Plant Biotechnology Applications Research Articles

Stem cutting: A novel introduction site for transporting water-insoluble particles into tomato (Solanum lycopersicum) seedlings

The introduction of exogenous particles into plants has promising applications in agriculture and biotechnology. Nanoparticles can be transported into plants through foliar application or root uptake. However, both methods have limitations in terms of the size of the particles (<40 nm) that can be transported due to the barriers of the cell wall and cuticle. In the present study, we proposed a novel method to deliver particles of up to 110 nm into plants by cutting the stem of tomato seedlings. We demonstrated for the first time, using water-insoluble silica colloids, that not only nanoparticles but also submicron particles can be transported toward the leaves when the plant stem is used as the entry point of particles. Thirty-five-day-old tomato seedlings were used as the target plants. When the cut stem seedlings were immersed in the colloidal particle suspension for up to 24 h, significant particle accumulation was observed in the nodes and leaves. The relatively low particle concentrations (10 mg/L) allowed effective transport throughout the plants. Silica particles with average diameters of 10 nm and 110 nm were both well transported and moved through the stem. Even after the particles entered the plant, adventitious roots were formed, resulting in the formation of whole plants with roots, stems, and leaves. This method can be applied not only to tomatoes but also to other food crops for various applications in plant biotechnology.

The "Duckweed Dip": Aquatic Spirodela polyrhiza Plants Can Efficiently Uptake Dissolved, DNA-Wrapped Carbon Nanotubes from Their Environment for Transient Gene Expression.

Duckweeds (Lemnaceae) are aquatic nongrass monocots that are the smallest and fastest-growing flowering plants in the world. While having simplified morphologies, relatively small genomes, and many other ideal traits for emerging applications in plant biotechnology, duckweeds have been largely overlooked in this era of synthetic biology. Here, we report that Greater Duckweed (Spirodela polyrhiza), when simply incubated in a solution containing plasmid-wrapped carbon nanotubes (DNA-CNTs), can directly uptake the DNA-CNTs from their growth media with high efficiency and that transgenes encoded within the plasmids are expressed by the plants─without the usual need for large doses of nanomaterials or agrobacterium to be directly infiltrated into plant tissue. This process, called the "duckweed dip", represents a streamlined, "hands-off" tool for transgene delivery to a higher plant that we expect will enhance the throughput of duckweed engineering and help to realize duckweed's potential as a powerhouse for plant synthetic biology.

Exploring the Sustainable Exploitation of Bioactive Compounds in Pelargonium sp.: Beyond a Fragrant Plant.

This review article aims to present an overview regarding the volatile compounds in different scented species of Pelargonium and their biological activities, immunomodulatory activity, cytotoxic activity, high larvicidal activity and ethnopharmacological uses. Although the Pelargonium genus includes many species, we focused only on the scented ones, with the potential to be used in different domains. Pelargonium essential oil showed great properties as antioxidant activity, antibacterial activity (against K. pneumonie, S. aureus or E. coli strains) and antifungal activity (against many fungi including Candida sp.), the responsible compounds for these properties being tannins, flavones, flavonols, flavonoids, phenolic acids and coumarins. Due to the existence of bioactive constituents in the chemical composition of fresh leaves, roots, or flowers of Pelargonium sp. (such as monoterpenoid compounds-citronellol, geraniol, linalool, and flavonoids-myricetin, quercetin and kaempferol), this species is still valuable, the bio-compounds representing the base of innovative substitutes in food processing industry, nutraceuticals, or preventive human or veterinary medicine (substitute of antibiotics). Highlighting the volatile chemical composition and properties of this scented plant aims to rediscover it and to emphasize the vast spectrum of health-promoting constituents for a sustainable approach. Future research directions should point to the application of plant biotechnology with a significant role in conservation strategy and to stimulate commercial interest.

Application of Plant Biotechnology in Forestry

Forests, often referred to as the lungs of our planet, stand as a testament to the incredible diversity and significance of our terrestrial ecosystems [...]

Transcriptomic Profiling of Embryogenic and Non-Embryogenic Callus Provides New Insight into the Nature of Recalcitrance in Cannabis.

