In situ embryo rescue for generation of wide intra- and interspecific hybrids of Panicum virgatum L.

Wide crosses have been used for decades as a method for transferring novel genetic material and traits in plant breeding. Historically, many products of wide crosses require tedious and inefficient surgical embryo rescue prior to embryo abortion to recover single plantlets. We have utilized transgenic switchgrass (Panicum virgatum L. cv Alamo) as a pollen donor in conjunction with antibiotic or herbicide selection for recovery of intra-and interspecific F crosses by using developing ovules from the female parent and selecting for embryogenic cultures derived from the in situ immature embryo. Using this approach, several intravarietial crosses were generated between transgenic Alamo and the switchgrass varieties Kanlow, Blackwell and Cave-in-Rock as well as an interspecific cross with Atlantic coastal panicgrass. This procedure selected F embryogenic callus produced from the developing embryo contained within isolated immature ovules. Several clonal plants were successfully regenerated from each cross. Southern blot, PCR, phenotypic analyses and genomic analysis confirmed F hybrids. Using genotyping-by-sequencing shows the hybridization of the recovered plants by determining the ratio of transgressive markers to total compared markers between parents and their potential offspring. The ratio of transgressive markers to total compared markers was significantly lower between parents and their predicted offspring than between parents and offspring unrelated to them. This approach provides the possibility to move useful transgenes into varieties that are recalcitrant to direct transformation which can be optionally segregated thus useful to create new hybrids, as well as recovery of wide crosses that are either difficult or impossible using traditional techniques.