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APPLICATION OF GENOMIC AND GENETIC ENGINEERING TOOLS FOR IMPROVEMENT OF GRAPEVINES.

Byline: A. Mwamahonje, Z. Maseta, F. Mlalila and T. Feyissa

Key words: Biotechnology, Breeding, Germplasm, Molecular Marker, QTL, Trait.

INTRODUCTION

Grape(Vitis vinifera) is a valuable horticultural fruit crop in the world. Historically, cultivation of domesticated Vitis vinifera species started 6,000-8,000 years ago from wild progenitor Vitis vinifera subsp. sylvestris(Myles et al., 2011). It was further expanded to the south and western side of the Fertile Crescent, the Jordan Valley, Anatolia, Syria and Egypt 5,000 years ago and later spread to the rest of the world(Pierozzi and Moura, 2016). Selection and early improvement of grape were based on several parameters like yield, disease resistance, sprouting ability, quality of fruits and wine. Advances in genetic techniques have simplified screening and improvement of health qualities of grape and grape-derived products. China ranks the top producer(13,160,788 tonnes annually) worldwide, followed by Italy and United States producing 7,169,745 and 6,679,211 tonnes per year respectively(FAOSTAT, 2017). V. vinifera is in the genus Vitis(2n=38 chromosomes).

The crop has about 60 species in the genus Vitis, family Vitaceae(Villano, 2015). Botanically, some grape species are hermaphrodite and others are monoecious. Grape is classified from the largest rank of domain Eukarya to the smallest which is genus Vitis. The genus Vitis is common in both wild and domesticated grape species. The genus includes Vitis vinifera L., Vitisacerifolia Raf, Vitisaestivalis Michx and Vitisamurensis Rupr. However, the species Vitis vinifera is the most cultivated in the world followed by Vitis labrusca(Villano, 2015). Grape is used for making wine, table grapes, juice, raisin and spirit(Villano, 2015). It is good source of protein, minerals, vitamin C, K, iron, potassium, zinc, calcium and manganese(Sousa, 2014). It plays an important role in the regulation of aging processes because of antioxidant produced by the fruits' resveratrol. It helps to increase efficiency of digestion and prevent infection in the body.

It contains phenolic compounds like tannins, phenolic acids, anthocyanins which maintain human health(Yilmaz and Toledo, 2004). This review describes the genomic and genetic engineering techniques for grape improvement including conventional and molecular breeding methods.

Current Cultivation of Grape: Grape is grown in temperate and sub-tropical climate with high and erratic rainfall(Vivier and Pretorius, 2000). The crop generally reaches at full production after 5 to 8 years from planting. The life cycle is 50-100 years depending on the regular agronomic management and ecology. The rich genetic diversity of grapes provides wide adaptation in multi-environmental conditions across the world. The crop can be developed in the tissue culture laboratory using parts of the grapevine or can be propagated by grafting technology. The convenient pH for viticulture ranges from 5.5-7.5 depending on the variety and ecology. Organic and inorganic fertilizers are recommended for grapes depending on the nutrient deficiency in vineyard. Pruning is done to control number of fruits, vigour and nutrient content. Weeds, diseases and pests are controlled by sustainable use of chemicals and other practices.

Genetic Diversity: The centre of origin for genetic diversity of grape reported in Anatolia since 8000 BC(Vouillamo et al., 2006). The diversity spread into North Africa in 5th millennium BC and 1st millennium BC in Europe and later spread all over the world including Central Asia and United States of America(Vouillamo et al., 2006). Most studies using morphology and molecular markers(RAPDs, AFLPs, SSRs, ISSRs and SNPs) revealed high genetic diversity among wild and cultivated grape varieties(Bowers et al., 1999; Riaz et al., 2013; Mwamahonje et al., 2015; Da Costa et al., 2017). However, Myles et al.(2011) proposed that, the domestication of grape reduces the genetic diversity giving example of recent 950 genotyped Vitis vinifera and 59 Vitissylvestris accessions using SNPs markers. The decrease of genetic diversity is contributed by cultivating the same varieties for a long period of time without releasing new cultivars.

