Regeneration of lycopene beta-cyclase (lcyb) mutants in bananas (Musa acuminata cv. Cavendish) using gene editing techniques and their LCYB expression levels

Regeneração de mutantes do gene licopeno betacliclase (lcyb) em bananas (Musa acuminata cv. Cavendish) utilizando técnicas de edição genética e seus níveis de expressão de LCYB

Abstract

Banana (Musa acuminata cv. Cavendish) is one of the most widely cultivated and consumed banana species in Indonesia, but little has been studied regarding the potential content of vitamin A and beta carotene in its fruit or leaves. This study aimed to regenerate banana mutants using the pRGEB32-sgLCYB vector and analyze the expression of the LCYB gene in the protoplasts of the recombinant mutant pRGEB32-sg lcyb. These findings indicate that the Cavendish banana mutant was successfully regenerated using the pRGEB32-sg LCYB vector, it can produce transgenic mutant protoplasts and there is low suppression of LCYB gene expression, especially in the transgenic mutant protoplasts when compared to the wild type or control. Electrophoresis results showed that there are two pRGEB32-LCYB plasmid DNA bands formed above and below 10,000 bp. This indicated that the isolation of the pRGEB32-LCYB plasmid was successful in this study. The sgRNA construction using gene editing successfully regenerated lcyb mutant plants and the results of expression analysis of lcyb mutant protoplasts showed a much lower expression level compared to the control. In summary, the Cavendish banana mutant was successfully regenerated using the pRGEB32-sg LCYB vector so that it could produce transgenic mutant protoplasts and there was low suppression of LCYB gene expression (-5 fold change) especially in transgenic mutant protoplasts when compared to the wild type or control.

Keywords:
beta-carotene; gene editing; lcyb; Musa acuminata cv. Cavendish; mutant

Resumo

A banana (Musa acuminata cv. Cavendish) é uma das espécies mais amplamente cultivadas e consumidas na Indonésia, mas pouco se estudou sobre o potencial teor de vitamina A e betacaroteno em seus frutos ou folhas. Este estudo teve como objetivo regenerar mutantes de banana utilizando o vetor pRGEB32-sgLCYB e analisar a expressão do gene LCYB nos protoplastos do mutante recombinante pRGEB32-sgLCYB. os resultados indicam que o mutante de banana Cavendish foi regenerado com sucesso usando o vetor pRGEB32-sg LCYB, sendo capaz de produzir protoplastos mutantes transgênicos e apresentando baixa supressão da expressão do gene LCYB, especialmente nos protoplastos mutantes transgênicos quando comparados ao tipo selvagem ou controle. Os resultados da eletroforese mostraram a presença de duas bandas de DNA plasmidial pRGEB32-LCYB formadas acima e abaixo de 10.000 pb. Isso indicou que o isolamento do plasmídeo pRGEB32-LCYB foi bem-sucedido neste estudo. A construção de sgRNA usando edição gênica regenerou com sucesso plantas mutantes lcyb, e os resultados da análise de expressão dos protoplastos mutantes lcyb mostraram um nível de expressão muito mais baixo em comparação ao controle. Em resumo, o mutante de banana Cavendish foi regenerado com sucesso utilizando o vetor pRGEB32-sgLCYB, o que permitiu a produção de protoplastos mutantes transgênicos e houve baixa supressão da expressão do gene LCYB (variação de -5 vezes), especialmente nos protoplastos mutantes transgênicos quando comparados ao tipo selvagem ou controle.

Palavras-chave:
betacaroteno; edição genética; lcyb; Musa acuminata cv. Cavendish; mutante

1. Introduction

Vitamin A deficiency is a serious public health problem affecting populations worldwide, including in Indonesia (Fendiyanto et al., 2023a). It can cause adverse health effects across different age groups, including children, adults, and pregnant women. Inadequate consumption of foods containing provitamin A carotenoids is one of the factors contributing to vitamin A deficiency. Vitamin A plays an important role in metabolism, vision, immune function, and cellular growth. Therefore, adequate intake of vitamin A and provitamin A carotenoids through fruits and vegetables, including bananas, is important for maintaining health (Fendiyanto et al., 2023b). Vitamin A also has antioxidant-related functions and is associated with protection against oxidative stress and several chronic health conditions (Pinazo et al., 2014).

