• 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.

• Plant Biotechnology Reports
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• v.5 no.3
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• pp.245-254
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• 2011

This study describes an efficient protocol for Agrobacterium tumefaciens-mediated transformation of two subgroups of genotype AAA bananas (Musa acuminata cv. Pei Chiao and Musa acuminata cv. Gros Michel). Instead of using suspension cells, cauliflower-like bud clumps, also known as multiple bud clumps (MBC), were induced from sucker buds on MS medium containing $N^6$-Benzylaminopurine (BA), Thidiazuron (TDZ), and Paclobutrazol (PP333). Bud slices were co-cultivated with A. tumefaciens C58C1 or EHA105 that carry a plasmid containing Arabidopsis root-type ferredoxin gene (Atfd3) and a plant ferredoxin-like protein (pflp) gene, respectively. These two strains showed differences in transformation efficiency. The EHA105 strain was more sensitive in Pei Chiao, 51.3% bud slices were pflp-transformed, and 12.6% slices were Atfd3-transformed. Gros Michel was susceptible to C58C1 and the transformation efficiency is 4.4% for pflp and 13.1% for Atfd3. Additionally, gene integration of the putative pflp was confirmed by Southern blot. Resulting from the pathogen inoculation assay, we found that the pflp transgenic banana exhibited resistance to Fusarium oxysporum f. sp. cubense tropical race 4. This protocol is highly advantageous to banana cultivars that have difficulties in setting up suspension cultures for the purpose of quality improvement through genetic transformation. In addition, this protocol would save at least 6 months in obtaining explants for transformation and reduce labor for weekly subculture in embryogenic cell suspension culture systems.

• 한국생물공학회:학술대회논문집
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• 2000.04a
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• pp.75-77
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• 2000

Somatic embryoginic cells of valuable medicinal plants were cultured in MS (Murashige and Skoog) liquid medium by subculture at 2 week intervals. The embryogenic cells could be proliferated with maintenance of identical embryogenesis. The cell clumps developed to somatic embryos of uniform sizes of torpedo stage after $4{\sim}5$ weeks of culture. The culturing for a period about $10{\sim}15$ days led the somatic embryos to the development of seedlings which could be utilized as materials for health foods or providing useful components.

• Journal of Plant Biology
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• v.39 no.1
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• pp.71-77
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• 1996

Embryogenic callus was selected from callus induced from hypocotyl segment cultures of Daucus carota seedlings on MS basal medium supplemented with 1.0 mg/L 2,4-D. Cell clumps prepared from the embryogenic callus were transferred to MS medium without 2,4-D for somatic embryo development. Cotyledonary abnormalities were frequently observed on somatic embryos developed after the incubation of cell clumps in MS basal medium with 1.0 mg/L 2,4-D for one week and then subculture in the same medium but without 2.4-D for two weeks. The percentage of abnormalities was as follows: 5% one cotyledon, 21% three cotyledons, 6% four cotyledons, 5% five cotyledons, 0.2% six cotyledons and 1% trumpet-like cotyledons. On the other hand, the normal somatic embryo with two cotyledons appeared at 63%. The germination rate of somatic embryos was higher in two cotyledon somatic embryos than in multicotyledonary embryos. Trumpet-like somatic embryos did not germinate normally showing limited elongation and enlargement of roots and cotyledons without shoot development. From anatomical examination circular procambium in the root of somatic embryo began to branch around the middle regions of the hypocotyl which extended into the cotyledons through the cotyledonary nodes and the number of branched procambial strands in hypocotyl was equal to the number of cotyledons. Monocotyledonous somatic embryo always had larger cotyledon than that of somatic embryos with multicotyledons and had horseshoe-like cotyledons where the procambium was of the same structure.ucture.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2003.04a
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• pp.61-62
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• 2003

