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

Abstract

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.

An efficient <i>Agrobacterium</i>-mediated transformation method for switchgrass genotypes using Type I callus

“…Transgenic plants were obtained in all tested callus lines of two lowland cultivars, ' Alamo' and 'Kanlow' , at comparably higher efficiency than that achieved in previous reports using Type I callus. The transformation efficiency of one ' Alamo' line, A11-01, was as high as 59.2%, which was comparable to the previously reported efficiency for Type II callus of ' Alamo' at 56.6% by Li and Qu (2011) and 72.8% by Liu et al (2015). In addition, we successfully transformed the upland cultivar 'Trailblazer' using the present method.…”

“…Albino shoot formation from Type II callus was also reported by Ramamoorthy and Kumar (2012). Very recently, Liu et al (2015) reported efficient induction and transformation methods of Type II calli in coupled with the discovery of "shell-core" structure. They achieved 72.8% of transformation efficiency for a lowland cultivar ' Alamo' , and successfully obtained first transgenic plants of an upland cultivar 'Dacotah' .…”

“…Song et al (2012) reported that herbicide-or hygromycin-resistant calli were induced from inoculated seedling segments of 'Cavein-Rock' but no transgenic shoots were regenerated. Very recently, Liu et al (2015) observed "shell-core" structure within a clump of caryopsis-derived callus of switchgrass cultivars. They induced Type II calli from pre-embryogenic core structures and successfully transformed one of the upland cultivar 'Dacotah' at the 8.0% of efficiency.…”

“…Frequent albino formation was reported in transformation methods using Type II calli derived from caryopses (Li and Qu 2011) and seedlings (Ramamoorthy and Kumar 2012). A highly-efficient transformation method of Liu et al (2015) has been used freshly established 4-month-old Type II calli for transformation experiments and relationships between subculture period and albino formation as well as transformation efficiency were not described. Considering the availability of explants and prevalence of formation, caryopsis-derived callus is the most useful material for switchgrass transformation.…”

“…Type I callus could be maintained for at least 2 years with relatively high regeneration ability and with low albino shoot formation rate (Ogawa 2015). Friable embryogenic callus, termed Type II, shows a high growth rate and regeneration ability and is used in more recent reports (Burris et al 2009;Li and Qu 2011;Liu et al 2015;Ramamoorthy and Kumar 2012). Li and Qu (2011) reported a highthroughput transformation protocol for Type II callus and achieved more than 44.6% transformation efficiency, whereas 100% of long-term subcultured calli of ' Alamo' formed albino shoots.…”