Callus Lines Research Articles

ВНУТРИКАЛЛУСНАЯ И МЕЖКАЛЛУСНАЯ МОРФОЛОГИЧЕСКАЯ ИЗМЕНЧИВОСТЬ УДВОЕННЫХ ГАПЛОИДОВ РИСА, ПОЛУЧЕННЫХ В АНДРОГЕНЕЗЕ INVITRO

Intracallus and intercallus morphological variability of doubled haploids of rice Oryza sativa L. obtained in androgenesis in vitro on callus lines with multiple regeneration was studied. Regenerants from hybrids of the second generation F2 were used: Don 4237 × (Szarvasi 70 × Heilunjiang)—D × S × H, plant no. 8; Romanika × (Darius 122 × Krasnodar 9167)—R × D × 67, plant no. 15; Kitaets × (VNIIR 3223 × Kenzo)—K × 23 × K, plant nos. 26 and 28. Seed offspring of doubled rice haploids of the first generation R1 were sown on a plot in 1.54 m2 vessels filled with field soil. In total, 1383 plants of 144 lines of doubled haploids of eight callus lines were analyzed. Analysis of variance found statistically significant differences between doubled haploids of two callus aggregates of the same anther and different anthers of the same hybrid. A correlation dependence (p < 0.05) of a number of biometric parameters on plant height was found: panicle length (r = 0.72), grain mass of the main panicle (r = 0.80), plant grain mass (r = 0.59), mass of 1000 grains (r = 0.74). Thus, tall plants turned out to be more productive than low and medium-sized ones. After an initial assessment of the lines of doubled haploids by biometric parameters, the seed offspring of the most productive plants of one callus line were combined to increase the sample volume and its rapid reproduction. This step will make it possible to move more quickly from considering the productivity of individual plants to the field assessment of productivity.

Получение и характеристика каллусных культур клеток Alhagi persarum Boiss. et Buhse - продуцентов изофлавоноидов

The physiological characteristics of the callus cell cultures of Alhagi persarum Boiss et Buhse, a member of the legume family, widely used in folk medicine, have been studied. It was shown that the source of the explant was an important factor in the initiation of callusogenesis: more intense callusogenesis (almost 100%) was observed for explants from various organs of sterile seedlings, rather than intact plants (less than 30%). As a result, more than 20 lines of morphologically different callus cell cultures were obtained, and the growth parameters for the 5 most intensively growing lines were determined. The composition of fatty acids (FA) of total lipids and secondary metabolites in the most physiologically stable callus line Aр-207 was analyzed. Using capillary gas-liquid chromatography with mass spectrometric detection (GLC-MS), 19 individual C12--C24 FAs were identified, the main fraction of which were palmitic (~ 23%), stearic (~ 22%), linoleic (~ 14%) and α-linolenic (~ 33%) acids. The established atypical ratio of FAs (a simultaneous high content of both saturated FAs and polyunsaturated α-linolenic acid) is possibly due to the adaptation of cells to in vitro growth conditions. Phytochemical analysis of the secondary metabolites was carried out using ultra-performance liquid chromatography with electrospray ionization mass spectrometric detection (UPLC MS). Compounds belonging to different structural groups of isoflavones were found. Aglycones (calycosin, formononetin and afrormosin isomer), glucosides (formononetin glucoside), as well as esters of glucosides (malonylglycosides of calicosin, formononetin, afrormosin isomers, glycitein and genistein) were detected. These secondary metabolites are widespread in plants of the Fabaceae family; however, isoflavones are rare in representatives of the Alhagi genus. The presence of malonylated isoflavone glycosides in Alhagi spp. was shown for the first time. endemic plant species, Alhagi, in vitro cell culture, callus cell culture, isoflavones, fatty acids All studies were carried out using the equipment of the "Experimental Biotechnological Facility" and the "All-Russian Collection of Cell Cultures of Higher Plants" of IРР RAS. This work was supported by the Russian Foundation for Basic Research (RFBR), contract no.18-54-06021 (Az_a), and the Government of the Russian Federation, Megagrant Project no. 075-15-2019-1882.