Differential gene expression profiles of various cannabis calli including non-embryogenic and embryogenic (i.e., rooty and embryonic callus) were examined in this study to enhance our understanding of callus development in cannabis and facilitate the development of improved strategies for plant regeneration and biotechnological applications in this economically valuable crop. A total of 6118 genes displayed significant differential expression, with 1850 genes downregulated and 1873 genes upregulated in embryogenic callus compared to non-embryogenic callus. Notably, 196 phytohormone-related genes exhibited distinctly different expression patterns in the calli types, highlighting the crucial role of plant growth regulator (PGRs) signaling in callus development. Furthermore, 42 classes of transcription factors demonstrated differential expressions among the callus types, suggesting their involvement in the regulation of callus development. The evaluation of epigenetic-related genes revealed the differential expression of 247 genes in all callus types. Notably, histone deacetylases, chromatin remodeling factors, and EMBRYONIC FLOWER 2 emerged as key epigenetic-related genes, displaying upregulation in embryogenic calli compared to non-embryogenic calli. Their upregulation correlated with the repression of embryogenesis-related genes, including LEC2, AGL15, and BBM, presumably inhibiting the transition from embryogenic callus to somatic embryogenesis. These findings underscore the significance of epigenetic regulation in determining the developmental fate of cannabis callus. Generally, our results provide comprehensive insights into gene expression dynamics and molecular mechanisms underlying the development of diverse cannabis calli. The observed repression of auxin-dependent pathway-related genes may contribute to the recalcitrant nature of cannabis, shedding light on the challenges associated with efficient cannabis tissue culture and regeneration protocols.

A Common Molecular Signature Indicates the Pre-Meristematic State of Plant Calli.

In response to different degrees of mechanical injury, certain plant cells re-enter the division cycle to provide cells for tissue replenishment, tissue rejoining, de novo organ formation, and/or wound healing. The intermediate tissue formed by the dividing cells is called a callus. Callus formation can also be induced artificially in vitro by wounding and/or hormone (auxin and cytokinin) treatments. The callus tissue can be maintained in culture, providing starting material for de novo organ or embryo regeneration and thus serving as the basis for many plant biotechnology applications. Due to the biotechnological importance of callus cultures and the scientific interest in the developmental flexibility of somatic plant cells, the initial molecular steps of callus formation have been studied in detail. It was revealed that callus initiation can follow various ways, depending on the organ from which it develops and the inducer, but they converge on a seemingly identical tissue. It is not known, however, if callus is indeed a special tissue with a defined gene expression signature, whether it is a malformed meristem, or a mass of so-called "undifferentiated" cells, as is mostly believed. In this paper, I review the various mechanisms of plant regeneration that may converge on callus initiation. I discuss the role of plant hormones in the detour of callus formation from normal development. Finally, I compare various Arabidopsis gene expression datasets obtained a few days, two weeks, or several years after callus induction and identify 21 genes, including genes of key transcription factors controlling cell division and differentiation in meristematic regions, which were upregulated in all investigated callus samples. I summarize the information available on all 21 genes that point to the pre-meristematic nature of callus tissues underlying their wide regeneration potential.

Unveiling the Role of Sorghum RPAP3 in the Function of R2TP Complex: Insights into Protein Assembly in Plants.

The chaperone R2TP has multiple subunits that assist in the proper folding, assembly, and stabilization of various protein complexes in cells and its study can offer valuable insights into the regulation and maintenance of protein assemblies in plant systems. The 'T' component of R2TP is Tah1 in yeast, consisting of 111 residues, while its counterpart in humans is RPAP3, with 665 residues. RPAP3 acts as a co-chaperone of Hsp90 and facilitates interactions between RUVBL proteins and other complex components, enhancing the recruitment of client proteins by the R2TP complex. These facts further underscore the relevance of studying this complex in different organisms. The putative gene corresponding to the RPAP3 in Sorghum bicolor, a monocotyledon plant, was cloned, and the protein (396 residues) purified for biochemical characterization. SbRPAP3 exists as a folded monomer and has a RPAP3 domain, which is present in human RPAP3 but absent in yeast Tah1. SbRPAP3 retains its functional capabilities, including binding with RUVBLs, Hsp90, and Hsp70. By elucidating the role of RPAP3 in plant R2TP complex, we can further comprehend the molecular mechanisms underlying plant-specific protein assembly and contribute to advancements in plant biology and biotechnological applications.

Silver Nanoparticles for Enhancing the Efficiency of Micropropagation of Banana (Musa acuminata L.).

