Identification of salt-induced transcripts by suppression subtractive hybridization and their expression analysis under the combination of salt and elevated CO<Subscript>2</Subscript> conditions in <Emphasis Type="Italic">Salicornia brachiata</Emphasis>

Soil salinity is a major abiotic stress that affects global agricultural productivity. Exploring the mechanisms that halophytes employ to thrive and flourish under saline environments is essential to increase the salt tolerance in sensitive crop species. Of the three halophytes used in this study Salicornia brachiata and Suaeda maritima belong to the same family Chenopodiaceae, while Sesuvium portulacastrum, a mangrove-associated halophyte, belongs to the family Aizoaceae. Assuming that halophytes of same family share similar salt tolerance mechanisms, we generated a suppression subtractive hybridization (SSH1) cDNA library from salt-treated leaf tissues of S. brachiata as tester and that of S. maritima as driver to identify salt-responsive genes unique to S. brachiata. To elucidate the difference in salt-tolerance mechanisms, and to identify salt-tolerance mechanisms amongst different families of halophytes, SSH2 library was generated from salt-treated leaf tissue of S. brachiata as tester and that of S. portulacastrum as driver. Totally, 87 and 49 EST clones representing unique genes were obtained from SSH1 and SSH2 libraries, respectively. Examination of the expression patterns of 17 (SSH1) and 15 (SSH2) differentially expressed genes using semi-quantitative RT-PCR confirmed up-regulation of these genes in shoots in response to salt treatment and elevated CO₂ condition, but to a different extent. This study has provided insights into the molecular responses of S. brachiata to salt stress and elevated CO₂ conditions.     

Cell culture system versus adventitious root culture system in Asian and American ginseng: a collation

Panax ginseng and Panax quinquefolius of Panax genus are valuable as health foods as well as pharmaceuticals for the treatment of cancer, diabetes and ageing as these plants possess saponins. In the current study, Cell and adventitious root cultures of P. ginseng and P. quinquefolius were investigated for the biomass, cell division, saponin content and ginsenosides profile from four lines namely P. quinquefolius (AM), P. ginseng mountain (Mt.) Baekdu line, P. ginseng Cheong-sol line (CS) and P. ginseng CBN line (CBN) with the objective of comparing cell and adventitious root systems to check their efficacy for the production of ginseng saponins. Additionally, genes related to ginsenoside biosynthesis were also analyzed concerning to cell and adventitious root lines. The results indicated that various cell lines were better in multiplication and growth compared to adventitious root lines. However, adventitious root lines showed higher accumulation of dry biomass (1.5–2 fold) than that of cell lines. CS adventitious root line showed higher saponin content and ginsenoside productivity (10.48 mg·g^?1 DW, 12.88 mg·L^?1, respectively) than that of CS cell line (9.50 mg·g^?1 DW, 2.39 mg·L^?1, respectively). Especially, Rd ginsenoside productivity of CS adventitious root line recorded fourfold higher than CS cell line. Genes which are related to ginsenoside biosynthesis such as P. ginseng squalene synthase (PgSS2), P. ginseng squalene epoxidase (PgSE2), P. ginseng protopanaxadial synthase (PgPPDS) and P. ginseng protopanaxatriol synthase (PgPPTS) were analyzed by real time quantitative polymerase chain reaction to support ginsenoside production. The adventitious root culture system described in this study is useful system for biomass and ginsenoside production.     

Diversity and antifungal activity of endophytic bacteria associated with <Emphasis Type="Italic">Panax ginseng</Emphasis> seedlings

Ginseng (Panax ginseng C.A. Meyer) is a medicinal crop that requires a long culture time before it is ready to harvest, thus generating high economic and environmental costs. Symbiotic bacteria that live within the plant provide the host plant with many advantages in terms of metabolism and disease resistance. Here, we isolated endophytic bacteria from various tissues of P. ginseng seedlings using a culture-dependent method and we compared their tissue distribution. In addition, their antimicrobial activity against two fungal pathogens was investigated. Based on 16S rRNA sequencing, we identified 21 bacterial strains from ginseng seedlings. Leaves and rhizomes showed higher bacterial species diversity than root bodies and tails. While Bacillus strains were detected in all tissues, Xanthomonas and Micrococcaceae strains were specifically isolated from rhizome and leaf tissues, respectively. Fourteen bacterial strains showed antimicrobial activities against Cylindrocarpon destructans and/or Botrytis cinerea, with different activities. Among them, two strains (PgKB29 and PgKB35) showed strong antimicrobial activities against both fungi. Taken together, these results provide a better understanding of endophytic bacteria in P. ginseng seedlings and suggest the possibility of biological control of fungal pathogens using endophytic bacteria.     