Biolistic Transformation of Japonica Rice Varieties.

Genetic improvement of rice is crucial to achieve global food security as rice is an important staple crop for more than half of the global population. One of the methodologies for genetic improvement is biolistic delivery of genetic components into plant cells. In this chapter, we describe steps involved in introducing plasmid DNA carrying gene of interest into rice mature embryos using Biolistic® PDS-1000/He particle delivery system. We also provide information required for recovery of transformed plants and production of transgenic seed for next generation analysis. Using this protocol, typical 50-70 putative independent transgenic callus lines can be generated from 100 bombarded embryos. Transgenic rice plantlets can be produced within 2months after the initiation of seed germination for transformation.

Producing the sulfur-containing metabolite asparaptine in Asparagus calluses and a suspension cell line.

Asparaptine, a conjugate of L-arginine and asparagusic acid, was found in green asparagus (Asparagus officinalis) using ultrahigh-resolution metabolomics for sulfur-containing metabolites (S-metabolites), called S-omics. Asparaptine has been shown to inhibit the activity of angiotensin-converting enzyme. Larger amounts of this S-metabolite are therefore required for further analysis; however, there are limitations that asparagus is a perennial plant and its spears, wherein asparaptine accumulates, can be mainly harvested at the spring to summer season. In order to overcome these, we prepared a callus and suspension cell line from green asparagus. Untargeted metabolome analysis using liquid chromatography-tandem mass spectrometry was performed in the materials as well as spears and three calluses derived from wild type Asparagus. The analysis demonstrated that the amount of asparaptine in the callus derived from the green asparagus was more than the others per mg dry weight. The suspension cell line treated with methyljasmonate showed the induction of asparaptine, suggesting that the asparaptine production is modifiable under appropriate culture conditions. The described materials can be utilized for the production of asparaptine and in integrated metabolomics to study the biosynthesis of this S-metabolite, which is currently unknown.

Ginsenoside and phenolic compounds in hydromethanolic extracts of American ginseng cell cultures and their antioxidant properties

The present study was performed to investigate the antioxidant properties of callus and suspension culture extracts of &lt;em&gt;Panax quinquefolium&lt;/em&gt; (American ginseng). The ginsenoside content and the total phenolic content (TPC) in these cultures were also examined. The total amount of the nine studied saponins was found to be 2.08, 1.69, and 0.202 mg g&lt;sup&gt;−1&lt;/sup&gt; dry weight in red callus line (RCL), green callus line (GCL), and suspension cultures, respectively, by HPLC analysis. The TPC was estimated using the Folin–Ciocalteu method. The TPC of the suspension culture extracts was approximately 36.7% and 17.6% higher than that of the RCL and GCL, respectively. The antioxidant activity of the extracts was evaluated using the in vitro ABTS [2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)] and FRAP (ferric reducing antioxidant power) assays; the methanolic &lt;em&gt;P. quinquefolium&lt;/em&gt; suspension culture extracts demonstrated stronger antioxidant activity than that of the callus cultures.

Effect of Explant Source, Culture Media, and Growth Regulators on Callogenesis and Expression of Secondary Metabolites of Curcuma Longa

ABSTRACTThe effects of explant source, medium composition, and growth regulators were examined in callus lines of Curcuma longa. Callus cultures were induced from leaf sheath, leaf lamina, leaf base, apical buds, and lateral buds. The growth potential of established callus lines varied with a combination of phytohormones and culture medium. The greatest callus induction (79%) after 21 d was observed with MS medium supplemented with 3 mg L−1 2,4-D and 0.8 mg mL−1 kinetin. Phytochemical and chromatographic analysis of callus extract after 6th sub-culture revealed the presence of 53.6 mg g−1 of total polyphenols and 36.1 mg g−1 curcumin dry wt. with higher in-vitro anti-oxidant activity compared to turmeric rhizome extract. The lateral bud callus of C. longa may be used as a starting material for the synthesis of secondary metabolites of pharmaceutical interest.