Silver nanoparticles (AgNPs) have numerous applications in plant biotechnology. The unique biological activities of AgNPs in reducing microbial contamination and promoting in vitro plant growth have encouraged their use in the development of novel culture systems for the in vitro cultivation of several plant species. In this study, the influence of (80 nm-100 nm) AgNPs on the micropropagation of banana was examined by incorporating AgNPs into shoot multiplication and rooting media at concentrations of 3 mg/L-15 mg/L. Biometric parameters for shoot multiplication (number of shoots/explant, shoot length and leaf surface area) and root development (number of roots/explant and root length) were analysed. In addition, shoot chlorophyll content, proline content and the possible impact of lipid peroxidation on membrane stability of plantlets were estimated. The results showed that all concentrations of AgNPs stimulated shoot growth and enhanced root development. The highest response was observed in media supplemented with 12 mg/L AgNPs. This optimal level of AgNPs caused a threefold increase in shoot growth parameter and a similar increase in root numbers/shoot and root length. Treatment with AgNPs at 12 mg/L also increased chlorophyll and proline content of shoots by 25% and 120% over control, respectively. Although the application of AgNPs increased the level of lipid peroxidation in shoots, it however, had a limited influence on membrane stability index. These results suggested that the administration of AgNPs to culture media can be effectively utilised for the enhancement of banana micropropagation with minimal toxic effects.

Stepwise protein targeting into plastoglobules are facilitated by three hydrophobic regions of rice phytoene synthase 2.

Plastoglobules (PGs) are plastidial lipid droplets enclosed by a polar monolayer born from the thylakoid membrane when plants require active lipid metabolism, including carotenogenesis, under the environmental stress and during plastid transition. Despite the fact that many proteins are reported to target PGs, their translocation mechanism has remained largely unexplored. To elucidate this process, we studied the influence of three hydrophobic regions (HR)-HR1 (1-45th aa), HR2 (46-80th aa), and HR3 (229-247th aa)-of rice phytoene synthase 2 (OsPSY2, 398 aa), which has previously shown to target PGs. As results, HR1 includes the crucial sequence (31-45th aa) for chloroplast import and the stromal cleavage occurs at a specific alanine site (64th aa) within HR2, verifying that a N-terminal 64-aa-region works as the transit peptide (Tp). HR2 has a weak PG-targeting signal by showing synchronous and asynchronous localization patterns in both PGs and stroma of chloroplasts. HR3 exhibited a strong PG-targeting role with the required positional specificity to prevent potential issues such as non-accumulation, aggregation, and folding errors in proteins. Herein, we characterized a Tp and two transmembrane domains in three HRs of OsPSY2 and propose a spontaneous pathway for its PG-translocation with a shape embedded in the PG-monolayer. Given this subplastidial localization, we suggest six sophisticated tactics for plant biotechnology applications, including metabolic engineering and molecular farming.

Nanotechnology and CRISPR/Cas9 system for sustainable agriculture.

The clustered regularly interspaced short palindromic repeats (CRISPR-Cas9), a genome editing tool, has gained a tremendous position due to its therapeutic efficacy, ability to counteract abiotic/biotic stresses in plants, environmental remediation and sustainable agriculture with the aim of food security. This is mainly due to their potential of precised genome modification and numerous genetic engineering protocols with versatility as well as simplicity. This technique is quite useful for crop refinement and overcoming the agricultural losses and regaining the soil fertility hampered by hazardous chemicals. Since CRISPR/Cas9 has been widely accepted in genome editing in plants, however, their revolutionised nature and progress enable genetic engineers to face numerous challenges in plant biotechnology. Therefore, nanoparticles have addressed these challenges and improved cargo delivery and genomic editing processes. Henceforth, this barrier prevents CRISPR-based genetic engineering in plants in order to show efficacy in full potential and eliminate all the barriers. This advancement accelerates the genome editing process and its applications in plant biotechnology enable us to sustain and feed the massive population under varying environments. Genome editing tools using CRISPR/Cas9 and nanotechnology are advantageous that produce transgenic-free plants that overcome global food demands. Here, in this review, we have aimed towards the mechanisms/delivery systems linked with CRISPR/Cas9 system. We have elaborated on the applications of CRISPR/Cas9 and nanotechnology-based systems for sustainable agriculture. Moreover, the challenges and limitations associated with genome editing and delivery systems have also been discussed with a special emphasis on crop improvement.