Age stages in the ontogeny of cultivated <Emphasis Type="Italic">Panax ginseng</Emphasis> C.A. Mey

The ontogeny of perennial polycarpic herb Panax ginseng C.A. Mey. (Araliaceae) under plantation conditions was described. Three periods (latent, pregenerative, and generative) and eight age stages have been identified in the ontogeny of cultivated P. ginseng. The generative period of this species is the longest ontogenetic period, which determines the timing of its cultivation in plantations.     

Development of interspecies hybrids to increase ginseng biomass and ginsenoside yield

Key message

Interspecific hybrids between Panax ginseng and P. quinquefolius results in hybrid vigor and higher ginsenoside contents.

Abstract

Ginseng is one of the most important herbs with valued pharmaceutical effects contributing mainly by the presence of bioactive ginsenosides in the roots. However, ginseng industry is impeded largely by its biological properties, because ginseng plants are slow-growing perennial herbs with lower yield. To increase the ginseng yield and amounts of ginsenosides, we developed an effective ginseng production system using the F₁ progenies obtained from the interspecific reciprocal cross between two Panax species: P. ginseng and P. quinquefolius. Although hybrid plants show reduced male fertility, F₁ hybrids with the maternal origin either from P. ginseng or P. quinquefolius displayed heterosis; they had larger roots and higher contents of ginsenosides as compared with non-hybrid parental lines. Remarkably, the F₁ hybrids with the maternal origin of P. quinquefolius had much higher ginsenoside contents, especially ginsenoside Re and Rb1, than those with the maternal origin of P. ginseng. Additionally, non-targeted metabolomic profiling revealed a clear increase of a large number of primary and secondary metabolites including fatty acids, amino acids and ginsenosides in hybrid plants. To effectively identify the F₁ hybrids for the large-scale cultivation, we successfully developed a molecular marker detection system for discriminating F₁ reciprocal hybrids. In summary, this work provided a practical system for reciprocal hybrid ginseng production, which would facilitate the ginseng production in the future.

     

A Comparative Analysis of Genetic Variability and Differentiation in <Emphasis Type="Italic">Panax vietnamensis</Emphasis> Ha et Grushv. and <Emphasis Type="Italic">P. ginseng</Emphasis> C.A. Meyer Using ISSR Markers

A comparative analysis of the genetic variability and differentiation of rare medicinal ginseng species, Panax vietnamensis Ha et Grushv. and P. ginseng C.A. Meyer, was carried out using inter-simple sequence repeat markers. It was demonstrated that all the genetic diversity parameters of Vietnamese ginseng were high and considerably exceeded those of P. ginseng. On the contrary, the level of genetic differentiation was higher in true ginseng. It is suggested that the differences in the levels of genetic variability and differentiation of the two ginseng species were influenced by the demographic history, peculiarities of the reproductive system, and human activity.     

Molecular characterization of 5-chlorophyll a/b-binding protein genes from Panax ginseng Meyer and their expression analysis during abiotic stresses

The chlorophyll _a/b-binding protein (CAB) serves in both photosystems (PS), I and II, as a coordinator of antenna pigments in the light-harvesting complex (LHC). The CABs constitute abundant and important proteins in the thylakoid membrane of higher plants. In our study, five CAB genes, which contained full-length cDNA sequences from the 4-year-old ginseng leaves (Panax ginseng Meyer), were isolated and named PgCAB. Phylogenetic comparison of the members of the subfamily between ginseng and higher plants, including Arabidopsis, revealed that the putative functions of these ginseng CAB proteins were clustered into the different family of Arabidopsis CABs; two PgCABs in LHCII family and three PgCABs in LHCI family. The expression analysis of PgCABs consistently showed dark-dependent inhibition in leaves. Expression analysis during abiotic stress identified that PgCAB genes responded to heavy metal, salinity, chilling, and UV stresses differently, suggesting their specific function during photosynthesis. This is the first comprehensive study of the CAB gene family in P. ginseng.     