A molecular system to investigate the effects of compound heterozygous mutations on multimeric pathogenic proteins

The RolA protein belongs to the RolB class of plant T-DNA oncogenes, and shares structural similarity with the papilloma virus E2 DNA-binding domain. It has potentially as an inducer of plant secondary metabolism, although its role in biotechnology has yet to be realised. In this investigation, a Rubia cordifolia callus culture transformed with the rolA plant oncogene for more than 10 years was analysed. Expression of the rolA gene in the callus line was stable during long-term cultivation, and growth parameters were both elevated and stable, exceeding those of the non-transformed control culture. The rolA-transformed calli not only demonstrated remarkably stable growth, but also the ability to increase the yield of anthraquinones (AQs) in long-term cultivation. After ten years of cultivating rolA callus lines, we observed an activation of AQ biosynthesis from 200 mg/l to 874 mg/l. The increase was mainly due to activation of ruberitrinic acid biosynthesis. The expression of key AQ biosynthesis genes was strongly activated in rolA-transgenic calli. We compared the effects of the rolA gene with those of the rolB gene, which was previously considered the most potent inducer of secondary metabolism, and showed that rolA was more productive under conditions of long-term cultivation.

Resveratrol supported on magnesium DiHydroxide (Resv@MDH, REVIFAST) represents an improved formulation of resveratrol with fast absorption and bioavailability in humans

The RolA protein belongs to the RolB class of plant T-DNA oncogenes, and shares structural similarity with the papilloma virus E2 DNA-binding domain. It has potentially as an inducer of plant secondary metabolism, although its role in biotechnology has yet to be realised. In this investigation, a Rubia cordifolia callus culture transformed with the rolA plant oncogene for more than 10 years was analysed. Expression of the rolA gene in the callus line was stable during long-term cultivation, and growth parameters were both elevated and stable, exceeding those of the non-transformed control culture. The rolA-transformed calli not only demonstrated remarkably stable growth, but also the ability to increase the yield of anthraquinones (AQs) in long-term cultivation. After ten years of cultivating rolA callus lines, we observed an activation of AQ biosynthesis from 200 mg/l to 874 mg/l. The increase was mainly due to activation of ruberitrinic acid biosynthesis. The expression of key AQ biosynthesis genes was strongly activated in rolA-transgenic calli. We compared the effects of the rolA gene with those of the rolB gene, which was previously considered the most potent inducer of secondary metabolism, and showed that rolA was more productive under conditions of long-term cultivation.

Activation of anthraquinone biosynthesis in long-cultured callus culture of Rubia cordifolia transformed with the rolA plant oncogene

The RolA protein belongs to the RolB class of plant T-DNA oncogenes, and shares structural similarity with the papilloma virus E2 DNA-binding domain. It has potentially as an inducer of plant secondary metabolism, although its role in biotechnology has yet to be realised. In this investigation, a Rubia cordifolia callus culture transformed with the rolA plant oncogene for more than 10 years was analysed. Expression of the rolA gene in the callus line was stable during long-term cultivation, and growth parameters were both elevated and stable, exceeding those of the non-transformed control culture. The rolA-transformed calli not only demonstrated remarkably stable growth, but also the ability to increase the yield of anthraquinones (AQs) in long-term cultivation. After ten years of cultivating rolA callus lines, we observed an activation of AQ biosynthesis from 200 mg/l to 874 mg/l. The increase was mainly due to activation of ruberitrinic acid biosynthesis. The expression of key AQ biosynthesis genes was strongly activated in rolA-transgenic calli. We compared the effects of the rolA gene with those of the rolB gene, which was previously considered the most potent inducer of secondary metabolism, and showed that rolA was more productive under conditions of long-term cultivation.

