A leaf-based regeneration and transformation system for maize (Zea mays L.)

Abstract

Efficient methods for in vitro propagation, regeneration, and transformation of plants are of pivotal importance to both basic and applied research. While being the world's major food crops, cereals are among the most difficult-to-handle plants in tissue culture which severely limits genetic engineering approaches. In maize, immature zygotic embryos provide the predominantly used material for establishing regeneration-competent cell or callus cultures for genetic transformation experiments. The procedures involved are demanding, laborious and time consuming and depend on greenhouse facilities. We have developed a novel tissue culture and plant regeneration system that uses maize leaf tissue and thus is independent of zygotic embryos and greenhouse facilities. We report here: (i) a protocol for the efficient induction of regeneration-competent callus from maize leaves in the dark, (ii) a protocol for inducing highly regenerable callus in the light, and (iii) the use of leaf-derived callus for the generation of stably transformed maize plants.

Morphogenic Regulators Baby boom and Wuschel Improve Monocot Transformation

“…When starting with leaf-base explants, successful reports of transformation in maize have all relied on an intermediate callus-culture step to produce the transformable explant. For example, leaf bases in germinating maize seedlings have been used to produce callus for later transformation (Sidorov et al, 2006;Ahmadabadi et al, 2007). Importantly, we demonstrated here that mesophyll cells from maize seedlings can be directly transformed using Agrobacterium-mediated delivery of Bbm/ Wus2 and that those cells divide and appear to be the source of the embryogenic, regenerable callus produced in our experiments.…”

“…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.…”

“…There have been no previous reports of directly transforming differentiated cells in explants such as mature maize embryos or leaf segments and directly stimulating dedifferentiation and subsequent callus formation. 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).…”

Improving in vitro leaf disk regeneration system of sugarcane (Saccharum officinarum L.) with concurrent shoot/root induction from somatic embryos

“…Agrobacterium tumefaciens-mediated gene transformation has been established both for dicot (Block et al, 1984) and monocot (Hiei, 1994) plants and is hence being used for different crops such as wheat (Jones, 2005), maize (Ahmadabadi, 2007), rice (Hiei et al, 1994), and sugarcane (Enriquez et al, 2000). Poor regeneration of transformants is still a major barrier in crop improvement through genetic engineering (Popelka and Altpeter, 2003).…”

Callus induction and plant regeneration from immature zygotic embryos of various maize genotypes (Zea mays L.)

“…옥수수 미성 숙 배로부터 식물체 재분화가 Green and Philips (1975)에 의 해 처음 보고된 이후로 다양한 품종의 미성숙 배로부터 식 물체 재분화 연구가 지속적으로 보고되었다 (Armstrong and Green 1985;Duncan et al 1985;Songstad et al 1988;Bohorova et al 1995;Carvalho et al 1997;El-Itriby et al 2003;Wang et al 2007;Binott et al 2008;Ombori et al 2008;Rakshit et al 2010;Anami et al 2010;De-yi et al 2011;González et al 2012;Ali et al 2014). 미성숙 배 외에도 성숙 배 (Wang 1987;Huang and Wei 2004;Al-Abed et al 2006;Abebe et al 2008;Jia et al 2008;Zhao et al 2008), 중배축(mesocotyl) 조직 (Torne et al 1980), 약 (Ting et al 1981), 유묘의 마디부분 (Santos et al 1984;Sidorov et al 2006), 잎 (Conger et al 1987;Ahmadabadi et al 2007), 영 (glume) (Suprasanna et al 1986), 화서(미성숙 tassel과 ears) 절 단 조직 (Pareddy and Petolino 1990;Songstad et al 1992), shoot-tip 분열 조직 (Li et al 2004)에서 캘러스 형성 및 식물체 재분화가 이루어졌다. 이렇게 옥수수의 여러 다양한 조직 으로부터 식물체 재분화가 이루어졌으나 지금까지 미성숙 배를 사용하였을 때 재분화 효율이 가장 높은 것으로 알려 져 왔다.…”