Humans cannot synthesize vitamin A de novo and therefore depend entirely on dietary intake. Vitamin A is obtained either as preformed retinoids (retinol and retinyl esters), found mainly in liver, fish oil, eggs, and dairy products, or as provitamin A carotenoids found in fruits and vegetables, which are enzymatically converted to retinol in the body (lutein, zeaxanthin, lycopene, and β-carotene) to prevent chronic disease (Boeing et al., 2012; Azzahroh et al., 2024; Vardhana et al., 2024). Of the approximately 50 carotenoids that possess provitamin A activity, β-carotene is the most important vitamin A precursor. β-carotene is commonly found in yellow fruits and vegetables such as mangoes, bananas, papayas, carrots, and beets (Singh et al., 2017). Increasing vitamin A content can be achieved through analysis of the lycopene beta-cyclase (LCYB) gene expression. The LCYB gene plays a role in the carotenoid formation process. Analyzing LCYB gene expression allows us to understand how to increase carotenoid content in fruit, thereby providing better nutritional value for the community. Based on metabolomics studies, β-carotene metabolite content and/or LCYB gene expression in banana is higher compared to rice (Fendiyanto et al., 2024), Mukia javanica (Pratami et al., 2025), Cucumis melo (Pratami et al., 2025), and Jatropha curcas (Setiawan et al., 2025).

Cavendish bananas (Musa acuminata) are one of the most widely cultivated and consumed banana varieties in Indonesia. Data from the Central Statistics Agency (BPS) from 2020 to 2021 shows that banana production in Indonesia increased from 8,182,756 tons to 8,741,147 tons (BPS, 2021). In addition to their sweet taste and soft texture, bananas are also an important source of carbohydrates for many people. However, despite its many benefits, the nutritional value of bananas still needs to be improved, particularly in terms of vitamin A. Vitamin A is an important factor in maintaining eye health, strengthening the immune system, and aiding cell growth. Therefore, this research focuses on improving the nutritional value, particularly vitamin A levels, of bananas, which are a source of national food security.

National food security in Indonesia is a critical issue, especially with the increasing population and the challenges of climate change. In addressing this issue, improving the nutritional quality of local food sources such as bananas is one strategy that can help achieve sustainable food security goals. The existence of biotechnology, including genetic breeding techniques and gene editing, can assist in analyzing the expression of the LCYB gene. By editing or modifying this gene, it is hoped that the carotenoid content in bananas can be increased, while maintaining the characteristics and original properties of the variety. The results of this research are expected to make a significant contribution to the development of more nutritious and quality banana varieties that can provide benefits to public health and support national food security. The objectives of this study were to regenerate banana mutants using the pRGEB32-sgLCYB vector and analyze the expression of the LCYB gene in protoplasts of the recombinant pRGEB32-sg lcyb mutant.

2. Materials and Methods

2.1. Materials

The materials used were samples of Cavendish banana leaves, Escherichia coli carrying the recombinant plasmid pRGEB32 and LCYB gene, sgLCYB reverse primer, M13 R forward primer (5’-CAGGAAACAGCTATGAC-3’), Murashige and Skoog (MS) Medium, 2-(N-morpholino) ethanesulfonic acid (MES), NaCl, CaCl₂, KCl, mannitol, MgCl₂, polyethylene glycol (PEG), Genomic RNA Mini Kit (Plant), SensiFAST cDNA Synthesis Kit, β-mercaptoethanol, absolute ethanol, RNase-Free Water, liquid nitrogen, 70% ethanol, Agarose, TAE buffer, DNA stain greensafe, DNA ladder 1 kb, distilled water.

2.2. Construction and Verification of the sgLCYB Insert

The sgLCYB insert was analyzed using the sequences 5’-AGAATTGAGTTGCTCCACCG-3’ and 5’-TGG-3’ PAM (Fendiyanto et al., 2023b). The sgLCYB insert was checked in the constructed pRGEB32 vector using a PCR machine. The primers used were LCYB-forward: 5’-AACTCCTCGAGCTTGTTCCA-3’ and LCYB-reverse: 5’-CCCATCGCTGCAAATCAAGA-3’.

2.3. Protoplast isolation and transformation

Protoplast isolation was performed following the protocol (Wang et al., 2022). The isolation source used was Cavendish banana leaves. The leaves were ground with liquid nitrogen until they were powder-like, then transferred using a spatula into a 2 ml microtube. 500 µl of 1% cellulose solution (Merck, USA) was added to the microtube. The sample was then spun down and allowed to stand for 1 hour to release the protoplasts. The resulting protoplasts were then observed under a microscope at 40x magnification.

Transfection of recombinant plasmids containing the desired genes into the protoplasts was performed using polyethylene glycol (PEG)-mediated transformation. After observing the protoplasts under the microscope, 190 µl of the protoplast sample was pipetted and centrifuged at 1,500 rpm for 10 minutes to obtain a pellet. Mannitol solution (13% w/v) was added as much as 200 µl, then added 10 µl of recombinant plasmid LCYB. PEG 4000 (20% w/v, Merck, USA)) was added as much as 200 µl. The sample was incubated overnight at room temperature in a dark room, so that transgenic mutant protoplasts would be obtained. The protoplasts to be isolated were protoplast DNA and protoplast RNA. Protoplast DNA used an additional CTAB of 500 µl and protoplast RNA used an additional GENEzol (Geneaid Biotech Ltd, TWN).

