Both genetic and environment factors effect callus formation and plant regeneration in cereal anther culture (Kasha et al. 1990; Rahimbaev et al. 1992; Raina 1997). Recent reports of increase in cereal anther culture results have been based mainly upon one or few responsive genotypes, while other genotypes show a wide range of response to culture conditions and media components. These strong genotypic differences are yet to be resolved for breeding purposes in most of the cereals. Anther culture ability in wheat can be divided into three independently inherited components: callus induction, plant regeneration and green plant formation, usually governed by more than one gene (Lazar et al. 1984; Deaton et al. 1987; Agache et al. 1989; Szakacs et al. 1988). Genotype seems to be one of the major determinants of callus and embryoid production in this work with durum wheat, since wide range of variations were observed among them for callus initiation (0-11.78%) and haploid plant formation (0-3.89%).
For many years, 2,4-D has been considered as an essential growth hormone for callus induction in cereal tissue culture. However, for direct regeneration, i.e. without transferring calli or embryos to regeneration media, the replacement of 2,4-D by IAA or NAA has been important (Ouyang 1986; Liang et al. 1987). During present investigation, modified N6NB supplemented with NAA and BA was less effective for callus induction as compared to N6 DK with 2,4-D and kinetin.
Most media used for wheat anther culture contain high sugar level between 6 and 10 percent. During present investigation as compared to sucrose, maltose increased the androgenesis considerably. Similar findings have been obtained for bread wheat (Last and Brettell 1990; Orshinsky et al. 1990; Zhou et al. 1991, 1992; Trottier et al. 1993; Novaro-Alvarez et al. 1994; Orshinsky and Sadasivaiah 1994). The mode of action of maltose is still unknown, however, it has been suggested that the slow hydrolysis of maltose could provide a carbon source for a longer period than sucrose, which is rapidly hydrolyzed (Orshinsky et al. 1990). Furthermore, a break down product of sucrose hydrolysis, fructose is also believed to inhibit androgenesis in vitro (Last and Brettell 1990). In addition, maltose could be more efficient than sucrose as its hydrolysis rate that is closer to the rate of utilization of glucose by embryos (Robert-Oehlschlager et al. 1990).
Both genetic and environmental factors effect callus formation and plant regeneration in cereal anther culture. Interactions among these factors are often significant. This usually makes comparison of work from various laboratories difficult as genotypes, media and culture conditions differ considerably. The results of current study indicate the importance of considering such interactions in the development of protocols with locally adopted genotypes and methodology.
For the maintenance of androgenic calli, it is desirable to have levels of hormones that provide good callus growth and do not hinder subsequent regeneration. Only few reports of successful maintenance of androgenic calli are available on barley with low levels of 2,4-D (Tiwari et al. 1990 a, b). During our preliminary experiments with durum wheat androgenic calli 2,4-D concentrations higher than 3 mg/l and other growth regulators such as NAA and IAA resulted in very low regeneration potential and therefore, were found to be unsuitable for long-term maintenance study. Silver nitrate, an ethylene inhibitor has shown to increase embryo production in cereals (Lentini et al. 1995; Ghaemi et al. 1994). During present investigation, silver nitrate was efficiently used to maintain cultures in combination with 2,4-D as compared to auxin alone. This effect was probably due to low callus proliferation with increased embryogenic response.
In conclusion, it was shown that anther culture response of recalcitrant species T. durum can be improved by selecting responsive genotypes and suitable culture medium. However, if compared with many cereals, the present results are still very low and suggest that the medium currently used for anther culture of durum wheat may not be optimal for green plant production. Therefore, adjustments of medium components such as concentration and type of growth hormones, sugars, gelling agents etc. should be made prior to culture.
Acknowledgment
Financial support was given by Department of Science and Technology, Ministry of Science and Technology, Government of India, New Delhi.