Robinsons Fruit Shoot Juiced Strawberry and Raspberry, 6 x 200ml
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Robinsons Fruit Shoot Juiced Strawberry and Raspberry, 6 x 200ml
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Strawberry plants produce runners. These stolons are horizontal stems that run above the ground and produce new clone plants at nodes spaced at varying intervals. Since strawberry plants possess stolons, they are considered “stoloniferous.” The long, leafless stems between the mother plant, plant-growing nodes, and growing tip of the stolon are called “internodes.” Adventitious Roots on a Strawberry Runner
Biswas MK, Dutt M, Roy UK, Islam R, Hossain M. Development and evaluation of in vitro somaclonal variation in strawberry for improved horticultural traits. Sci Hortic. 2009;22:409–16. The phytohormone ABA plays crucial roles in plant growth, development, and stress responses [ 56]. The ripening of non-climacteric fruits, such as strawberry, has been revealed to be ABA-dependent [ 34]. Identification and functional definition of the core elements in ABA biosynthesis and signal transduction pathway have expanded our understanding of the mechanistic roles of ABA-mediated regulation of non-climacteric fruit ripening. The transcript levels of MTA and MTB increased significantly upon ripening initiation of strawberry fruit (Fig. 5c–e), which may account for the m 6A hypermethylation in the CDS region. It should be noted that the m 6A methyltransferase genes in tomato express stably and no obviously global m 6A hypermethylation was observed during fruit ripening [ 36]. By contrast, it is the m 6A demethylase SlALKBH2 that positively regulates tomato fruit ripening through mediating the mRNA stability of SlDML2, a key DNA demethylase gene determining the DNA methylation patterns during ripening [ 36]. Due to the pivotal role of DNA methylation in the regulation of fruit ripening in both climacteric and non-climacteric fruits, it is reasonable to assume that m 6A modification may modulate strawberry fruit ripening by modulating the DNA methylation machinery. Borkowska B. Morphological and physiological characteristics of micropropagated strawberry plants rooted in vitro or ex vitro. Sci Hortic. 2001;89:195–206. Nishi S, Oosawa K. Mass propagation method of virus free strawberry plants through meristem callus. Jpn Agric Res Q. 1973;7:189–94.
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Munir M, Iqbal S, Baloch JUD, Khakwani AA. In vitro explant sterilization and bud initiation studies of four strawberry cultivars. J Appl Hortic. 2015;17:192–8. Wang X, Zhao BS, Roundtree IA, Lu Z, Han D, Ma H, et al. N 6-methyladenosine modulates messenger RNA translation efficiency. Cell. 2015;161(6):1388–99. https://doi.org/10.1016/j.cell.2015.05.014.
Most plants have a root system that consists of a primary root or primary roots with root branches forming and growing from the primary root. Strawberry plants have this arrangement for the majority of their root system. However, they also have a special advantage: adventitious root formation at the nodes of their stolons. To explore the possibility that MTA and MTB in strawberry function in the form of heterodimer as METTL3 and METTL14 in mammals [ 12, 13], we subsequently analyzed the interactions between MTA and MTB using the yeast two-hybrid (Y2H) system. As shown in Fig. 5f, the yeast cells co-expressing AD-MTA and BD-MTB, but not the negative controls, displayed normal growth on the selective SD/-Leu-Trp-His (-LWH) and SD/-Leu-Trp-His-Ade (-LWHA) solid medium and turn to blue with the addition of X-α-gal, indicating that MTA interacts with MTB. The interactions between MTA and MTB were further verified by the split luciferase complementation imaging (LCI) assay, in which the luciferase activity was detected when MTA-nLUC and cLUC-MTB were co-expressed in N. benthamiana leaves (Fig. 5g). It should be noted that, compared with the MT-A70 domain of MTB, the full-length MTB protein exhibit relatively weaker combining capacity with MTA (Fig. 5g). Subcellular localization analysis by transiently expressing MTA-mCherry and MTB-eGFP fusion proteins in N. benthamiana leaves showed that MTA is present in both the nucleus and cytoplasm, while the MTB protein is specifically localized in the nucleus (Fig. 5h). Interestingly, when