• Plant Biotechnology Reports
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• v.5 no.3
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• pp.265-271
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• 2011

The cultivated potato as well as its tuber-bearing relatives are considered model plants for cell and tissue culture, and therefore for exploiting the genetic variation induced by in vitro culture. The association between molecular stability and tissue culture in different genetic backgrounds and ploidy levels has already been explored. However, it still remains to be ascertained whether somaclonal variation differs between callus-derived chromosome-doubled and undoubled regenerants. Our research aimed at investigating, through amplified fragment length polymorphism (AFLP) markers, the genetic changes in marker-banding patterns of diploid and tetraploid regenerants obtained from one clone each of Solanum bulbocastanum Dunal and S. cardiophyllum Lindl (both 2n = 2x = 24) and tetraploids from cultivated S. tuberosum L. (2n = 4x = 48). Pairwise comparisons between the banding patterns of regenerants and parents allowed detecting considerable changes associated to in vitro culture both at diploid and tetraploid level. The percentages of polymorphic bands between diploid and tetraploid regenerants were, respectively, 57 and 69% in S. bulbocastanum and 58 and 63% in S. cardiophyllum. On average, the frequencies of lost parental fragments in regenerants were significantly higher than novel bands both in S. bulbocastanum (48 vs. 22%) and S. tuberosum (36 vs. 18%) regenerants. By contrast, in S. cardiophyllum, a similar incidence of the two events was detected (32 vs. 29%). Our results revealed that structural changes after tissue culture process strongly affected the genome of the species studied, but diploid and tetraploids regenerated plants responded equally.

• Korean Journal of Plant Resources
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• v.36 no.6
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• pp.588-596
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• 2023

Long-term culture of cell lines is an important issue in in vitro culture and in plant science. In this study, the regeneration ability and ex vitro acclimatization of regenerants were evaluated. The ploidy level of regenerants derived from long-term cultured cell lines was measured in Imperata cylindrica 'Rubra', Poaceae. Adventitious buds (shoots) were successfully induced from five-year-cultured calli on MS medium containing 0.1 mg/L BA or 2.0 mg/L TDZ, combined with 0.01 mg/L auxins (IAA, IBA, NAA and 2,4-D), respectively. Adventitious roots were also induced on MS medium containing 0.01 mg/L auxins (IBA, NAA and 2,4-D), respectively. Interestingly, regenerants with both red and green leaf were successfully obtained when regenerants were cultured on MS medium with 9% sucrose. Regenerants derived from long-term cultured calli were transferred to pots using an optimal acclimatization process and successfully adapted to both pot and soil conditions. Moreover, the ploidy level was measured using calli and regenerants that had been kept on MS medium containing various kinds of plant growth regulators (PGRs).

• Korean Journal of Plant Resources
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• v.36 no.5
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• pp.496-507
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• 2023

The in vitro organogenesis is one of important issues in plant embryology, and somaclonal variations are existing in calli and/or regenerants induced from a process of the organogenesis with in vitro circumstances. In this study, expressions of organogenesis-related genes were evaluated and genetic stability of regenerants derived from the process of in vitro organogenesis were measured using ISSR markers in Imperata cylindrica 'Rubra', Poaceae. The expressions of organogenesis-related genes were detected all of regenerants at the process of the organogenesis. All ISSR markers produced with an average of 71 bands per in vitro-cultured regenerants, and the scorable bands were varied from two to eight with an average of 5.14 bands per a primer. The polymorphism rates of the in vitro regenerants were higher than that of mother plants (1.4%), showing 4.1% (pot-cultured regenerants), 4.3% (field-cultured regenerants), 4.2% (in vitro-cultured regenerants), 5.6% (calli with green shoots) and 1.4% (calli), respectively. The genetic similarity matrix (GSM) among all accessions ranged from 0.747 to 1.0 with a mean of 0.868. GSM of the regenerants showed differences (from 0.972 to 1.00) compared with that of mother plants (0.991). According to the clustering analysis, two independent groups were divided into; the one is mother plants and regenerants cultured at room and open field, the other is regenerants cultured in vitro. The results give a new insight for understanding the dynamics of organogenesis in monocot plant.

