Biolistic Research Articles

Over expression of modified Isomaltulose Synthase Gene II (ImSyGII) under single and double promoters drive unprecedented sugar contents in sugarcane.

Sugarcane has been grown all around the world to meet sugar demands for industrial sector. The current sugar recovery percentage in sugarcane cultivars is dismally low which demands scientific efforts for improvements. Multiple approaches were adopted to enhance sugar contents in commercial sugarcane plants in contrast to conventional plant breeding methods. The exploitation of biotechnological methods and exploration of isomaltulose synthetic genes presented a promising solution to increase the existing low level of sugar recovery percentage in Saccharum officinarum L. Isomaltulose synthase gene II was employed and integrated into plant expression vector driven under the leaf and stem specific promoters terminated by nopaline synthase gene in a cloning strategy shown in the present study. Three gene constructs were developed in various combinations driven under promoters Zea mays ubiquitin and Cestrum Yellow Leaf Curl virus in the single and double combined stacked system. The transformation was executed in multiple formats with single transformed events, double promoter transformation events and triple construct stacked promoters in sugarcane induced calli via the particle gene gun. The transformation of ImSyGII in sugarcane genotype HSF-240 was confirmed by molecular gene analysis while expression quantification was determined through Real Time PCR. Furthermore, HPLC was also done to harvest the increased amounts of Isomaltulose in transgenic sugarcane juice. The present work upheld the enhanced ImSyGII expression in leaves owing to the exploitation of ubiquitin, while the Cestrum Yellow Leaf Curl virus promoter enhanced gene expression in sugarcane stems. The employment of three gene constructs collectively produced elite sugar lines producing more than 78% enhancements in whole sugar recovery percentage. The mature internode proved highly efficient and receptive regarding the production of isomaltulose. Quantifications and sugar contents evaluations upheld an increased Brix ratio of transgenic sugarcane lines than control lines.

TRANSGENE MANUFACTURING: FROM TOOLS TO APPLICATIONS

Genetic modification generation is an important intersection of genetic engineering and biotechnology and involves the integration of foreign genes into the genome of organisms to produce specific proteins or repair genetic abnormalities. The machine has many applications in medicine, agriculture and biotechnology. The evolution of genetically modified production technologies from traditional methods to advanced genetic engineering tools demonstrates their evolution. This review discusses various methods incorporating modern and advanced technologies and explores their applications and future prospects. Early models of genetic modification generally involved organisms modified to produce human insulin, followed by the evolution of animals and plants. Traditional methods such as microinjection, retrovirus-mediated gene transfer and embryonic stem cell -mediated gene transfer are important for the production of transgenic animals. Similarly, Agrobacterium-mediated transformation and biolistic transformation methods are used to produce genetically modified plants. Although useful, these methods often result in synergistic and differential gene expression. Made with genetic modification. This technology allows modification of the target with minimal impact on the target, thus increasing the predictability and efficiency of genetic modification. Additionally, CRISPR/Cas9's ability to alter multiple genes simultaneously and adaptability to various organisms expands its applications in medicine and agriculture. Synthetic biology continues to advance genetic engineering by designing and building new biological materials and systems, enabling innovations such as genetic engineering, metabolic processes, and minimal genomes. Viral vectors such as adeno-associated virus (AAV), lentivirus, and adenovirus have unique advantages and clinical challenges. Nonviral vectors, including lipid nanoparticles, electroporated, and polymeric vectors, provide alternative delivery methods with varying efficacy and specificity. Painkiller. It can improve crop growth, livestock and disease resistance in agriculture. Applications of biotechnology include biofuels, biopharmaceuticals, and bioremediation. Future directions aim to improve delivery, expand applications, explore synthetic genomics, advance personalized medicine, and develop regenerative agriculture.In summary, with the influence of advances in genetic engineering and technology, genetically modified production has become a complex field with many applications. Its future promises transformative solutions to global challenges in health, food security and environmental sustainability. To reach its full potential, continuous innovation and ethical thinking are essential.

Painted flowers: Eluta generates pigment patterning in Antirrhinum.