MTA-mCherry was co-expressed with MTB-eGFP, the two proteins tend to colocalize in the nucleus (Fig. 5i). m 6A methyltransferases positively regulate strawberry fruit ripening Fleshy fruits, which are enriched with nutrients, such as flavor compounds, fiber, vitamins and antioxidants, represent one of the commercially valuable structures of horticultural crops. As an important component of diets, fleshy fruits are indispensable for human health [ 26]. The ripening of fleshy fruits, which is characterized by dramatic changes in color, texture, flavor and aroma compounds [ 27], is a complex, genetically programmed process that impacts fruit nutritional quality and shelf life. Fruit ripening is regulated by both environmental and internal cues, including light, temperature, phytohormones, and developmental genes [ 28, 29]. Based on the different ripening mechanisms, fruits are classified into two groups: climacteric (e.g., tomato, apple, banana, and avocado) and non-climacteric (e.g., strawberry, grape, and citrus) [ 30]. Phytohormone ethylene is essential for the ripening of climacteric fruits [ 31, 32], and substantial insights have been made toward ethylene biosynthesis, ethylene perception and signal transduction, and downstream gene regulation [ 33]. In comparison, the ripening of non-climacteric fruits is thought to be abscisic acid (ABA)-dependent [ 32, 34], although the regulation of ABA pathway is poorly understood. A comprehensive understanding of the common regulatory mechanisms underlying ripening in climacteric and non-climacteric species has great potential for improving fruit quality and maintaining shelf-life.Naing AH, Chung JD, Park IS, Lim KB. Efficient plant regeneration of the endangered medicinal orchid, Coelogyne cristata using protocorm-like bodies. Acta Physiol Plant. 2011;33:659–66. Whitehouse AB, Govan CL, Hammond KJ, Sargent DJ, Simpson DW. Meristem culture for the elimination of the strawberry crown rot pathogen Phytophthora cactorum. J Berry Res. 2011;1:129–36. Wang X, Lu Z, Gomez A, Hon GC, Yue Y, Han D, et al. N 6-methyladenosine dependent regulation of messenger RNA stability. Nature. 2014;505(7481):117–20. https://doi.org/10.1038/nature12730.
The 1608 hypermethylated m 6A peaks were highly enriched (83.04%) in the CDS region, whereas the 865 hypomethylated m 6A peaks were mainly distributed around the stop codon (40.66%) or within the 3′ UTR (58.59%) (Fig. 3e; Additional file 1: Figure S6). This is in accordance with the dynamic m 6A distribution pattern (Fig. 2), showing that the percentage of m 6A peaks locating in the CDS region increased sharply, while that around the stop codon or within the 3′ UTR declined, from S6 to RS1. Interestingly, of the 1608 hypermethylated m 6A peaks, 1424 (88.56%) fell into the newly generated peaks, which represents ripening-specific peaks (Additional file 11: Table S10). For the differential m 6A peaks identified after ripening initiation, both the hypermethylated and hypomethylated m 6A peaks were highly enriched (over 90%) around the stop codon or within the 3′ UTR (Fig. 3e). To explore the biological significance of genes with dynamic m 6A modification, we performed Gene Ontology (GO) analysis on genes containing differential, non-differential, and ripening-specific m 6A peaks. In line with the progression of fruit development and ripening, genes harboring ripening-specific m 6A peaks were mostly annotated to developmental pathways, including response to hormone stimulus, developmental process, histone modification, small molecular biosynthetic process, and protein transport (Additional file 1: Figure S7a). Similar enrichment was observed for genes covering differential m 6A peaks at the initiation stage of ripening (from S6 to RS1) (Additional file 1: Figure S7b). In contrast, genes with non-differential m 6A peaks were enriched in multiple biological processes in addition to developmental pathways (Additional file 1: Figure S7c). These results suggest that dynamic changes in m 6A modification are responsible for those genes to exert their functions during fruit development and ripening. Bertero A, Brown S, Madrigal P, Osnato A, Ortmann D, Yiangou L, et al. The SMAD2/3 interactome reveals that TGFβ controls m 6A mRNA methylation in pluripotency. Nature. 2018;555(7695):256–9. https://doi.org/10.1038/nature25784.