• Korean Journal of Medicinal Crop Science
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• v.3 no.3
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• pp.207-216
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• 1995

This study was conducted to establish technologies for plug seedling production using somatic embryos-derived regenerants and their seeds in Bupleurum falcatum L. Among distilled water, GA (0.1mg/l) and putrescine (0.lmg/l) treated to regenerants for acclimatization, GA was most effective to develop shoots and roots, 1/2X MS medium and NAA 0.1mg/l + BA 0. 5mg/l enhanced the growth rates of the regenerants and increased dry weight. Activated charcoal effected to grow markedly leaves and roots of the regenerants at the level of 0.4 %. Regenerants increased their plant height, root length and dry weight at $30^{\circ}C$. Plug seedlings originated from seeds of the tissue culture regenerants showed the maxium growth on the mixture of peatmoss soil (2) and mountain sand (1) .Root length, leaf area and dry weight of plug seedlings increased significantly when No.1, 2 and 3 of Wondergrow solution were mixed in the ratio of 1.3 - 0.9 - 0.1. Light supplement (4%) and high tem­perature $(30^{\circ}C)$ promoted the growth of plug seedlings as well as dry weight. Ninety days seedlings were more vigorous and adaptable for transplanting than other seedlings.

• Journal of Plant Biotechnology
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• v.6 no.3
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• pp.157-163
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• 2004

An efficient system for the regeneration of adventitious shoots from in vitro cultured leaf sections of Lycium chinense Miller was developed and silent somaclones from the regenerants detected with RAPD method. Among the eight media tested (B5, SH, N&N, 1/2MS, MS, 3/2MS, GD and WPM), and four cytokinins (BA, kinetin, 2ip and zeatin) with different concentrations (1, 5, 10, 20, 30 and 40 $\mu{M}$), 1/2 MS medium supplemented with 20 and 30 $\mu{M}$ zeatin showed the best regeneration frequency (100% and 93.7%) and higher average number of shoots (9.0 and 9.4). All regenerants easily elongated after subculturing on 1/4MS without growth stimulants and produced spontaneous adventitious roots from their basal parts. With phenotypically normal 40 regenerants, RAPD analysis with 15 different random primers was performed to examine the cryptic somaclonal variants. No substantial differences in banding patterns were found in the amplified polymorphic DNAs implying no DNA changes during dedifferentiation into adventitious shoots. However, one (OPF-4) of the 15 primers detected silent somaclonal variation in one regenerant in which two different polymorphic bands did not appear when compared with the rest regenerants. The results indicate that regenerantion via intervening callus phase can be used to establish true-to-type planting stocks for homogeneous population.

• Plant Breeding and Biotechnology
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• v.6 no.4
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• pp.426-433
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• 2018

Plant tissue culture is a technique that has invariably been used for various purposes such as obtaining transgenic plants for crop improvement or functional analysis of genes. However, this process can be associated with a variety of genetic and epigenetic instabilities in regenerated plants, termed as somaclonal variation. In this study, we investigated mutation spectrum, chromosomal distributions of nucleotide substitution types of single-nucleotide polymorphisms (SNPs) and insertions/deletions (InDels) by whole genome re-sequencing between Dongjin and Nipponbare along with regenerated plants of Dongjin from different induction periods. Results indicated that molecular spectrum of mutations in regenerated rice against Dongjin genome ranged from $9.14{\times}10^{-5}$ to $1.37{\times}10^{-4}$ during one- to three-month callus inductions, while natural mutation rate between Dongjin and Nipponbare genomes was $6.97{\times}10^{-4}$. Non-random chromosome distribution of SNP and InDel was observed in both regenerants and Dongjin genomes, with the highest densities on chromosome 11. The transition to transversion ratio was 2.25 in common SNPs of regenerants against Dongjin genome with the highest C/T transition frequency, which was similar to that of Dongjin against Nipponbare genome.

