• Journal of Plant Biotechnology
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• v.32 no.2
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• pp.123-128
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• 2005

A series of experiments were performed to establish regeneration system through friable embryogenic callus (FFC) of Lilium longiflorum 'Nellie White'. Only hard and regular callus was induced from bulb scales on medium containing 2.0 mg/L dicamba and $30{\sim}90$ g/L sucrose. The induced hard callus was subcultured on medium with 2.0 mg/L dicamba and 30 g/L sucrose, and used as a material for induction of FEC. In order to induce FEC, induced hard and regular callus was chopped into $1{\sim}2\;mm$ segments, and re-cultured on medium with 2.0 mg/L dicamba and 90 g/L sucrose. FEC was induced from chopped hard calli by the subcultures of two months interval. The induction rate of FEC was enhanced when hard callus was subcultured on same medium. FEC was proliferated more than 5 times on medium with $1.0{\sim}2.0\;mg/L$ dicamba and 90 g/L sucrose. Bulblet differentiation from FEC was very favorable on MS medium supplemented with 0.1 mg/L BA, 1.0 mg/L NAA and 30 g/L maltose, but many differentiated bulblets were changed to vitrificated ones. The differentiation of normal bulblets was most effective on medium containing $0.5{\sim}1.0\%$ activated charcoal and 30 g/L sucrose.

• The Journal of the Convergence on Culture Technology
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• v.7 no.3
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• pp.605-608
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• 2021

Lily is one of the most important 5 cut flowers in international flower market and lilies are distributed in Asia, Eurasia and North America. To develop a new lily cultivar, in addition to hybridization, mutation and selection methods, biotechnological techniques including tissue culture are also required. Establishment of tissue culture system is one of the requirement for the breeding program in Lily. Among many fields of plant tissue culture, establishment of regeneration system via embryogenic calluses are studied in many crops. In this study, research was carried out to decide the proper concentration of picloram which is used for the induction of embryogenic calluses. As a result, 3 different types of callused were observed after 3-4 weeks. They were CEC (compact embryogenic callus), FEC (friable embryogenic callus) and white callus type. 1.0 mg /l of picloram showed the best result for the production of embryogenic callus, however, due to its higher rate of browning in this concentration, 0.75 mg/l of picloram was selected as a proper concentration of picloram for the induction of CEC and FEC in Lily. These results can be contributed to the establishment of both regeneration system and mass propagation in lily in the future.

• Journal of Plant Biotechnology
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• v.47 no.2
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• pp.164-171
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• 2020

Transgenic Alstroemeria plants resistant to Alstroemeria mosaic virus (AlMV) were generated through RNA-mediated resistance. To this end, the friable embryogenic callus (FEC) of Alstroemeria was induced from the leaf axil tissue and transformed with a DNA fragment containing the coat protein gene and 3'-nontranslated region of AlMV through an improved particle bombardment system. The bar gene was used as a selection marker. More than 300 independent transgenic FEC lines were obtained. Among these, 155 lines resistant to phosphinothricin (PPT) were selected under low stringent conditions. After increasing the stringency of PPT selection, 44 transgenic lines remained, and 710 somatic embryos from these lines germinated and developed into shoots. These transgenic shoots were then transferred to the greenhouse and challenged with AlMV. In total, 25 of the 44 lines showed some degree of resistance. PCR analysis confirmed the presence of the viral sequence. Virus resistance was observed at various levels. Establishment of an efficient transformation system for Alstroemeria will allow inserting transgenes into this plant to confer resistance to viral and fungal pathogens. Accordingly, this is the first report on the production of a transgenic virus-resistant Alstroemeria and lays the foundation for alternative management of viral diseases in this plant.

• Journal of Plant Biotechnology
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• v.50
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• pp.11-18
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• 2023