2.4. DNA isolation

We performed DNA isolation using CTAB methods with slightly modification (Miftahudin et al., 2021). Leaves were extracted using liquid N2, added CTAB solution. CTAB solution was added to the pellet and then centrifuged again at 1000 rpm for 5 minutes and the supernatant was discarded. Solution A (Cell Resuspension) was added as much as 200 µl to the pellet, then resuspended using a vortex. Solution B (Cell Lysis) was added as much as 200 µl, then the microtube was inverted for 10 times. Solution C (Neutralization) was also added as much as 200 µl, then the microtube was inverted for 10 times. The suspension was centrifuged at 10,000 rpm at room temperature for 5 minutes. The supernatant portion was taken as much as 500 µl and transferred to a new microtube. As much as 1x volume of C:I was added to the supernatant and resuspended using a vortex. The suspension was centrifuged at 10,000 rpm at room temperature for 5 minutes. 400 µl of the upper phase was removed and transferred to a new microtube. 40 µl of 2M NaOAC solution (Merck, USA) was added, followed by 1,000 µl of 100% EtOH (v/v) and inverted 10 times. The sample was then incubated overnight at -20 °C in a freezer.

The incubated sample was centrifuged at 10,000 rpm at 4 °C for 25 minutes. The supernatant was discarded, leaving only the pellet. 500 µl of 70% EtOH was added to the pellet, then centrifuged at 10,000 rpm at 4 °C for 5 minutes, and the supernatant was discarded. The sediment or pellet in the microtube was dried by removing the cap and inverting the microtube on a prepared tissue. The microtube was inverted for 10 minutes. Next, 20 µl of ddH₂O was added, then resuspended.

2.5. RNA Isolation and cDNA synthesis

RNA isolation using the Genomic RNA Mini Kit (Plant) Protocol (Geneaid Biotech Ltd, TWN). The sample was cut using sterile scissors and ground using liquid nitrogen, then the sample was put as much as 50 mg into a 1.5 mL microtube. 500 µL of RB Buffer or PRB Buffer and 5 µL of β-mercaptoethanol (Merck, USA) were added to the 1.5 mL microtube and then vortexed. Next, incubation was carried out at 60 °C for 5 minutes and every 5 minutes was inverted. Then the filter column was placed in a 2 mL collection tube and the sample supernatant was put into the filter column, then centrifuged at a speed of 1,000 x g for 1 minute. The filter column was discarded and the supernatant was transferred into a 1.5 mL microtube. ½ volume of absolute ethanol was added from the total volume of the filtrate or supernatant and then stirred. The RB Column was placed in a 2 mL collection tube, and the sample supernatant was added to the RB Column, then centrifuged at 14-16,000 x g for 1 minute. The supernatant was discarded, and the RB Column was placed back in the 2 mL collection tube.

Next, 400 µL of W1 buffer was added to the RB Column and centrifuged at 14-16,000 x g for 30 seconds. The supernatant was discarded, and the RB Column was placed back in the 2 mL collection tube. 600 µL of wash buffer was added to the RB Column, then centrifuged at the same speed for 1 minute, and the supernatant was discarded. The RB Column was placed back in the 2 mL collection tube and centrifuged again at the same speed for 3 minutes to dry, and the supernatant was discarded. The RB column was transferred to a sterile 1.5 mL microtube, and 50 µL of RNase-Free Water was added to the center of the column. The column was allowed to stand for 2 minutes, then centrifuged at 14-16,000 x g for 1 minute to obtain pure RNA.

The isolated RNA was then quantified by diluting 2 µL of total RNA in 0.01% ddH2O-DEPC to yield 398 µL. RNA concentration was calculated using a nanodrop (Nanodrop One/One C Microvolume, Thermoscientific, USA) at wavelengths of 260 and 280 nm. The purity of the isolated total RNA was determined using the 260/280 nm ratio. The quality of the isolated RNA was then tested by electrophoresis on a 1.5% agarose gel for 60 minutes using 1x TAE buffer.

cDNA is DNA that is complementary to mRNA. cDNA was synthesized for use as a template in RT-PCR reaction amplification. cDNA synthesis was carried out through reverse transcription using the RevertAid First Strand cDNA Synthesis kit protocol from Thermo Scientific, USA. cDNA synthesis used actin and LCYB primers, according to the procedure from (Miftahudin et al., 2021) to amplify specific genes. The identified regions correspond to the sequence similarity of LCYB with M. acuminata (accession number KP406755.1). The primers used were from (Fendiyanto et al., 2023a), LCYB-forward: 5’-AACTCCTCGAGCTTGTTCCA-3’ and LCYB-reverse: 5’-CCCATCGCTGCAAATCAAGA-3’. These primers can produce an amplified product of 460 bp.