• Korean Journal of Plant Resources
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• v.26 no.2
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• pp.320-327
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• 2013

This study was carried out to address an efficient in vitro regeneration system from seed-derived callus of Phragmites communis, and to evaluate genetic variations of the regenerants using ISSR markers. Shoot regeneration via calli was greatly influenced by N6 medium compared with MS medium, and plant regeneration frequency was 90% in N6 supplemented with BA 0.25 mg/L and BA 0.5 mg/L. According to ISSR analysis of the thirty regenerants, out of 94 loci detected overall, 16 were identified to be polymorphic with a rate (PR) of 17.0%. The mean gene diversity (h) of different in vitro condition was 0.03 and ranged from 0.008 for N6 with BA 5 mg/L, to 0.040 for MS with IAA 0.1 mg/L+kinetin 2 mg/L. The results indicate that the regenerants have a low genetic variation, and ISSR analysis is effective to detect genetic variation of regenerants.

• Proceedings of the Korean Society of Crop Science Conference
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• 2017.06a
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• pp.176-176
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• 2017

Hydropriming had positive effects on the time for germination to reach 50%, the germination index, the time to final germination percentage, and the number of uniform seedlings with enlarged cotyledons in in vitro germination of small watermelon. In addition, the highest shoot initiation was obtained from hydroprimed cotyledonary nodes ($95{\pm}6%$), followed by non-primed cotyledonary nodes ($78{\pm}6%$), hydroprimed cotyledons ($72{\pm}4%$), and non-primed cotyledons ($48{\pm}4%$). Meanwhile, no shoots were initiated from hypocotyls. The total number of shoots that initiated from cotyledonary nodes and cotyledon explants was insignificant, indicating that both cotyledons and cotyledonary node were good sources for the in vitro culture. Choosing explant sources that favor tetraploidy should be the key for producing higher polyploidy plants; a total of 10.5% of tetraploid regenerants were entirely identified from cotyledon explants. Cotyledons with highly differentiated cells might show higher variations than cotyledonary nodes with more preexisting meristematic cells. Cells of cotyledon tissue might undergo changes in ploidy level during differentiation of the culture, or it might be that some of the variations were already present in the tissues of the donor plant. Morphological changes in fruit length of tetraploid regenerants are genotype-dependent.

• Korean Journal of Plant Tissue Culture
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• v.27 no.2
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• pp.125-131
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• 2000

Protoplasts of Arabidopsis thaliana were easily isolated from the shoot-forming (SF) suspension-cultured cell clusters with 4 hours-shaking condition (40 rpm) on CPD enzyme solution containing 1% cellulase R-10, 0.25% pectolyase Y-23 and 0.5% driselase. Protoplasts were cultured on liquid KAO medium supplemented with 1 mg/L 2,4-D, 0.5 mg/L kinetin, 200 mg/L spermidine and 68 g/L glucose. Also, protoplasts were cultured on 0.2 $\mu$M membrane filter placed onto CP solid medium containing the suspension cells as feeder cells in the dark at $25^{\circ}C$ for 4 weeks. Protoplast-derived-SF calli were cultured on MS medium containing 0.05 mg/L IAA, 7 mg/L 2 ip and 30 g/L sucrose under the continuous illumination for four weeks. The frequency of shoot formation was about 60%. The regenerants were transferred into potting soil to grow mature plants. The regenerants formed the silques with seeds after 8 weeks of cultures.

• Journal of Plant Biotechnology
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• v.5 no.1
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• pp.25-31
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• 2003

As a consequence of tissue culture of rice, RAPD analysis was peformed to determine whether extended culture periods as undifferentiated calli affected the subsequent genetic constancy, and whether any resulting DNA rearrangements could be detected between sibling plants produced from the same callus. Somaclonal variation was induced at the initial stage of tissue culture and it increased with the length of culture maintenance. Of the 192 total bands, the number of polymorphic bands was 22 (11.5%), 33 (17.2%), and 49 (25.5%) in the callus of 1,3, and 6 months culture, respectively. A significantly higher level of genotypic polymorphisms between regenerants from two different somaclones was also detected, although all the regenerants were derived from a single genotype. In comparison of DNA polymorphisms between regenerants from non-irradiated and from irradiated calli, a scope of variation spectrum by gamma ray irradiation was larger than that by tissue culture. Consideration must be given to this genomic variation where attempts are to be made to use desirable somaclonal variants for plant breeding purpose and in genetic engineering program.