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JXB Advance Access published online on July 7, 2007
Journal of Experimental Botany, doi:10.1093/jxb/erm099
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© 2007 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)

RESEARCH PAPER

Overexpression of HBK3, a class I KNOX homeobox gene, improves the development of Norway spruce (Picea abies) somatic embryos

Mark F. Belmonte1, Muhammad Tahir1, Dana Schroeder2 and Claudio Stasolla1,*

1Department of Plant Science, University of Manitoba, Winnipeg, R3T 2N2, Manitoba, Canada
2Department of Botany, University of Manitoba, Winnipeg, R3T 2N2, Manitoba, Canada

* To whom correspondence should be addressed. E-mail: stasolla{at}ms.umanitoba.ca

In order to investigate the effects of HBK3, a spruce gene member of the class I KNOX family, during somatic embryogenesis, sense (HBK3-S) and antisense (HBK3-A) Norway spruce (Picea abies) lines were generated. Somatic embryos produced from these lines were then analysed at morphological and structural levels. Compared with control, differentiation of immature somatic embryos from pro-embryogenic masses (PEMs) was accelerated in lines overexpressing HBK3 (HBK3-S). Such immature embryos showed enlarged embryogenic heads and were able to produce fully developed cotyledonary embryos at higher frequency. Furthermore, HBK3-S embryos had enlarged shoot apical meristems (SAMs) and enlarged expression pattern of PgAGO, a molecular marker gene specific to meristematic cells. Lines in which HBK3 (HBK3-A) was down-regulated had reduced ability to produce immature somatic embryos from PEMs and were not able to complete the maturation processes. To assess the function of HBK3 in comparison with that of angiosperm KNOX genes, this gene was ectopically expressed in Arabidopsis plants. As observed for spruce, Arabidopsis embryos overexpressing HBK3 had enlarged meristems and enlarged expression pattern of SHOOTMERISTEMLESS, a SAM molecular marker gene. In addition, transformed embryos were able to germinate at a higher rate and the resulting plants showed a variety of phenotypic aberrations, including abnormal leaves and reduced apical dominance. Overall, these data confirm the importance of KNOTTED genes during development and reveal the participation of HBK3 in conifer embryogeny. Furthermore, the results show redundant functions of this gene during embryonic growth of spruce and Arabidopsis, but not during post-embryonic growth.

Key words: Embryo, HBK3, Picea abies, shoot apical meristem, spruce somatic embryogenesis, transformation

Received 6 December 2006; Revised 15 March 2007 Accepted 17 April 2007


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