Somatic embryogenesis and in vitro plant regeneration from various explants of the halophyte Leymus chinensis (Trin.)

Yan Lin Sun,홍순관 한국식물생명공학회 2009 식물생명공학회지 Vol.36 No.3

The halophyte Leymus chinensis (Trin.) is a perennial rhizome grass (tribe Gramineae) that is widely distributed throughout China, Mongolia and Siberia. This study was conducted to investigate an optimal condition for plant regeneration from mature seeds, leaf base segments, and root segments in L. chinensis. Plant growth regulators affecting embryogenic callus induction and plant regeneration were investigated by four-factor-three-level [L9 (34)] orthogonal test in this study. The effects of explants types (mature seeds, leaf base segments and root segments), callus types, medium types were examined in this study. Wild type (WT) and Jisheng No. 1 plants (JS) were used for primary callus induction. A clear explants difference was seen during callus induction; mature seeds were considered as the preferred explants; and the highest frequency of callus induction was obtained in Medium 6 using mature seeds as explants in WT. Plant regeneration ability was evaluated by frequencies of green callus forming, shooting, rooting, and shooting with roots. Effect of α-naphthalene acetic acid (NAA) on shoot regeneration was remarkable with the highest frequency of 70.8% in WT after 2-month culture. The medium with 0.2- 0.5 mg/L NAA was found to have the highest shoot induction. All regenerated shoots were successfully rooted when transferred on half-strength Murashige and Skoog (MS) basal medium. The acclimatized plantlets were grown to mature with flowering and seeds setting in green house conditions.

Genetic diversity of Kalopanax pictus populations in Korea based on the nrDNA ITS sequence

Yan-Lin Sun,Hak-Bong Lee,김남영,박완근,홍순관 한국식물생명공학회 2012 식물생명공학회지 Vol.39 No.1

Kalopanax pictus is a long-lived deciduous perennial tree in the family Araliaceae mainly distributed in the East Asia. In Korea, this species is of ecological and medical importance. Because typical populations of this species are small and distributed in patches, K. pictus has been considered as a narrow habitat species. To understand the genetic diversity and population structure of this species, the sequence variation of the nuclear ribosomal DNA (nrDNA) internal transcribed spacer (ITS) region was analyzed among 18 different K. pictus populations in the present investigation. The nrDNA ITS sequences of Korean populations investigated in this study showed identical of 616 bp in length, and no any nucleotide variation was found in the entire nrDNA ITS region sequence. This result suggested that the K. pictus populations in Korea might belong to the same isolate, and no mutation was found in the nrDNA ITS region. Compared with other known ITS sequence sources from K. pictus populations, only four variable nucleotide sites were found within the entire ITS region. Very narrow genetic diversity appearing in the population level of K. pictus makes us hypothesize that their relatively isolated habitats. The long-lived traits might be one main reason. However, another probability was that the nr- DNA ITS region might be noneffective in classifying populations of K. pictus. Thus, to further understand the phylogenetic relationship of K. pictus populations, more samplings should be performed based on more DNA sequences.

Molecular identification of medicinal herbs, Oldenlandia diffusa and Oldenlandia corymbosa based on nrDNA ITS region sequence

Yan-Lin Sun,Dong Wang,Myung-Hun Yeom,Duck-Hee Kim,Han-Gon Kim,홍순관 한국식물생명공학회 2011 식물생명공학회지 Vol.38 No.4

The medicinal herb Oldenlandia diffusa is known as a folk medicine for the treatment of hepatitis, sore throat, appendicitis, malignant tumors and urethral infection in Southern China and Korea. Another species O. corymbosa,is also used for the therapy of the similar conditions,however, only O. diffusa is referred to the medicinal herb by Chinese Pharmacopoeia. Due to their similar morphology,O. diffusa and O. corymbosa are often misidentified. To easily identify O. diffusa from O. corymbosa, the phylogenetic utility of nuclear ribosomal DNA (nrDNA) internal transcribed spacers (ITS) were investigated among different O. diffusa and O. corymbosa populations in Korea. The nrDNA ITS sequence of O. diffusa contained 791 bp, with GenBank accession number of JF837601-JF837602. The nrDNA ITS sequence of O. corymbosa was 785-786 bp, with GenBank accession number of JF837603-JF837611. The results showed that there are some certain divergences in the ITS region sequence between both species, even among different populations of the same species. Particularly, O. corymbosa ST-4 population showed the highest dissimilarity of the ITS region sequence with other nine populations of O. corymbosa and two populations of O. diffusa. This consequence makes us further understand the molecular diversification between O. corymbosa and O. diffusa, and help to promote the correct use and safety.

