Abstract A protocol for preserving grape embryogenic cultures indefinitely has been defined, and through re- current cycles of secondary embryogenesis, Vitis rupes- tris Scheele cultures are still regenerating after 10 years. The morphogenic competence of a sample of 1,204 so- matic embryos with such a long history has been evalu- ated. Within a 15-month-long culture, secondary embry- ogenesis regeneration reached an average efficiency of 23%, proving that morphogenic competence is retained during prolonged culture times. While the culture main- tenance described here was one of the crucial aspects of our genetic transformation protocol, several V. rupestris plants with various exogenous genes have been regener- ated during these last 10 years. Keywords Grape · Microscopy · Morphogenic competence · Secondary somatic embryogenesis · Vitis rupestris Abbreviations IAA: Indole-3-acetic acid · IBA: Indole-3-butyric acid · SEM: Scanning electron microscope Introduction Somatic embryogenesis was first attempted in grapes in the 1970s (Gresshoff and Doy 1974).The first com- mercial planting of vines from somatic embryos was es- tablished in the spring of 1977 (Vitis sp. cv. Seyval, Maryland, USA; Krul and Mowbray 1984), and in 1985 a protocol for grape somatic embryogenesis was patent- ed in the United States (Krul 1985). Since then this tech- nique has been significantly improved in several geno- types and from various explants (Gray 1995; Perl and Eshdat 1998; Martinelli and Gribaudo 2001); however, somatic embryogenesis in grape is, at this time, not a routine procedure, as reproducibility and efficiencies in the most agronomically important genotypes need to be improved. Once induced and established in the laboratory grape embryogenic cultures are precious material. Therefore, several strategies have been adopted in order to keep in- definitely the embryogenic competence of the cultures and thus obtain a long-term source of somatic embryos (Krul and Worley 1977; Lebrun and Branchard 1986; Gray and Mortensen 1987; Stamp and Meredith 1988; Matsuta and Hirabayashi 1989; Martinelli et al. 1993; Perl et al. 1995). In some cases, culture maintenance has been reported to last for some years (Gray 1989; Torregrosa 1998). Both embryogenic callus storage (Moriguchi et al. 1988) and the re-initiation of embryogenic callus from somatic embryos (secondary embryogenesis) have been used for preserving the embryogenic potential of the cultures (Martinelli et al. 1993; Kuksova et al. 1997). Mozsar and Viczian (1996) demonstrated a genotype effect on the morphogenic competence of somatic em- bryos. Secondary embryogenesis has been successfully applied in genetic transformation experiments where whole somatic embryos (Martinelli and Mandolino 1994, 2001; Martinelli et al. 1996, 2000; Scorza et al. 1996) or sections (Mullins et al. 1990) thereof were deemed to be suitable explants for co-culture with Agrobacterium tumefaciens. In our laboratory, embryogenic cultures of Vitis ru- pestris S. are still propagating after 10 years of recurrent secondary embryogenesis induction and are still being employed for various studies, in particular for regenerat- ing transgenic plants. Here we report on a study that evaluated the morphogenic competence of these cultures with such a long history. Communicated by H. Lörz L. Martinelli ( ✉ ) · E. Candioli · D. Costa · V. Poletti Istituto Agrario, 38010 San Michele all’Adige (TN), Italy e-mail: Lucia.Martinelli@mail.ismaa.it Tel.: +39-0-461-615231, Fax: +39-0-461-615288 N. Rascio Dipartimento di Biologia, Università degli Studi di Padova, via U. Bassi 58B, 35131 Padova, Italy Plant Cell Rep (2001) 20:279–284 DOI 10.1007/s002990100339 CELL BIOLOGY AND MORPHOGENESIS L. Martinelli · E. Candioli · D. Costa · V. Poletti N. Rascio Morphogenic competence of Vitis rupestris S. secondary somatic embryos with a long culture history Received: 21 November 2000 / Revision received: 29 January 2001 / Accepted: 13 March 2001 / Published online: 12 May 2001 © Springer-Verlag 2001