Molecular characterization and expression analysis of <Emphasis Type="Italic">pathogenesis related</Emphasis> protein 6 from <Emphasis Type="Italic">Panax ginseng</Emphasis>

Panax ginseng Meyer is one of the important medicinal plants in the world, particularly in Asian countries. Ginseng encounters many stress exposure during its long cultivation period. However, the molecular mechanism of stress resistance is still poorly understood in spite of its importance. In this study, pathogenesis-related protein 6 (PR6), also called proteinase inhibitor (PI), was isolated from ginseng embryogenic callus, named PgPR6. The small size of PR6, containing an open reading frame of 219 bp encoding 72 amino acids, the typical characteristic of PR6 protein, shares the highest sequence similarity to PR6 of Theobroma cacao (69% identity). Sequence and structural analysis indicated that PgPR6 belongs to class Kunitz-type PI family. This is the first report pertaining to the identification of PR6 gene from the P. ginseng genome. The high-level expression of PgPR6 was observed in root as revealed by quantitative real-time PCR. The temporal expression analysis demonstrated that PgPR6 expression was highly up-regulated by signaling molecules, heavy metals, mechanical wounding, chilling, salt, sucrose, and mannitol stress, indicating that PgPR6 may play an important role in the molecular defense response of ginseng to a various range of environmental stresses.     

Integrated analysis of leaf morphological and color traits in different populations of Chinese cabbage (<Emphasis Type="Italic">Brassica rapa</Emphasis> ssp. <Emphasis Type="Italic">pekinensis</Emphasis>)

Key message

QTLs and candidate gene markers associated with leaf morphological and color traits were identified in two immortalized populations of Brassica rapa, which will provide genetic information for marker-assisted breeding.

Abstract

Brassica rapa is an important leafy vegetable consumed worldwide and morphology is a key character for its breeding. To enhance genetic control, quantitative trait loci (QTLs) for leaf color and plant architecture were identified using two immortalized populations with replications of 2 and 4 years. Overall, 158 and 80 QTLs associated with 23 and 14 traits were detected in the DH and RIL populations, respectively. Among them, 23 common robust-QTLs belonging to 12 traits were detected in common loci over the replications. Through comparative analysis, five crucifer genetic blocks corresponding to morphology trait (R, J&U, F and E) and color trait (F, E) were identified in three major linkage groups (A2, A3 and A7). These might be key conserved genomic regions involved with the respective traits. Through synteny analysis with Arabidopsis, 64 candidate genes involved in chlorophyll biosynthesis, cell proliferation and elongation were co-localized within QTL intervals. Among them, SCO3, ABI3, FLU, HCF153, HEMB1, CAB3 were mapped within QTLs for leaf color; and CYCD3;1, CYCB2;4, AN3, ULT1 and ANT were co-localized in QTL regions for leaf size. These robust QTLs and their candidate genes provide useful information for further research into leaf architecture with crop breeding.

     

Fungal endophytes of turmeric (<Emphasis Type="Italic">Curcuma longa</Emphasis> L.) and their biocontrol potential against pathogens <Emphasis Type="Italic">Pythium aphanidermatum</Emphasis> and <Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

Endophytic fungi have been isolated from the healthy turmeric (Curcuma longa L.) rhizomes from South India. Thirty-one endophytes were identified based on morphological and ITS–rDNA sequence analysis. The isolated endophytes were screened for antagonistic activity against Pythium aphanidermatum (Edson) Fitzp., and Rhizoctonia solani Kuhn., causing rhizome rot and leaf blight diseases in turmeric respectively. Results revealed that only six endophytes showed >?70% suppression of test pathogens in antagonistic dual culture assays. The endophyte T. harzianum TharDOB-31 showed significant in vitro mycelial growth inhibition of P. aphanidermatum (76.0%) and R. solani (76.9%) when tested by dual culture method. The SEM studies of interaction zone showed morphological abnormalities like parasitism, shriveling, breakage and lysis of hyphae of the pathogens by endophyte TharDOB-31. Selected endophytic isolates recorded multiple plant growth promoting traits in in vitro studies. The rhizome bacterization followed by soil application of endophyte TharDOB-31 showed lowest Percent Disease Incidence of rhizome rot and leaf blight, 13.8 and 11.6% respectively. The treatment of TharDOB-31 exhibited significant increase in plant height (85 cm) and fresh rhizome yield/plant (425 g) in comparison with untreated control under greenhouse condition. The confocal microscopy validates the colonization of the TharDOB-31 in turmeric rhizomes. The secondary metabolites in ethyl acetate extract of TharDOB-31 were found to contain higher number of antifungal compounds by high resolution liquid chromatograph mass spectrometer analysis. Thereby, endophyte T. harzianum isolate can be exploited as a potential biocontrol agent for suppressing rhizome rot and leaf blight diseases in turmeric.