In the early 1900s, Erwin Baur established Antirrhinum majus as a model system, identifying and characterising numerous flower colour variants. This included Picturatum/Eluta, which restricts the accumulation of magenta anthocyanin pigments, forming bullseye markings on the flower face. We identified the gene underlying the Eluta locus by transposon-tagging, using an Antirrhinum line that spontaneously lost the nonsuppressive el phenotype. A candidate MYB repressor gene at this locus contained a CACTA transposable element. We subsequently identified plants where this element excised, reverting to a suppressive Eluta phenotype. El alleles inhibit expression of anthocyanin biosynthetic genes, confirming it to be a regulatory locus. The modes of action of Eluta were investigated by generating stable transgenic tobacco lines, biolistic transformation of Antirrhinum petals and promoter activation/repression assays. Eluta competes with MYB activators for promoter cis-elements, and also by titrating essential cofactors (bHLH proteins) to reduce transcription of target genes. Eluta restricts the pigmentation established by the R2R3-MYB factors, Rosea and Venosa, with the greatest repression on those parts of the petals where Eluta is most highly expressed. Baur questioned the origin of heredity units determining flower colour variation in cultivated A. majus. Our findings support introgression from wild species into cultivated varieties.

Improved biolistic transformation and genome editing in wheat by using trehalose for high osmotic treatment.

The tissue culture process is usually involved in gene transfer and genome editing in plants. Like other species, there is enormous variation among wheat genotypes in tissue culture response. In the rapidly advancing system of CRISPR/Cas9 for genome editing, particle bombardment has received increasing attention as a delivery method for a large amount of nucleic acids and RNA-protein complexes. However, the efficiency of transformation by particle bombardment has been low in wheat, and only a limited number of varieties have been transformed. In this study, replacement of maltose with trehalose as an osmolyte for high osmotic treatment for the protection of tissues from physical impacts improved callus formation in immature wheat embryos and efficiency of transformation and genome editing in varieties that are relatively poor in tissue culture response. The range of varieties amenable to biolistic transformation and genome editing may be expanded by this modification.

CRISPR-based gene editing in plants: Focus on reagents and their delivery tools

Introduction: CRISPR-Cas9 technology has revolutionized plant genome editing, providing precise and efficient methods for genetic modification. This study focuses on the advancements and delivery of CRISPR-Cas9 in plant gene editing. Methods: A comprehensive search in scientific databases, including PubMed, ScienceDirect, and Google Scholar, was conducted to gather information on CRISPR-Cas9 gene editing and its delivery in precise gene modification in plants. Results: The evolving landscape of CRISPR nucleases has led to the development of innovative technologies, enhancing plant research. However, successful editing is contingent on efficient delivery of genome engineering reagents. CRISPR-based gene editing in plants utilizes diverse delivery methods: Agrobacterium-mediated transformation for bacterial transfer, biolistic transformation for physical gene insertion, electroporation for direct gene entry, expression of developmental regulators for gene expression modulation, and tobacco rattle virus as a viral vector, each offering distinct advantages for precise and efficient genetic modification in plants. Conclusion: CRISPR-Cas9 gene editing stands as a pivotal advancement in plant genetics, offering precise gene manipulation with applications in agriculture and biotechnology. The continuous refinement of reagent delivery tools reinforces CRISPR-Cas9's transformative role in plant genome editing, with significant implications for broader scientific applications.

Mitochondrial Transformation in Baker's Yeast to Study Translation and Respiratory Complex Assembly.

Baker´s yeast Saccharomyces cerevisiae has been widely used to understand mitochondrial biology for decades. This model has provided knowledge about essential, conserved mitochondrial pathways among eukaryotes, and fungi or yeast-specific pathways. One of the many abilities of S. cerevisiae is the capacity to manipulate the mitochondrial genome, which so far is only possible in S. cerevisiae and the unicellular algae Chlamydomonas reinhardtii. The biolistic transformation of yeast mitochondria allows us to introduce site-directed mutations, make gene rearrangements, and introduce reporters. These approaches are mainly used to understand the mechanisms of two highly coordinated processes in mitochondria: translation by mitoribosomes and assembly of respiratory complexes and ATP synthase. However, mitochondrial transformation can potentially be used to study other pathways. In the present work, we show how to transform yeast mitochondria by high-velocity microprojectile bombardment, select and purify the intended transformant, and introduce the desired mutation in the mitochondrial genome.