Consumers' preferences have an impact on the genetic diversity of grapes for instance religious ceremonies have specific varieties for wine making(Migicovsky et al., 2017). Lack of consistent genebank for grape species conservation accelerates loss of diversity(Zhou et al., 2017). On the other hand, the grapes have long life cycle which takes many years to release new varieties. Climate change has contribution to decrease of genetic diversity such that, some varieties lose their performance due to changes in temperature, pH of the soil and photoperiod. Genetic diversity plays a major role in the grape breeding by providing broad base of breeding materials. This increases the chances of exploiting the traits of preference which meet demands(Da Costa et al., 2017). Genetic diversity can be modified through a number of ways like breeding contrasting traits to introduce new varieties. It can be enhanced by mutation, plant tissue culture and germplasm collection(This et al., 2006; Da Costa et al., 2017).

Improvement Strategies: Grape improvement aims to address various constraints like diseases, low yield, sugar content, drought and pests. This is achieved by breeding improved varieties in relation to diseases and pests, drought tolerance, sugar content, taste and early maturity(Pierozzi and Moura, 2016). The methods used include conventional and molecular breeding methods. Conventionally, it is possible by breeding based on phenotyping only in the field. The molecular method is achieved using molecular markers, marker assisted selection and genetic engineering tools and phenotyping field data for accurate results. Both approaches entail backcrossing during incorporation of the traits of interest to the new varieties. Molecular breeding method is assisted by molecular markers. The method of traits transfer can be done using genetic engineering for instance Agrobacterium tumefaciens and gene gun(Laimer, 2007).

Through this approach new varieties with high yield, disease resistance, sugar content, early maturity, standard antioxidants and other traits have been modified(Laimer, 2007). Use of disease free seedlings at young stage is recommended. Increase of wine, juice and raising industries will provide the opportunities for expansion of acreage of grapes production.

Traditional Breeding and Limitation in Grape: Traditional breeding of grapes involves crossing of the varieties with contrasting traits. It exploits traits of interest from one variety to another. Some of the traits which are considered for improvement include yield, diseases and pests resistance and biochemical content. These parameters help plant breeders to screen the useful traits for incorporation into new variety. Conventional plant breeding method may take 6-20 years to release new grape variety depending of the heritability of traits. This is due to long juvenile phase that takes 5-6 years from planting(McClure et al., 2014). In spite of grape to undergo out crossing still, the produced varieties lack genetic differences as evidenced in Cocoa(Schnell et al., 2007). Utilization of wild grape species in grape breeding provides potential traits which can improve the domesticated grape. It can be achieved through conventional breeding.

For example, foxy smell in wild grape species V. labrusca shares the same ancestor with V. vinifera over 20 years ago(McClure et al., 2014). Hence, crossing to susceptible varieties may produce new improved varieties with combination of traits.

Role of Biotechnology in Grape Breeding: Use of biotechnology improves quality of grape products. It helps to introduce new varieties with combination of traits such as drought tolerance, diseases and pests' resistance and high yield from one genotype to another through gene transformation(Gascuel et al., 2017; Hvarleva et al., 2009). The technology is achieved using molecular markers for traits and QTLs identification which ultimately can be introgressed into potential variety through marker assisted backcrossing and genetic engineering(Migicovsky and Myles, 2017). Genetic engineering enhances rapid breeding programmes as it increases efficiency, performance and quality grapes produced within a short period of time. The technology involves both laboratory screening using markers and field phenotyping which together strengthen selection of suitable heritability(Gascuel et al., 2017). Through this technology, the variety Chancellor has been introduced.

