A highly efficient Agrobacterium-mediated transformation system for Lilium × formolongi was established by modifying the medium used for inoculation and co-cultivation. Meristematic nodular calli of Lilium were inoculated with an overnight culture of A. tumefaciens strain EHA101 containing the plasmid pIG121-Hm harboring an intron-containing β-glucuronidase (GUS), hygromycin phosphotransferase, and neomycin phosphotransferase II genes. The effects of ten different types of media and carbohydrates (sucrose, d-glucose, and l-arabinose) in both inoculation and co-cultivation media were evaluated. Interestingly, a dramatic increase in the frequency of transformation (25.4%) was observed when Murashige and Skoog (MS) medium containing sucrose and lacking KH2PO4, NH4NO3, KNO3, and CaCl2 was used. Hygromycin-resistant transgenic calli were obtained only in medium supplemented with sucrose. The effects of this modified medium were also investigated for Lilium cultivars ‘Acapulco’, ‘Casa Blanca’, and ‘Red Ruby’. The highest frequency of transformation (23.3%) was obtained for cv. Acapulco. Hygromycin-resistant calli were successfully regenerated into plantlets on plant growth regulator-free MS medium. Transgenic plants were confirmed by GUS histochemical assay, polymerase chain reaction (PCR), and Southern blot analyses.

Macro elements in inoculation and co-cultivation medium strongly affect the efficiency of Agrobacterium-mediated transformation in Lilium

Lilium spp. is a bulb flower with worldwide distribution and unique underground organs. The lack of an efficient genetic transformation system for Lilium has been an international obstacle. Because existing model plants lack bulbs, bulb-related gene function verification studies cannot be carried out in model plants. Here, two stable and efficient genetic transformation systems based on somatic embryogenesis and adventitious bud regeneration were established in two Lilium species. Transgenic plants and T-DNA insertion lines were confirmed by β-glucuronidase (GUS) assay, polymerase chain reaction (PCR) and Southern blot. After condition optimization, transformation efficiencies were increased to 29.17% and 4% in Lilium pumilum DC. Fisch. and the Lilium longiflorum ‘White Heaven’, respectively. To further verify the validity of these transformation systems and apply the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9) technology in Lilium, the LpPDS gene in the two Lilium species was knocked out. Completely albino, pale yellow and albino–green chimeric mutants were observed. Sequence analysis in the transgenic lines revealed various mutation patterns, including base insertion, deletion and substitution. These results verified the feasibility and high efficiency of both transformation systems and the successful application of the CRISPR/Cas9 system to gene editing in Lilium for the first time. Overall, this study lays an important foundation for gene function research and germplasm improvement in Lilium spp.

https://www.mdpi.com/1422-0067/20/12/2920/pdf

Establishment of Efficient Genetic Transformation Systems and Application of CRISPR/Cas9 Genome Editing Technology in Lilium pumilum DC. Fisch. and Lilium longiflorum White Heaven.

A review on factors affecting the Agrobacterium-mediated genetic transformation in ornamental monocotyledonous geophytes

Slow-growing compact calluses were initiated from bulb scales of Lilium longiflorum cv. Nellie White that had been cultured for at least 6 months on Murashige and Skoog (MS) medium with 9 μM dicamba. To develop a reliable selection system, the sensitivity of nontransformed calluses and in vitro plants to different selective agents such as phosphinothricin, kanamycin, geneticin, paromomycin, and hygromycin was tested when grown on MS medium. Nontransformed calluses showed high sensitivity to 0.5 mg·L -1 phosphinothricin, 25 mg·L -1 geneticin, and 5 mg·L -1 hygromycin. Nontransformed plants grown in vitro died on either 2 mg·L -1 phosphinothricin or 75 mg·L -1 hygromycin. Plants did not die when grown on either 200 mg-L ' kanamycin or 100 mg·L -1 geneticin, and 100 mg·L -1 paromomycin stimulated plant growth. Transformation was achieved using biolistics on callus bombarded with either the bar-uidA fusion gene under control of the CaMV 35S promoter or npt II and uidA under control of the CaMV 35S promoter. One week after biolistic bombardment, callus bombarded with the bar-uidA fusion gene was cultured for 1 month on MS medium supplemented with 9 μM dicamba and 0.1 mg·L -1 phosphinothricin and then transferred to 0.2 mg·L -1 phosphinothricin for 1 month followed by 1.0 mg·L -1 for the next 4 months. Regenerating shoots and well-established plants were cultured on MS medium lacking hormones and with either 0.2 mg·L -1 or 2.0 mg·L -1 phosphinothricin, respectively. Callus bombarded with the npt II gene was cultured on MS medium with 50 mg-L ' geneticin until shoots regenerated. Regenerated shoots were cultured on MS medium lacking hormones. Under optimal conditions, 10 transgenic plants were selected from seven plates of callus bombarded with the bar-uidA fusion gene using phosphinothricin for selection. Both Southern hybridization of genomic DNA and polymerase chain reaction analysis verified the presence of the transgene in transformed 'Nellie White' plants. Transgenic plants were phenotypically normal, and they were crossed with nontransformed plants of L. longiflorum cvs. Sakai, Yin tung, Sakai, and Flavo. The presence of the bar gene in 41% of the Tl progeny plants confirmed stable integration of the transgene into the genomic DNA of transgenic lily plants. β-glucuronidase expression resulting from the uidA gene was demonstrated in leaves and roots of some of the transgenic lily plants by histochemical staining, determination of the specific activity of the β-glucuronidase enzyme, and Northern hybridization.

