Agrobacterium rhizogenes-mediated transformation of Hypericum sinaicum L. for the development of hairy roots containing hypericin

Hypericum sinaicum L. is an endangered Egyptian medicinal plant of high importance due to the presence of naphthodianthrones (hypericins), which have photodynamic properties and pharmaceutical potential. We sought to assess H. sinaicum ability to develop hairy roots that could be cultured in contained conditions in vitro and used as a source for hypericin production. We used four A. rhizogenes strains differing in their plasmids and chromosomal backgrounds to inoculate excised H. sinaicum root, stem and leaf explants to induce hairy root development. Additionally, inoculum was applied to shoots held in Rockwool cubes supporting their stand after removal of the root system. All explant types were susceptible to A. rhizogenes although stem explants responded more frequently (over 90%) than other explant types. The A4 and A4T A. rhizogenes strains were highly, and equally effective in hairy root induction on 66-72% of explants while the LBA1334 strain was the most effective in transformation of shoots. Sonication applied to explants during inoculation enhanced the frequency of hairy root development, the most effective was 60 s treatment doubling the percentage of explants with hairy roots. However, shoot transformation was the most effective approach as shoots developed hairy roots within 10 days after inoculation. Molecular analyses confirmed that the established hairy root cultures in vitro were indeed obtained due to a horizontal gene transfer from bacteria. These cultures grew fast and the hypericin content in hairy roots was about two fold higher than in H. sinaicum plants as determined by HPLC.

Comparison of haploid and doubled haploid sugar beet clones in their ability to micropropagate and regenerate

Background: Haploid plant material is considered as recalcitrant to organogenesis, propagation, and maintenance in vitro. However, sugar beet (Beta vulgaris L.) breeders utilizing doubled haploid (DH) technology in their breeding programs indicate that sugar beet haploids may be cultured in vitro as well as diploids. Thus in this paper the in vitro performance of haploid and the doubled haploid sugar beet of various origin was evaluated. The DHs were derived from haploids by diploidization and twelve such haploid and corresponding DH clone pairs were obtained thus the comparison included haploid and DH clones that had identical allelic composition and differed only in their ploidy level. Results: The genotypes differed in shoot morphology and susceptibility to blackening during culture in vitro, but no significant differences were observed between the haploids and DHs. The micropropagation rate was, on average, higher for the haploids than DHs. Viability of the midrib and petiole explants after a 6-week culture was highly genotype dependent, but not affected by explant ploidy level. However, regeneration efficiency depended on both the genotype and ploidy level. The explants of several haploids regenerated more frequently and developed more adventitious shoots than the corresponding DHs thus overall efficiency was higher for haploids. Conclusions: The results obtained indicate that most of the haploids used in the comparison performed similar to or even better than DHs. This suggests that sugar beet haploid material can be successfully used not only for the production of DHs, but also maintained in vitro and utilized in projects requiring haploid tissues as the source material.