Germplasm Biodiversity and Conservation

Germplasm Diversity: Germplasm is validated regularly by adding new identified or collected cultivars. The grape germplasm contains wild and domestic species(Leao et al., 2011). Both have useful information for diversity studies. Some cultivars are originated by spontaneous or induced mutation. The diversity studies of grape germplasm use different tools including ampelography. Molecular markers are used in the study of DNA gene differences among germplasm(Leao et al., 2011). Genotyping helps to correct misnaming accessions by ampelography. The accessions may be the same but different name in different geographical location. The correcting naming of accessions can be achieved by using molecular markers like ISSR and SSR. These markers can identify the genetic differences and similarities within and between cultivars(Emanuelli et al., 2013). Both ampelography and molecular markers assist grape breeders to find individuals for grape improvement.

Through this approach, drought tolerant, disease resistant and sugar content germplasm have been screened(Leao et al., 2009). Similar recommendations have been reported by a number of studies(Martinez et al., 2006). Despite the efforts made to maintain germplasm, the speed of releasing new grape varieties is slow perhaps there is a need for further optimization of protocols used in each molecular marker.

Grape Cultivars Characterization and Phylogeny: Grape cultivars are characterized based on physiological, biochemical and molecular markers. In ampelography, grape cultivars are characterized using descriptor lists. It entails number of autochthonous and hybrid cultivars which are involved in assessing the genetic variation within and among cultivars(Ates et al., 2011). The characterization of grape cultivars provides wide range of exploiting traits for improvement(Ates et al., 2011). Mutations have contribution in the formation of new cultivars that may or may not be useful for direct adoption. The best cultivars developed by mutations are selected and kept in the genetic resources centres for future research(Sefc et al., 2001). Each marker has advantage and drawback. Therefore, it depends on the objective of study. For instance, RAPD is less costy, easy to apply, time saving for genetic diversity studies in grape genotypes and rootstock variability(Sefc et al., 2001).

Nevertheless, RAPD is not versatile to the environmental conditions, depend on quality DNA, it is difficult to standardize results in the laboratory. AFLP can show only one single band difference. SSR markers play a major role in the genetic diversity studies in grapes due to its ability to identify genetic similarities and pedigree construction(Mwamahonje et al., 2015). It is locus specific, co-dominant and reproducible. SSR cannot sometimes identify the diversity at intra-variety level; the cost is high and it is a time consuming tool during construction and screening of genomic libraries, design and optimization of PCR primers(Doulati-Baneh et al., 2013). Alternatively, ISSR markers can usually identify the diversity at intra-variety level and among grape accessions(Pierozzi and Moura, 2016). The knowledge of genetic diversity studies helps plant breeders to find the best combinations in breeding programs. SSR markers are commonly used in many genetic diversity study of grape.

SNPs markers have well distribution in the grape genome, high resolution and powerful in confirming the parentage of crops genotypes(Myles et al., 2011). They can identify the genetic differences among genotypes to single base level(Bautista et al., 2008). Through SNPs, it is possible to identify traits which are compatible to high yield, disease resistant, drought tolerant, fruit quality and quality wine(Bautista et al., 2008). Emphasis is given by grape breeders to keep the new varieties and cultivars in the gene bank for future application. The genetic resources conservation of grape accessions remains unstable. Only few traits have been exploited from the point of origin. In the future, most of them may be depleted if not conserved. Wild species which are highly influenced by natural situation should properly be genotyped(Emanuelli et al., 2013).

Genetic Resources and Conservation Approaches of Grape: The conservation of grapes involves both domesticated and wild species. Genetic resources maintain the genetic diversity which is a key for using grape accessions for future traits exploitation. The advancement of good yield, quality, and disease resistance depends on availability of germplasm in the genetic resource conservation centres. It offers wide range of choosing the suitable cultivars for wine, juice and raisins processed products(Villano, 2015). Germplasm conservation involves both ex situ and in situ conservation methods. Conservation can be in the genebank and plant tissue culture laboratory(Tehrim and Saji, 2011). Distribution of germplasm to farmers helps to ensure stability of germplasm so that, in case and damage and loss from genebank, the germplasm can be recollected from the farmers' field(FAO, 2014), for instance, Genetic Identification and Conservation of Local Turkish Grapevine(Vitis vinifera L.).

