# Rye sprout as a host for the rapid transient production of a recombinant protein using a geminivirus DNA-containing expression vector

## TL;DR

This paper explores using rye sprouts to quickly produce recombinant proteins, achieving yields comparable to other efficient plant hosts.

## Contribution

The study identifies rye sprouts as a viable alternative host for rapid recombinant protein production using geminivirus vectors.

## Key findings

- Raitaro and Ryokuhiyo rye sprouts produced up to 1.8 mg/g of EGFP, comparable to radish sprouts and N. benthamiana.
- Protein production was localized to leaf tips and stomatal guard cells, suggesting limitations in Agrobacterium entry or T-DNA transfer.
- Microneedling enhanced EGFP production at wounded sites, indicating potential for improving yield through physical methods.

## Abstract

The agroinfiltration technique using sprouts as a host is one of the most cost-effective, efficient, and rapid methods for producing recombinant proteins. We previously reported that radish sprouts were the best host for this purpose. To find suitable alternative sprouts comparable to radish sprouts, we investigated rye sprouts using a wheat dwarf virus (a geminivirus) DNA-containing expression vector. Various rye cultivars were tested, and Raitaro and Ryokuhiyo sprouts exhibited the highest enhanced green fluorescent protein (EGFP) productivity. When agroinfiltrated after a 5-d cultivation period, including 1 day of seed imbibition, approximately 1.8 mg of EGFP was produced per gram fresh weight of leaf in areas exhibiting EGFP fluorescence. This yield is comparable to that of mature leaves from Nicotiana benthamiana and radish sprouts. However, only a limited number of leaves produced the protein, and production was confined to areas near the leaf tips. Elevated production levels were observed in the guard cells of stomata and at wounded sites via microneedling, suggesting that the limiting factors for protein production may involve the entry of Agrobacterium into the leaves and/or the subsequent transfer of T-DNA into the plant cells.