Paulownia - Bioenergy Crop Plants - page 683

been reported in P. elongata (Ipecki and Gozukirmizi 2003, 2004). Direct somatic embryogenesis
was induced on leaf and internodal explants of P. elongata and synthetic seeds were also produced
(Ipecki and Gozukirmizi 2003). For large-scale production of elite plants, this technology has great
relevance because the process can be easily scaled-up using a bioreactor. Advances in somatic
embryogenesis have brought mass clonal propagation of the top commercial trees closer to reality,
and efficient gene transfer systems have been developed for a number of conifers and hardwoods.
27.6.2 g EnEtic t ranSformation
Advances in technology for in vitro propagation and genetic transformation have accelerated
the development of genetically engineered trees during the past 15 years. Targeted traits include
herbicide tolerance, pest resistance, abiotic stress tolerance, modified fiber quality and quantity,
and altered growth and reproductive development. Commercial potential has been demonstrated
in the field for a few traits, in particular herbicide tolerance, insect resistance, and altered lignin
content. Now that commercial implementation is feasible, at least for the few genotypes that can
be efficiently transformed and propagated, environmental concerns have become the main obstacle
to public acceptance and regulatory approval. Ecological risks associated with commercial release
range from transgene escape and introgression into wild gene pools, to the effect of transgene
products on other organisms and ecosystem processes. Evaluation of those risks is confounded by
the long life span of trees and by the limitations of extrapolating results from small-scale studies to
larger-scale plantations.
Preliminary experiments using in vitro shoots to establish a transformation protocol were
carried out using Agrobacterium tumifaciens (strains 542, A281, or C58) and A. rhizogenes (strain
R1601). Opine analyses demonstrated the expression of the introduced gene in proliferating galls
or hairy roots (Bergmann et al. 1999). In a separate study, A. tumifaciens (LBA4404) harboring
binary vector pBI121 (Clontech Laboratories, Inc., Mountain View, CA) was used to transform
P. fortunei using in vitro grown petiole segments (Mohri et al. 2003). Successful transformation
was confirmed by histochemical analysis of β-glucuronidase (GUS) activity in kannamycin-
resistant calli; however, the frequency of shoot regeneration was very low. Transformation studies
coupled with molecular techniques helped in establishing the role of transcription factor PkMADS1
isolated from P. kawakamii (Prakash and Kumar 2002). In this study, it was seen that the antisense
suppression of PkMADS1 resulted in gross morphological changes such as a change in phyllotaxy.
Leaf explants obtained from the antisense PkMADS1 transgenic plants showed an almost 10-fold
decrease in adventitious shoot formation compared with the explants from the sense transgenic lines
or the wild-type plants. In a recent study (Castellanos-Hernandez et al 2009), a biolistic protocol for
stable genetic transformation was developed using leaf explants. Regenerated plants exhibited the
integration of the transgenes as stable expression was demonstrated by GUS assay, determination of
NPTII activity, and polymerase chain reaction analysis.
PWB disease, caused by PWB phytoplasma, is one of the most devastating diseases of this genus.
PWB seriously slows down tree growth and is even capable of causing seedling death. Introduction
of the shiva-1 gene that encodes an antibacterial peptide using A. tumifaciens -mediated gene transfer
resulted in plants with fewer phytoplasma and less symptoms in plants (Du et al. 2005). Further
analysis of transgenic plants suggested that breeding shiva-1 Paulownia is an effective strategy
to control PWB disease. Developing reproducible transformation systems will be of great help in
developing fast-growing lignocellulosic feedstock in the future.
Radical alterations in the quantity and quality of lignin in wood have been shown to be possible in
softwoods and hardwoods through identification of naturally occurring mutants and by engineering
the lignin biosynthetic pathway with transgenes. The potential environmental and social impacts of
the release of transgenic trees have become an increasingly contentious issue that will require more
attention if we are to use these technologies to their full advantage.