Клітинна селекція калюсних культур Triticum aesticum L. на стійкість до збудника базального бактеріозу L. N. Butsenko, J. V. Kolomiiets

The increase in the number and severity of bacterial diseases in modern conditions of growing crops makes it necessary to use in the production of wheat varieties, which are characterized by high-quality yield for high resistance to phytopathogenic bacteria. The purpose of research. Select by the method of direct cell selection callus cultures of Triticum aesticum L., which are characterized by high growth index values and regeneration potential under the conditions of bacterial stress caused by Рseudomonas syringae pv. atrofaciens. Research methods. The material for the selection work was the seeds of winter wheat of the varieties Dark-skinned, Favorite, Metropolitan, Podolyanka. Preparation of nutrient media, introduction to culture and subculturing were carried out using traditional techniques. Results. It has been established that cell selection for resistance to the causative agent of basal bacteriosis can be carried out for the Podolyanka and Stolichnaya varieties with a sublethal concentration of 0,8 % IR P. syringae pv. atrofaciens 9400, Favoritka varieties – 0,6 %, Smuglyanka varieties – 0,4 %. The method of cell selection obtained resistant to P. syringae pv. atrofaciens, regenerative callus lines of wheat varieties Podolyanka, Stolichnaya, Favoritka and Smuglyanka.

In Vitro Cell Selection of Sugar Beet for Resistance to Culture Filtrate of the Fungus Fusarium oxysporum

A multistep in vitro cell selection was carried out on a well-proliferating callus tissue of sugar beet capable of morphogenesis. A 20-day culture filtrate of the fungus Fusarium oxysporum was used as a selective agent. The general features of the behavior of sugar beet callus cells cultivated under stress conditions on increasing concentrations of the culture filtrate, 5, 10, 15, and 20%, have been established. It was found that a low concentration of CF (culture filtrate) of the pathogen (5%) significantly stimulated the process of callusogenesis, while high concentrations (10–20%) had an inhibitory effect. As a result of cell and tissue selection, calluses that retained the ability for normal growth in the presence of sublethal concentrations of the selective agent were selected. By the method of multistep selection, 82 callus lines characterized by resistance to the toxic effects of CF were selected. From selected callus lines, 47 regenerated plants adapted to the soil were obtained. Only 24 plants passed the test for resistance to the fungus Fusarium oxysporum under in vivo conditions.

Factors Affecting the Regeneration, via Organogenesis, and the Selection of Transgenic Calli in the Peach Rootstock Hansen 536 (Prunus persica × Prunus amygdalus) to Express an RNAi Construct against PPV Virus.

Prunus spp. is one of the most recalcitrant fruit tree species in terms of in vitro regeneration and transformation, mostly when mature tissues are used as explants. The present study describes the in vitro regeneration via indirect organogenesis, and Agrobacterium tumefaciens-mediated transformation of the peach rootstock Hansen 536 (Prunus persica × Prunus amygdalus) through the use of meristematic bulks (MBs) as starting explants. Efficient adventitious shoot regeneration was obtained when Hansen 536 MBs were cultured on an optimized medium consisting of modified McCown Woody Plant medium (WPM) enriched with 4.4 μM 6-Benzyladenine (BA), 0.1 μM 1-Naphthaleneacetic acid (NAA) and 6.0 g L−1 plant agar S1000 (B&V). MB slices were used later as starting explants for Agrobacterium-mediated transformation to introduce an RNAi construct “ihp35S-PPV194” against PPV virus. Transgenic events were identified by both green fluorescent protein (GFP) screening and kanamycin selection at different concentrations (0, 17 or 42 μM). GFP-fluorescent proliferating callus lines were selected and confirmed to stably express the ihp35S-PPV194::eGFP gene construct by molecular analysis. Although shoot regeneration from these transgenic calli has not been obtained yet, this represents one of the few examples of successful attempts in peach genetic transformation from somatic tissues, and also serves as a useful in vitro system for future gene functional analysis in peach.