The emerging role of nanotechnology in plant genetic engineering

Genetic engineering to improve the capabilities of plants is essential given climate change and population growth pressures. Current manipulation methods are laborious and species dependent, which limits advances in agriculture and molecular farming. Therefore, new approaches and tools are needed to broaden the range of transformable species and increase the throughput at which transformation is achieved. Nanotechnology has revolutionized delivery, sensing and imaging in microbial and animal systems, but its application in plants remains scant. However, reports of nano-mediated delivery for the genetic manipulation of plants have emerged, including direct germline editing as well as plastid and mitochondrial genome modification. Here, we review the application of nanotechnology to plant genetic manipulation, including the development of nanocarriers for the delivery of genetic cargos and advances in nano-mediated plant regeneration. Particular focus is given to understanding structure–function relationships for the rational design of nanocarriers, and how these developments can catalyse progress in nucleic acid and protein delivery for plant biotechnology applications. Current methods for the genetic manipulation of plants have low throughput and are amenable to a limited range of species. This Review discusses advances in the development of nanotechnology tools and the understanding of structure–function relationships to overcome these issues.

Analysis of Transgenic Cotton Plants Containing Universal Stress Protein (GaUSP-1, GaUSP-2) and Zinc Finger Transcriptional Factor (GaZnF) Genes under Drought Stress.

Water is the most limiting factor for plant growth and crop productivity. Drought stress adversely affects crop yield throughout the world. Up to 50% of crop yield in Pakistan is severely affected by the shortage of water. Cotton is an important cash crop for Pakistan known as "white gold." It accounts for 8.2% of the value added in agriculture and about 3.2% of GDP. Besides, being the world's fourth-largest cotton producer, our yield per acre ranks 13th in the world. If we look at the Pakistan scenario, water deficiency is one of the major yield-limiting factors. Limitations related to conventional breeding and the advancements in plant genomics and biotechnology applications have opened new horizons to plant improvements. Therefore, in the current study, we carry out a comparative analysis to evaluate the morphological, physiological biochemical and molecular parameters in transgenic plants containing GaUSP-1, GaUSP-2 and GaZinc Finger genes under different drought stress conditions. Data showed that transgenic plants showed more tolerance as compared to non-transgenic plants. Transgenic and non-transgenic assist us in our better understanding of the drought-responsive mechanism and its effect on different plant growth traits, so, in this way, we would be able to explore drought tolerance mechanism and this will open the doors for the identification of drought-related genes.

Phenolic Profile and Antioxidant Properties in Extracts of Developing Açaí (Euterpe oleracea Mart.) Seeds.

We investigated changes in the phenolic profile and antioxidant properties in the extracts of developing seeds of açaí (Euterpe oleracea). Four developmental stages were evaluated, with earlier stages displaying higher antioxidant activity and polyphenols content, while mass spectrometry analysis identified procyanidins (PCs) as the major components of the extracts in all stages. B-type PCs varied from dimers to decamers, with A-type linkages in a smaller number. Extracted PCs decreased in average length from 20.5 to 10.1 along seed development. PC composition indicated that (-)-epicatechin corresponded to over 95% of extension units in all stages, while (+)-catechin presence as the starter unit increased from 42 to 78.8% during seed development. This variation was correlated to the abundance of key enzymes for PC biosynthesis during seed development. This study is the first to report PC content and composition variations during açaí seed development, which can contribute to studies on the plant's physiology and biotechnological applications.

High-efficiency retron-mediated single-stranded DNA production in plants.

Retrons are a class of retroelements that produce multicopy single-stranded DNA (ssDNA) and participate in anti-phage defenses in bacteria. Retrons have been harnessed for the overproduction of ssDNA, genome engineering and directed evolution in bacteria, yeast and mammalian cells. Retron-mediated ssDNA production in plants could unlock their potential applications in plant biotechnology. For example, ssDNA can be used as a template for homology-directed repair (HDR) in several organisms. However, current gene editing technologies rely on the physical delivery of synthetic ssDNA, which limits their applications. Here, we demonstrated retron-mediated overproduction of ssDNA in Nicotiana benthamiana. Additionally, we tested different retron architectures for improved ssDNA production and identified a new retron architecture that resulted in greater ssDNA abundance. Furthermore, co-expression of the gene encoding the ssDNA-protecting protein VirE2 from Agrobacterium tumefaciens with the retron systems resulted in a 10.7-fold increase in ssDNA production in vivo. We also demonstrated clustered regularly interspaced short palindromic repeats-retron-coupled ssDNA overproduction and targeted HDR in N. benthamiana. Overall, we present an efficient approach for in vivo ssDNA production in plants, which can be harnessed for biotechnological applications. Graphical Abstract.

