The following procedure for isolating embryos exploits differences in both size and density between developing embryos and other tissues of the caryopsis. It requires about 30 minutes to recover immature (coleoptilar and older) embryos from 3 to 7 ears. Embryos prepared in this fashion have been used in our lab to study uptake of abscisic acid, and as a source of tissue for isolation of mRNA, enzymes, and pigments.
Husk leaves, tassels, and insect- or disease-damaged sections are cleaned from each ear of corn. These ears are rinsed in cold tap water and kept on crushed ice until shelled. With a serrated paring knife, each row of kernels is cut carefully at the base and lifted from the cob. The kernels may be collected in a beaker or released directly into the shallow pan which contains 800 ml of collection medium (20%, w/v, sucrose in 0.05M potassium phosphate buffer, pH 6.5 to 7.0). About 1500 kernels may be collected and processed at one time. While the shallow pan can accommodate twice as many kernels, the capacity of sieves (8" dia.) is only about 1500 kernels. Additional sieves could be used if additional capacity is required.
The kernels should be one layer thick in the pan. The shallow pan is 2 cm deep, with enough width to permit the rolling pin to easily rest along the edges and still leave a 2 to 3 min clearance between the bottom of the pan and the nearest surface of the rolling pin. This clearance is adequate for embryos up to 2 min in their largest dimension. For larger embryos, greater clearance can be achieved by adding suitable strips of wood to raise the edges of the pan appropriately.
The seeds are firmly crushed with a single pass of the rolling pin over the kernels. The entire solution is poured into the stacked sieves (stainless steel, USA standard testing #8 [2.36 mm], #18 [18 mm], #30 [0.60 mm], #80 [0.18 mm]) with largest pore size on top, decreasing in size to the smallest pore size at the bottom. The kernels are rapidly rinsed with cold tap water to wash through the embryos which were released from the kernels. The kernels are then returned to the shallow pan, mixed with 800 ml of fresh collection medium, and crushed again with the rolling pin. Several cycles are needed to open all kernels. It is important to remove the embryos from the broken kernels before crushing the kernels again as the rolling pin will damage the freed embryos. Rinsing the kernels in the stack sieves is best accomplished in a sink where excess solutions rapidly drain away.
After the last crushing, the kernels are thoroughly rinsed with cold tap water, with gentle stirring to ensure the passage of embryos into the lower sieves. Broken kernels retained on the largest pore sieve are discarded. Each subsequent sieve is rinsed until reaching the sieve which retains the majority of the embryos. With a gentle stream of water, trapped embryos and other tissues are moved to one side of the sieve and transferred to a large beaker (500 ml). The majority of the very light chaff is removed by filling the beaker with water and pouring off the chaff after the embryos sink.
The remaining tissue is primarily embryos and endosperm contaminated with a small amount of chaff and silks. This suspension is increased in sucrose concentration until density separation is achieved. Variations among inbred lines and age of embryo affect the exact density of embryos and endosperm tissue.
Once the sucrose density is reached for maximum separation of embryos and endosperm, the test tube is capped and placed horizontally for about 60 seconds to permit rapid separation of the tissues (Figure 1). Then the tube is very gently raised to a 45-degree position, cap end up. The tube is gently agitated to facilitate the migration of the tissues to their new positions. Finally, the tube is raised to a fully upright position. By starting in the horizontal position, there is minimal interference between the tissues as they move to their final positions. Embryos will float to the top of the tube and can be readily recovered by pouring that fraction into a petri dish.
For embryos to be used in physiological or ultrastructural studies which require intact embryos, a single flotation separation is adequate to remove the majority of nonembryonic tissues. This preparation then can be transferred to a petri dish and the intact embryos manually sorted from debris with a flat spatula or a pasteur pipette. Generally, about 70% of the embryos are intact with a typical preparation of stage one embryos (12 to 15 days after pollination). The efficiency of recovery of undamaged embryos increases as they develop beyond coleoptilar stages (3).
With very young embryos 5 to 9 days after pollination, it is necessary to use a dissecting microscope to pick out the embryos with a pasteur pipette. Since the differences in density between the embryos and endosperm are not as great in these younger kernels, the primary enrichment for these embryos occurs at the sieving steps. With embryos from mature and nearly mature caryopses with desiccated endosperm, it is very difficult to break the pericarp open without damaging the embryo with the rolling pin. This procedure is most effective for immature embryos when endosperm tissues are still soft and pliable.
Figure 1. Top: Embryos floating away from nonembryo tissues in sucrose solution. Bottom: Embryos isolated from commercial sweet corn; bar = 5 mm.
Franklin Fong and J. D. Smith
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