Genetic transformation of cauliflower (Brassica oleracea var. botrytis) by direct DNA uptake into mesophyll protoplasts

Abstract

Mesophyll protoplasts of Brassica oleracea var. botrytis were successfully transformed using polyethylene glycol (PEG). The success of plant transformation depended on both gene transfer and plant regeneration. Parameters, such as PEG and vector concentrations and heat shock conditions were tested in experiments on transient expression of the beta-glucuronidase (EC 3.2.1.31) gene and the most suitable conditions for DNA uptake were determined. Two antibiotic resistance marker genes for neomycin phosphotransferase (EC 2.7.1.95) and hygromycin phosphotransferase (EC 2.7.1.104), and three vector plasmids with different lengths were used to obtain stable transformants.

Nuclear and plastid transformation of Brassica oleracea var. botrytis (cauliflower) using PEG-mediated uptake of DNA into protoplasts

“…The absolute transformation frequency (ratio of resistant colonies/shoots per the number of treated protoplasts) ranged from (0.3 to 1.3) Â 10 À5 . This is lower than reported for cauliflower hypocotyl protoplasts [29,30] but much higher than previously obtained for cauliflower mesophyll protoplasts (5 Â 10 À7 [32]). A range of GUS expression levels was evident in calli and leaves from different transformants (Fig.…”

“…Nuclear transformation of cauliflower has been achieved via Agrobacterium (e.g. [27,28]) and direct DNA uptake into hypocotyl [29,30] or mesophyll protoplasts [31,32].…”

Regeneration from leaf explants and protoplasts of Brassica oleracea var. botrytis (cauliflower)

“…Kik and Zaal, 1993) and comparable to the broccoli cultivar Green Comet (Robertson and Earle, 1986). With a division frequency reliably around 70% for several cultivars including Thalassa, using the modified protocol in combination with vented vessels, we have a sound basis for improved transgenesis from protoplasts of cauliflower, compared to earlier studies (Radchuk et al, 2002;Nugent et al, 2006).…”

“…Bhalla and Smith, 1998), Agrobacterium rhizogenes (David and Tempé , 1988) and through direct DNA uptake into protocols from hypocotyls (e.g. Mukhopadhyay et al, 1991;Xue et al, 1997) or mesophyll cells (Radchuk et al, 2002;Nugent et al, 2006). Apart from transient expression data in broccoli (Puddephat et al, 1999) there are no reports of nuclear transformants of Brassica oleracea via biolistics.…”

Transformação genética de maracujá amarelo visando resistência à Xanthomonas axonopodis pv. passiflorae

“…Bluggoe (Sági et al, 1995), Agrostis palustris (Lin et al, 1997;Asano et al, 1998), Stylosanthes guianensis (Quecini et al, 2001;, Ipomoea batata (Okada et al, 2001) e Brassica oleracea (Radchuk et al, 2002 flavicarpa, (Manders et al, 1991;D'Utra Vaz et al, 1993;Dornelas et al, 1993) P. amethystina, P. cincinata, P. alata, P. giberti e P.…”