J . Sci. Food Agrie. 1982, 33, 419420

The Purification of Immature Barley Endosperm

Michael O’Dell and Richard D. Thompson

Cytogenei’ics Department, Plant Breeding Institute, Maris Lane, Cambridge CB2 2LQ

(Manuscript received 8 May 1981)

A procedure for the efficient and rapid isolation of 100-g quantities of immature endosperm has been developed using a roller device. The material produced has been used successfully for the isolation of messenger ribonucleic acid and deoxyribonucleic acid.

1. Introduction

The devt:loping endosperm in the Hordeue is the site of the regulated expression of many genes affecting grain quality.

The molecular biology of this tissue is currently receiving intensive scrutiny. One objective is the characterisation of mRNAs and their regulation.

In barley (Hordeurn vulgure), mRNAs encoding storage proteins are in highest concentration 14-24 days after anthesis.l At this time, the endosperms may be harvested by squeezing them out of the husk and fused pericarp/testa after manual dissection of grains from a spike.

The accumulation of sufficient endosperms for mRNA isolation using this method is laborious and inefficient. This paper describes a small mangle which greatly accelerates the operation.

2. Method and discussion

The roller apparatus consists of two 20 cm long hard rubber photographic rollers (J. Blishen, 75 Kilburn Lane, London WlO), fixed parallel, in contact with each other, in an aluminium frame

Figure 1. The endosperm mangle. 40 mm

0022-5142/82/0500-0419 $02.00 0 1982 Society of Chemical Industry 419

420 M. O’Dell and R. D. Thompson

(Figure 1). A handle attached to the lower roller rotates the upper roller by friction when turned. Immature ears, on a short stalk, are fed through the mangle stalk-first, by slowly turning the handle. This motion compresses the grain and expels the endosperms to either side of the ear, which passes through. The released endosperms are collected by being swept with a sterilised 2.5 cm paint brush along the rollers through a recess in the frame and into a flask of liquid nitrogen. Endosperms are recovered from the liquid nitrogen with a vegetable sieve and can be stored frozen at - 80°C for up to 1 year without any apparent deterioration in the quality of the mRNA.

The apparatus shown in Figure 1 has proved most satisfactory for isolating endosperms from two-row barley varieties. Wheat (Triricurn aestivurn) endosperms are more difficult to harvest efficiently because of the arrangement of grains in opposite spikelets on the spike. However, the apparatus has been modified by incorporating a cog-drive to prevent slippage of the rollers and screws to adjust the pressure applied. These modifications increase extraction efficiency and make this design more suitable for wheat.

Studies of biochemical components of immature cereal endosperms demand large quantities of freshly harvested tissue. The technique described here has been used to accumulate several batches in excess of lOOg which have been used as the source of mRNA for in-vitro translation2 and cDNA cloning (Thompson, R. D.; Forde, B., unpublished).

Acknowledgements This research was supported by the Agricultural Research Council (UK). The authors thank P. Bavister for contructing the mangles.

References 1 .

2.

Shewry, P. R.; Pratt, H. M.; Leggatt, M. M.; Miflin, B. J. Protein metabolism in developing endosperms of high-lysine and normal barley. Cereal Chern. 1979, 56, 1 1 0 - 1 17. Matthews, J. A,; Miflin, B. 5. In vitro synthesis of barley storage proteins. Planra 1980, 149, 262-268.