Image_1_Influence of foliar spray and post-harvest treatments on head yield, shelf-life, and physicochemical qualities of broccoli.pdf

Rapid senescence is the key factor in the deterioration of post-harvest shelf-life in broccoli heads. This study evaluates the head yield and its related traits, and physicochemical attributes of broccoli under four foliar sprays of mineral nutrients (B, Zn, Mo, and B + Zn + Mo) with control. The interaction effects of shelf-life and physicochemical attributes of broccoli for these five pre-harvest and five post-harvest storage treatments (LDP bag, HDP vacuum pack, 2% eggshell powder solution, 2% ascorbic acid, and control) both at cold storage and room temperature were evaluated with three replications. The significantly higher marketable head yield of 28.02 t ha−1, maximum gross return [(Bangladesh Taka (BDT 420300 ha−1)], net return (BDT 30565 ha−1), and maximum benefit–cost ratio (BCR) of 3.67 were obtained from the pre-harvest foliar application of B + Zn + Mo in broccoli. Pre-harvest foliar spray of combined nutrient B + Zn + Mo and post-harvest treatment high-density polyethylene (HDP, 15 μm) vacuum packaging efficiently improve post-harvest physicochemical attributes, viz., compactness, green color, texture, carbohydrates, fats, energy, antioxidants, vitamin C, and total phenols in broccoli head compared to the rest of the treatment combinations. In addition, this treatment combination also confirmed a maximum shelf-life of 24.55 days at cold storage [relative humidity (RH) 90–95% and 4°C] and 7.05 days at room temperature (RH 60–65% and 14–22°C) compared to the rest of the treatment combinations. Therefore, we recommend a pre-harvest foliar spray of combined nutrient elements B + Zn + Mo and an HDP (15 μm) vacuum post-harvest packaging for the maximum benefits for both farmers and consumers to get the best head yield, anticipated physicochemical attributes, and maximum shelf-life of broccoli.</p

Data_Sheet_1_Influence of foliar spray and post-harvest treatments on head yield, shelf-life, and physicochemical qualities of broccoli.docx

Rapid senescence is the key factor in the deterioration of post-harvest shelf-life in broccoli heads. This study evaluates the head yield and its related traits, and physicochemical attributes of broccoli under four foliar sprays of mineral nutrients (B, Zn, Mo, and B + Zn + Mo) with control. The interaction effects of shelf-life and physicochemical attributes of broccoli for these five pre-harvest and five post-harvest storage treatments (LDP bag, HDP vacuum pack, 2% eggshell powder solution, 2% ascorbic acid, and control) both at cold storage and room temperature were evaluated with three replications. The significantly higher marketable head yield of 28.02 t ha−1, maximum gross return [(Bangladesh Taka (BDT 420300 ha−1)], net return (BDT 30565 ha−1), and maximum benefit–cost ratio (BCR) of 3.67 were obtained from the pre-harvest foliar application of B + Zn + Mo in broccoli. Pre-harvest foliar spray of combined nutrient B + Zn + Mo and post-harvest treatment high-density polyethylene (HDP, 15 μm) vacuum packaging efficiently improve post-harvest physicochemical attributes, viz., compactness, green color, texture, carbohydrates, fats, energy, antioxidants, vitamin C, and total phenols in broccoli head compared to the rest of the treatment combinations. In addition, this treatment combination also confirmed a maximum shelf-life of 24.55 days at cold storage [relative humidity (RH) 90–95% and 4°C] and 7.05 days at room temperature (RH 60–65% and 14–22°C) compared to the rest of the treatment combinations. Therefore, we recommend a pre-harvest foliar spray of combined nutrient elements B + Zn + Mo and an HDP (15 μm) vacuum post-harvest packaging for the maximum benefits for both farmers and consumers to get the best head yield, anticipated physicochemical attributes, and maximum shelf-life of broccoli.</p

Near-diploid male cell lines highly expressing <i>L1CAM</i>, besides SK-N-BE(2), may serve as platforms for the <i>L1CAM</i> assay.

(A) Representative dot plots obtained in this analysis. The CHP-134, LA-N-5, and TGW neuroblastoma cell lines and the HCT116 colon cancer cell line were transfected with ex26-1 or ex26-2 nuclease, a vector control, or none. The cells were then stained as indicated at the top of the panels and analyzed applying FCM settings noted along the X- and Y-axes of dot plots. Percentages of Alexa Fluor 488-negative cells are denoted in the plots. (B) Graphical summary of the experimental results, with representatives shown in (A). The results of FCM analyses conducted without staining cells are omitted from the graphs. Data represent the mean and SEM values from three independent experiments.</p

Alexa Fluor 488 positivity in the <i>L1CAM</i> correction assay represents the reversion of <i>L1CAM</i> mut-2 to a wild-type sequence.

