Figure 1. Protein accumulation in leaves and peeled storage roots of transgenic cassava expressing zeolin under control of the patatin and 35S promoters.

A) Protein content in storage roots as quantified by Bradford assay after 7 months of growth in pots in the greenhouse. Non-transgenic cassava cv. 60444 (dark green bar) and transgenic plant line with empty gene vector only (pCambia2300) (light green bar) show total protein content of less than 3% dw, while all seven transgenic lines expressing zeolin under control of the patatin promoter (blue bars) accumulate total protein at 9.5% to 10.3% dw. Storage roots from plants expressing zeolin under control of the 35S promoter (orange bars) do not accumulate protein at levels above controls. B) Accumulation of total protein in peeled storage roots of pot grown transgenic plants expressing zeolin under control of the patatin promoter with time, as assessed by Bradford assay. Total protein content of non-transgenic cassava cv. 60444 (green bar) shows no increase over a 7 month cultivation period in the greenhouse, while the three transgenic events studied accumulated protein at slightly differing rates, but all reached approximately 10% dw by the end of the 7 month period. C) SDS-PAGE of crude protein extracted from leaves and roots of in vitro transgenic cassava lines expressing the zeolin transgene. Forty micrograms of protein extract were loaded as follows: lane 1: protein ladder; lanes 2 and 3: non-transgenic cassava cv. 60444 leaves and roots respectively; lanes 4 and 5: leaves and roots of transgenic cassava line transformed with pILTAB601 in which 35S drives expression of zeolin; and lanes 6 and 7: leaves and roots transgenic cassava line transformed with pILTAB600 in which the patatin promoter drives expression of zeolin. Presence of distinct band at 65 kDa in transgenic plants, but not in the control, indicates accumulation of zeolin in leaves of plants transgenic for 35S-zeolin and roots of plants transgenic for patatin-zeolin, but not vice versa. D) Western blot analysis of leaves (L) and peeled storage roots (R) harvested from greenhouse soil beds at 7 months of age. Total protein was isolated, 100 µg loaded in each lane and detected with specific anti-zein antibodies. Presence of zeolin protein was detected in all tissues, but accumulated at 3.5 times greater amounts in roots when expressed under the control of patatin versus the 35S promoter with the reverse pattern seen in leaves. E) Immuno-printing of 50–100 µm thick sections of cassava storage roots from transgenic line pILTAB600-25 in which the patatin promoter drives zeolin. Plants were grown in pots in the greenhouse and harvested from one to sevens months of age with non-transgenic cv. 60444 used as a control (top right panel). Presence of zeolin is clearly seen in roots of all ages in the transgenic, but not the control root sections, with significant accumulation in the core (xylem parenchyma) as well as the outer peel layer. Bars = 1 cm.