Establishment of a novel plant regeneration system from suspensionderived callus in the halophytic Leymus chinensis (Trin.)

Sun Yan-Lin,홍순관 한국식물생명공학회 2010 JOURNAL OF PLANT BIOTECHNOLOGY Vol.37 No.2

The establishment of cell suspension culture and plant regeneration of the halophytic Leymus chinensis (Trin.) are described in this study for the first time. Callus induction solid medium containing Murashige and Shoog (MS) basic salt, 2.0 mg l-1 2,4-dichlorophenoxyacetic acid (2,4-D), and 5.0 mg l-1 L-glutamic acid with 30.0 g l-1 sucrose and 4.0 g l-1 gelrite for solidification induced the highest rate of cell division in Type 1 callus among calli of various types. Liquid medium with the same hormone distribution was therefore, used for cell suspension culture from Type 1 callus. Over a 30 d suspension culture at 100 rpm, great amounts of biomass were accumulated, with 71.07% average daily increment and 22.32-fold total fresh weight increment. Comparison of before and after suspension culture, the distribution of different size callus pieces and the maintenance of callus type were basically unaltered, but a slight increase in relative water contents was observed. To induce the potential of plant regeneration, the directly transferring on plant regeneration solid medium containing MS basic salt, 0.2 mg l-1 α-naphthalene acetic acid (NAA), 2.0 mg l-1 kinetin (Kn), and 2.0 g l-1 casamino acid and indirectly transferring were simultaneously performed. Even now growth rates of suspension-derived callus on solid medium were approximately half of those of Type 1 callus, but faster somatic embryogenesis was observed. Rooting of all regenerated shoots was successfully performed on halfstrength MS medium. All plants appeared phenotypically normal.

Establishment of a novel plant regeneration system from suspension-derived callus in the halophytic Leymus chinensis (Trin.)

Sun, Yan-Lin,Hong, Soon-Kwan The Korean Society of Plant Biotechnology 2010 식물생명공학회지 Vol.37 No.2

The establishment of cell suspension culture and plant regeneration of the halophytic Leymus chinensis (Trin.) are described in this study for the first time. Callus induction solid medium containing Murashige and Shoog (MS) basic salt, $2.0\;mg\;l^{-1}$ 2,4-dichlorophenoxyacetic acid (2,4-D), and $5.0\;mg\;l^{-1}$ L-glutamic acid with $30.0\;g\;l^{-1}$ sucrose and $4.0\;g\;l^{-1}$ gelrite for solidification induced the highest rate of cell division in Type 1 callus among calli of various types. Liquid medium with the same hormone distribution was therefore, used for cell suspension culture from Type 1 callus. Over a 30 d suspension culture at 100 rpm, great amounts of biomass were accumulated, with 71.07% average daily increment and 22.32-fold total fresh weight increment. Comparison of before and after suspension culture, the distribution of different size callus pieces and the maintenance of callus type were basically unaltered, but a slight increase in relative water contents was observed. To induce the potential of plant regeneration, the directly transferring on plant regeneration solid medium containing MS basic salt, $0.2\;mg\;l^{-1}$ $\alpha$-naphthalene acetic acid (NAA), $2.0\;mg\;l^{-1}$ kinetin (Kn), and $2.0\;g\;l^{-1}$ casamino acid and indirectly transferring were simultaneously performed. Even now growth rates of suspension-derived callus on solid medium were approximately half of those of Type 1 callus, but faster somatic embryogenesis was observed. Rooting of all regenerated shoots was successfully performed on half-strength MS medium. All plants appeared phenotypically normal.