Agrobacterium and biolistic mediated genetic transformation of mungbean cultivar Samrat using embryogenic explant

Agrobacterium and biolistic mediated genetic transformation of mungbean cultivar Samrat using embryogenic explant

Combined biolistic and cell penetrating peptide delivery for the development of scalable intradermal DNA vaccines

Physical-based gene delivery via biolistic methods (such as the Helios gene gun) involve precipitation of nucleic acids onto microparticles and direct transfection through cell membranes of exposed tissue (e.g. skin) by high velocity acceleration. The glycosaminoglycan (GAG)-binding enhanced transduction (GET) system exploits novel fusion peptides consisting of cell-binding, nucleic acid condensing, and cell-penetrating domains, which enable enhanced transfection across multiple cell types. In this study, we combined chemical (GET) and physical (gene gun) DNA delivery systems, and hypothesized the combination would generate enhanced distribution and effective uptake in cells not initially transfected by biolistic penetration. Physicochemical characterization, optimization of bullet contents and transfection experiments in vitro in cell monolayers and engineered tissue demonstrated these formulations transfected efficiently, including DC2.4 dendritic cells. We incorporated these formulations into a biolistic format for gene gun by forming fireable dry bullets obtained via lyophilization (freeze drying). This system is simple and with enhanced scalability compared to conventional methods to generate bullets. Flushed GET bullet contents retained their ability to mediate transfection (17-fold greater and 13-fold greater reporter gene expression than standard spermidine bullets in the absence and presence of serum, respectively). Fired GET bullets in vitro (in cells and collagen gels) and in vivo (mice) showed increased reporter gene transfection compared to untreated controls, whilst maintaining cell viability in vitro and having no obvious toxicity in vivo. Lastly, a SARS-CoV-2 plasmid DNA vaccine with spike (S) protein-receptor binding domain (S-RBD) was delivered by gene gun using GET bullets. Specific T cell and antibody responses comparable to the conventional system were generated. The non-physical and physical combination of GET‑gold-DNA carriers using gene gun shows potential as an alternative DNA delivery method that is scalable for mass deployable vaccination and intradermal gene delivery.

CRISPR/Cas9-Mediated Genome Editing in Indica Rice (Oryza sativa L. subsp. indica var. CR-5272).

Tissue culture optimization protocols limit indica rice breeding. Such a challenge is vital because emergent techniques still rely on tissue culture methods and could allow the breeding of new varieties with higher production and toleration of adverse environmental effects caused by climate change. Genome editing technology, using CRISPR/Cas9, is a fast and precise method for accelerated plant breeding. It limited its use in indica subspecies because of the recalcitrant response to in vitro culture methods. This chapter describes a protocol for CRISPR/Cas9 editing in indica subspecies, specifically in the CR-5272 variety derived from parental lines IR-822, using Agrobacterium tumefaciens and biolistic transformation.

Biolistic Transformation of Cryptococcus neoformans.

Biolistic transformation of Cryptococcus neoformans is used as a molecular tool to genetically alter or delete targeted genes. The DNA is introduced into the yeast on DNA-coated gold beads by a helium shock wave produced using a biolistic particle system. The procedure often involves insertion of a dominant selectable marker into the desired site by homologous recombination. To increase the likelihood of homologous recombination, large fragments of overlapping DNA are used. The two most used dominant selectable markers are nourseothricin and Geneticin. With the need to generate multiple gene deletions in the same strain, there are recyclable marker systems, such as the bacteriophage P1 Cre-loxP system or CRISPR that provide additional useful molecular tools. While newer strategies exist to generate deletions and introduce markers and other gene modifications, biolistic transformation has remained a viable tool to facilitate the construction of genetically modified yeast strains. This chapter provides a working protocol on how to delete and restore a gene in C. neoformans.

Targeted Gene Editing of Nuclear-Encoded Plastid Proteins in Phaeodactylum tricornutum via CRISPR/Cas9.

Genome modifications in microalgae have emerged as a crucial and indispensable tool for research in fundamental and applied biology. In particular, CRISPR/Cas9 has gained significant recognition as a highly effective method for genome engineering in these photosynthetic organisms, enabling the targeted induction of mutations in specific regions of the genome. Here, we present a comprehensive protocol for generating knock-out mutants in the model diatom Phaeodactylum tricornutum using CRISPR/Cas9 by both biolistic transformation and bacterial conjugation. Our protocol outlines the step-by-step procedures and experimental conditions required to achieve successful genome editing, including the design and construction of guide RNAs, the delivery of CRISPR/Cas9 components into the algae cells, and the selection of the generated knockout mutants. Through the implementation of this protocol, researchers can harness the potential of CRISPR/Cas9 in P. tricornutum to advance the understanding of diatom biology and explore their potential applications in various fields.