The SK-N-BE(2)-derived mut-2 reporter clone was transfected with sgRNA #4 and #6 coupled with Cas9 (H840A) and Donor-L1CAM to correct the L1CAM mutation via TPN. The L1CAM protein on the surface of transfected cells was labeled with Alexa Fluor 488, and cells were subjected to FCM-based sorting to isolate Alexa Fluor 488-positive and -negative populations. PCR was then performed to amplify a genomic region spanning the mut-2 site within L1CAM exon 14 in the Alexa Fluor 488-positive and -negative populations. The amplified PCR products were cloned into a plasmid, and multiple plasmids containing the PCR products as inserts were isolated and sequenced. (A) DNA sequences of PCR products amplified from Alexa Fluor 488-positive cells. Sequences are shown in alignment with a wild-type control derived from parental SK-N-BE(2) cells displayed at the top. Arbitrary numbers placed above the aligned sequences indicate the relative positions of nucleotides. Blue shading indicates a wild-type sequence resulting from mut-2 reversion. Green letters indicate substituted nucleotides. (B) Representative sequencing chromatogram obtained in the analysis shown in (A). (C) DNA sequences of PCR products amplified from Alexa Fluor 488-negative cells displayed in a manner similar to (A). Red shading indicates the mut-2 nonsense mutation. (D) Representative sequencing chromatogram obtained in the analysis shown in (C). (E) L1CAM genotypes in Alexa Fluor 488-positive and -negative cells determined based on the experimental results shown in (A)–(D). 1-bp substitutions shown in (A) and (C), probably introduced during genome editing or by PCR errors, are not considered in genotyping L1CAM, because they are located within an intronic sequence distant from the exon–intron boundary. In (A)–(D), the vertical dotted lines in red indicate a genomic site nicked by Cas9 (H840A) coupled with sgRNA #4 or #6. WT, wild-type; mut, mutant. (PDF)</p

DNA sequences surrounding the mutated genomic sites at <i>L1CAM</i> exon 14 in the SK-N-BE(2)-derived mut-1 and mut-2 reporter clones (top) and the corresponding wild-type sequence in the Donor-<i>L1CAM</i> plasmid (bottom).

Target sequences of Cas9 nucleases (A and B, brown) and Cas9 nickases (#1–#6, green) are color-shaded, with darker shading on the neighboring PAMs. Brown and green numbers are arbitrary values showing the relative positions of cleavage by Cas9 nucleases and nickases, respectively. Nucleotides indicated by bold letters with pink and blue shading represent mut-1 and mut-2 truncating mutations in the reporter clones and their corresponding wild-type sequences in Donor-L1CAM, respectively. Uppercase and lowercase letters in DNA sequences indicate exonic and intronic sequences, respectively. (PDF)</p

Potential off-target sites for the sgRNAs used in this study.

Potential off-target sites for the sgRNAs used in this study.</p

Correction of <i>L1CAM</i> mutations via prime editing can be quantified using FCM.

(A) Schematic diagrams describing the correction of mut-1 (top) and mut-2 (bottom) L1CAM mutations via prime editing. Nucleotide sequences in black and green indicate gDNA and pegRNAs, respectively. Nucleotides shown by red and blue bold letters represent mutated genomic positions in SK-N-BE(2)-derived reporter clones and corresponding wild-type sequences in pegRNAs, respectively. Although not delineated in the diagrams, pegRNAs were either modified at their 3′ ends by appending “tmpknot” or “tevopreQ1” pseudoknot motifs [29], or left unmodified; i.e., three different types of pegRNAs for mut-1 and mut-2, respectively, were used in the assay. PAM, protospacer adjacent motif. See S1 Fig for the sequences of respective pegRNAs. (B) The efficiencies of mut-1 (top) and mut-2 (bottom) correction via prime editing. Prime editing was conducted using one of three pegRNAs (tmpknot, tevopreQ1, and unmodified) in combination with one of two prime editor proteins (PE2 and PEmax), and gene correction efficiencies achieved under the respective experimental settings were compared. PEmax is an enhanced version of the PE2 protein in which (i) additional amino acid changes were introduced in the Cas9 (H840A) domain to improve its nicking activity, (ii) the reverse transcriptase domain was codon-optimized for better expression in human cells, and (iii) additional nuclear localization signals were incorporated [30]. Data represent the mean and SEM values from three independent experiments. tmpknot, a trimmed pseudoknot from Moloney murine leukemia virus; tevopreQ1, a modified and trimmed prequeosine1-1 riboswitch aptamer [29].</p

A 4,825-bp-long DNA sequence of Donor-<i>L1CAM</i>.

Yellow and blue shading indicates a 1,922-bp-long donor DNA fragment and L1CAM exon 14 within the donor sequence, respectively. Letters with purple and red backgrounds represent wild-type sequences corresponding to mut-1 and mut-2 sites, respectively. Nucleotide sequences without shading represent those from pBluescript II KS (+). See S6 and S7 Figs for the sequences of mut-1 and mut-2. (PDF)</p

Sequence chromatograms showing mutated genomic sites in the mut-1 and mut-2 reporter clones and the corresponding genomic sites in the parental SK-N-BE(2) cell line.

Letters with pink and blue shading indicate mutated and wild-type sequences, respectively. (PDF)</p

<i>L1CAM</i> disruption can be quantified using FCM.

(A) Representative dot plots obtained from FCM analyses. SK-N-BE(2) cells were transfected with a plasmid listed at the top, stained with primary and secondary antibodies shown on the left, and FCM-analyzed. The percentages of Alexa Fluor 488-negative cells are denoted in the plots. In the figures shown hereafter, abbreviations are used as follows: ex26-1, a Cas9 nuclease targeted to the boundary of L1CAM intron 25 and exon 26; V.C., vector control; 5G3, anti-L1CAM monoclonal antibody 5G3; 488, Alexa Fluor 488-conjugated secondary antibody. (B) Graphical summary of experimental results, with representatives shown in (A). Only the results obtained using 5G3 and 488 antibodies are summarized. (C) SK-N-BE(2) cells were transfected with the indicated amounts of ex26-1 nuclease plasmid and analyzed similarly to (A) and (B). (D) The ratios of L1CAM-positive and -negative SK-N-BE(2) cells within the cell mixture over a 15-day incubation period. SK-N-BE(2) cells comprising L1CAM-positive and -negative populations at six different intermixing ratios (shown by six polygonal lines) were analyzed using FCM at four sequential time points (indicated at the bottom) during a 15-day incubation period. No significant differences in the percentages of L1CAM-positive cells were found among data obtained at four different time points (one-way repeated measures ANOVA).</p