Improvement and Innovation of Cryopreservation and In Vitro Methods in Plant Resource Protection.

Plant genetic resources (PGRs) are perhaps the most precious gift of nature to humanity: they provide food, shelter, medicines, and many goods of high economic value, not to mention their key importance for healthy ecosystems and their aesthetic value [...

Critical Role of Regrowth Conditions in Post-Cryopreservation of In Vitro Plant Germplasm.

Cryopreservation is an effective option for the long-term conservation of plant genetic resources, including vegetatively propagated crops and ornamental plants, elite tree genotypes, threatened plant species with non-orthodox seeds or limited seed availability, as well as cell and root cultures useful for biotechnology. With increasing success, an arsenal of cryopreservation methods has been developed and applied to many species and material types. However, severe damage to plant material accumulating during the multi-step cryopreservation procedure often causes reduced survival and low regrowth, even when the optimized protocol is applied.

Optimization of a Cryopreservation Method for the Endangered Korean Species Using a Systematic Approach: The Key Role of Ammonium and Growth Regulators.

Cryopreservation provides a secure long-term conservation option for rare and endangered plant species with non-orthodox or limitedly available seeds. Wide application of cryopreservation to biobank wild flora is hampered by the need to re-optimize nearly all protocol steps for every new species. We applied a systematic approach to simplify optimization of a multi-stage droplet-vitrification method for the endangered wetland Korean species, .

Influence of Different Carbohydrates on Flavonoid Accumulation in Hairy Root Cultures of Scutellaria baicalensis.

Carbohydrate sources play important roles in energy and growth of plants. Therefore, in this study, we investigated the optimal carbohydrate source in hairy root cultures (HRCs) of Scutellaria baicalensis infected with Agrobacterium rhizogenes strain R1000. The hairy roots were cultured in half-strength B5 liquid medium supplemented with seven different carbohydrates sources (sucrose, fructose, glucose, galactose, sorbitol, mannitol and maltose), each at a concentration of 100 mM, in order to identify the best carbon sources for the production of major flavones, such as wogonin, baicalin and baicalein.

Frozen beauty: The cryobiotechnology of orchid diversity.

Orchids (Orchidaceae) are one of the most diverse plant groups on the planet with over 25,000 species. For over a century, scientists and horticulturalists have been fascinated by their complex floral morphology, pollinator specificity and multiple ethnobotanical uses, including as food, flavourings, medicines, ornaments, and perfumes. These important traits have stimulated world-wide collection of orchid species, often for the commercial production of hybrids and leading to frequent overexploitation.

Elimination of chrysanthemum stunt viroid and chrysanthemum chlorotic mottle viroid from infected chrysanthemum by cryopreservation.

Chrysanthemum morifolium 'Borami' and 'Secret Pink' showing symptoms of stunt disease caused by chrysanthemum stunt viroid (CSVd) and 'Yellow Cap' showing chlorotic mottle disease caused by chrysanthemum chlorotic mottle viroid (CChMVd) were confirmed to be infected by the respective viroids by using reverse transcription polymerase chain reaction (RT-PCR). Real-time PCR results showed that the viroid concentrations in the infected cultivars varied between the different regions of origin (Chilgok, Gumi, and Gyeongsan). We applied a cryopreservation protocol for elimination of CSVd from naturally infected 'Borami' collected from Gumi, showing the lowest concentration of CSVd, by varying several factors such as plant vitrification solutions (PVS2 and PVS3), duration of exposure to liquid nitrogen, shoot-tip size, and low-temperature treatment.

Accumulation of phenylpropanoids and correlated gene expression in hairy roots of tartary buckwheat under light and dark conditions.

Differential expression patterns of flavonoid biosynthetic pathway genes in the hairy roots of tartary buckwheat cultivars "Hokkai T8" and "Hokkai T10" were studied over a time course of the light-dark cycle. The Agrobacterium rhizogenes-mediated transformation system was applied for inducing hairy roots. Further, a total of six phenolic compounds and two anthocyanins were analyzed in the hairy roots which were exposed to both light and dark conditions, and their amounts were estimated by HPLC.