Mobilization of Plasmids From Bacteria Into Diatoms by Conjugation Technique.

Diatoms serve as a source for a variety of compounds with particular biotechnological interest. Therefore, redirecting the flow to a specific pathway requires the elucidation of the gene's specific function. The most commonly used method in diatoms is biolistic transformation, which is a very expensive and time-consuming method. The use of episomes that are maintained as closed circles at a copy number equivalent to native chromosomes has become a useful genetic system for protein expression that avoids multiple insertions, position-specific effects on expression, and potential knockout of non-targeted genes. These episomes can be introduced from bacteria into diatoms via conjugation. Here, we describe a detailed protocol for gene expression that includes 1) the gateway cloning strategy and 2) the conjugation protocol for the mobilization of plasmids from bacteria to diatoms.

METHODS OF GENETIC TRANSFORMATION: MAJOR EMPHASIS TO CROP PLANTS

Advancements in gene transfer technology have indeed opened up exciting possibilities for more effectively manipulating the genetic makeup of live organisms, ranging from microorganisms to plants and animals. Direct and indirect transformations are the two basic types of gene transfer techniques. Indirect method comprises Agrobacterium mediated method as it involves intermediate host between gene of interest and target and this method is most opted one out of all present. Direct gene transformation methods, on the other hand, do not involve the use of an intermediate host organism. Instead, they rely on physical means to transfer genes between cells. Biolistic transformation uses high-velocity particles to deliver DNA into target cells, while microinjection and macroinjection involve the direct injection of DNA into cells. Protoplast fusion combines the genetic material of two different cells by fusing their protoplasts. Natural methods for gene transfer encompass mechanisms that occur naturally in various organism, includes transposition, conjugation, phage and retroviral transductions and bacterial transformation. Chemical techniques utilize chemical agents to facilitate gene transfer, such as calcium phosphate-mediated transformation, polyethylene glycol (PEG)-mediated transformation, DEAE (Diethylethanolamine)-Dextran-mediated transformation. Genes can be also being transferred using electrical techniques such as electroporation and electrofusion. Crop improvement and trait improvement are now being hastened by the fast-rising number of sequenced plant genomes, information from functional genomics data to understand gene function, innovative gene cloning, and tissue culture techniques. Despite being indispensable, its progress is still hindered by the fact that many plant species and agricultural genotypes exhibit low transformability or are resistant to established tissue culture and regeneration conditions. Here, we review the techniques employed in plant transformation and provide a concise overview of their evolution in agricultural crops, from their first inception to present time.

Advancing gene editing: multiplex mutagenesis in hexaploid triticale

The presence of several sets of chromosomes in polyploid crops is a serious problem for the application of gene and genome editing systems. Efficient CRISPR/Cas-based mutagenesis of series of genes involved in the grain starch biosynthesis of hexaploid triticale has been developed. Triticale (×Triticosecale),is a hybrid of rye (Secale) and wheat (Triticum) and consists of three subgenomes. Four genes were targeted and to ensure efficient editing of all subgenomes, a trio of guide RNAs for each target genes were designed. To enable simultaneous editing of 36 genetic loci at once (three sgRNAs × four genes × three subgenomes), an expression cassette was constructed, assembled as an array of twelve sgRNAs. The polysitron vector was delivered to morphogenic calli using a gene gun [1] together with a vector encoding Cas9 nuclease [2] to induce mutations. A number of transgenic plants of spring and winter triticale carrying both Cas9 and sgRNAs inserts have been generated. The efficiency of native gene editing varied depending on the target gene and sgRNA activity. Using a trio of sgRNAs for each target gene, we successfully mutated all three subgenome copies, thereby modifying seed starch synthesis. It can be expected that the described approach will make an important contribution to the future breeding of polyploid crops to produce various combinations of new genetic alleles encoding desired traits.
 The study is supported by Kurchatov Genomics Center of All-Russia Research Institute of Agricultural Biotechnology, agreement No. 075-15-2019-1667.

