Cascade huckleberry, mountain huckleberry, and oval-leaf bilberry belong to the genus Vaccinium that contains ≈400 species distributed worldwide except Antarctica and Australia (Vander Kloet, 1988) and is typically characterized as woody perennial vines or shrubs producing moderate-sized fleshy, and more-or-less edible fruit. Plants may be terrestrial or epiphytic and are generally found on acidic, sandy, peaty, or organic soils. Huckleberries and bilberries belong to the section Myrtillus of this genus and are native to the northwestern United States and western Canada. Mountain huckleberry (black huckleberry, thin-leaf huckleberry, or mountain bilberry) and cascade huckleberry (cascade bilberry or blue huckleberry) are tetraploid species rich in aroma and flavor chemicals (Fellman et al., 1998). The oval-leaf bilberry (oval-leaf blueberry or alaska blueberry) also is a tetraploid species [occasionally diploid (Vander Kloet, 1988)] and is rich in antioxidant compounds (Lee et al., 2004; Taruscio et al., 2004) creating opportunities for value-added nutritional products. These berries are harvested from wild stands and used in fresh, frozen, and processing markets.
In vitro methods complement traditional breeding programs to introduce new traits into selected plants, to multiply clonal plants, and to develop suitable cultivars in a minimum time. The ability to regenerate plants is crucial to the successful application of in vitro methods (Cao and Hammerschlag, 2000; Qu et al., 2000). Additionally, a shoot regeneration system can be used to identify and/or induce somaclonal variants and to develop transgenic plants following genetic transformation of plant cells.
To produce superior Vaccinium clones more rapidly, we began in vitro techniques at our research center in St. John's, NL, Canada, in 1999 (Debnath, 2000). A successful system for cloning cascade and mountain huckleberries and oval-leaf bilberries from axillary shoot meristems was developed so that superior germplasm could be more rapidly introduced to the Vaccinium industry (Barney et al., 2007). Generating shoots reliably from somatic plant tissues, such as leaves or stem segments, would permit the genetic manipulation of these species. Although adventitious shoot regeneration by cascade and mountain huckleberries and by oval-leaf bilberry was absent, shoot proliferation from nodal explants of these species has been reported (Barney et al., 2007) and adventitious shoot regeneration also has been reported in other Vaccinium species, including lowbush blueberry [V. angustifolium (Debnath, 2009, 2011)], highbush blueberry [V. corymbosum (Cao and Hammerschlag, 2000)], southern highbush blueberry [predominantly V. corymbosum germplasm with germplasm from V. darrowii, V. ashei, and/or V. tenellum (Liu et al., 2010; Meiners et al., 2007)], half-high blueberry [V. corymbosum × V. angustifolium (Graham et al., 1996)], cranberry [V. macrocarpon (Qu et al., 2000)], bilberry [V. myrtillus (Shibli and Smith, 1996)], ohelo [V. pahalae (Shibli and Smith, 1996)], and lingonberry [V. vitis-idaea (Debnath, 2003, 2005; Debnath and McRae, 2002)].
Cytokinins play an important role in the regulation of plant growth and morphogenesis (Miller, 1961). They induce cell division and shoot differentiation (Skoog and Miller, 1957). Zeatin and its derivatives are the most abundant cytokinins in plants (Letham and Palni, 1983). Zeatin was found effective for shoot regeneration in lingonberry (Debnath and McRae, 2002). The objective of the study is to obtain plantlets using leaves and stem segments derived from micropropagated shoots. Hormonal, physical, and genetic experimental factors were investigated to optimize plant regeneration from the leaves and stem segments of a cascade huckleberry clone, a mountain huckleberry clone, and of an oval-leaf bilberry clone.
Barney, D.L., Lopez, O.M. & King, E. 2007 Micropropagation of cascade huckleberry, mountain huckleberry, and oval-leaf bilberry using woody plant medium and Murashige and Skoog medium formulations HortTechnology 17 279 284
Callow, P., Haghighi, K., Giroux, M. & Hancock, J. 1989 In vitro shoot regeneration on leaf tissue from micropropagated highbush blueberry HortScience 24 373 375
Compton, E.C. 1994 Statistical methods suitable for the analysis of plant tissue culture data Plant Cell Tissue Org. Cult. 37 217 242
Debnath, S.C. 2000 Combined application of classical and biotechnological techniques in the development of small fruits Can. J. Plant Sci. 80 233 (abstr.).
