Ovule Development and Transformation of Maudiae by -Mediated Ovary-Injection.

In this paper, the development of the Maudiae embryo sac at different developmental stages after pollination was assessed by confocal laser scanning microscopy. The mature seeds of Maudiae consisted of an exopleura and a spherical embryo, but without an endosperm, while the inner integument cells were absorbed by the developing embryo. The Maudiae embryo sac exhibited an type of development. The time taken for the embryo to develop to a mature sac was 45-50 days after pollination (DAP) and most mature embryo sacs had completed fertilization and formed zygotes by about 50-54 DAP. transformation was achieved by injection of the ovaries by , resulting in 38 protocorms or seedlings after several rounds of hygromycin selection, corresponding to 2, 7, 5, 1, 3, 4, 9, and 7 plantlets from -mediated ovary-injection at 30, 35, 42, 43, 45, 48, 50, and 53 DAP, respectively. Transformation efficiency was highest at 50 DAP (2.54%), followed by 2.48% at 53 DAP and 2.45% at 48 DAP. Four randomly selected hygromycin-resistant plants were GUS-positive after PCR analysis. Semi-quantitative PCR and quantitative real-time PCR analysis revealed the expression of the gene in the leaves of eight hygromycin-resistant seedlings following -mediated ovary-injection at 30, 35, 42, 43, 45, 48, 50, and 53 DAP, while expression was not detected in the control. The best time to inject Maudiae ovaries with is 48-53 DAP, which corresponds to the period of fertilization. This protocol represents the first genetic transformation protocol for any species and will allow for expanded molecular breeding programs to introduce useful and interesting genes that can expand its ornamental and horticulturally important characteristics.