• Journal of Plant Biotechnology
• /
• v.47 no.2
• /
• pp.164-171
• /
• 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
• /
• v.37 no.4
• /
• pp.511-516
• /
• 2010

The process to acquire intron-GUS gene-expressed transformants from somatic embryos (including embryogenic calli) of Rosa hybrida cv. 'Sweet Yellow' using Agrobacterium-meditated transformation method was reported in this study. Somatic embryos including embryogenic calluses were infected with Agrobacterium tumefaciens AGL1 strain (O.D = 0.7~1.6) including intron-GUS gene for 30 min, and were co-cultured for 3 days. After co-cultivation, they were cultured on embryo germination medium (EGM) supplemented with $250\;mg{\cdot}L^{-1}$ cefotaxim at $4^{\circ}C$ for 7 days. Then, transient GUS gene expression was observed. Shoots were regenerated from the shoot primodia induced from the intron-GUS gene-transferred either somatic embryos or embryogenic calli cultured on EGM supplemented with both cefotaxim $250\;mg{\cdot}L^{-1}$ and ppt $2\;mg{\cdot}L^{-1}$. Before induction of rooting from shoots cultured on shoot growing medium supplemented with both cefotaxim $250\;mg{\cdot}L^{-1}$ and ppt $2\;mg{\cdot}L^{-1}$, the shoots were cultured on multi-shoot induction medium supplemented with both cefotaxim $250\;mg{\cdot}L^{-1}$ and ppt $2\;mg{\cdot}L^{-1}$ to induce multi-shoots. When expression of the gene from a part of the multi-shoots was identified by GUS transient assay, the putative transgenic multishoots were transferred to rooting medium supplemented with cefotaxim $250\;mg{\cdot}L^{-1}$. After the formation of healthy roots, transgenic plantlets were transferred to the greenhouse after acclimatization. The expression rate of the intron-GUS gene in the multi-shoots was 100%.

• Journal of Plant Biotechnology
• /
• v.39 no.3
• /
• pp.196-204
• /
• 2012

This study was carried out to develop reliable systems for somatic embryogenesis in oil palm tree (Elaeis guineensis Jacq.), and to verify the somaclonal variants by RAPD analysis. Embryogenic callus was induced successfully on modified half-strength MS medium containing $NaH_2PO_4{\cdot}2H_2O$ and casein. Embryogenic callus was further developed to somatic embryo mass (SEM), which is very hard and bonded tightly each other. Plantlets were proliferated when SEM was cultured on modified MS medium containing half strength $NH_4NO_3$, casein and L-ascorbic acid. Plantlets were transplanted into pots containing artificial soils. When RAPD analysis was conducted using randomly selected 95 in vitro plantlets and 19 random primers, somaclonal variation was detected using BNR35 primer. There was missing band around 1 kb in #22, #28, #35, and #77 plantlets. In addition, bands obtained from #28, #35, and #77 was much stronger than other normal bands. The blast results at NCBI revealed that somaclonal variation observed in this study was related to chloroplast genome of oil palm. The results also revealed that oil palm reproduction system through somatic embryogenesis is quite reliable and early detection of somaclonal variants seem to be possible at in vitro stage by RAPD analysis.

• KSBB Journal
• /
• v.7 no.3
• /
• pp.216-221
• /
• 1992

The physiological changes of somatic embryogenesis in cultured pepper cells (Capsicum annuum L. cv Shinhong) were investigated. The somatic embryogenesis was induced by cultivating the callus in hormone-free MS medium. The peroxidase patterns in the somatic embryogenic cells and the culture medium was revealed three and two of cathodic and anodic bands by isoelectric focusing respectively. Activity of peroxidase released into culture medium was 4 times higher than that of 12th day cultured cells. At the heart stage, the isozyme patterns of the MDH and esterase were found to be changed, which were showed by starch gel electrophoresis. It means these isozymes can be used as markers for studying somatic embryogenesis and differentiation.

• Journal of Ginseng Research
• /
• v.38 no.3
• /
• pp.220-225
• /
• 2014

An efficient in vitro protocol has been established for somatic embryogenesis and plantlet conversion of Korean wild ginseng (Panax ginseng Meyer). Wild-type and mutant adventitious roots derived from the ginseng produced calluses on Murashige and Skoog (MS) medium supplemented with 0.5 mg/L 2,4-dichlorophenoxyacetic acid and 0.3 mg/L kinetin; 53.3% of the explants formed callus. Embryogenic callus proliferation and somatic embryo induction occurred on MS medium containing 0.5 mg/L 2,4-dichlorophenoxyacetic acid. The induced somatic embryos further developed to maturity on MS medium with 5 mg/L gibberellic acid, and 85% of them germinated. The germinated embryos were developed to shoots and elongated on MS medium with 5 mg/L gibberellic acid. The shoots developed into plants with well-developed taproots on one-third strength Schenk and Hildebrandt basal medium supplemented with 0.25 mg/L 1-naphthaleneacetic acid. When the plants were transferred to soil, about 30% of the regenerated plants developed into normal plants.