Genetic diversity and phylogenetic analysis of genus Paeonia based on nuclear ribosomal DNA ITS sequence

Yan-Lin Sun,홍순관 한국식물생명공학회 2011 식물생명공학회지 Vol.38 No.3

The genus Paeonia belongs to the family Paeoniaceae having significant medicinal and ornamental importance. The present investigation was undertaken with an aim to understand phylogenetic relationships of three Paeonia species (P. lactiflora, P. obovata, and P. suffruticosa) that are widely distributed in China, Korea, and Japan, using nuclear ribosomal DNA (nrDNA) internal transcribed spacer (ITS) sequence and to compare the phylogeny results with investigations reported earlier using existed sequences of the same species. The size variation obtained among sequenced nrDNA ITS region was narrow and ranged from 722 to 726 bp. The highest interspecific genetic distance (GD) was found between P. lactiflora and P. suffruticosa or P. obovata. The phylogram obtained using our nrDNA ITS sequences showed non-congruence with previous hypothesis of the phylogeny between section Paeonia and section Moutan of genus Paeonia. This result was supported by the phylogenetic relations showed in the phylogram constructed with existed sequences in NCBI. The present study suggested that P. obovata belonging to section Paeonia was phylogenetically closer to P. suffruticosa representing section Moutan of genus Paeonia than P. lactiflora belonging to section Paeonia. The main reason of the paraphyly of section Paeonia is thought to be nucleotide additivity directly caused by origin hybridization. This study provides more sequence sources of genus Paeonia, and will help for further studies in intraspecies population, and their phylogentic analysis and molecular evolution.

Exogenous proline mitigates the detrimental effects of saline and alkaline stresses in Leymus chinensis (Trin.)

Sun, Yan-Lin,Hong, Soon-Kwan The Korean Society of Plant Biotechnology 2010 식물생명공학회지 Vol.37 No.4

Proline accumulates in plants under environmental stresses including saline stress and alkaline stress. Here, we investigated the responses to two different stresses, saline stress (200 mM NaCl) and alkaline stress (100 mM $Na_2CO_3$) in two Leymus chinensis (Trin.) genotypes, LcWT07 and LcJS0107, and effects of exogenous proline on the activities of antioxidant enzymes. Both saline stress and alkaline stress significantly induced the accumulation of proline in leaves of the two genotypes after 96 h, and alkaline stress caused a transient and significant increase in LcJS0107 plants at 6 h. A reduction in the activities of catalase (CAT, EC 1.11.1.6) and ascorbate peroxidase (APX, EC 1.11.1.11), but not in the activity of superoxide dismutase (SOD, EC 1.15.1.1), was detected in plants exposed to saline and alkaline stresses. Remarkable decrease in relative water contents (RWC) was found in 144 h stressed plants. However, lipid peroxidation estimated by malonyldialdehyde (MDA) content in leaves remained relatively stable. With the addition of exogenous proline, it did not cause changes of proline levels in two genotypes, but combined with saline or alkaline stress, the exogenous application of proline significantly induced proline accumulation after even short treatment periods. Combined with salt stress, the exogenous application also increased the activities of CAT and APX. These results indicated that exogenous proline not only increases proline levels in vivo as a osmotic adjustment under stress, but mitigates the detrimental effects of saline and alkaline stresses by increasing the activities of antioxidant enzymes.

Somatic embryogenesis and in vitro plant regeneration from various explants of the halophyte Leymus chinensis (Trin.)

Sun, Yan Lin,Hong, Soon-Kwan The Korean Society of Plant Biotechnology 2009 식물생명공학회지 Vol.36 No.3