Debnath, S.C. 2003 Improved shoot organogenesis from hypocotyls segments of lingonberry (Vaccinium vitis-idaea L.) In Vitro Cell. Dev. Biol. Plant 39 490 495
Debnath, S.C. 2005 A two-step procedure for adventitious shoot regeneration from in-vitro-derived lingonberry leaves: Shoot induction with TDZ and shoot elongation using zeatin HortScience 40 189 192
Debnath, S.C. 2009 A two-step procedure for adventitious shoot regeneration on excised leaves of lowbush blueberry In Vitro Cell. Dev. Biol. Plant 45 122 128
Debnath, S.C. 2011 Adventitious shoot regeneration in a bioreactor system and EST PCR based clonal fidelity in lowbush blueberry (Vaccinium angustifolium Ait.) Sci. Hort. 128 124 130
Debnath, S.C. & McRae, K.B. 2001a An efficient in vitro shoot propagation of cranberry (Vaccinium macrocarpon Ait.) by axillary bud proliferation In Vitro Cell. Dev. Biol. Plant 37 243 249
Debnath, S.C. & McRae, K.B. 2001b In vitro culture of lingonberry (Vaccinium vitis-idaea L.): The influence of cytokinins and media types on propagation Small Fruits Rev. 1 3 19
Debnath, S.C. & McRae, K.B. 2002 An efficient adventitious shoot regeneration system on excised leaves of micropropagated lingonberry (Vaccinium vitis-idaea L.) J. Hort. Sci. Biotechnol. 77 744 752
Fellman, J., Thorngate, J. & Barney, D. 1998 Identification of western huckleberry flavor components Proc. Northwest Ctr. Small Fruit Res. 7 62 63
George, E.F. 1993 Plant propagation by tissue culture. Part 1. The technology. Exegetics, Edington, United Kingdom.
George, E.F. & Sherrington, P.D. 1984 Plant propagation by tissue culture. Exegetics, Reading, United Kingdom.
Huetteman, C.A. & Preece, J.E. 1993 Thidiazuron: A potent cytokinin for woody plant tissue culture Plant Cell Tissue Org. Cult. 33 105 119
Lee, J., Finn, C.E. & Wrolstad, R.E. 2004 Anthocyanin pigment and total phenolic content of three Vaccinium species native to the Pacific Northwest of North America HortScience 39 959 964
Liu, C., Callow, P., Rowland, L.J., Hancock, J.F. & Song, G. 2010 Adventitious shoot regeneration from leaf explants of southern highbush blueberry cultivars Plant Cell Tissue Org. Cult. 103 137 144
Marcotrigiano, M., McGlew, S.P., Hackett, G. & Chawla, B. 1996 Shoot regeneration from tissue-cultured leaves of the American cranberry (Vaccinium macrocarpon) Plant Cell Tissue Org. Cult. 44 195 199
Marks, T.R. & Simpson, S.E. 1994 Factors affecting shoot development in apically dominant Acer cultivars in vitro J. Hort. Sci. 69 543 551
Meiners, J., Schwab, M. & Szankowski, I. 2007 Efficient in vitro regeneration systems for Vaccinium species Plant Cell Tissue Org. Cult. 89 169 176
Minocha, S.C. 1987 Plant growth regulators and morphogenesis in cell and tissue culture of forest trees, p. 50–66. In: J.M. Bonga and D.J. Durzan (eds.). Cell and tissue culture in forestry, Vol. I. Martinus Nijhoff Publishers, Dordrecht, The Netherlands.
Preece, J.E., Huetteman, C.A., Ashby, W.C. & Roth, P.L. 1991 Micro- and cutting propagation of silver maple. I. Results with adult and juvenile propagules J. Amer. Soc. Hort. Sci. 116 142 148
Qu, L., Polashock, J. & Vorsa, N. 2000 A highly efficient in vitro cranberry regeneration system using leaf explants HortScience 35 948 952
Rowland, L.J. & Ogden, E.I. 1992 Use of a cytokinin conjugate for efficient shoot regeneration from leaf sections of highbush blueberry HortScience 27 1127 1129
Shibli, R.A. & Smith, M.A.L. 1996 Direct shoot regeneration from Vaccinium pahalae (ohelo) and V. myrtillus (bilberry) leaf explants HortScience 31 1225 1228
Skoog, F. & Miller, C.O. 1957 Chemical regulation of growth and organ formation in plant tissue cultured in vitro Symp. Soc. Exp. Biol. 11 118 30
Taruscio, T.G., Barney, D.L. & Exon, J. 2004 Content and profile of flavonoid and phenolic acid compounds in conjunction with the antioxidant capacity for a variety of Northwest Vaccinium berries J. Agr. Food Chem. 52 3169 3176
Vander Kloet, S.P. 1988 The genus Vaccinium in North America. Agr. Can. Publ. 1828.