of bar, the potato proteinase inhibitor II gene, and gus in transgenic R,, R,, and R, plants were confirmed. Localized expression of the actin 1-CUS protein in the R, and R, plants was extensively analyzed by histochemical and fluorometric assays.The shoot tip, or shoot apex, consists of the shoot apical meristem, a region in which lateral organ primordia form, a subapical region of cell enlargement, and severa1 leaf primordia (Steeves and Sussex, 1989). The meristem region contains apical initial cells and subepidermal cells from which the gametes are derived (Medford, 1992). Theoretically, there are two possibilities for recovering transgenic plants via transfer of DNA into the shoot apical meristem. One possibility is that transgenic progeny may be directly produced via transformation of the subepidermal germline cells followed by the development of a partially transgenic reproductive organ. In this case, the primary transformants will always be chimeric. An alternative possibility is to multiply transgenic apical meristem cells and/or germ-line cells, which can be reprogrammed in the developmental direction under in vitro conditions. Transgenic plants can be regenerated from these cells with or without selection. Our previous research on maize (Zea mays L.) morphogenesis demonstrated that the maize meristem is morphogenetically plastic and can be manipulated to produce multiple shoots, somatic embryos, tassels, or ears in a relatively genotype-independent manner by simple variation of in vitro culture conditions (Zhong et al., 1992a(Zhong et al., , 1992b. Based on this concept, we transformed maize meristems via microprojectile bombardment with a series of chimeric genes, including bar, pin2, and gus.In this paper, we report the efficient recovery of fertile transgenic maize plants via a shoot-multiplication system after microprojectile bombardment of shoot tips. Maize shoot apices were transformed with a plasmid incorporating bar driven by the CaMV 35s promoter and pin2 with the wound-inducible pin2 promoter (Fig. l), either alone or in combination with another plasmid containing gus driven by the 5' region of Actl (Fig. 1). The co-integration and co-inherita...

Morphogenic Regulators Baby boom and Wuschel Improve Monocot Transformation

“…However, to date, the closest researchers have come to directly using mature seed tissues for transformation in monocots has been a report in which a brief preculture period (1 to 5 d) was used before Agrobacterium-mediated DNA delivery in japonica rice (Toki et al, 2006). The remaining reports of "mature seed transformation" in monocots have all progressed through an intermediate culture step, using either embryogenic callus (Hiei et al, 1997;Chen et al, 1998;Dai et al, 2001;Sidorov et al, 2006;Ahmadabadi et al, 2007), organogenic callus (O' Connor-Sánchez et al, 2002) or proliferating meristem cultures (Zhong et al, 1996;Zhang et al, 2002), and finally using the proliferating cultured cells as the transformation target. Here, using the transcription factors Bbm and Wus2, we demonstrated that cells in the exposed embryo axis of mature maize seed can reproducibly produce transgenic callus and regenerate healthy, fertile T0 plants.…”

“…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.…”

Competence of oat (Avena sativa L.) shoot apical meristems for integrative transformation, inherited expression, and osmotic tolerance of transgenic lines containing hva1

“…Samples were immersed in GUS substrate mixture and incubated at 37°C as described elsewhere (Jafferson et al 1987). Tissues were prepared using free-hand cross sections and were examined under a Zeiss SV8 stereomicroscope according to Zhong et al (1996).…”

Resourceful and High Efficiency Agrobacterium Mediated Transformation of Maize (Zea mays L.) Using Coleoptilar Nodal Explants

“…By using above optimized parameters an average transformation frequency of 3.25% was achieved. Comparatively, explants derived from the mature embryos (Cao et al, 2014;Huang and Wei, 2005;Zhong et al, 1996) yielded less frequencies of transformation than the immature embryos (Ishida et al, 1996). All these conditions of different parameters would help us in developing a congenial high throughput method of Agrobacterium mediated genetic transformation in maize.…”