2.6. qRT-PCR

LCYB Gene Expression Analysis in Cavendish banana cultivar using two-way real-time PCR. The analysis was carried out by amplifying cDNA from RT-PCR results as a template using primers from the LCYB gene. The amount of cDNA was normalized using the amplified housekeeping gene in bananas, namely actin (Pandey et al., 2016). This housekeeping gene functions as a control gene that is usually found in every plant. Housekeeping gene expression analysis is usually used to produce normal gene expression data. The LCYB gene amplifying primer is the forward primer 5’-AAC-TCC-TCG-AGC-TTG-TTC-CA-3’ and the reverse primer 5’-CCC-ATC-GCT-GCA-AAT-CAA-GA-3’. Meanwhile, the actin gene is used as an internal control or reference gene. The actin gene was amplified using the forward primer 5’-GAA-GGA-TGC-CTA-TGT-TGC-TGA-3’ and the reverse primer 5’-CTT-CAT-AGA-TTG-GCA-CGG-TGT-3’. Further quantification of the Ct value was performed to determine the level of LCYB gene expression using 2^-∆∆Ct according to the following Equation 1 (Miftahudin et al., 2021):

$$\begin{array}{l}\Delta Ct=Ct\text{LCYB}-Ct\text{Actin}\\ \Delta \Delta Ct=\Delta Ct\text{LCYB}\text{.Var}-\Delta Ct\text{LCYB}\text{. control}\\ LCYB-\exp =2^{-\Delta \Delta Ct}\end{array}$$ (1)

where: LCYB-exp: Relative expression of LCYB genes in mutants; ∆Ct LCYB.Var: The intensity of the LCYB gene expression in mutants; ∆Ct LCYB.control: The intensity of the LCYB gene expression in control; ∆Ct: Ct value of housekeeping gene (Actin); x: Cavendish banana control.

2.7. Data analysis

The data generated from this study used visualization images of PCR results, LCYB gene sequences from Basic Local Alignment Search Tool (BLAST) analysis. The analysis method in this study also used T-test visualized with R Studio software. Meanwhile, LCYB gene expression data in Cavendish banana was displayed in the form of a box plot graph.

3. Results

3.1. Isolation of the pRGEB32-LCYB plasmid

The isolated pRGEB32-LCYB plasmid can be visualized using electrophoresis to determine the quality of the isolated plasmid. The electrophoresis results were viewed using a UV transilluminator (Figure 1). The arrow indicates a marker with a size of 10,000 base pairs (bp). The electrophoresis results showed that the presence of pRGEB32-LCYB plasmid DNA was indicated by the light-colored band formed above 10,000 bp and a darker band indicating the presence of supercoiling. This indicates that the isolation of the pRGEB32-LCYB plasmid was successful in this study.

Figure 1
Construction of Lcyb mutants using CRISPR/Cas 9 technique in Escherichia coli dh5𝛂 and Agrobacterium tumefaciens LBA4404 bacteria. Recombinant lcyb plasmid vector (A). Isolation results of recombinant pRGEB32-LCYB plasmid viewed through a UV transilluminator. M: Lambda marker; M1-M3: Plasmid pRGEB32-LCYB isolated from the 1st, 2nd, and 3rd colonies; C2: Positive control of non-recombinant pRGEB32 vector. Propagation of recombinant E. coli sgLCYB bacteria (B). The media used was Luria-Bertani agar + 50 ppm kanamycin. Negative control: non-recombinant E. coli, LCYB: recombinant E. coli sgLCYB. Verification of sg-LCYB insertion in the CRISPR/Cas 9 vector (C). M13Rfix: M13 forward primer 1, M13R test: M13 forward primer 2, sgRNA LCYB R: lcyb reverse primer 1, Oligo 2R: lcyb reverse primer 2. Triparental mating assay to generate recombinant A. tumefaciens-sgLCYB (D). E1: E.coli recombinant sgLCYB; E2: E.coli strain DH1; AT: A. tumefaciens strain LBA4404; MX: mixed of all bacteria (E1, E2, and AT).

3.2. Verification of sg-LCYB gene insertion FROM recombinant Agrobacterium tumefaciens plasmid pRGEB32

Verification of the sg-LCYB gene insertion was carried out by PCR, and qualitative analysis was performed by electrophoresis to determine the band size. The electrophoresis results were viewed using a UV transilluminator (Figure 1). The arrow indicates a marker with a size of 1000 basepairs (bp). The electrophoresis results showed that there was an sg-LCYB gene insertion in the A. tumefaciens Recombinant Plasmid pRGEB32. This can be seen from the size of the resulting band in Figure 1. The PCR results of the sample using the M13R fix primer and sgRNA LCYB Reverse obtained a band measuring approximately 425 basepairs (bp). Meanwhile, the PCR results of the sample using the M13 Reverse fix primer and Oligo 2R were approximately 250 basepairs (bp). The PCR results of the sample using the M13 Reverse test primer and sgRNA LCYB Reverse obtained a band measuring approximately 425 basepairs (bp). Meanwhile, the PCR results of the sample using the M13 Reverse test primer and Oligo 2R were approximately 250 basepairs (bp).

