Figure 1.
Construction of transgenic plants overexpressing A. niger phyA or E. coli appA.
A and B: Schematic representation of binary vector pBI121-phyA (A) and pBI121-appA (B) for B. napus plant transformation. Phytase genes (A. niger phyA and E. coli appA) under the control of the CaMV 35S promoter and nos-terminator were modified for extracellular secretion by inclusion of an extracellular targeting sequence from the carrot extensin (ex) gene. The construct has a selectable marker for the NPT?? gene. C and D: Southern blotting analysis of ex::phyA (C) and ex::appA (D) transgenic lines, using phyA and appA probes, respectively. Genomic DNA was digested by HindIII and EcoRI. C, M, Marker; WT, wild-type canola; 1–4,T3 transgenic plants of P3 line; 5–6, T3 transgenic plants of P11 line; D, 1–4, T3 transgenic plants of a18 line. E and F: Northern blotting analysis of phyA (E) and appA (F) expression in roots of transgenic lines using phyA and appA probe, respectively.
Figure 2.
Phytase activity in leaf (A) and root (B) extracts of transgenic lines when grown in different P conditions.
HP: plants were grown in high P (250 µM KH2PO4) nutrient solution for 18 days; LP: plants were grown in low P (5 µΜ KH2PO4) nutrient solution for 18 days; Phy-P: plants were grown in 250 µM P in the form of phytate for 18 days. Each column is the mean of four replicates with SD. Different letters represent significant differences at the p<0.05 level.
Figure 3.
Phytase activity (A) and acid phosphatase activity (B) exuded from roots of transgenic lines expressing ex::phyA/appA and WT.
Plants were grown in different P treatment solutions. Each column is the mean of four replicates with SD. Different letters represent significant differences at the p<0.05 level.
Figure 4.
Growth response of transgenic plants and WT plants in sand culture.
A: Phenotype of transgenic lines (P3, P11 and a18) and WT plants grown for 60 d in quartz sand culture with different P sources. B: Leaf number and size of transgenic lines and WT plants when grown in quartz sand with phytate as the sole P source. The leaves arranged from left to right ranged from the oldest to youngest according to the growth order in the plant.
Table 1.
Shoot biomass, P concentration and P accumulation of transgenic lines and WT grown in quartz sand.
Table 2.
Effects of different P sources on seed yield, P concentration, P accumulation, phytase activity and phytic acid content of WT and transgenic lines grown in P deficient soil.
Figure 5.
Phytase activity (A) and phytic acid content (B) in seeds of transgenic T3 lines and WT.
Each column is the mean of four replicates with SD. Different letters represent significant differences at the p<0.05 level. The seeds were harvested from the field by self-cross. n.d., means not detectable.