English

Production and use of double haploids is important for attainment of systematic genetic gains, and indeed several plant breeding programmes have benefited from it. Gynogenesis, the in vitro culture of unfertilized ovules and/or embryos has specifically been exploited in several economically important crops but not cassava. In this study, we examined possibilities of generating doubled haploids (DH) in cassava through gynogenesis, by bagging female flowers of selected varieties to prevent pollination. A total of 2,466 flowers across 32 elite cassava varieties were bagged for a period of one-to-three days. Early embryo rescue and ovule culture were done at 7 to 42 days after anthesis. Consequently, 517 fruits (21%) were harvested and dissected to obtain 97 seeds from which 47 unique embryos and 18 callus lines were obtained in vitro. Only six of the rescued embryos (12.8%) regenerated into plantlets. Upon undertaking ploidy analysis and single nucleotide polymorphism (SNP) genotyping, it was established that all samples analyzed (regenerated plants and calli) were diploid. SNP genotyping revealed increased homozygosity (up to 85.7%), but no doubled haploids were noticed. The knowledge generated is a significant contribution towards understanding cassava flowering biology and thus a foundation to on-going efforts towards developing protocols for generation of cassava DH. Key words: Anthesis, embryo rescue, gynogenesis, homozygosity, ploidy.

Establishment of cell suspension culture of Lonicera japonica Thunb and analysis its major secondary metabolites

Lonicera japonica Thunb, as a species of honeysuckle native to Asia (including China, Korea, and Japan), is often used for physical fitness due to its extensive biological activity and pharmaceutical properties. The goal of this work was to establish the cell suspension culture of L. japonica as well as to identify and quantify its major secondary metabolites. After sterilized, L. japonica buds, the best explant compared with young stems and leave, can be induced to form yellow friable callus on the MS medium supplemented with 1.5 mg L−1 NAA, 1.0 mg L−1 2,4D, 0.75 mg L-1 KIN and 0.15 mg L−1 BA. The induced callus was transferred on MS medium containing BA 1.5 mg L−1 + NAA 0.2 mg L−1 + 2,4D 0.1 mg L−1, and then sub-cultured three or four times to generate homogenous callus line. A cell suspension system was successfully established, with characteristic of a “S” growth curve and healthy morphology. 3,5-di-O-caffeoylquinic acid, 3-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid and 3,4-di-O-caffeoylquinic acid were the main secondary metabolites in the suspension culture cells. The total content of these chlorogenic acids was 22.7 mg g-1 in the suspension cells.

Variability of Rice Haploids Obtained From In Vitro Anther Culture

Morphological variability of haploid plants and doubled haploids of rice obtained from one callus line in in vitro anther culture has been studied. The study was performed on rice (Oryza sativa L. subsp. japonica Kato) plants of the Kaskad varietу. Regenerants of the same callus line derived from one rice anther (four lines in total) were divided into 2–3 groups of 20–30 plants (depending on the sample size) in the order of their differentiation on a callus and transplantation on the rooting medium. Two callus lines (15.1 and 18.1) generated both haploids and doubled haploids (in equal proportion), while two other callus lines (5.1 and 7.2) formed numerous haploids. In the case of the 5.1 and 7.2 lines, regenerants with a larger ordinal number were characterized by decreased size indices of derived plants (plant height, number of flowers on the main panicle, and the number of panicles). No statistically significant difference has been revealed between the haploids and doubled haploids originating from the 15.1 and 18.1 lines. To induce the doubling of a chromosome number in haploid regenerants by colchicine or other antitubulin compounds, it is recommended to use the earliest plants developing on a callus. Using the Hotelling’s T2 criterion calculated for the whole complex of biometric indices, a statistically significant (p = 0.001) difference was revealed between haploids of four callus lines and doubled haploids of two callus lines. Haploids of different lines differed by 3–4 traits, while doubled haploids differed by three of five traits (panicle length, productive tilling capacity, and plant height). Rice varieties interesting for breeders may be improved using in vitro anther cultures.