3.3. Recombinant Agrobacterium infection in M. acuminata

The plantlet part used for transformation was the Cavendish banana (M. acuminata) tuber. Infection was performed by soaking the plantlets in recombinant Agrobacterium tumifaciens carrying the LCYB gene for 15 minutes, then placing them on Murashige and Skoog (MS) medium. A 15-minute incubation is the optimal time for transformation using the A. tumefaciens vector. The next step was co-cultivation, incubating the transformed plantlets for three days in a dark room with no light. Co-cultivation was carried out to ensure optimal transformation and the LCYB gene insertion into the plantlets. After the co-cultivation stage was completed, several banana tubers showed excessive Agrobacterium growth, often referred to as overgrowth. This was indicated by a thick, white, slimy fluid surrounding the transformed banana tubers (Figure 1).

Banana stems that have completed the co-cultivation stage are then rinsed with sterile distilled water for 20-30 seconds, then planted on MS selection media with the addition of 50 ppm of kanamycin antibiotics. The banana stems that are rinsed with sterile distilled water aim to reduce excessive growth of Agrobacterium (overgrowth). Comparison of results was carried out by transferring other banana stems that have completed the co-cultivation stage without rinsing with sterile distilled water and directly planted on MS selection media with the addition of 50 ppm of kanamycin and hygromycin antibiotics (Figure 2).

Figure 2
M. acuminatalcyb mutant containing pRGEB32-sgLCYB: Transformation of Agrobacterium tumefacien in M. acuminata (A). M. acuminata mutant in co-cultivation phase (B). M. acuminata rinsed with distilled water on Murashige and Skoog (MS) selection medium + hygromycin for 30 days after transplantation (C). M. acuminata on Murashige and Skoog (MS) selection medium + hygromycin for 60 days after transplantation (D). Verification of lcyb mutant in M. acuminata cv. Cavendish (E). A1, A2, A3: recombinant A. tumefaciens colonies with sgRNA-lcyb primers; M1-M7: mutant M. acuminata cv. Cavendish plants containing hpt gene insertion (encoding hygromycin); C1: non-mutant plant control; C2: ddH₂O control (non-template PCR). A. tumefaciens colonies used for lcyb insert colony PCR (F). A1, A2, A3: recombinant A. tumefaciens colonies with sgRNA-lcyb primers.

The results of the selection media rinsed with distilled water showed no white mucus-like fluid around the banana corm, indicating that there was no excessive growth of A. tumefaciens.. Furthermore, there was no brownish color on the banana corm, indicating that the tissue in the banana corm was still alive (Figure 2). Meanwhile, the results of the selection media not rinsed with distilled water showed a white mucus-like fluid around the banana corm, indicating that there was excessive growth of the transformant vector, A. tumefaciens. This excessive growth can be a factor in the malfunctioning of plant tissue, causing the plant to wilt and die, which is indicated by the brownish color of the banana corm. On the other hand, this study showed that the banana mutant plants were successfully regenerated and showed positive results when confirmed with hygromycin primers (Figure 2). The protoplasts of the successfully created mutant plants were then isolated to test the level of lcyb gene expression.

3.4. Isolation and transformation of mutant protoplasts of M. acuminata

The isolated M. acuminata protoplast samples consisted of RNA and DNA derived from Cavendish banana (M. acuminata) leaves. The isolated protoplast RNA and DNA were then transformed using recombinant A. tumefaciens carrying pRGEB32-LCYB. The transformed M. acuminata RNA and DNA protoplasts were viewed using a light microscope at 4x, 10x, and 4x magnifications (Figure 3). The red arrow in Figure 3 indicates successfully transformed protoplasts, as indicated by their round, brightly colored appearance.

Figure 3
Relative Lycopene β-cyclase (LCYB) Gene Expression in Musa acuminata protoplasts using qRT-PCR (A) and Amplification curve plot showing the Ct value to determine the level of LCYB gene expression (B). Isolation of protoplasts in M. acuminata lcyb mutants (C). MP1, MP2, MP3: Mutant protoplasts 1, 2, and 3. Sequence of mutant M. acuminata and its wild type/ control (D). Columns and bars represent the mean value and standard error. The bar can also represent that there is a significant difference between control M. acuminata protoplasts and Lycopene β-cyclase (LCYB) mutant M. acuminata protoplasts. Biological replications: 12, and technical replications: 3, respectively. The ∆Rn value is obtained from the Rn value in the experimental reaction minus the Rn value in the baseline signal generated from the instrument. Isolation of protoplasts in M. acuminata lcyb mutants. MP1, MP2, MP3: Mutant protoplasts 1, 2, and 3. Asterisk (*) represents significantly difference based on T-Test (p=0.0032, p <0.05).

