Efficient regeneration of fertile barley plants from callus cultures of several Nordic cultivars

摘要

Barley is the fourth important cereal crop worldwide (after wheat, rice and maize). Therefore genetic engineering of barley is of utmost importance. Attempts to establish a routine protocol for barley transformation have been quite unsuccessful (Iyer et al. 2000). Most reports on transgenic barley plants are based on the cultivar Golden Promise. However, the cell culture conditions established for Golden Promise (Wan and Lemaux 1994) are not suitable for many economically important cultivars, especially due to the low plant regeneration frequencies (Bregitzer et al. 1998a). It has been shown that several cultivars are also directly recalcitrant to the transformation process (Ritala et al. 1994; Koprek et al. 1996). The latter is caused, besides other factors, by the gradual loss of embryogenic and regenerative potential as well as by the increase of albino plants during the selection procedure of transformed tissues (Jiang et al. 1998). Thus, in order to elaborate a successful genetic transformation protocol for barley, it is important to fulfill at least two conditions for the corresponding cell cultures: 1) regeneration potential has been increased; 2) prolonged regeneration period is used (and thus the number of individuals plants regenerated from each callus is increased) in parallel with the reduced rates of somaclonal variation.The increase of regeneration potential of economically important barley cultivars has been addressed in several papers (Dahlen 1995; Bregitzer et al. 1998a; Castillo et al. 1998; Cho et al. 1998; Jiang et al. 1998; Nuutila et al. 2000). In all these works it was emphasized that higher concentrations of CuSO4 are important for the increase of regeneration potential. Castillo et al. (1998) specifically studied the regeneration potential of different barley cultivars after prolonged cell culture periods (3 to 4 months), i.e. periods close to that required for the selection of transgenic callus lines. They compared three different auxins to each other and concluded that Dicamba is better than 2,4D or Picloram for the induction and maintenance of embryogenic callus and thus also for the achievement of higher regeneration potential. At the same time it has been proposed that Dicamba increases the somaclonal variation when compared to 2,4D (Bregitzer et al. 1998b). Jiang et al. (1998) showed that the optimal ratios between 2,4D and BAP help to maintain the regeneration potential of green plantlets better than combinations of Dicamba and BAP. Hagio et al. (1995) also used 2,4D and BAP for the induction of barley callus. Indeed, they did not observe the regeneration of albino plants after the selection of the transgenic callus with hygromycin. Cho et al. (1998) have demonstrated that when 2,4D or Dicamba alone are used in callus induction medium, even short culture periods on the medium containing 2,4D, BAP and elevated levels of copper can increase considerably the regeneration rates of green plants after prolonged callus cultures.The concentration of NH4+ has been considered as an important factor in order to raise the regeneration potential of barley cell cultures (Kasha et al. 1990). Several groups (Wan and Lemaux 1994; Cho et al. 1998; Jiang et al. 1998) have successfully used FHG medium for the barley regeneration from embryogenic callus. This medium is characterized by the content of NH4NO3 ten times lower than that of MS medium (Murashige and Skoog 1962) as well as by the high maltose concentration (60 g l?1) and addition of glutamine as the organic source of the nitrogen. The latter was more deeply investigated while studying the regeneration potential of barley variety Kymppi (Nuutila et al. 2000). It was demonstrated that the higher concentrations of organic nitrogen are required during the early embryogenesis, while in the stage of shoot development the inorganic nitrogen in the form of nitrates is more beneficial.The aim of the current study was to determine the optimal cell culture conditions