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 F1 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 F1 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 F1 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.

Inside out: high-efficiency plant regeneration and Agrobacterium-mediated transformation of upland and lowland switchgrass cultivars.

KEY MESSAGE: Selection of pre-embryogenic callus from a core structure from mature seed-derived callus is the key for high-efficiency plant regeneration and transformation of switchgrass different cultivars. Switchgrass (Panicum virgatum L.) has been identified as a dedicated biofuel crop. For its trait improvement through biotechnological approaches, we have developed a highly efficient plant regeneration and genetic transformation protocol for both lowland and upland cultivars. We identified and separated a pre-embryogenic "core" structure from the seed-derived callus, which often leads to development of highly regenerative type II calluses. From the type II callus, plant regeneration rate of lowland cultivars Alamo and Performer reaches 95%, and upland cultivars Blackwell and Dacotah, 50 and 76%, respectively. The type II callus was also amenable for Agrobacterium-mediated transformation. Transformation efficiency of 72.8% was achieved for lowland cultivar Alamo, and 8.0% for upland cultivar Dacotah. PCR, Southern blot and GUS staining assays were performed to verify the transgenic events. High regenerative callus lines could be established in 3 months, and transgenic plants could be obtained in 2 months after Agrobacterium infection. To our knowledge, this is the first report on successful plant regeneration and recovery of transgenic plants from upland switchgrass cultivars by Agrobacterium-mediated transformation. The method presented here could be helpful in breaking through the bottleneck of regeneration and transformation of lowland and upland switchgrass cultivars and probably other recalcitrant grass crops.