Vol.:(0123456789) 1 3 Plant Cell Tiss Organ Cult DOI 10.1007/s11240-017-1214-7 ORIGINAL ARTICLE Old methods rediscovered: application and improvement of two direct transformation methods to hybrid poplar (Populus tremula × P. alba) Julia Nietsch 1  · Tobias Brügmann 1  · Dirk Becker 2  · Matthias Fladung 1   Received: 5 October 2016 / Accepted: 9 April 2017 © Springer Science+Business Media Dordrecht 2017 can be considered suitable for transferring cisgenes directly into poplar. Keywords Biolistic · PEG transformation · Protoplast · Regeneration · Gfp · dsRed · Cisgenic poplar Introduction Since the beginning of the 20th century, the economy of the industrial and emerging countries has been based on the use of fossil fuels such as coal, oil and natural gas. How- ever, the carbon dioxide released by these fuels is consid- ered an important cause of global warming and the asso- ciated climate change. Therefore, over the past 20 years, the portion of renewable energy (solar, wind, hydro power, geothermal and biomass) has been increased worldwide. The advantage of biomass energy against the other forms of renewable energies is that biogas and biofuels can be stored and are available when they are needed. Smith et al. (2000) have shown that in Europe, of all options examined, bioenergy crops, e.g., maize, rapeseed or potato show the greatest potential for carbon mitigation. However, there are increasing complaints about the competition for land between food and energy crops (“food-fuel discussion”). Trees represent the most obvious source of biomass. To reduce the pressure on native forests, plantation forestry is used as a very efcient source for wood. Forty million ha of plantations (about 1.2% of the global forest area) deliver 25% of global wood fbres. The cultivation of fast-growing tree species such as poplar on marginal land, i.e., on land that is unsuitable for agricultural crops, could be one pos- sibility to refute the “food-fuel discussion”. But to achieve a high biomass yield of poplar on marginal land means that trees have to be further optimized towards a more efcient Abstract Two direct DNA transfer methods, biolistic transformation and a protoplast transformation approach using the INRA-clone 717 1B4 (Populus tremula × P. alba), are applied to poplars and compared. Both the in vitro cul- ture and the transformation parameters were optimized to receive a maximum quantity of transformed cells to achieve a stable transformation. For the frst time, the stable inte- gration of gfp and dsred in the poplar genome and their expression as visual reporter genes in regenerated plant- lets can be shown. For biolistic transformation, stem seg- ments cut lengthwise and incubated for 10 days on a callus induction medium revealed the highest number of transient Gfp- and dsRed signals. After optimization of the in vitro culture parameter, Gfp and dsRed-expressing transgenic poplars were regenerated, proven by PCR and Southern blot analysis. For protoplast transformation, the focus was initially on the development of a highly efcient protoplast isolation and plant regeneration system. Using an enzyme solution consisting of 1.0% cellulase R10 and 0.24% mac- erozyme, 1 × 10 7 protoplasts were obtained from 1 g fresh weight leaves. Following incubation of the protoplasts in 600 mOsm culture medium, a high number of microcalli were obtained, from which plantlets were regenerated. The parameters for isolation and regeneration were then com- plemented by an efcient protoplast transformation proto- col with 40% PEG 1500 . The results of this study confrm that both the biolistic and the protoplast transformation methods * Julia Nietsch julia.nietsch@thuenen.de 1 Thünen Institute of Forest Genetics, Sieker Landstraße 2, 22927 Großhansdorf, Germany 2 Biozentrum Klein Flottbek, MPG, Hamburg University, Ohnhorststraße 18, 22609 Hamburg, Germany