New Somatic Hybrid Mandarin Tetraploids Generated by Optimized Protoplast Fusion and Confirmed by Molecular Marker Analysis and Flow Cytometry

Six mandarin cultivars, Ponkan (Citrus reticulata), Willowleaf (Citrus deliciosa), Kinnow (Citrus nobilis × C. deliciosa), Murcott (purported C. reticulata × Citrus sinensis), W. Murcott [purported (C. reticulata × C. sinensis) × C. reticulata)], and Snack (purported C. reticulata hybrid), were used in protoplast fusion with different parental combinations to generate somatic hybrids. Sixty-five somatic regenerants were obtained using optimized formulation of enzymes and molecular weight of polyethylene glycol for improved protoplast yield and heterokaryon fusion rate, respectively. Flow cytometry was used to determine the ploidy level of somatic regenerants, and nuclear expressed sequence tag–simple sequence repeat (EST-SSR) markers to determine their parental source. Of the 65 somatic regenerants, 46 were identified as autotetraploids, 18 allotetraploids, and one undefined. The EST-SSR markers also revealed that some ‘W. Murcott’ embryogenic callus lines that were presumed to be of nucellar origin were actually derived unexpectedly from individual ovules of zygotic origin. These mandarin-derived tetraploids are valuable as potential breeding parents for interploid crosses with an aim at seedlessness and easy-peeling traits.

Variability of rice haploids obtained in anther culture in vitro

Morphological variability of haploid plants and doubled haploids of rice obtained on one callus line in anther culture in vitro was studied. The work was carried out on rice plants Oryza sativa L. subspecies japonica Kato varietу Cascade. Regenerant plants of one callus line obtained from one rice anther (four in total) were divided into two or three groups of 20-30 plants, depending on the sample size in order of their differentiation on callus and transplantation on the rooting medium. Two callus lines (15.1 and 18.1) formed half of the haploids, half of the doubled haploids, and two other callus lines (5.1 and 7.2) numerous haploids. On callus lines with numerous haploids (5.1 and 7.2), as the regenant number increases, the size of plants decreases (plant height, number of flowers on the main panicle, number of panicles). On the lines 15.1 and 18.1 between groups of haploids and between groups of doubled haploids statistically significant differences absent. In breeding purposes for the induced doubling of the number of chromosomes in haploid regenerants with antitubulin substances such as colchicine, it is advisable to use plants that form on callus among the first. Between haploids of four callus lines and doubled haploids of two callus lines, statistically significant differences (at p=0.001) were revealed using the Hotelling's T2-criterion, calculated for the whole complex of biometric features. Haploids of different lines differed in three or four of them, doubled haploids on three of the five signs (length of panicle, productive bushiness and plant height). Varieties of interest to breeders may be improved by anther culture in vitro.

Dual-targeting by CRISPR/Cas9 leads to efficient point mutagenesis but only rare targeted deletions in the rice genome

The present study investigated the efficiency of CRISPR/Cas9 in creating genomic deletions as the basis of its application in removing selection marker genes or the intergenic regions. Three loci, representing a transgene and two rice genes, were targeted at two sites each, in separate experiments, and the deletion of the defined fragments was investigated by PCR and sequencing. Genomic deletions were found at a low rate among the transformed callus lines that could be isolated, cultured, and regenerated into plants harboring the deletion. However, randomly regenerated plants showed mixed genomic effects, and generally did not harbor heritable genomic deletions. To determine whether point mutations occurred at each targeted site, a total of 114 plants consisting of primary transgenic lines and their progeny were analyzed. Ninety-three plants showed targeting, 60 of which were targeted at both sites. The presence of point mutations at both sites was correlated with the guide RNA efficiency. In summary, genomic deletions through dual-targeting by the paired-guide RNAs were generally observed in callus, while de novo point mutations at one or both sites occurred at high rates in transgenic plants and their progeny, generating a variety of insertion–deletions or single-nucleotide variations. In this study, point mutations were exceedingly favored over genomic deletions; therefore, for the recovery of plant lines harboring targeted deletions, identifying early transformed clones harboring the deletions, and isolating them for plant regeneration is recommended.