Development of vitrification protocol in Rubia akane (nakai) hairy roots using a systematic approach.

Background: A solution-based vitrification protocol is a process of sequentially changing-solutions from which both influx of cryoprotectants (loading) and efflux of water (dehydration) were accomplished before cryo-exposure. Hence, we need to properly control the concentration /composition of the cryoprotectant solutions.

Objective: The study was, using a systematic approach, to develop a protocol for Rubia akane hairy roots, a very sensitive material to cytotoxicity of vitrification solutions.

Cloning and characterization of a flavonol synthase gene from Scutellaria baicalensis.

Flavonols are the most abundant of all the flavonoids and play pivotal roles in a variety of plants. We isolated a cDNA clone encoding flavonol synthase from Scutellaria baicalensis (SbFLS). The SbFLS cDNA is 1011 bp long, encodes 336 amino acid residues, and belongs to a family of 2-oxoglutarate-dependent dioxygenases.

Comparative analysis of flavonoids and polar metabolites from hairy roots of Scutellaria baicalensis and Scutellaria lateriflora.

Baicalin, baicalein, and wogonin were accumulated in hairy roots derived from Scutellaria lateriflora and Scutellaria baicalensis. The levels of baicalein and baicalin were 6.8 and 5.

Effect of the successive steps of a cryopreservation protocol on the structural integrity of Rubia akane Nakai hairy roots.

In this work, we studied the impact of the successive steps of the droplet-vitrification protocol technique employed for cryopreservation of Rubia akane hairy roots on the features of cortical, pericycle and endoderm cells of apical and central root segments, using histology techniques and combining qualitative and quantitative observations. In apical segments, plasmolysis (22-71 %, depending on cell type) was observed only after the loading treatment and did not increase after the following steps of the protocol. By contrast, in central segments, plasmolysis (39-45 %) was already observed after the sucrose pretreatment; it increased to 54-68 %, depending on cell type, after the loading treatment, but no further changes were noted after treatment with the vitrification solution.

MYB transcription factors regulate glucosinolate biosynthesis in different organs of Chinese cabbage (Brassica rapa ssp. pekinensis).

In this study, we investigated the expression of seven MYB transcription factors (a total of 17 genes that included Dof1.1, IQD1-1, MYB28, MYB29, MYB34, MYB51, and MYB122 and their isoforms) involved in aliphatic and indolic glucosinolate (GSL) biosynthesis and analyzed the aliphatic and indolic GSL content in different organs of Chinese cabbage (Brassica rapassp. Pekinensis).

Differential stress-response expression of two flavonol synthase genes and accumulation of flavonols in tartary buckwheat.

Flavonoids are ubiquitously present in plants and play important roles in these organisms as well as in the human diet. Flavonol synthase (FLS) is a key enzyme of the flavonoid biosynthetic pathway, acting at the diverging point into the flavonol subclass branch. We isolated and characterized a FLS isoform gene, FtFLS2, from tartary buckwheat (Fagopyrum tataricum).

Characterization of genes for a putative hydroxycinnamoyl-coenzyme A quinate transferase and p-coumarate 3'-hydroxylase and chlorogenic acid accumulation in tartary buckwheat.

Tartary buckwheat ( Fagopyrum tataricum Gaertn.) contains a high level of flavonoid compounds, which have beneficial and pharmacological effects on health. In this study, we isolated full-length cDNAs encoding hydroxycinnamoyl-coenzyme A quinate hydroxycinnamoyltransferase (HQT) and p-coumarate 3'-hydroxylase (C3H), which are involved in chlorogenic acid (CGA) biosynthesis.

Ethylene inhibitors enhance shoot organogenesis of gloxinia (Sinningia speciosa).