Polish Achievements in Bioactive Compound Production From In Vitro Plant Cultures

AbstractPlant cell and organ cultures are potential sources of valuable secondary metabolites that can be used as food additives, nutraceuticals, cosmeceuticals, and pharmaceuticals. Phytochemical biosynthesis in various in vitro plant cultures, in contrast to that in planta, is independent of environmental conditions and free from quality fluctuations.Pharmaceutical application of plant biotechnology is of interest to almost all departments of the Faculty of Pharmacy and Institute of Pharmacology in Poland with a botanical profile (Pharmaceutical Botany, Pharmacognosy, and Pharmacology).This study discusses the advances in plant biotechnology for the production of known metabolites and/or biosynthesis of novel compounds in plant cell and organ in vitro cultures in several scientific centers in Poland.

Hibiscus Rosa Sinensis Linn.

Hibiscus Rosa Sinensis Linn. (Malvaceae) is a popular medicinal plant found in tropical and sub-tropical region of world. All plant parts of Hibiscus Rosa Sinensis Linn are most widely used to treat several ailments like anti-Tumor, anti-fertility, anti-ovultory, anti-implantation, anti-Inflammatory, analgesic, anti-estrogenic, anti-pyretic, anti-spasmodic, anti-viral, anti-fungal, anti-bacterial, hypoglycemic, spasmolytic, CNS depressant, hypertensive and juvenoid Activity. Due to its multidimensional pharmacological approach it is certain to emerge as a effficient player in the flourising field of herbal supplements, medicines and health care system. This review aims to present recent details on botany, ethnomedicinal uses, photochemistry, pharmacological effects, toxic effects, with the purpose to find research gaps demanding for upcoming research and investigation of Hibiscus Rosa Sinensis Linn. Principal constituents reported in  Hibiscus Rosa Sinensis Linn are flavones, containing quercetin-3-sophorotrioside, kaempferol-3xylosylglucoside,quercetin-3-diglucoside, quercetin-3,7-diglucoside, cyaniding-3,5-diglucoside, cyaniding-3-sophoroside-5-glucoside and other constituent are cyanidin chloride, cyclopeptide alkaloid, ascorbic acid, riboflavin, thiamine, hentriacontane, taraxeryl acetate, ß-sitosterol, malvalic acids and cyclic acids sterculic. Complete  literature was probed via websites,online databases, thesis and texts. The available reports was portray physicochemical parameters, nutritional aspects and phytochemical analysis of bioactive plant parts. Friendly holistic conservation approaches offered by plant biotechnology applications are also discussed. 

Unidentified N-glycans by N-glycosidase A were Identified by Nglycosidase F under Denaturing Conditions in Plant Glycoprotein.

The identification of N-glycans in plant glycoproteins or plant-made pharmaceuticals is essential for understanding their structure, function, properties, immunogenicity, and allergenicity (induced by plant-specific core-fucosylation or xylosylation) in the applications of plant food, agriculture, and plant biotechnology. N-glycosidase A is widely used to release the Nglycans of plant glycoproteins because the core-fucosylated N-glycans of plant glycoproteins are hydrolyzed by N-glycosidase A but not by N-glycosidase F. However, the efficiency of Nglycosidase A activity in plant glycoproteins remains unclear. The aim of the study was to elucidate the efficient use of N-glycosidases to identify and quantify the N-glycans of plant glycoproteins; it aimed at identification of released N-glycans by Nglycosidase F and assessment of their relative quantities with a focus on unidentified N-glycans by N-glycosidase A in plant glycoproteins, Phaseolus vulgaris lectin (PHA) and horseradish peroxidase (HRP). Liquid chromatography-tandem mass spectrometry was used to analyze and compare the N-glycans of PHA and HRP treated with either N-glycosidase A or F under denaturing conditions. The relative quantities (%) of each N-glycan (>0.1%) to the total N-glycans (100%) were determined. N-glycosidase A and F released 9 identical N-glycans of PHA, but two additional corefucosylated N-glycans were released by only N-glycosidase A, as expected. By contrast, in HRP, 8 N-glycans comprising 6 core-fucosylated N-glycans, 1 xylosylated N-glycan, and 1 mannosylated N-glycan were released by N-glycosidase A. Moreover, 8 unexpected N-glycans comprising 1 corefucosylated N-glycan, 4 xylosylated N-glycans, and 3 mannosylated N-glycans were released by Nglycosidase F. Of these, 3 xylosylated and 2 mannosylated N-glycans were released by only Nglycansodase F. These results demonstrate that N-glycosidase A alone is insufficient to release the Nglycans of all plant glycoproteins, suggesting that to identify and quantify the released N-glycans of the plant glycoprotein HRP, both N-glycosidase A and F treatments are required.