Modification of agricultural traits in cultivated varieties of barley and wheat

CRISPR/Cas technology makes it possible to induce mutations at defined positions. In breeding-oriented research, this opens up exciting opportunities for the targeted improvement of many agricultural crops. Wheat and barley are among the most important cereals in the world. However, the transformation poses a particular challenge for cereals and is strongly genotype dependent. This is because agrobacteria, which is mostly used for delivering the CRISPR/Cas system, have a limited compatibility with these non-host plants. Transformation of wheat is additionally difficult due to the large genome size and polyploidy.
 Besides obtaining improved genotypes, the object of the current study was to optimize the method of genomic editing based on the CRISPR/Cas system using particle bombardment for non-model varieties of barley and wheat. In barley, we targeted theNudgene that controls hulled/naked phenotype of the grain. Since the regeneration rate remains an issue for the cultivated cultivars, we used the JD633 vector that carries theGRF4-GIF1chimera to increase the efficiency of regeneration. We obtained five T0plants, carrying mutations. In wheat, targetingPpd-1genes that control photoperiod-dependent floral induction results in Cas9-induced mutations in 52 of 210 T0plants. The developed collection of wheat plants with different new alleles ofPpd-D1andPpd-B1genes is being studied for the expression under short day conditions and the effect on the vegetation period.
 Thus, we have obtained plants of the cultivated varieties of barley and wheat with edited agronomically important genes, using the improved protocols of biolistic transformation.
 This work was done within the framework of State Assignment Kurchatov Genomic Center of ICG SB RAS (No. 075-15-2019-1662).

Biolistic Delivery of Photosensitizer‐Loaded Porous Si Carriers for Localized Photodynamic Therapy

AbstractAmong numerous approaches for treating cancer, clinically approved photodynamic therapy (PDT) is considered a promising non‐invasive therapeutic strategy for solid tumors. While PDT has distinct advantages over conventional cancer treatments, systemic exposure to the photosensitizer and its stability are some of the limitations of clinical PDT. Herein, a therapeutic strategy for highly localized focal PDT is introduced based on direct biolistic delivery of photosensitizer‐loaded carriers to cancerous tumors. Degradable porous silicon microparticles (PSiMPs) are used as efficient carriers for the photosensitizer, meso‐tetrahydroxy‐phenylchlorin (mTHPC), and its conjugates with gold nanoparticles (AuNP‐mTHPC conjugates). The loaded PSiMP carriers are successfully bombarded using a pneumatic gene gun to breast cancer cells in vitro and into tumor xenografts in vivo, and subsequent uptake of the released photosensitizer payload is demonstrated. PDT irradiation is commenced after 24 h based on the release profile, resulting in complete cell death in vitro and substantial inhibition of tumor growth in vivo. Notably, using empty PSiMP carriers also leads to some tumor growth inhibition, ascribing to its intrinsic photosensitizing activity. Treatment with AuNP‐mTHPC‐loaded PSiMPs exhibits a superior therapeutic effect in comparison to bombarded mTHPC‐loaded carriers and administration of free mTHPC. This biolistic delivery scheme may be advantageous for precision photodynamic therapy.

DNA Vaccine Co-Expressing Hemagglutinin and IFN-γ Provides Partial Protection to Ferrets against Lethal Challenge with Canine Distemper Virus.

Canine distemper (CD), caused by canine distemper virus (CDV), is a highly contagious and lethal disease in domestic and wild carnivores. Although CDV live-attenuated vaccines have reduced the incidence of CD worldwide, low levels of protection are achieved in the presence of maternal antibodies in juvenile animals. Moreover, live-attenuated CDV vaccines may retain residual virulence in highly susceptible species and cause disease. Here, we generated several CDV DNA vaccine candidates based on the biscistronic vector (pIRES) co-expressing virus wild-type or codon-optimized hemagglutinin (H) and nucleocapsid (N) or ferret interferon (IFN)-γ, as a molecular adjuvant, respectively. Apparently, ferret (Mustela putorius furo)-specific codon optimization increased the expression of CDV H and N proteins. A ferret model of CDV was used to evaluate the protective immune response of the DNA vaccines. The results of the vaccinated ferrets showed that the DNA vaccine co-expressing the genes of codon-optimized H and ferret IFN-γ (poptiH-IRES-IFN) elicited the highest anti-CDV serum-neutralizing antibodies titer (1:14) and cytokine responses (upregulated TNF-α, IL-4, IL-2, and IFN-γ expression) after the third immunization. Following vaccination, the animals were challenged with a lethal CDV 5804Pe/H strain with a dose of 105.0 TCID50. Protective immune responses induced by the DNA vaccine alleviated clinical symptoms and pathological changes in CDV-infected ferrets. However, it cannot completely prevent virus replication and viremia in vivo as well as virus shedding due to the limited neutralizing antibody level, which eventually contributed to a survival rate of 75% (3/4) against CDV infection. Therefore, the improved strategies for the present DNA vaccines should be taken into consideration to develop more protective immunity, which includes increasing antigen expression or alternative delivery routes, such as gene gun injection.