Shoot organogenesis and plant regeneration in Sinningia speciosa were improved using ethylene inhibitors. The leaf explants were cultured on initial shoot regeneration media (MS media with BAP at 2 mg/L + NAA at 0.1 mg/L) supplemented with different concentrations of aminoethoxyvinylglycine (AVG), cobalt chloride (CoCl₂), and silver thiosulphate (STS).

'Personalisation' of droplet-vitrification protocols for plant cells: a systematic approach to optimising chemical and osmotic effects.

Although an appropriate cryopreservation protocol is a prerequisite for basic studies and large-scale implementation as well as further cryopreservation studies, the process relies on trial and error. Among the vitrification-based cryopreservation techniques, droplet-vitrification produces high post-cryopreservation recovery. However, the protocol itself cannot solve the problems engaged in plant cryopreservation, prominently due to dehydration with cytotoxic vitrification solutions.

Improved cryopreservation of chrysanthemum (Chrysanthemum morifolium) using droplet-vitrification.

A droplet-vitrification protocol has been established for cryopreserving Chrysanthemum morifolium cv. Peak using axillary shoot tips and apical shoots of in vitro plants. In the optimized procedure, explants were submitted to a step-wise preculture in liquid sucrose-enriched medium (0.

Analysis of carotenoid accumulation and expression of carotenoid biosynthesis genes in different organs of Chinese cabbage (Brassica rapa subsp. pekinensis).

The relationship between carotenoid accumulation and expression of carotenoid biosynthesis genes was investigated in the flowers, stems, young leaves, old leaves, and roots of Chinese cabbage (Brassica rapa subsp. pekinensis). Quantitative real-time PCR analysis showed that the mRNA levels of BrPSY, BrPDS, BrZDS, BrLCYB, BrLCYE, BrCHXB, and BrZEP leading to the production of carotenoids were highest in the flowers or the leaves and lowest in the roots of Chinese cabbage.

Carotenoid accumulation and characterization of cDNAs encoding phytoene synthase and phytoene desaturase in garlic (Allium sativum).

Phytoene synthase (PSY) and phytoene desaturase (PDS), which catalyze the first and second steps of the carotenoid biosynthetic pathway, respectively, are key enzymes for the accumulation of carotenoids in many plants. We isolated 2 partial cDNAs encoding PSY (AsPSY-1 and AsPSY-2) and a partial cDNA encoding PDS (AsPDS) from Allium sativum. They shared high sequence identity and conserved motifs with other orthologous genes.

Cryopreservation of hairy roots of Rubia akane (Nakai) using a droplet-vitrification procedure.

An efficient protocol for the cryopreservation of madder (Rubia akane Nakai) hairy root cultures was developed using droplet-vitrification and alternative loading and vitrification solutions formulated previously in our laboratory. Among eight preculture treatments tested, the highest post-cryopreservation regeneration was obtained for explants incubated in liquid half-strength MS medium with progressively increased sucrose concentration (0.3 M for 54 h, then 0.

Cryopreservation of protocorm-like bodies of the hybrid orchid Bratonia (Miltonia flavescens × Brassia longissima).

In this study, cryopreservation of Bratonia (Miltonia flavescens (Lindl.) Lindl. × Brassia longissima (Reichb.

Molecular cloning and characterization of phenylalanine ammonia-lyase and cinnamate 4-hydroxylase in the phenylpropanoid biosynthesis pathway in garlic (Allium sativum).

The cDNAs encoding phenylalanine ammonia-lyase (PAL) and cinnamate 4-hydroxylase (C4H) were cloned from garlic (Allium sativum) using reverse transcription-polymerase chain reaction (RT-PCR) with degenerate primers and 5' and 3' rapid amplification of cDNA ends (RACE) PCR. Amino acid sequence alignments showed that AsPAL and AsC4H have more than 70% amino acid identity with their homologues in other plants. The expression of AsPAL and AsC4H transcripts was highest in the roots but surprisingly low in the bulbils, where phenylpropanoid compounds are most concentrated.