Pentacyclic triterpenoids and polyphenols accumulation in cell suspension culture of Chaenomeles japonica (Thunb.) Lindl. ex Spach

Summary Introduction: Callus and cell suspension cultures are widely applied in investigation of production of high-value secondary metabolites, which may be used as cosmeceuticals, nutraceuticals and pharmaceuticals. Plant cell cultures are promising alternative to intact plant sources for the production of plant-derived drugs of industrial importance. Objective: The aim of the study was to (i) initiate the cell suspension culture of Chaenomeles japonica from homogenous and uniform callus, (ii) stabilize the selected line and (iii) verify its ability to produce the desired groups of secondary metabolites – pentacyclic triterpenoids and polyphenols. Methods: To establish a cell suspension culture, stabilized and homogeneous callus was selected. Cell cultures were systematically passaged every 2 weeks to fresh liquid medium with the same composition. Biomass from cultures at the growth phase and stationary phase was designated for phytochemical research. UHPLC-DAD-MS analyzes were performed. At the same time, their macroscopic and microscopic observations were carried out. Results: Cells of suspension culture line A2 were characterized by the intense divisions. Cell culture extracts (both from the growth phase and stationary phase) contained pentacyclic triterpenoids. In addition, phe-nolic compounds (chlorogenic acid and proanthocyanidins type B) and in a small amount also epicatechin are present in the extract of the cells harvested from the growth phase. In the present studies, three pentacyclic triterpenoids were detected and quantified in the extracts of cell suspensions and callus line A2. Ursolic and oleanolic acids were the main triterpenoids in the studied extracts. The cell suspension culture from the growth phase exhibited the highest content of ursolic, oleanolic, and betulinic acid (separately and together). Conclusion: The cell suspension culture of Chaenomeles japonica is a promising source of pentacyclic triterpenoids.

Expression and active testing of VP7 from GCRV (Grass carp reovirus) fused with cholera toxin B subunit in rice calli

Grass carp reovirus (GCRV) is one of the most serious pathogens threatening grass carp (Ctenopharyngodon idellus) production and results in high mortality in China. VP7 from GCRV is involved in viral infection and could be suitable for developing vaccines for the control of GCRV infection. To obtain a genetically engineered vaccine and a plant-based oral vaccine and to evaluate their immune efficacy as an oral vaccine against GCRV, cholera toxin B subunit (CTB) of Vibrio cholerae fused to VP7 (CTB-VP7) was transformed into BL21(DE3) for expression. SDS-PAGE and Western blotting showed that the purified CTB-VP7 fusion protein (rCTB-VP7) was approximately 49.0 kDa. Meanwhile, CTB-VP7 was transformed into rice callus cells by Agrobacterium tumefaciens-mediated gene transformation. CTB-VP7 was integrated into the nuclear genome by PCR, and mRNA transcripts of CTB-VP7 were detected. ELISA and Western blot analyses revealed that the CTB-VP7 fusion protein (CTB-VP7) could be expressed in rice callus lines. The level of expression was determined to be 1.54% ± 0.43 of the total soluble protein. CTB-VP7 showed a binding affinity for monosialoganglioside(GM1), a receptor for CTB. CTB-VP7 showed a higher affinity towards GM1 compared to rCTB-VP7. CTB-VP7 bonded to GM1 with different affinities under different temperatures. Maximum binding of CTB-VP7 to GM1 was reported to occur within 2 h at 37 °C, and approximately half of the binding affinity remained at 25 °C. Our results suggest that CTB-VP7 could be produced in rice calli, increasing the possibility that edible plants can be employed in mucosal vaccines for protection against GCRV in aquaculture.