Clonal propagation of high-value forest trees through somatic embryogenesis (SE) has the potential to rapidly capture the benefits of breeding or genetic engineering programs and to improve raw material uniformity and quality. A major barrier to the commercialization of this technology is the low quality of the resulting embryos. Several factors limit commercialization of SE for Corsican pine, including low initiation rates, low culture survival, culture decline causing low or no embryo production, and inability of somatic embryos to fully mature, resulting in low germination and reduced vigour of somatic seedlings. The objective was to develop a Corsican pine maturation medium that would produce cotyledonary embryos capable of germination. Treatments were arranged in a completely randomized design. Data were analyzed by analysis of variance, and significant differences between treatments determined by multiple range test at P=0.05. Corsican pine (Pinus nigra var. maritima) cultures were initiated on modified !P6 medium. Modifications of the same media were used for culture multiplication and maintenance. Embryogenic cultures were maintained on the same medium semi solidified with 2.5 g/l Gelrite. A maturation medium, capable of promoting the development of Corsican pine somatic embryos that can germinate, is a combination of iP6 modified salts, 2% maltose, 13% polyethylene glycol (PEG), 5 mg!l abscisic acid (ABA), and 2.5 g/l Gelrite. After initiation and once enough tissue developed they were grown in liquid medium. Embryogenic cell suspensions were established by adding 0.951.05 g of 10- to 14-day-old semisolid-grown embryogenic tissue to 9 ml of liquid maintenance media in a 250ml Erlenmeyer flask. Cultures were then incubated in the dark at 2022$^{\circ}$C and rotated at 120 rpm. After 2.53 months on maturation medium, somatic embryos were selected that exhibited normal embryo shape. Ten embryos were placed horizontally on 20 ml of either germination medium ($\frac{2}{1}$strength Murashige and Skoog (1962) salts with 2.5 g/l activated charcoal) or same medium with copper sulphate adjusted to 0.25 mg/1 to compensate for copper adsorption by activated carbon. 2% and 4% maltose was substituted by 7.5% and 13% PEG respectively to improve the yield of the embryos. Substitution of' maltose with PEG was clearly beneficial to embryo development. When 2% of the maltose was replaced with 7.5% PEG, many embryos developed to large bullet-shaped embryos. At latter stages of development most embryos callused and stopped development. A few short, barrel-shaped cotyledonary embryos formed that were covered by callus on the sides and base. When 4% of the maltose was removed and substituted with 13% PEG, the embryos developed further, emerging from the callus and increasing yield slightly. Microscopic examination of the cultures showed differing morphologies, varying from mostly single cells or clumps to well-formed somatic embryos that resembled early zygotic embryos only liquid cultures with organized early-stag. A procedure for converting and acclimating germinants to growth in soil and greenhouse conditions is also tested. Seedling conversion and growth were highly related to the quality of the germinant at the time of planting. Germinants with larger shoots, longer, straighter hypocotyls and longer roots performed best. When mature zygotic embryos germinate the root emerges, before or coincident with the shoot. In contrast, somatic embryos germinate in reverse sequence, with the cotyledons greening first, then shoot emergence and then, much later, if at all, the appearance of the root. Somatic seedlings, produced from the maturation medium, showed 100% survival when planted in a field setting. Somatic seedlings showed normal yearly growth relative to standard seedlings from natural seed.

• Korean Journal of Medicinal Crop Science
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• v.9 no.4
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• pp.310-317
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• 2001

The regenerated-bulblets placed in liquid free media resulted in good formation of roots and bulblets. On 1/4 MS free medium, roots and bulblets were predominantly induced. The 1/4 MS liquid medium supplemented with plant growth regulators was the best suitable condition for elongation of leaves and roots. Somatic embryos were frequently developed from embryogenic callus in liquid media with 2,4-D 1mg/ l . On free liquid media, the viability of callus reduced. As the salt strength of MS media reduces, the viability of callus reduced significantly. However, Leaves were induced from several callus clumps. When leaves, roots and bulb-scale segments were placed on MS media containing NAA 1mg/ l or 2,4-D 1mg/ l and various sucrose concentration, the best result about the differentiation, growth of leaf and the differentiation of leaf was obtained on MS media added 1.5% sucrose and 2,4-D 1mg/ l, 3% sucrose and NAA 1mg/ l, and 1.5% sucrose and NAA 1mg/ l, respectively. Also the better result differentiation, growth of root and differentiation of bulb was obtained on MS media with 6% sucrose and NAA 1mg/ l. Spermidine promoted the growth of leaf and the differentiation of bulb. However, spermine promoted the differentiation of leaf, the differentiation and the growth of root in MS solid media. On the MS liquid media, both spermine and spermidine stimulated organogenesis from bulb-scale segments. Regenerated plantlets were acclimatizated and grown in greenhouse in vermiculite + perlite (1 : 1 by volume) well. The optimal soil condition of rooting for plantlets regenerated was in peat moss.