ORIGINAL PAPER Ectopic expression of class 1 KNOX genes induce adventitious shoot regeneration and alter growth and development of tobacco (Nicotiana tabacum L) and European plum (Prunus domestica L) C. Srinivasan • Zongrang Liu • Ralph Scorza Received: 15 September 2010 / Revised: 20 December 2010 / Accepted: 21 December 2010 / Published online: 7 January 2011 Ó Springer-Verlag (outside the USA) 2011 Abstract Transgenic plants of tobacco (Nicotiana taba- cum L) and European plum (Prunus domestica L) were produced by transforming with the apple class 1 KNOX genes (MdKN1 and MdKN2) or corn KNOX1 gene. Transgenic tobacco plants were regenerated in vitro from transformed leaf discs cultured in a medium lacking cyto- kinin. Ectopic expression of KNOX genes retarded shoot growth by suppressing elongation of internodes in trans- genic tobacco plants. Expression of each of the three KNOX1 genes induced malformation and extensive lobbing in tobacco leaves. In situ regeneration of adventitious shoots was observed from leaves and roots of transgenic tobacco plants expressing each of the three KNOX genes. In vitro culture of leaf explants and internode sections excised from in vitro grown MdKN1 expressing tobacco shoots regenerated adventitious shoots on MS (Murashige and Skoog 1962) basal medium in the absence of exoge- nous cytokinin. Transgenic plum plants that expressed the MdKN2 or corn KNOX1 gene grew normally but MdKN1 caused a significant reduction in plant height, leaf shape and size and produced malformed curly leaves. A high frequency of adventitious shoot regeneration (96%) was observed in cultures of leaf explants excised from corn KNOX1-expressing transgenic plum shoots. In contrast to KNOX1-expressing tobacco, leaf and internode explants of corn KNOX1-expressing plum required synthetic cytokinin (thidiazuron) in the culture medium to induce adventitious shoot regeneration. The induction of high-frequency regeneration of adventitious shoots in vitro from leaves and stem internodal sections of plum through the ectopic expression of a KNOX1 gene is the first such report for a woody perennial fruit trees. Keywords Adventitious shoot regeneration Á Tobacco Á Plum Á Corn and apple KNOX1 genes Á Altered leaf morphology Introduction Adventitious shoot regeneration from in vitro-cultured explants is an important requirement for producing trans- genic plants of most crop species. While most herbaceous species regenerate adventitious shoots from cultured explants when exposed to synthetic plant growth regula- tors, explants from perennial fruit trees are generally recalcitrant to regenerate adventitious shoots (Scorza 2001; Srinivasan et al. 2005). In these recalcitrant species a dif- ferent strategy, one that utilizes the ectopic expression of genes involved in meristem and shoot apical meristem (SAM) formation, and development may provide a more successful approach. Work with Arabidopsis thaliana has revealed that shoot apical meristem formation and main- tenance are regulated by a number of key genes, including the meristem identity genes, such as the class I KNOX (KNOTTED-1-like HOMEOBOX) genes, which belong to a homeobox gene family consisting of STM, KNAT1, KNAT2 and KNAT6 (Hake et al. 2004), the ortholog KNOX1 gene in corn (Vollbrecht et al. 1991), and OSH1 gene from rice (Matsuoka et al. 1993). The KNOX genes are versatile regulators of plant development and diversity (Hake et al. 2004; Hay and Communicated by P. Lakshmanan. C. Srinivasan (&) Á Z. Liu Á R. Scorza United States Department of Agriculture, Agricultural Research Service, Appalachian Fruit Research Station, 2217 Wiltshire Road, Kearneysville, WV 25430, USA e-mail: Chinnathambi.Srinivasan@ars.usda.gov 123 Plant Cell Rep (2011) 30:655–664 DOI 10.1007/s00299-010-0993-7