Safflower, Carthamus tinctorius L. - Bioversity International

38 Safflower. Carthamus tinctorius L.
mediated transformations and regenerations of transgenic safflower are also being
undertaken, using the cultivar Centennial (Ying et al. 1992). Efficient callus formation
was achieved from cotyledon, stem and leaf explants. Shoot buds were regenerated
from 26% of leaf-derived calluses on callus induction medium. Transformation
and stable integration of transgenes was confirmed by the use of GUS assay
and DNA hybridization in kanamycin-resistant calluses and GUS assay in regenerated
shoots. A protocol was established for the transformation and regeneration of
safflower, based on co-cultivation of explants on induction medium, transferral to
shoot formation medium containing carbenicilin, followed by transfer to the same
medium containing kanamycin (Orlikowska et al. 1995). After regeneration of the
leafy structures, transfer to elongation medium containing geneticin follows; after
elongation, shoots are detached from the original explant tissue and transferred to
the same medium, with only transformed shoots remaining healthy and being transferred
to rooting medium. Root regeneration, while successful, is still at too low a
percentage for this system to be a practical breeding tool (J.W. Bergman, pers. comm.,
1996). In India, a protocol for easy and efficient regeneration of plantlets with welldeveloped
root systems was developed using the cultivars A-1 and Manjira
(Tejovathi and Anwar 1993). Cotyledons excised from 2- to 3-day-old seedlings and
cultured on MS medium supplemented with 0.1 mg NAA +0.5 mg
benzylaminopurine/L gave the greatest shoot bud formation, inducing 10-12 shoot
buds/explants. Transferring these regenerated shoots to the MS medium containing
1 or 2 mg 2,4,5-trichlorophenoxy propionic acid/L induced rooting.
In Freiburg, Germany, the team of U. Matern and R.E. Kneusel attempted to use
genetic engineering techniques for safflower to introduce resistance to leaf blight
caused by Alternaria spp. (Matern and Kneusel 1993). This group used molecular
methods to identify the macrolide, brefeldin A, as the phytotoxin from A. carthami
which suppresses the plant’s defence response and is thus identified as a virulence
factor of the fungal pathogen. The phytotoxin is inactivated in a one-step hydrolysis
by a strain of Bacillus subtilis, and incorporation of the DNA directing manufacture
of the enzyme responsible into the safflower genome was proposed as an effective
means to protect safflower from alternaria leaf spot disease. The gene was
identified, cloned and successful regenerations have been carried out using tester
strains of Agrobacterium tumefaciens, but efforts at transformations of safflower with
the isolated brefeldin A-esterase gene were not successful and the team has disbanded
(U. Matern, pers. comm., 1996).