It also combines the essentials of multivitamins. Their Juiced range of Fruit Shoot is the combination of water and juice in the same proportion. They are pressed from juicy, sweet, ripe fruit from the seasonal crops. Popescu AN, Isac VS, Coman MS, Radulescu MS. Somaclonal variation in plants regenerated by organogenesis from callus culture of strawberry ( Fragaria × Ananassa). Acta Hortic. 1997;439:89–96. For subcellular localization analysis, the coding sequence of MTA and MTB was amplified from the cDNAs of diploid woodland strawberry and then inserted into the pCambia2300-mCherry and pCambia2300-eGFP plasmids to generate 35S:: MTA-mCherry and 35S:: MTB-eGFP vectors, respectively. The resulting constructs were separately transformed into A. tumefaciens strain GV3101. The agrobacteria were subsequently infiltrated into N. benthamiana leaves for the individual expression of mCherry-tagged MTA (MTA-mCherry) and eGFP-tagged MTB (MTB-eGFP) or the co-expression of the two fusion proteins. After culture for 36 h, the mesophyll protoplasts were isolated from N. benthamiana leaves as previously reported [ 91] and observed under a Leica confocal microscope (Leica DMI600CS). Protoplasts expressing eGFP or mCherry were used as negative controls. The primers used for vector constructions are listed in Additional file 18: Table S17. Agroinfiltration-mediated transient transformation in strawberry fruit Here we show that m 6A methylation displays a dramatic change at ripening onset of strawberry, a classical non-climacteric fruit. The m 6A modification in coding sequence (CDS) regions appears to be ripening-specific and tends to stabilize the mRNAs, whereas m 6A around the stop codons and within the 3′ untranslated regions is generally negatively correlated with the abundance of associated mRNAs. We identified thousands of transcripts with m 6A hypermethylation in the CDS regions, including those of NCED5, ABAR, and AREB1 in the abscisic acid (ABA) biosynthesis and signaling pathway. We demonstrate that the methyltransferases MTA and MTB are indispensable for normal ripening of strawberry fruit, and MTA-mediated m 6A modification promotes mRNA stability of NCED5 and AREB1, while facilitating translation of ABAR. Conclusion Transient transformation of strawberry fruit mediated by agroinfiltration was performed as previously described [ 54]. To construct the RNA interference (RNAi) vectors, a ~ 300-bp fragment targeting the coding sequence region of MTA or MTB was cloned and inserted into the pCR8 plasmid, and then restructured into the pK7GWIWGD (II) plasmid by using the Gateway LR Clonase TM Enzyme Mix (Invitrogen, 11791-020). To construct the overexpression (OE) vectors, the coding sequence of MTA and MTB was amplified and ligated into the pCambia2300-eGFP plasmid to generate 35S:: MTA-eGFP and 35S:: MTB-eGFP vector, respectively. The resulting constructs were separately transformed into the A. tumefaciens strain GV3101. The agrobacteria were cultured at 28 °C overnight in LB liquid medium supplemented with 50 μg mL −1 kanamycin, 50 μg mL −1 gentamycin, and 50 μg mL −1 rifampicin, and then diluted 1:100 in 100 mL of fresh LB medium to continue culturing for approximately 8 h. The agrobacteria cells were subsequently collected by centrifugation at 5,000 g for 5 min and resuspended in the infiltration buffer (10 mM MES, pH 5.6, 10 mM MgCl 2, and 100 μM acetosyringone) to a final OD 600 of 0.8. After being kept at room temperature for 2 h without shaking, the suspensions were injected into the octoploid strawberry fruit at large green (LG) stage by using a 1 mL syringe. The infiltrated fruits were cultured for 5–7 days in a growth room with the following conditions: 23 °C, 80 % relative humidity, and a 16/8-h light/dark photoperiod with a light intensity of 100 μmol m −2 s −1. The experiment was performed with more than three independent biological replicates, and each group contained at least fifteen fruits. The primers used for vector constructions are listed in Additional file 18: Table S17. mRNA stability assay