The halophyte Leymus chinensis (Trin.) is a perennial rhizome grass (tribe Gramineae) that is widely distributed throughout China, Mongolia and Siberia. This study was conducted to investigate an optimal condition for plant regeneration from mature seeds, leaf base segments, and root segments in L. chinensis. Plant growth regulators affecting embryogenic callus induction and plant regeneration were investigated by four-factor-three-level [L9 (34)] orthogonal test in this study. The effects of explants types (mature seeds, leaf base segments and root segments), callus types, medium types were examined in this study. Wild type (WT) and Jisheng No. 1 plants (JS) were used for primary callus induction. A clear explants difference was seen during callus induction; mature seeds were considered as the preferred explants; and the highest frequency of callus induction was obtained in Medium 6 using mature seeds as explants in WT. Plant regeneration ability was evaluated by frequencies of green callus forming, shooting, rooting, and shooting with roots. Effect of α-naphthalene acetic acid (NAA) on shoot regeneration was remarkable with the highest frequency of 70.8% in WT after 2-month culture. The medium with 0.2- 0.5 mg/L NAA was found to have the highest shoot induction. All regenerated shoots were successfully rooted when transferred on half-strength Murashige and Skoog (MS) basal medium. The acclimatized plantlets were grown to mature with flowering and seeds setting in green house conditions.

Establishment of Cell Suspension Cultures and Plant Regeneration in White Dandelion (Taraxacum coreanum NAKAI.)

Sun, Yan-Lin,Kim, Jae-Hak,Hong, Soon-Kwan The Plant Resources Society of Korea 2011 한국자원식물학회지 Vol.24 No.3

In this study, we established a novel somatic embryogenesis and plant regeneration system through cell suspension culture of white dandelion (Taraxacum coreanum NAKAI.). Embryogenic calli could be initiated from leaf and root explants of sterile seedlings on solid Murashige and Skoog (MS) medium supplemented with 1.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) after 3-week cultures. To proliferate embryogenic calli rapidly, cell suspension culture was performed with transferred to liquid MS medium with various combinations of plant growth regulators (PGRs) including 2,4-D, ${\alpha}$-naphthalene acetic acid (NAA), indole-3-acetic acid (IAA), $N^6$-benzylamino purine (BAP), thidiazuron (TDZ), and kinetin. During suspension cultures, embryogenic calli not only greatly proliferated, but shoot organogenesis also simultaneously occurred from the surface of somatic embryos. Among them, TDZ at lower concentration, 0.1 mg/L produced the highest efficiency of somatic embryo formation and shoot organogenesis. Rooting of embryogenic calli with adventitious shoots was done on solid MS medium containing 0.1 mg/L NAA and 0.3% activated carbon. Nearly 80% of embryogenic calli with shoot organogenesis could be rooted normal. Well-rooted plantlets were transferred into pots under a greenhouse condition, and plants derived from this system appeared phenotypically normal.

Genetic Diversity and Phylogenetic Relationships between Chinese Cabbages [B. campestris (syn. rapa) L.] and Cabbages (B. oleracea L.) in Korea

Sun, Yan-Lin,Zheng, Shi-Lin,Park, Kyong-Cheul,Choi, Ki-Young,Kang, Ho-Min,Hong, Soon-Kwan Korean Society of Horticultural Science 2016 원예과학기술지 Vol.34 No.2

Members of the genus Brassica, which are known as oil crops or cruciferous vegetables, are widely cultivated in Canada, Australia, Asian and Europe. Because Brassica species have high yields, are well adapted to their environments, and are self-incompatible, the germplasm is abundant. Previous studies have reported abundant genetic diversity even within Brassica subspecies. In Korea, fresh cabbage leaves are eaten with roast meat, and to meet the current popular demand, new varieties are being increasingly bred. To determine the genetic diversity and relationships among the cabbage vegetables in Korea, we evaluated the genetic variation of 18 accessions based on 5S and 18S ribosomal RNA (rRNA) gene sequences. We detected many variable nucleotide sites, especially in the 5S rRNA gene sequences. Because the length of the 18S rRNA gene might influence the dissimilarity rate statistics, we used both the 5S and 18S sequences to analyze the phylogenetic relationships. S7 (B. oleracea) showed the most distant phylogenetic relationship with the other Brassica species. Interestingly, B2 (B. oleracea), B15, and B18 (B. campestris) have three different types of leaf profiles, and were divided into one group, and the other Brassica species formed another group. Statistical analysis of interspecies and intraspecies genetic distances revealed that B. campestris L. showed higher genetic diversity than B. oleracea L. This work provides additional data that facilitates the evaluation of the genetic variation and relationships among Brassica species. The results could be used in functional plant breeding programs to improve Brassica crops.