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

Sep 30, 2026

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Agroinfiltration using sprouts as hosts offers a highly cost-effective, efficient, and rapid route to produce recombinant proteins. Using a geminivirus wheat dwarf virus DNA-containing expression vector, rye sprouts were evaluated as alternatives to radish sprouts. Cultivars Raitaro and Ryokuhiyo achieved the highest EGFP productivity, producing about 1.8 mg EGFP per gram fresh weight in GFP-fluorescent areas after a 5-day cultivation (plus 1-day seed imbibition). Protein expression was limited to few leaves and mainly near tips, with elevated levels in stomatal guard cells and wounded sites, implying constraints in Agrobacterium entry and/or subsequent T-DNA transfer.

LanguageEnglish

CategoryHealthcare

FormatPDF

Pages9

Reading Time23 min

AccessFree to download

Introduction

Expression vectors and geminivirus DNA replication

Limitations of Nicotiana benthamiana cultivation

Rationale for using sprouts

Materials and Methods

Sprout selection criteria

Prior radish sprout findings

Agroinfiltration and harvesting schedule

Results

EGFP productivity across rye cultivars

Localization of protein expression

Plant Biotechnology 42, 449–457 (2025) DOI: 10.5511/plantbiotechnology.25.0515a Original Paper Rye sprout as a host for the rapid transient production of a recombinant protein using a geminivirus DNA-containing expression vector Sakihito Kitajima1, *, Shigeto Morita2,3, Kohki Natsuhara1 1 Department of Applied Biology, Kyoto Institute of Technology, Matsugasaki Sakyo-ku, Kyoto, Kyoto 606-8585, Japan; 2 Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Kyoto, Kyoto 606-8522, Japan; 3 Biotechnology Research Department, Kyoto Prefectural Agriculture, Forestry and Fisheries Technology Center, Seika, Kyoto 619-0244, Japan *E-mail: [sakito@kit.ac.jp](sakito@kit.ac.jp) Tel: +81-75-724-7791 Received March 5, 2025; accepted May 15, 2025 (Edited by M. Takemura) 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 ofT-DNA into the plant cells. Key words: agroinfiltration, amplicon, rye (Secale cereale), sprout, wheat dwarf virus. Introduction Mature leaves of Nicotiana benthamiana are widely recognized as effective hosts for the transient production of recombinant proteins, primarily due to their high infection and expression efficiencies with theagroinfiltration technique. The use of recently developed expression vectors containing geminivirus genomic DNA fragments significantly enhances protein production by enabling DNA replication following transfer into the plant nucleus via the Agrobacterium T-DNA system. This approach can yield up to 3.7 mg of enhanced green fluorescent protein (EGFP) per gram of fresh weight (FW) of leaf (Yamamoto et al. 2018; for other recent studies, see references cited in Kim et al. 2024) . A notable constraint of this system is that N. benthamiana requires approximately five weeks of growth after germination before agroinfiltration can be initiated. This extended cultivation period requires a substantial growing area and specialized equipment to enable continuous mass production of recombinant proteins. Using sprouts as a host in a transient production system significantly shortens the cultivation period, making it one of the most cost-effective, efficient, and rapid methods for producing recombinant proteins. The following criteria should be considered when selecting plant species as hosts for sprouts: they should demonstrate high gene transfer efficiency and protein productivity; there should be a large supply of seeds available year-round at a low cost; germination and subsequent growth should be rapid and synchronous; and the biomass produced per cultivated area should be high. We previously tested the sprouts of three species from the Fabaceae family, three species from the Brassicaceae family, and six cultivars of radish (Raphanus sativus). Our findings indicated that radish sprouts

FAQs

Why are rye sprouts evaluated as hosts for transient recombinant protein production?

Rye sprouts are assessed to find alternatives to radish sprouts that can deliver comparable transient production efficiency while using sprouts to shorten cultivation time.

Which rye cultivars showed the highest EGFP productivity?

Raitaro and Ryokuhiyo sprouts exhibited the highest enhanced green fluorescent protein (EGFP) productivity among the rye cultivars tested.

What factors are suggested to limit recombinant protein production in rye sprouts?

The study suggests limiting factors may involve the entry of Agrobacterium into leaves and/or the subsequent transfer of T-DNA into plant cells.

Sep 30, 2026

Uploaded by Bulrr