Factors influencing Agrobacterium-mediated transformation of switchgrass cultivars

Abstract

To optimize Agrobacterium tumefaciens-mediated transformation, factors influencing gene delivery, selection of transformed cells, and plant regeneration were investigated using two major switchgrass cultivars, including a lowland tetraploid cultivar Alamo and an upland octoploid cultivar Cave-in-Rock (CIR). Transient expression studies monitored by histochemical b-glucuronidase assay in seedling segments indicated that A. tumefaciens strain EHA105 was more effective in gene delivery than LBA4404 or GV3101. Of three major selectable genes, the bialaphos resistance (bar) gene and the hygromycin phosphotransferase (hpt) allowed effective selection of transformed cells using 2 mg l -1 glufosinate ammonium herbicide and 50 mg l -1 hygromycin, respectively; whereas the neomycin phosphotransferase II gene did not yield effective selection using 100 mg l -1 kanamycin. Herbicideor hygromycin-resistant calluses were induced from seedling segments after 2-3 months of selection. Transformants of 'Alamo' with the bar or hpt were obtained 3-4 weeks after the resistant calluses were transferred onto regeneration medium; in contrast, no regenerant was produced from the calluses of 'CIR'. Most of transformants showed normal growth in the greenhouse. Low percentages of mature seeds ranging from 1.7 to 8.7% of husks were obtained from open pollinated plants. Southern blot analysis confirmed stable integration of the bar in selected T0 transformants. Reverse transcription PCR and herbicide/hygromycin tolerance tests indicated expression of transgenes. The optimized transformation protocol using basal parts of seedling as explants shortened the process by 4-5 weeks, and it has potential use for transformation of other switchgrass cultivars.

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

“…So far no genetic transformation of upland cultivars has been reported. Even callus regeneration is very difficult to achieve (Song et al 2012;Nageswara-Rao et al 2013). Thus, establishment of a highly efficient and amenable plant regeneration and transformation system of switchgrass is still in urgent need.…”

Long-term maintenance of high regeneration ability of switchgrass embryogenic callus

“…Prolonged subculture of callus, especially using the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D), turns embryogenic cells into non-embryogenic cells resulting in the loss of regeneration ability, and also leads to somaclonal variations, such as albinism (Karp 1994;Vasil and Vasil 1994). Although both Type I and Type II embryogenic callus cultures of switchgrass have been established from various explant sources, i.e., mature caryopsis, young leaf, immature inflorescence and young seedling (Alexandrova et al 1996;Denchev and Conger 1994;Song et al 2012), reports on the longevity of embryogenic cells and the occurrence of somaclonal variations are limited. Burris et al (2009) reported that inflorescence-derived Type II callus could be maintained for more than 6 months, while the embryo viability of Type I callus was limited to less than 2 months.…”

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

“…Seo et al (2010) induced embryogenic callus of 'Blackwell GR63' , although the frequency of shoot regeneration was low (10.9% of calli inoculated). 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.…”

“…Different genotypes within a cultivar affect the transformation efficiency (Ogawa et al 2014;Somleva et al 2002) and potentially affect characteristics of transgenic plants that carry the same transgene(s). Seedling-derived callus has been induced and used for transformation (Song et al 2012), although these callus lines also comprise different genotypes. Inflorescences can be obtained from vegetatively propagated plants for callus induction, and thus are a suitable explant source from which to induce callus and transformants derived from a single genotype.…”