Joanna Cerazy, Karolina Malec, Aurelia ��lusarkiewicz-Jarzina, Aleksandra Ponitka, Stanis��aw Je�¼owski and Tomasz Pniewski
Posters-Accepted Abstracts: J Fundam Renewable Energy Appl
Miscanthus is a genus perennial giant grass with great potential for biomass production, which can be used as a renewable
feedstock for bioenergy or bioethanol. Biotechnological methods, including in vitro cultures and plant transformation
can substantially complement projects aimed to improve miscanthus biomass yield or quality. Several genotypes of M. sinensis
(lines MS1, MS16 and MS17), M. x giganteus (line D-116 and MG3) and M. sacchariflorus (‘Robustus’) were subjects of studies
on plant regeneration. Among young spikelets, spike axles, inflorescence receptacles and nodes, the first appeared appropriate
initial explants for all genotypes. Callus was induced 2-3 weeks on MS medium with 5.0 mg/l 2,4-D and 0.5 mg/l BAP, while
plants were regenerated within 2 months on MS medium + 2.0 mg/l BAP. The efficiency of obtaining callus and plants depended
on the genotype and the highest regeneration rate was noted for M. sinensis line 17 and M. x giganteus D-116, where 659.0 and
517.9 calluses and 2454.4 and 1811.9 plants per 100 explants were obtained, respectively, but only 192.0 calluses and 78.4 plants
for M. sacchariflorus. Callus induction and plant regeneration were approx. 1.3 and 2.0 times improved by using respectively, C17
medium with 5.0 mg/l 2,4-D and 0.5 mg/l BAP and 190-2 medium with NAA and KIN, each 0.5 mg/l. Developed regeneration
procedure was partially adopted for Agrobacterium-mediated miscanthus transformation trials. All used Agrobacterium
tumefaciens strains carried pCAMBIA1201 vector with T-DNA containing hygromycin phosphotransferase marker gene (hpt)
and GUS reporter gene, both under control of 35S RNA CaMV promoter. Embryogenic 10-week-old calli (50 per variant)
were inoculated with Agrobacterium hypervirulent strains: EHA105, AgL0 and AgL1. Callus was induced on MS medium
with L-proline (0.5 mg/l), L-glutamine (0.5 mg/l), casein (1 mg/l) and 2,4-D (2 mg/l) but plants were regenerated as described
above. All media were supplemented with 2.5 mg/l of hygromycin as a selection agent. Finally 12 putative transgenic plants, 5
for MS17 and 7 for MG, were obtained, then micropropagated up to 5 clones, rooted and transferred to soil. Probable T-DNA
genomic integration was confirmed by PCR in all clones for 5 transformants, hence the transformation efficiency was about 1
to 2%. Further plant analyses as assay of GUS or hpt activity, etc. are in progress.