Observation of the protoplasts at 4x magnification revealed a round shape, but the resulting bright color was less visible. Therefore, further observation was performed at 10x magnification. Observation of the protoplasts at 10x magnification revealed a round, brightly colored protoplast, making them clearly visible under a light microscope. Meanwhile, observations of protoplasts at 40x magnification, have almost the same results as at 10x magnification, showing a round protoplast shape and bright color, so that it can be seen clearly using a light microscope.

3.5. Quantification of RNA and DNA of M. acuminata protoplasts

Quantification of M. acuminata protoplast RNA and DNA was performed using nanodrop. The control protoplast RNA nanodrop results had a nucleic acid concentration of 29.60 ng/µL, while the transformed protoplast RNA had a nucleic acid concentration of 68.80 ng/µL. The control protoplast DNA nanodrop results had a nucleic acid concentration of 95.00 ng/µL, while the transformed protoplast DNA had a nucleic acid concentration of 285.0 ng/µL (Table 1).

Table 1
The RNA and DNA quantification of Musa acuminata protoplasts transformed with recombinant plasmid Agrobacterium tumefaciens.

No	Sample	Concentration (ng/ µL)	A260	A280	A260/280	A260/280 *
1	RNA Protoplast Control	29.60	0.74	0.54	1.37	1.81
2	RNA Protoplast Mutant	68.80	1.72	1.40	1.22	1.90
3	DNA Protoplast Control	95.00	1.90	1.30	1.46	2.00
4	DNA Protoplast Mutant	285.00	5.70	4.72	1.20	1.92

• Note: asterisk (*) represents value after purification.

3.6. Relative LCYB gene expression in lcyb protoplast mutants

LCYB Relative Gene Expression was performed using real-time polymerase chain reaction (PCR). The highest LCYB gene expression results came from protoplasts that were not given transformation treatment (control). Meanwhile, the lowest LCYB gene expression results came from protoplasts that were given transformation treatment (LCYB mutant). The level of LCYB relative gene expression showed that there was a significant difference between protoplasts that were not given treatment (control) and those that were given treatment (LCYB mutant) (Figure 3). The LCYB relative gene expression value based on the amplification curve and ∆Rn value was obtained by comparing the actin gene as a housekeeping gene and the negative control.

4. Discussion

Protoplasts derived from Cavendish bananas (M. acuminata) can be regenerated by genetic engineering, such as transformation methods. The vector used is the recombinant plasmid pRGEB32 LCYB, which has been transformed into A. tumefaciens. pRGEB32 is also often used in CRISPR/Cas9 genome editing technology (Fendiyanto et al., 2025a), because it has a U3 snoRNA promoter (U3p) that can express sgRNA and a rice ubiquitin promoter (UBIp) with a conserved 5' untranslated region that can express Cas9 (Xie et al., 2015; Fendiyanto et al., 2025b). Furthermore, the recombinant plasmid pRGEB32-LCYB, which has been transformed into A. tumefaciens, was used to transform into the M. acuminata protoplast genome. The protoplast transformation process was mediated using polyethylene glycol (PEG) 4000. Transformed protoplasts can be quantified by measuring their concentration and purity.

The present study evaluated a CRISPR/Cas9-mediated transformation approach targeting the LCYB gene in Cavendish banana (M. acuminata). The pRGEB32-based construct was selected because this vector contains regulatory elements for sgRNA and Cas9 expression, providing a suitable platform for targeted genome modification. The use of a CRISPR/Cas9-based system is particularly relevant for LCYB because modification of this gene may influence carotenoid biosynthesis and consequently alter the metabolic characteristics of banana tissues. In the present study, both protoplast-mediated transformation using polyethylene glycol (PEG) and Agrobacterium tumefaciens-mediated transformation were evaluated as potential approaches for introducing the CRISPR/Cas9 construct (Figures 1 and 2).

DNA and RNA purity can be assessed using the A260/A280 absorbance ratio. The RNA isolated from the transformed protoplasts showed an A260/A280 ratio of 1.22, indicating relatively low RNA purity and suggesting the possible presence of contaminants. In general, an A260/A280 ratio close to 2.0 is considered indicative of relatively pure RNA (Fendiyanto et al., 2021). Therefore, additional purification may be required to improve RNA quality and remove potential contaminants. Methods such as DNase or RNase treatment, ethanol precipitation, or re-purification using chloroform alcohol (CI) may be employed depending on the type of contamination (Table 1). An A260/A280 ratio close to 2.0 is generally associated with relatively pure RNA, although this ratio should not be interpreted as evidence of complete absence of contamination (Fendiyanto et al., 2023a).

