Establishment of an efficient in vitro culture and particle bombardment-mediated transformation systems in Miscanthus sinensis Anderss., a potential bioenergy crop

Abstract

Plants belonging to the genus Miscanthus are considered promising bioenergy crops. Here, we report the establishment of tissue culture system through particle bombardment‐mediated transformation in Miscanthus sinensis Anderss. Callus was induced efficiently from mature seeds in a medium containing a combination of a relatively high level of 2,4‐dichlorophenoxyacetic acid (5 mg L−1) and a relatively low level of 6‐benzyladenine (BA) (0.01 mg L−1). Callus induction potential of 18 accessions of M. sinensis, which were collected from various sites in Japan, was compared. Significant correlation was detected between compact (embryogenic) callus induction frequency and average annual air temperature in collection sites. An accession from Tanegashima Island showed the highest production of compact callus. We found that a higher level of BA causes callus browning; the 2 mg L−1 BA is the optimal concentration for regeneration. Both compact and friable calli were suitable for particle bombardment transformation. Through selection under the presence of 50 mg L−1 hygromycin for 3 weeks and further selection under the presence of 150 mg L−1 for 1 month, hygromycin‐resistant calli survived, of which 72.2% had been entirely transformed. Plants were regenerated from calli in the presence of hygromycin; transcripts of the hpt and gfp genes, which were cobombarded to the calli, were detected in the regenerated plants. This is the first report on the establishment of the in vitro culture of M. sinensis using mature seeds, the variation of callus formation among accessions collected from various sites in Japan, and particle bombardment‐mediated transformation in the genus Miscanthus.

Agrobacterium-mediated genetic transformation of Miscanthus sinensis

“…Although we tried to develop a genetic transformation system of M. sinensis using the Geumo seeds, we failed to obtain transgenic plants with these seeds, probably due to genetic heterogeneity of the Geumo seeds. Previous reports also suggested the importance of genotypes in callus induction, plant regeneration and genetic transformation (Somleva et al 2008;Głowacka et al 2010;Wang et al 2011). Thus, we obtained seven germplasms of M. sinensis which were maintained at Seoul National University (SNU), and investigated the induction of embryogenic calli and their regeneration to find a good candidate for the genetic transformation.…”

“…It is known that an embryogenic callus is compact and friable, whereas soft and watery calli are not embryogenic (Wang et al 2011). Thus, we checked the types of induced calli, and found three embryogenic callus types: yellowish compact, whitish friable, and whitish compact types (Fig.…”

“…Transformation is an important tool for improving the performance of crop species and effective transformation techniques should allow for the incorporation of foreign genes that improve crop traits efficiently. In the case of Miscanthus species, there have been several reports related to tissue culture conditions for callus induction and plant regeneration (Głowacka and Je_ zowski 2009;Park et al 2009;Głowacka et al 2010;Kim et al 2010;Pła_ zek and Dubert 2010;Zhang et al 2012), but there has been only one recent report on success of genetic transformation of M. sinensis using particle bombardment (Wang et al 2011). However, particle bombardment-mediated transformation method has demonstrated certain disadvantages, including a low frequency of success and the insertion of multiple gene copies, which is thought to correlate with gene silencing, while the Agrobacterium-mediated transformation method can overcome some of these disadvantages, such as transgene copy number and cost (Dai et al 2001).…”

Molecular Breeding of Grasses by Transgenic Approaches for Biofuel Production

“…Later, transgenic M. sinensis plants were produced by particle bombardment methods (Wang et al, 2011); four transgenic plants containing a foreign hph gene with or without the gfp were recovered from 120 bombarded calli, and foreign gene expression was confirmed by reverse transcription-PCR analyses. So far, there are no other reports on Miscanthus transformation.…”

“…Mature seeds of 18 accessions from various sites in Japan were subjected to callus induction, and a combination of a relatively high 2,4-D concentration (5 mg L -1 ) and a relatively low BA concentration (0.01 mg L -1 ) efficiently induced regenerable calli that could be used to produce transgenic Miscanthus plants (Wang et al, 2011). Interestingly, there was a correlation between the average annual air temperature at accession collecting sites and the frequency of embryogenic callus induction; seeds collected from warmer regions formed a higher percentage of embryogenic calli (Wang et al, 2011). Most recently, Zhang et al (2011) germinated seeds collected in China and compared explants from the epicotyls, young leaves, and radicles.…”