Castor bean (Ricinus communis L.) is an oilseed found worldwide and commonly cultivated in tropical and subtropical regions. India, Mozambique, China, and Brazil are the major producers. India is responsible for about 80% of the world’s production . Castor oil is the most important product, with great value in industry, especially to produce lubricants, medicines, and cosmetics, as it contains high amounts of ricinoleic acid, a viscous and highly stable fatty acid .
Castor cake is the by-product generated after oil extraction. It is mainly used as a fertilizer and soil conditioner. However, it could also be used in animal feed because it contains high amounts of protein and essential amino acids. Nevertheless, it is extremely toxic due to the presence of the highly toxic/allergenic proteins, ricin and RCA120 [3, 4]. Ricin is a highly toxic ribosome-inactivating protein (RIP) present in castor seeds’ endosperm, formed by two chains. Chain B is a lectin that binds to glycoproteins/glycolipids present on the cell surface and that allows ricin to enter animal cells. Chain A inactivates the ribosomes by depurination of one adenosine in the conserved loop on the 28 S rRNA subunit, resulting in cell death . RCA120 is a strong hemagglutinin, composed of two A chains and two B chains, highly similar to ricin (90 and 84%, respectively) .
The demand for castor products has increased, leading to the need for increased production/yield . Castor crop yield is affected by several factors, such as weed management, which is a challenge, since castor is very sensitive to competition and initially takes time to grow, in contrast to weeds, which grow faster . To control weeds in large crops, several agronomic practices must be taken into account. However, mechanical control is very expensive, and the use of herbicides is more efficient, especially if herbicides with a distinct mode of action are used [6, 7].
We generated an RNA interference-mediated ricin-silenced castor bean event, named TB14S-5D . The vector pRicRNAi used to generate the event contained an intron hairpin cassette to silence ricin and the gus gene (coding for a β-glucuronidase) and the mutated Atahas gene (coding for an acetohydroxyacid synthase, with a mutation at position 653 bp resulting in a serine to asparagine substitution), which confers tolerance to imidazolinone herbicides . The T1 generation showed an undetectable amount of ricin in seeds and no hemagglutination activity . In addition, seed protein extracts were not toxic to both IEC-6 cells and mice. T1 progeny also revealed high expression of gus and in vitro tolerance to imazapyr. This class of herbicides inhibits the activity of the AHAS enzyme, impairing the biosynthesis of isoleucine, leucine, and valine .
This work aims to evaluate the tolerance of the transgenic event TB14S-5D to imidazolinone, as well as the stability of ricin/RCA120 silenced, and gus expression in subsequent generations.