Meanwhile, the DNA isolated from the transformed protoplasts showed an A260/A280 ratio of 1.20. Previous studies have reported that relatively pure DNA generally has an A260/A280 ratio of approximately 1.8-2.0 (Sambrook et al., 1989). Ratios below 1.8 may indicate contamination by proteins, phenol, or other compounds, whereas ratios above 2.0 may suggest possible RNA contamination (Li et al., 2014). Therefore, the A260/A280 ratio of 1.20 observed in the present study suggests that the DNA was not highly pure and may have contained residual contaminants. Further purification would therefore be necessary to improve DNA quality before downstream molecular analyses.

The quantification of the isolated DNA and RNA from the transgenic mutant protoplasts was then amplified using the actin primer pair and the LCYB primer pair. Prior to amplification, the isolated protoplast RNA was synthesized into cDNA. The resulting cDNA can be used directly as a template for PCR or qPCR. The synthesized cDNA was then amplified along with the isolated protoplast DNA. Protoplast amplification results were visualized by electrophoresis using a 1% agarose gel. The amplification results showed a faint band with an amplicon size of approximately 250 base pairs (bp) on the gel (Figure 3). Good DNA quality is considered good if the electrophoresis results show a bright and focused banding pattern (Fendiyanto et al., 2025a; Sihombing et al., 2025). DNA with a high concentration and purity of 1.8-2.0 is likely to produce a bright and focused banding pattern. Band intensity is also influenced by the distribution of primer attachment sites in the genome, the purity, and concentration of the genome in a reaction (Sindiya et al., 2024). The intensity of the amplified DNA band with each primer is strongly influenced by the purity and concentration of the template DNA and the primer attachment site on the template DNA (Satrio et al., 2024).

The primers used can also be a factor in successful DNA amplification. Non-specific primers can lead to mispriming or even complete failure of DNA amplification (Resmi et al., 2024). Furthermore, the success of an amplification process is determined by the annealing temperature used in a reaction. This is because the DNA elongation process begins with the primer (Satrio et al., 2025). Research by Ratnadewi et al. (2021) found that the primer attachment process to the target DNA or cDNA is significantly influenced by the temperature setting during the annealing phase. Commonly used annealing temperatures are 50-60 °C. Meanwhile, the optimal annealing temperature in this study was 55 °C. Based on the research, the faint bands seen in the amplification results are due to the low purity and concentration of the DNA, as well as the annealing temperature used.Amplified transgenic mutant protoplast samples will also be sent to genetic science for sequencing. Sequencing is performed to identify and verify the mutated bases in the desired target sequence.

The PCR results showed amplification of a fragment of approximately 250 bp, although the intensity of the band was relatively weak. Importantly, the detection of an amplicon of the expected size provided sufficient material for subsequent sequence analysis. The relatively weak amplification may have resulted from the low quality or concentration of the template DNA, as well as primer-template interactions and reaction conditions. The annealing temperature of 55 °C used in this study was within the commonly applied range for PCR amplification and was selected to facilitate specific primer binding. Therefore, optimization of template concentration, purification, primer concentration, and PCR conditions may improve amplification efficiency in future experiments. More importantly, the PCR product obtained in the present study enabled sequence-level evaluation of the targeted LCYB region.

Sequence analysis provided stronger molecular evidence than PCR alone for evaluating the genetic status of the regenerated plants. Comparison of the amplified LCYB target region with the corresponding wild-type sequence revealed a 6-bp deletion at the predicted CRISPR/Cas9 target region, immediately upstream of the protospacer adjacent motif (PAM) (Figure 3D). The location of this sequence variation in the predicted target region is consistent with the expected outcome of Cas9-mediated double-strand break repair through endogenous DNA repair mechanisms. Thus, the sequencing result strengthens the evidence that the regenerated plant material contained a sequence modification at the intended LCYB target region rather than merely demonstrating the presence of the amplified DNA fragment.

The nature of the detected 6-bp deletion is also relevant to the potential functional consequence of the modification (Figure 3). Because the deletion consists of six nucleotides, which is a multiple of three, a deletion occurring within a protein-coding region would not necessarily result in a frameshift but could remove two amino acids from the encoded protein. However, the functional consequence depends on the precise location of the deletion within the LCYB gene and whether the affected region contributes to an essential structural or functional domain of the protein. Therefore, sequence alignment alone demonstrates the presence of a sequence modification but does not establish its functional consequence. Additional analysis of the predicted protein sequence and functional characterization would be required to determine whether the detected deletion alters LCYB protein activity.

The LCYB expression analysis further showed relatively low transcript abundance in the transformed protoplasts compared with the control. This observation is potentially relevant to the sequence modification detected at the target locus. A reduction in transcript abundance may be associated with changes in gene regulation or transcript stability; however, the present data do not establish a direct causal relationship between the 6-bp deletion and the observed reduction in LCYB expression (Figure 3). The expression result should therefore be interpreted as molecular evidence of altered LCYB transcript abundance in the evaluated transformed material rather than as definitive evidence that the detected deletion alone caused the expression change. Additional experiments, including analysis of independent edited lines, sequencing of multiple clones or alleles, and functional characterization of LCYB expression and protein activity, would strengthen this interpretation.