A DNA-binding protein capture technology that purifies proteins by directly isolating the target DNA

DNA-protein interactions are critical to almost all cellular functions, and identification of the proteins that bind to an DNA site of interest (gene-centered approach) is an important investigation area. However, gene-centered methods are mainly based on DNA hybridization to isolate target proteins, which is complex and inefficient. Here, we built a gene-centered approach involving direct isolation of target DNA, termed protein capture based on biolistic transformation (PCaB). The target DNA was labeled with biotin and cyanine 3 (Cy3) at its 5′ and 3′ DNA ends, respectively, and introduced into the host plants through biolistic transformation. The DNA and its binding proteins were crosslinked using formaldehyde. The labeled DNAs were obtained using gel excision and biotin-Streptavidin affinity according to the indication of Cy3 fluorescence, which make harvest of target DNA with a low background. The DNA-binding proteins were identified using mass spectrometry analysis. The PCaB method allowed us to identify and confirm 16 putative upstream regulators of the BpERF3 gene from Betula platyphylla. Theoretically, PCaB could be adapted to all plant species that can be transformed using biolistic bombardment, and captures DNA-binding proteins quickly with a low background. Therefore, PCaB will provide a powerful tool to discover DNA-protein interactions.

Intracellular Localization during Blood–Brain Barrier Crossing Influences Extracellular Release and Uptake of Fluorescent Nanoprobes

To improve the efficacy of nanoparticles (NPs) and boost their theragnostic potential for brain diseases, it is key to understand the mechanisms controlling blood–brain barrier (BBB) crossing. Here, the capability of 100 nm carboxylated polystyrene NPs, used as a nanoprobe model, to cross the human brain endothelial hCMEC/D3 cell layer, as well as to be consequently internalized by human brain tumor U87 cells, is investigated as a function of NPs’ different intracellular localization. We compared NPs confined in the endo-lysosomal compartment, delivered to the cells through endocytosis, with free NPs in the cytoplasm, delivered by the gene gun method. The results indicate that the intracellular behavior of NPs changed as a function of their entrance mechanism. Moreover, by bypassing endo-lysosomal accumulation, free NPs were released from cells more efficiently than endocytosed NPs. Most importantly, once excreted by the endothelial cells, free NPs were released in the cell culture medium as aggregates smaller than endocytosed NPs and, consequently, they entered the human glioblastoma U87 cells more efficiently. These findings prove that intracellular localization influences NPs’ long-term fate, improving their cellular release and consequent cellular uptake once in the brain parenchyma. This study represents a step forward in designing nanomaterials that are able to reach the brain effectively.

PhCHS5 and PhF3'5'H Genes Over-Expression in Petunia (Petunia hybrida) and Phalaenopsis (Phalaenopsis aphrodite) Regulate Flower Color and Branch Number.

Flower breeders are continually refining their methods for producing high-quality flowers. Phalaenopsis species are considered the most important commercially grown orchids. Advances in genetic engineering technology have provided researchers with new tools that can be used along with traditional breeding methods to enhance floral traits and quality. However, the application of molecular techniques for the breeding of new Phalaenopsis species has been relatively rare. In this study, we constructed recombinant plasmids carrying flower color-related genes, Phalaenopsis Chalcone synthase (PhCHS5) and/or Flavonoid 3',5'-hydroxylase (PhF3'5'H). These genes were transformed into both Petunia and Phalaenopsis plants using a gene gun or an Agrobacterium tumefaciens-based method. Compared with WT, 35S::PhCHS5 and 35S::PhF3'5'H both had deeper color and higher anthocyanin content in Petunia plants. Additionally, a phenotypic comparison with wild-type controls indicated the PhCHS5 or PhF3'5'H-transgenic Phalaenopsis produced more branches, petals, and labial petals. Moreover, PhCHS5 or PhF3'5'H-transgenic Phalaenopsis both showed deepened lip color, compared with the control. However, the intensity of the coloration of the Phalaenopsis lips decreased when protocorms were co-transformed with both PhCHS5 and PhF3'5'H. The results of this study confirm that PhCHS5 and PhF3'5'H affect flower color in Phalaenopsis and may be relevant for the breeding of new orchid varieties with desirable flowering traits.