Regeneration and Agrobacterium-mediated transformation of multiple lily cultivars

Abstract

To pursue genetic improvement of lily, efficiency of both regeneration and transformation from callus cultures induced from different explants were evaluated in multiple cultivars. Thirty-five callus lines induced from filaments or styles and one control callus line derived from bulb scales of in total twenty lily cultivars representing Lilium longiflorum, Oriental 9 Trumpet and Longiflorum 9 Asiatic hybrids were maintained on a medium with 8.3 lM picloram (PIC). In this study, they were tested for their regeneration potential by transferring them onto a regeneration medium supplemented with 0.4 lM PIC and 0.044 lM 6-benzyladenine. Regeneration was obtained in all cultivars examined and the percentage varied from zero to 89 % in the 36 callus lines. Regeneration frequency was significantly influenced by the genotype (cultivar). Subculturing the calli every 4 weeks by refreshing the regeneration medium contributed positively to bulblet formation, when compared to an eight week subculture frequency. It was found that the regeneration ability generally decreased with an increasing age of the callus cultures for all cultivars. The origin of the callus (style or filament) did not lead to significant differences in regeneration frequency, but there was an interaction between callus origin and genotype. Calli of eight randomly chosen cultivars were co-cultivated with Agrobacterium tumefaciens strain AGL0 carrying binary vectors with the gus gene as reporter and putative transgenic plants were produced. GUS histochemical assays demonstrated transient and stable expression of the gus gene in both calli and regenerated lily plants. Transient expression frequencies ranged from 0.3 to 20.6 % while stable transformation was much lower, only 1.4 % as the maximum.

Agrobacterium-mediated transformation of embryogenic cell suspension cultures and plant regeneration in Lilium tenuifolium oriental × trumpet ‘Robina’

“…In most lily hybrids, the explants used for embryogenic callus induction have usually been taken from floral organs, bulb scales (Hoshi et al 2004;Wang et al 2012), leaves and seeds . We are aware of no research in Lilium species that has been done on stem axis explants.…”

“…For successful production of genetically modified lily plants, one prerequisite is the establishment of a transformation protocol based on the regeneration and transformation competence of cells (Wang et al 2012). Plant cell suspensions are becoming the favored model system for this due to their properties of straightforward transformation, rapid cell proliferation and good growth characteristics (Santos-Ballardo et al 2013).…”

Expression of a cystatin transgene can confer resistance to root lesion nematodes in Lilium longiflorum cv. ‘Nellie White’

“…Generally, the transformation efficiency of ornamental lilies is quite low making it very laborious to generate a population of transgenic plants, and this may be one reason why only a few transformation studies have used lilies. Wang et al (2012) compared the Agrobacterium-mediated transformation efficiencies of eight ornamental lily cultivars, and the highest was 1.4 % for callus regenerated from filaments of the cultivar 'Santander'. Azadi et al (2010Azadi et al ( , 2011 found a low 0.5 % transformation efficiency but were able to engineer expression of seven carotenoid genes.…”

Optimization of a protocol for the production of transgenic lily plants via particle bombardment

“…Structure of the T-DNA region of pCAMBIA3301:AtSIZ vector provided by Genomine INC (Pohang, Korea) (NPTII (neomycin phosphotransferase); NOS: nopaline synthase promoter; TNOS: 3' signal of nopaline synthase; 35S: Cauliflower Mosaic Virus (CaMV) 35S promoter 수행되어왔다 (Mercuri et al 2003;Nam and Kim 2004;Hoshi et al 2004;Ogaki et al 2008;Azadi et al 2010;Liu et al 2011;Wang et al 2012). 한편 유전자총을 이용한 형질전환 나리식물체 개발에 관한 연구에서는 1998년 최초로 Watad et al (1998) (Hoshino et al 2008;Kim et al 2007;Lin et al 2000), 히야신스 (Popowich et al 2007), 심비디움 (Roh et al 2011 and2013), 팔레놉시스 (Roh et al 2014)나 튤립 (Wilmink et al 1992) 등과 비교하면 형질전환연 구가 비교적 많이 수행되었음을 알 수 있다.…”