The integration of PCR, sequencing, and gene expression data provides a more comprehensive molecular characterization of the regenerated material (Figures 2 and 3). PCR demonstrated amplification of the target region, sequencing revealed a specific 6-bp deletion at the predicted CRISPR/Cas9 target site, and expression analysis indicated altered LCYB transcript abundance. Taken together, these results provide complementary evidence supporting CRISPR/Cas9-mediated modification of the targeted LCYB region and demonstrate the feasibility of the transformation approach. Nevertheless, the extent and stability of the detected modification should be further evaluated in additional independent regenerated plants and, where applicable, in subsequent generations.

LCYB gene expression pattern analysis was performed using genetic material from M. acuminata protoplasts. Relative gene expression was determined by comparing the amount of LCYB gene expression in the protoplasts with the amount of actin gene expression, a reference gene derived from the housekeeping gene. Based on the LCYB gene expression results, transformed protoplasts exhibited low expression. This is because the LCYB gene in the protoplasts has been edited. The gene expression results in this study are still preliminary, so further research is needed to confirm the effect of changes in protein function when LCYB gene mutations occur in transgenic mutant protoplasts.

To further verify the molecular status of the candidate transformed mutant plants, the PCR-amplified LCYB target region was subjected to sequencing and compared with the corresponding sequence from the wild-type plants. Sequence alignment revealed nucleotide differences at/around the CRISPR/Cas9 target site in the transformed plants compared with the wild type (Figure 3D). The identified sequence variations included 6 base pair (bp)- deletion at nucleotide position before protospacer adjacent motif (PAM) of LCYB, corresponding to the predicted Cas9 target region. The observed sequence variation at the target site provides additional molecular evidence supporting CRISPR/Cas9-mediated modification of the LCYB target region. However, the sequence data should be interpreted together with the PCR and LCYB expression results to evaluate the molecular characteristics of the regenerated plants.

Meanwhile, transformation was also used to directly transform explants. Transformation was carried out through A. tumefaciens. Transformation is carried out to introduce foreign genes into plants, allowing researchers to easily observe the processes of overexpression, knockdown, or knockout of gene expression in these plants (Miftahudin et al., 2021). Transformation mediated by A. tumefaciens is a method that can produce stable transformant plants, as a model for studying cellular level localization and interactions between proteins that are very important for functional gene discovery analysis (Miftahudin et al., 2021), so that the method can also be used to bring CRISPR/Cas9 into the plant genome. The role of A. tumefaciens uses a virulence mechanism that can induce tumors, so it can be used to bring T-DNA into host cells where the T-DNA will be integrated into the plant genome. The transformed plants were then selected to find out which plants were successfully inserted with foreign genes by growing them in Murashige and Skoog (MS) media with the addition of kanamycin antibiotics through tissue culture. However, verification of the LCYB gene has not been carried out on transformed plants, because it uses explants and requires a long time to make the part grow into a whole plant for verification.

Overall, the present findings demonstrate that the combination of CRISPR/Cas9-mediated transformation, PCR-based molecular screening, sequence analysis, and gene expression analysis can provide a useful framework for investigating targeted modification of LCYB in banana. The identification of a 6-bp deletion at the predicted target region provides a promising molecular indication of targeted sequence modification, while the observed change in LCYB transcript abundance suggests a potential molecular consequence that warrants further functional validation. These findings provide a foundation for subsequent development and validation of LCYB-targeted banana lines with potential applications in carotenoid-related metabolic improvement.

5. Conclusions

In summary, based on the research that has been conducted, it can be concluded that the banana mutant cv. Cavendish was successfully regenerated using the pRGEB32-sg LCYB vector so that it can produce transgenic mutant protoplasts and there is low suppression of LCYB gene expression, especially in transgenic mutant protoplasts when compared to the wild type or control.

Acknowledgements

The author(s) declare that financial support was received for the the National Research and Innovation Agency (BRIN/Badan Riset dan Inovasi Nasional) and LPDP (Ministry of Finance), through number 86/II/HK/2023, for their financial support of the Visiting Researcher (VR) research scheme to MHF and BRH, and Ministry of Defense, Republic of Indonesia for the Fiscal Year (FY) 2023-2025 grant to MHF.

Data Availability Statement

The dataset analyzed or produced in this study can be requested from the corresponding author.

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*

e-mail: [e-mail]; [e-mail]

Editor:

Takako Matsumura Tundisi

Publication Dates

• Publication in this collection
  12 Oct 2026
• Date of issue
  2026

History

• Received
  24 Apr 2026
• Accepted
  23 Aug 2026