Regulation of WOX11 Expression Represents the Difference Between Direct and Indirect Shoot Regeneration.

Somatic cells of higher plants possess the remarkable ability to regenerate new individuals via reestablishing apical meristems. Reconstitution of shoot meristem is the vital process and is required for application of plant biotechnology. Under in vitro culture condition, shoot meristem can be formed directly or indirectly, depending on the absence or presence of callus as the intermediate status. However, the difference of regulatory mechanisms between the two regeneration types remains unknown. In this study, we established a bi-directional system in which shoots regenerated directly from lateral root primordia (LRP) and indirectly from hypocotyl-derived callus simultaneously. The results based on this system revealed that regulation of WOX11 expression represents the difference between the two regeneration types in two aspects. Firstly, number of founder cells expressing WOX11 is tightly associated with regeneration types. Relatively more founder cells gave rise to callus and produce larger meristem, whereas less founder cells produce LRP that regenerate smaller meristem. Secondly, non-CG DNA methylation specifically regulated WOX11 transcription in LRP and promoted direct shoot regeneration, but had no influence on indirect regeneration. The results provide new insights for understanding the regulatory mechanisms of cell fate transition during de novo organogenesis.

Genome-wide characterization of i-motifs and their potential roles in the stability and evolution of transposable elements in rice.

I-motifs (iMs) are non-canonical DNA secondary structures that fold from cytosine (C)-rich genomic DNA regions termed putative i-motif forming sequences (PiMFSs). The structure of iMs is stabilized by hemiprotonated C-C base pairs, and their functions are now suspected in key cellular processes in human cells such as genome stability and regulation of gene transcription. In plants, their biological relevance is still largely unknown. Here, we characterized PiMFSs with high potential for i-motif formation in the rice genome by developing and applying a protocol hinging on an iMab antibody-based immunoprecipitation (IP) coupled with high-throughput sequencing (seq), consequently termed iM-IP-seq. We found that PiMFSs had intrinsic subgenomic distributions, cis-regulatory functions and an intricate relationship with DNA methylation. We indeed found that the coordination of PiMFSs with DNA methylation may affect dynamics of transposable elements (TEs) among different cultivated Oryza subpopulations or during evolution of wild rice species. Collectively, our study provides first and unique insights into the biology of iMs in plants, with potential applications in plant biotechnology for improving important agronomic rice traits.

Recent developments in breeding approaches of tomato (Solanum lycopersicum L)– a review

Tomato (Solanum lycopersicum L) is an important vegetable crop under family Solanaceae. It is a diploid plant having chromosome number 2n=2x=24. In tomato breeding great advances are achievable due to the possibility of hybridization within and among species in a controlled manner, the variability present in the species, the presence of self-pollination which further leads to the recessive mutation expression and the lack of gene duplication. In tomato, fruit yield, quality and resistance to pests and diseases are the general breeding goals. For improvement of the qualitative traits by gene transfer from wild species to the cultivated ones, tomatoes are believed to be a classical example. The achievements in breeding of tomato are based on traditional breeding-genetic methods till now. Also a change in the introduced cultivars possessing useful and desired traits, is not exploited yet. For enhancing the productivity in future, it is believed that conventional breeding would not help out. Different techniques are established using molecular genetics and molecular markers for significant progress. So altogether, the valuable tools for tomato breeding might be the combined application of traditional breeding and plant biotechnology including selection based on molecular markers and marker-assisted selection.