[Agricultural Production]

Start of experimental rearing of transgenic silkworms as ‘Type 1 Use’ in an isolated zone

Natuo Kômoto¹, Kenji Yukuhiro¹, Tetsuya Iizuka¹, Eiji Okada¹, Mai Tsuda², Yutaka Tabei³, Shuichiro Tomita¹, Makoto Kiuchi⁴, Hideki Sezutsu¹

¹Transgenic Silkworm Research Unit, ²Soybean Applied Genomics Research Unit, ³Research Promotion Section for Genetically Modified Organisms, ⁴Genetic Resources Conservation Research Unit

［Abstract］

We compared the mutation frequency in various Cas9, sgRNA constructs under the same experimental condition and established an efficient targeted mutagenesis system via CRISPR/Cas9 system in rice. Using the sgRNA designed on the conserved region, multiple paralogous genes were mutagenized by single sgRNA by on- and off-target cleavage. In addition, we revealed that extension of the culture period of rice calli expressing Cas9 and sgRNA is an effective approach for increasing mutation efficiency.

［Keywords］

Keywords: transgenic silkworms, Type 1 Use, biodiversity risk assessment, high function silk

［Background］

We are developing transgenic silkworms which produce silk with high value in order to establish new industries. However, introduction of genetically modified silkworm in sericultural farms requires ministerial approval after application with a biological diversity risk assessment report (Fig. 1). The silkworm, Bombyx mori, is an extremely domesticated animal, which is unable to survive or reproduce in nature. Appropriate assessment of the impact of possible crossing with a wild relative, B. mandarina, is expected. We reviewed the possibility of incidental crossing that may have occurred around sericultural farms. We have also started experimental rearing of transgenic silkworm producing green-fluorescent silk, with ministerial approval for the usage in an isolated zone in compliance with the ‘Type 1 Use’ of transgenic animals in Japan, in order to evaluate the impact on biodiversity.

［Results and Discussion］

1. The mitochondrial COI haplotypes of 3,633 B. mandarina moths collected in 37 areas in Japan from Hokkaido to Kumamoto are clearly different from those of 147 strains of B. mori, showing no signs of crossing between the two species.
   
2. The fifth-instar larvae of B. mori (800 in total) reared outdoor were completely captured by insects and birds and no cocoons were found.
   
3. There is no statistically-significant difference in walking distance of fifth-instar larvae between transgenic silkworms producing green-fluorescent silk and non-transgenic silkworms (Fig. 2A). The female moths of the transgenic strain laid eggs in smaller areas.
   
4. There is no statistically-significant difference in the impact of sericultural wastes on the germination and growth of broccoli and soil bacteria (Fig. 2B).
   
5. For the investigation of impact of transgenic silkworms on biodiversity in the condition of sericultural farms, we have started experimental rearing of silkworms producing green-fluorescent silk as ‘Type 1 Use’ in an isolated zone (Fig. 3). Monitoring around the zone yielded no hybrids between the transgenic silkworm and wild silkworm, B. mandarina.

1. The procedures presented in this study have made it possible to evaluate the impact of transgenic silkworm rearing on biological diversity from the perspective of behavioral characteristics and hazardous substance production. In addition, the data obtained by the experimental rearing in the isolated zone will encourage the approval of transgenic silkworm rearing in sericultural farms.

Fig. 1. Development of transgenic silkworms.

 

Fig. 2. Comparison of transgenic and non-transgenic silkworms. (A) At walking distance after 16 hours, there was no statistically significant difference between transgenic and non-transgenic silkworm larvae (thirty larvae each, 2nd day of 5th instar). (B) The effect on germination rate was analysed in broccoli seeds sown in soil mixed with larval body or feces of transgenic and non-transgenic silkworms. Five tests with 30 seeds were conducted and a statistically significant difference was not observed.

 

Fig. 3. Experimental rearing of transgenic silkworms which produce green-fluorescent silk for ‘Type 1 Use’ in the isolated zone. Left: Feeding silkworms with mulberry leaves. Right: Rearing house in the isolated zone.

 

[Reference]

1. Yukuhiro K, Iwata K, Kômoto N, Tomita S, Itoh M, Kiuchi M (2012) Nucleotide sequences of mitochondrial cytochrome C oxidase subunit I (COI) gene show clear differences between the domesticated silkmoth Bombyx mori and the wild mulberry silkmoth Bombyx mandarina from Japan Journal of Insect Biotechnology and Sericology 81 (1):29-35