Manihot esculenta Crantz, commonly known as cassava, is a staple aliment that is a significant source of revenue for farmers. The embryogenic callus is crucial in the genetic engineering of various crop species, including cassava. Four cultivar cassava landraces from East Java were assessed for their ability to produce friable embryogenic callus (FEC) for protoplast isolation. In this study, four cassava cultivars; (Kaspro, Kuning, Gajah, and Gendruwo); were used to obtain FEC, which involved the culture of immature leaf lobes (ILLs) and apical buds (ABs) media containing MS supplemented with 33 μM picloram and 2 μM of CuSO₄ (M1) or MS supplemented with 50 μM 2,4-D and 2 μM CuSO₄ (M2). The highest FEC induction efficiency ranged from 72% to 57%, and the highest FEC number ranged from 4.7 to 3.7 with AB explants in media containing MS + 33 μM pilocram and 2 μM CuSO₄ (M1). On the other hand, the efficiency of somatic embryogenesis induction ranged from 67% to 53%, and the number ranged from 4.4 to 3.4. The efficiencies of FEC induction ranged from 48% to 42%, and the number ranged from 3.1 to 2.6 with AB explants in media containing MS + 50 μM 2,4-D and 2 μM CuSO₄ (M2); the efficiency of FEC induction ranged from 56% to 50%, and the value ranged from 3.6 to 2.4 with ILL explants. The FEC induction of the Gendruwo cultivar, which was examined using AB and ILL explants, demonstrated the lowest efficiency. Nevertheless, all four cultivars showed the ability to generate FEC, even though their effectiveness differed depending on the explant genotype and the applied media.

• Journal of Plant Biotechnology
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• v.47 no.1
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• pp.66-72
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• 2020

Alstroemeria plants were transformed by using an improved particle-gun-mediated transformation system. Friable embryogenic callus (FEC) induced from the leaves with axil tissues of Alstroemeria plant was used as the target tissue. Also, FEC was transformed with the bar gene was used as a selectable marker. In the case of plasmid pAHC25, 7.5% of the twice-bombarded FEC clumps showed blue foci, whereas the clumps with single bombardment showed only 2.3%. Additionally, a 90° rotation with double bombardment led to a higher frequency (6 times) of luciferase gene expression in PBL9780 than the control treatment. After 8 weeks of bombardment, more than 60 independent transgenic lines were obtained for pAHC25 and nearly 150 independent transgenic lines were obtained for PBL9780, all of which were resistant to PPT and demonstrated either GUS or luciferase activity. Regarding effect of osmotic treatment (0.2 M mannitol) with 7 different periods, the highest transient gene expression was obtained in 8 h before and 16 h after transformation in both pAHC25 and PBL9780. Compared with the control, at least three times more GUS foci and photons were observed in this treatment. With respect to different combinations of mannitol and sorbitol with 8 h before and 16 h after transformation, high numbers of transient and stable transgene expressions were observed in both 0.2 M mannitol and 0.2 M sorbitol used in the osmotic pre-culture. This combination showed the highest transformation efficiency in both pAHC25 (8.5%) and PBL9780 (14.5%). In the control treatment, only 10% of the FEC clumps produced somatic embryos. However, by using 0.2 M mannitol and 0.2 M sorbitol, the frequency of somatic embryos increased to 36.5% (pAHC25) and 22.9% (PBL9780). Of the somatic embryos produced, at least 60% germinated. Approximately 100 somatic embryos from these 210 independent transgenic lines from 2 plasmids developed into shoots, which were then transferred to the greenhouse. PCR analysis confirmed the presence of the bar gene. This is the report on the production of transgenic Alstroemeria plants by using particle bombardment with a high efficiency, thereby providing a new alternative for the transferring of gene of interests in Alstroemeria in the breeding program in the future.

• Asian Journal of Turfgrass Science
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• v.18 no.3
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• pp.141-148
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• 2004

In this report, several factors such as infection time and concentration of bacterial suspension, influencing on transient gene expression in Agrobacterium-mediated transformation were evaluated. An appropriate concentration (O.D 600nm = 1.0-1.2) of bateria and 30 min of infection time showed a higher level of GUS expression. To improve transformation efficiency (TE), friable embryogenic calli (FEC) were bombarded by tungsten particles without plasmid DNA, and then co-cultivated with A. tumefaciens LBA4404 which contains pTOK233 super binary vector, carrying neomycin phosphotransferase (NPTII), hygromycin phosphotransferase (hpt) and$\beta-glucuronidase$ (GUS) genes. Three days after co-cultivation with A. tumefaciens and particle bombardment, FEC cultures were transferred to the selection medium (SM: MS medium supplemented with BA 1mg/l, hygromycin 100mg/l, cefotaxime 250 mg/l and vancomycin 200mg/l). They were cultured for 2 weeks and then transferred to the second SM containing hygromycin 50mg/l, cefotaxime 200 mg/l and vancomycin 100mg/l. Later, stable GUS expression was detected 4 to 6 weeks after transfer to the SM. Further, TE from Agrobacterium-mediated transformation after particle bombardment increased to about 3-folds compared with Agrobacterium-mediated transformation without particle bombardment. In the present study, we established an efficient transformation protocol of zoysiagrass by using A. tumefaciens in the combination with particle bombardment for the first time.