Doubled haploid plants are invaluable breeding tools but many crop species are recalcitrant to available haploid induction techniques. To test if haploid inducer lines can be engineered into crops, CENH3 −/− and CENH3:RNAi lines were complemented by AcGREEN-tailswap-CENH3 or AcGREEN-CENH3 transgenes. Haploid induction rates were determined following testcrosses to wild-type plants after independently controlling for inducer parent sex and transgene zygosity. CENH3 fusion proteins were localized to centromeres and did not cause vegetative defects or male sterility. CENH3:RNAi lines did not demonstrate consistent knockdown and rarely produced haploids. In contrast, many of the complemented CENH3 −/− lines produced haploids at low frequencies. The rate of gynogenic haploid induction reached a maximum of 3.6% in several hemizygous individuals when backcrossed as males. These results demonstrate that CENH3-tailswap transgenes can be used to engineer in vivo haploid induction systems into maize plants.

Maize is an important food and feed crop in many countries. It is also one of the most important target crops for the application of biotechnology. Currently, there are more biotech traits available on the market in maize than in any other crop. Generation of transgenic events is a crucial step in the development of biotech traits. For commercial applications, a high throughput transformation system producing a large number of high quality events in an elite genetic background is highly desirable. There has been tremendous progress in Agrobacterium-mediated maize transformation since the publication of the Ishida et al. (1996) paper and the technology has been widely adopted for transgenic event production by many labs around the world. We will review general efforts in establishing efficient maize transformation technologies useful for transgenic event production in trait research and development. The review will also discuss transformation systems used for generating commercial maize trait events currently on the market. As the number of traits is increasing steadily and two or more modes of action are used to control key pests, new tools are needed to efficiently transform vectors containing multiple trait genes. We will review general guidelines for assembling binary vectors for commercial transformation. Approaches to increase transformation efficiency and gene expression of large gene stack vectors will be discussed. Finally, recent studies of targeted genome modification and transgene insertion using different site-directed nuclease technologies will be reviewed.

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...

Agrobacterium tumefaciens caused tissue browning leading to subsequent cell death in plant transformation and novel anti-oxidative compounds enhanced Agrobacterium -mediated plant transformation by mitigating oxidative stress. Browning and death of cells transformed with Agrobacterium tumefaciens is a long-standing and high impact problem in plant transformation and the agricultural biotechnology industry, severely limiting the production of transgenic plants. Using our tomato cv. MicroTom transformation system, we demonstrated that Agrobacterium caused tissue browning (TB) leading to subsequent cell death by our correlation study. Without an antioxidant (lipoic acid, LA) TB was severe and associated with high levels of GUS transient expression and low stable transformation frequency (STF). LA addition shifted the curve in that most TB was intermediate and associated with the highest levels of GUS transient expression and STF. We evaluated 18 novel anti-oxidative compounds for their potential to enhance Agrobacterium-mediated transformation, by screening for TB reduction and monitoring GUS transient expression. Promising compounds were further evaluated for their effect on MicroTom and soybean STF. Among twelve non-antioxidant compounds, seven and five significantly (P < 0.05) reduced TB and increased STF, respectively. Among six antioxidants four of them significantly reduced TB and five of them significantly increased STF. The most efficient compound found to increase STF was melatonin (MEL, an antioxidant). Optimal concentrations and stages to use MEL in transformation were determined, and Southern blot analysis showed that T-DNA integration was not affected by MEL. The ability of diverse compounds with different anti-oxidative mechanisms can reduce Agrobacterium-mediated TB and increase STF, strongly supporting that oxidative stress is an important limiting factor in Agrobacterium-mediated transformation and the limiting factor can be controlled by these compounds at different levels.

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