Dormancy and germination of true potato (Solanum tuberosum L.) seeds : characterization of endo-β-mannanase genes Public Deposited

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  • True potato (Solanum tuberosum) seed (TPS) is used for preservation of variable genetic lines of wild and cultivated potatoes (Hawkes et al., 2000) and for propagation of food crops in some developing countries. TPS has advantages over seed potato tubers in storage and transportation and favors lower virus infection levels in fields. However, TPS has thermodormancy and will not readily germinate at 25°C and above (D'Antonio and McHale, 1988; Pallais, 1995a, b; Alvarado et al., 2000). TPS can be extremely unreliable when planted directly in fields due to poor emergence related to diseases and soil crusting. Germination tests were conducted with two lots of TPS derived from cvs. EB-8109 and All Blue, respectively, to study dormancy mechanisms. Seeds were germinated under four temperature regimes (10°C, 15°C, 20°C and 25°C). The two lots showed distinctly different germination characteristics. EB-8109 seeds showed only thermodormancy whereas All Blue seeds showed very deep dormancy. A carotenoid synthesis inhibitor, fluridone, which blocks abscisic acid (ABA) synthesis, effectively broke thermodormancy in EB-8109 TPS but did not break primary dormancy in All Blue seeds. Additional treatments, including pre-chilling and hormonal regimes, also failed to break All Blue deep dormancy. When the micropylar region of the endosperm (endosperm cap) was removed from seeds of both seed lots, radicle elongation was observed, suggesting that mechanical resistance from the endosperm cap restrains radicle protrusion, and that weakening of the endosperm cap is requisite for TPS germination. Endo-β-mannanase expression was measured to help characterize mechanism underlying the weakening of endosperm cap tissues. This enzyme is thought to permit radicle protrusion by degrading cell walls thereby weakening the tissues of the endosperm cap (Groot et al., 1988). The coding region of germination-specific mannanase was isolated from the potato genome by use of polymerase chain reaction (PCR) with primers specifically designed for the tomato germination-specific mannanase gene (LeMAN2, Nonogaki et al., 2000). The cDNA of the TPS mannanase was identical to that of LeMAN2. The expression of mannanase mRNA was detected in the endosperm cap of germinating TPS after 72 h of imbibition at 15°C, while no expression was detected at 25°C (thermodormant condition). Fluridone induced mannanase expression in the micropylar region of the endospem at 25°C. Thus, there was a correlation between induction of mannanase and dormancy breakage. A major increase in TPS post-germinative endo-β-mannanase activity was detected by use of gel diffusion assay. Two isoforms of mannanases were detected in the protein extracts of germinated TPS by activity staining of native polyacrylamide gel electrophoresis. The post-germinative mannanase was detected in the whole endosperm of germinated TPS by using tissue printing with the LeMAN1 (Bewley et al., 1997) RNA probe. These results suggest that, as with tomato, TPS also expresses post-germinative mannanase activity. The promoter region of a new tomato mannanase was isolated during this research. This promoter was shown to be involved in anther-specific expression of mannanase.
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