Establishment of regeneration and transformation protocols to create hypoalleregenic peanut (Arachis hypogaea) and mustard (Brassica juncea) through genome editing

Abstract

A stable and reproducible in vitro regeneration system is necessary for genetic engineering, however, explant responses vary widely between and within species and are highly dependent on the culture conditions. Despite decades of research, plant regeneration is still challenging especially with some plant species referred to as recalcitrant. In this study, we investigated the regeneration and genetic transformation capacities of different lines of peanut (Arachis hypogaea) that is considered recalcitrant and Brown mustard (Brassica juncea) in order to enable editing of the allergens Ara h 1 and Bra J I in peanut and mustard, respectively, via CRISPR/Cas9. Moreover, we demonstrated that, several of the first edited mustard lines displayed reduced or total absence of the Bra J I protein. Firstly, the factors affecting the adventitious shoot regeneration of both plant species were investigated using four peanut lines and six mustard lines from two geographical regions (Europe and India). In both species, shoot regeneration was significantly influenced by the explant type and the genetic make-up of the different lines tested. In mustard, the 5 days old cotyledon explants of all lines showed better responses than hypocotyls regarding adventitious shoot regeneration whereas in peanut the leaflet explants of 5-day-old seedlings exhibited superiority. The combinations of different types of cytokinins and auxins were tested on the explants of both plant species. In all peanut lines, the leaflet explants responded best on medium with 22.19 µM 6-benzylaminopurine (BAP) (+ 2.3 µM kinetin), which was reduced to 7.40 µM BAP after eight weeks, with regeneration rates of 10-89.1% and a mean shoot number per regenerating explant of 1-3.1 shoots. For mustard, the medium containing 8.88 µM BAP, 5.37 µM 1-naphthaleneacetic acid (NAA), and 9.95 µM Silver nitrate (AgNO3) resulted in the highest shoot regeneration rates (58-72% and 65-90% for the European and Indian lines, respectively) as well as the highest shoot numbers per regenerating explant (2.2-2.7 and 2.3-3.0). Furthermore, the effect of different light qualities on shoot regeneration from leaflet explants of the peanut lines was investigated in order to promote shoot induction and elongation. A strong effect of the culture temperature on the regeneration efficiency was observed as different light treatments were connected with different culture temperatures. However, red and blue LEDs could substitute tubular fluorescent lamps without affecting shoot regeneration. In the second step, Agrobacterium-mediated transformation of both plant species was investigated. Transgenic plants carrying large deletions of 566 up to 790 bp as well as indels in the targeted regions especially, indels in all four Bra j I alleles were obtained with the mustard lines tested. Seed viability was investigated in several transgenic mustard lines through in vitro and ex vitro germination. A decrease in seed viability and seed formation was observed in some edited lines, which indicated that the mutation of the major allergen Bra J I in mustard affected seed development. Part of the seeds exhibited aberrant phenotypes that resulted in the rupture of the testa already in the siliques. In contrast, the regenerated shoots from the different transformation experiments with various peanut lines exhibited a lack of transgenicity.