/pdf/biolistic-mediated-transformation-of-lilium-longiflorum-cv-g3iq0lk7lq.pdf

Biolistic-mediated Transformation of Lilium longiflorum cv. Nellie White

Alternative methods of in vitro cloning that involve both adventitious (direct) and callus intermediate (indirect) pathways were investigated for the endangered species Lilium pumilum. Plantlet regeneration was obtained from leaf explants, cultured on Murashige and Skoog (MS) basal medium supplemented with various combinations of auxins and cytokinins at different concentrations. About 30% of the explants directly formed adventitious shoots on MS medium containing 8.88 μM 6-benzyladenine (BA) and 2.69 μM α-naphthaleneacetic acid (NAA). For production of regenerable callus, callus formation followed by shoot induction was best when explants were initially cultured on MS medium supplemented with 9.05 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Regenerable calli were yellow or purple and readily regenerated shoots when subcultured onto MS medium containing 2.22 μM BA and 1.61 μM NAA. About 78% of the calli were able to produce adventitious shoots. Shoots were rooted on half-strength MS medium supplemented with 1.34 μM NAA and were successfully acclimatized to greenhouse conditions. This report describes an efficient method for the in vitro multiplication of whole plants from leaf explants of the endangered species L. pumilum.

Direct and indirect shoot and bulblet regeneration from cultured leaf explants of Lilium pumilum, an endangered species

Genetic transformation mediated by bombardment with microscopic metal particles carrying target genes is the preferred method for the introduction of foreign genes into monocotyledonous plants. The fact that most flower bulbs are monocotyle-donous and that almost all commercial cultivars are propagated vegetatively makes them good candidates for molecular breeding through microprojectile bombard-ment. We report here on a development of a reliable method for an efficient genetic transformation of both Lilium longiflorum and Ornithogalum dubium using a particle inflow gun to deliver gene constructs into the target plant tissue, followed by a prolonged selection in the dark in liquid medium supplemented with kanamycin. The system was first optimized for Lilium longiflorum 'Snow Queen'. Based on the level of transient GUS expression, liquidgrown cell clumps are more competent than leaves. Large cell clusters (2-10mm) maintain their organogenic potential while smaller clusters (<2mm) cease to grow and die. The liquid-grown tissue cultures have a level of competence for transformation about 50-70 times greater than that of solid-grown callus cultures, and compact cell clusters are more competent than loose clusters. The cells were bombarded with a pCAMBIA2301 vector, carrying nptII gene conferring kanamycin resistance and GUS reporter gene. Following selection for 4-6 months in a liquid medium supplemented with 80 mg l -1 kanamycin in the dark, the cell clusters were transferred to a regeneration medium in the light where hundreds of transgenic plantlets developed. The plants retained their stable transgenic state when grown in the greenhouse for two seasons. The transformation of O. dubium was similar in principle to that of L. longiflorum with three major differences: lily liquid-grown cultures grew more rapidly and had a higher potential for somatic embryo development. Ornithogalum cultures under selection took longer to develop into semi-organized cell clumps of sufficient size to allow continued shoot regeneration, were mostly organogenic, and the regenerated plantlets had higher rate of vitrification.

/pdf/efficient-genetic-transformation-of-lilium-longiflorum-and-33bwgy8yfw.pdf

Efficient Genetic Transformation of Lilium longiflorum and Ornithogalum dubium by Particle Acceleration Followed by Prolonged Selection in Liquid Medium

T. Arazi

Papers

Efficient Genetic Transformation of Lilium longiflorum and Ornithogalum dubium by Particle Acceleration Followed by Prolonged Selection in Liquid Medium

Particle bombardment-mediated transformation of ornithogalum dubium for ornithogalum mosaic virus resistance

Molecular breeding of ornithogalum for erwinia resistance