The m 6A-seq was performed according to the method described by Dominissini et al. (2013) [ 37]. Briefly, total RNAs were extracted from the woodland strawberry fruit at S6, RS1, and RS3 stages, and the RNAi- MTA and control fruit by the plant RNA extraction kit (Magen, R4165-02), and then 300 μg of intact total RNAs were used for mRNA isolation by the Dynabeads mRNA purification kit (Life Technologies, 61006). The purified mRNAs were randomly fragmented into ~ 100 nucleotide-long fragments by incubation at 94 °C for 5 min in the RNA fragmentation buffer (10 mM Tris-HCl, pH 7.0, and 10 mM ZnCl 2). The reaction was terminated with 50 mM EDTA, and then the fragmented mRNAs were purified by standard phenol-chloroform extraction and ethanol precipitation. For immunoprecipitation (IP), 5 μg of fragmented mRNAs was mixed with 10 μg of anti-m 6A polyclonal antibody (Synaptic Systems, 202003) and incubated at 4 °C for 2 h in 450 μL of IP buffer consisting of 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% NP-40 (v/v), and 300 U mL -1 RNase inhibitor (Promega, N2112S). After the addition of 50 μL Dynabeads protein-A (Life Technologies, 10002A), the mixture was incubated at 4 °C for another 2 h. The beads were subsequently washed twice with high-salt buffer containing 50 mM Tris-HCl, pH 7.4, 1 M NaCl, 1 mM EDTA, 1% NP-40 (v/v), and 0.1% SDS (w/v) and twice with IP buffer. The m 6A-containing fragments were eluted from the beads by incubation with 6.7 mM N 6-methyladenosine (TargetMol, T6599) in IP buffer at 4 °C for 2 h, followed by standard phenol-chloroform extraction and ethanol precipitation. Then, 50 ng of m 6A-containing mRNAs or pre-immunoprecipitated mRNAs (the input) were used for library construction by the NEBNext ultra RNA library preparation kit (NEB, E7530). High-throughput sequencing was conducted on the Illumina HiSeq X sequencer with a paired-end read length of 150 bp following the standard protocols. The sequencing was performed with three independent biological replicates, and each RNA sample was prepared from the mix of at least 60 strawberry fruits to avoid individual differences among fruits. Zhang C, Samanta D, Lu H, Bullen JW, Zhang H, Chen I, et al. Hypoxia induces the breast cancer stem cell phenotype by HIF-dependent and ALKBH5-mediated m 6A-demethylation of NANOG mRNA. Proc Natl Acad Sci U S A. 2016;113(14):E2047–56. https://doi.org/10.1073/pnas.1602883113. The m 6A-seq results were validated by independent m 6A immunoprecipitation followed by qPCR (m 6A-IP-qPCR) analysis. Three m 6A peak-containing transcripts, as well as three m 6A peak-free transcripts, were randomly selected and examined (Additional file 1: Figure S3a). As expected, m 6A enrichment was only observed in transcripts containing m 6A peaks, but not in those without m 6A peaks (Additional file 1: Figure S3b), indicating that our m 6A-seq data were accurate and robust.
Bhatt ID, Dhar U. Micropropagation of Indian wild strawberry. Plant Cell Tissue Organ Cult. 2000;60:83–8.
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