• Journal of Plant Biotechnology
• /
• v.29 no.3
• /
• pp.189-192
• /
• 2002

An efficient plant regeneration system of major cultivars of sweetpotato (Ipomoea batatas (L.) Lam.) in Korea via somatic embryogenesis was established. Embryogenic calli were formed from shoot apical meristems of sweetpotato cultivars when cultured on LS medium supplemented with 1 mg/L auxin (2,4-D, picloram, dicamba). Among three kinds of auxin, 1 mg/L 2,4-D showed the highest embryogenic calli induction rate. After 4 weeks of cultures on LS medium supplemented with 1 mg/L 2,4-D, embryogenic calli induction rates of Sinhwangmi, Zami, Yulmi, and White Star were 86%, 78%, 76%, and 80%, respectively. Upon transfer onto LS basal medium, most of somatic embryos developed into plantlets. Regenerated plantlets were transplanted to potting soil and grown to mature plants in a greenhouse.

• Plant Biotechnology Reports
• /
• v.2 no.3
• /
• pp.179-189
• /
• 2008

We established an efficient plant regeneration system for Catharanthus roseus L. (G.) Don through somatic embryogenesis. Embryogenic callus was induced from hypocotyl of seed germinated in vitro. Somatic embryogenesis in Catharanthus has been categorized into three distinct stages: (1) initiation and proliferation of embryo; (2) maturation, and; (3) germination or plantlet conversion. Beside plant growth regulators, various stages of embryogenesis were screened for their response to a wide variety of factors (pH, gelrite, light, sugar alcohols, polyethyleneglycol and amino acids), which affect embryogenesis. All of the tested factors had a small to marked influence on embryogeny and eventual conversion to plantlets. The plantlets were acclimatized successfully in a greenhouse. To our knowledge, this is the first report describing a detailed study of various cultural factors which regulate embryogenesis in C. roseus. The results discussed in this paper may be used in mass propagation to produce medicinal raw material, and the embryo precursor cells could be used in genetic modification programmes that aim to improve the alkaloid yield as well.

• Korean Journal of Plant Resources
• /
• v.10 no.1
• /
• pp.86-93
• /
• 1997

In order to induce somatic embryogenesis from the stem explants and anther of Kalanchoe daigremontiana, the explants were cultured on MS medium supplemented with auxin (2,4-D, IAA, NAA) and/or cytokinin (BAP) for 8 weeks. Callus from explants was induced most efficiently on MS medium containing. 2.0mg/L NAA and 0.2mg/L BAP. Somatic embryogenesis in stem callus was formed by transfering embryogenic callus from induction media containing growth regulators to medium without growth regulators and then to the medium containing auxin and cytokinin (0.1 mg/L IAA and 1.0mg/L BAP). Callus formation occurred actively in the anthers at early uninucleate stage, and by low temperature pretreatment at $4^{\circ}C$ for 3days. Somatic embryogenesis from the anther callus was induced on MS medium containing 1.0mg/L NAA and 1.0mg/L BAP, 2.0mg/L NAA and 0.2mg/L BAP. The tetraploid of 5.4% was obtained among plants regenerated from anthers.

• Journal of Plant Biotechnology
• /
• v.50
• /
• pp.89-95
• /
• 2023

In order to investigate the effect of carbon sources on somatic embryogenesis and cotyledon number variations in carrot, embryogenic callus were cultured in the medium supplemented with various concentrations of sucroseor glucose, and with combination of 2% sucrose and various concentrations of mannitol or sorbitol. The maximum number of somatic embryos formed per flask (1,555.70) was obtained in the medium supplemented with 2% sucrose rather than glucose alone or a combination of mannitol or sorbitol and 2% sucrose, and the number of somatic embryos was decreased with the increasing of mannitol or sorbitol concentration. The frequencies of somatic embryos with two cotyledons were 35.14% for sucrose, 19.88% for glucose, 32.55% for mannitol + 2% sucrose, and 38.59% for sorbitol + 2% sucrose, respectively, and the frequencies of abnormal somatic embryos having 3 or more cotyledons were 64.86% for sucrose, 80.12% for glucose, 67.44% for mannitol + 2% sucrose, and 61.41% for sorbitol + 2% sucrose, respectively. Particularly, the frequency of somatic embryos with two cotyledons (59.16%) was the highest in the 2% sucrose medium compared to the frequency of abnormal somatic embryogenesis with three or more cotyledons, and the frequency gradually decreased with increasing concentration of glucose, mannitol or sorbitol. According to these results, it was found that the ratio of abnormal somatic embryo was higher than the normal somatic embryo in carrot, and was shown that somatic embryogenesis and the cotyledon number was affected by the concentrations of sucrose, glucose as carbon source, and mannitol and sorbitol as osmotic agents in culture medium.

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