Genotypes on the edge of extinction must be considered in both in situ and ex situ germplasm conservation(Sabir et al., 2018). Grape varieties Cabernet franc, Semillon, Petit, Verelot, Carmenere and Cot have been achieved through this method(Roby et al., 2014).

Molecular Breeding Tool: Molecular breeding is a method of plant breeding which uses tools such as marker assisted selection, marker assisted backcrossing, genomic selection, bioinformatics, genome wide association and genetic engineering for improvement of new crop varieties. These tools shorten breeding cycles compared to conventional method(De Lima et al., 2006). The markers are designed based on the objective of the study and used for exploitation of traits of interest(Topfer et al., 2011). The tools are applied in grape to supplement conventional breeding. Grape breeders use molecular breeding tools to screen and evaluate the best breeding parents for different traits(Topfer et al., 2011). The common traits which are considered in grape improvement include yield, diseases and pest resistance, quality and maturity of the breeding materials(Sanchez-Mora et al., 2017).

In addition, the tools are applicable to detect the genes that are tolerance to drought due to rapid increase of the temperature that affects the crop.

Marker-assisted Selection: Marker assisted selection is an application of molecular markers to exploit traits of interest for improvement. The tool supplements conventional breeding method which uses many cycles to achieve new improved varieties(Mwamahonje et al., 2015). In grapes, marker assisted selection has been useful for addressing a number of problems(Dalbo et al., 2001). Powdery mildew, a fungal disease in grape, has been addressed using markers introgression which is able to identify QTLs associated with disease resistance(Dalbo et al., 2001). This is achieved through crossing contrasting traits thus, developing population for genotyping. The QTLs are introgressed to the susceptible varieties through marker assisted backcrossing. Marker assisted selection plays a major role in grape breeding. It helps to address different challenges pertaining crops for example, diseases, pests, low yield, drought and long juvenile phase.

Marker assisted selection shortens breeding programmes in crops(Edge-Garza et al., 2015). Markers which are tightly linked close to QTLs controlling traits of interest are mapped. The mapped QTLs can be developed through Recombinant Inbred Lines(RILs) by marker assisted backcrossing. Commonly used molecular markers for QTLs identification in crops include SSR and SNPs. These markers have high throughput, efficient resolution, polymorphism and are co-dominant which help to identify useful QTLs influencing traits parameters in crops(Viana et al., 2016). The point to note, marker assisted selection cannot work alone thus, depends on comparison with phenotypic data from the field. The QTLs for resistance to grapevine powdery mildew have been tested using SSR markers and identified the QTLs influencing resistance on the chromosome number 18(Migicovsky and Myles, 2017).

Proposing that, molecular markers SSR can be used to upgrade more powerful tools for high efficiency QTLs to pyramid new grape varieties for multiple disease resistance(Riaz et al., 2009). Pyramid resistance genes Run1 and Rpv1 for powdery mildew and down mildew respectively, have been achieved through backcrossing to susceptible grape varieties(Eibach et al., 2007). The efficiency of QTLs from different traits is proved by phenotyping of breeding materials in the field(Kicherer et al., 2017). The population which retains the traits from the donor parents are screened for further evaluation and finally released as the new varieties(Kicherer et al., 2017). Thus, marker assisted breeding and phenotyping depend on each other for disease control. Phenotyping with good trait heritability include leaves, shoot, rachis and berries(Riaz et al., 2011). The best way to confirm QTLs for powdery mildew using molecular markers is to use the reference of published mapped QTLs.

Powdery mildew is highly favoured in warm climate with temperature ranging from 25-30OC. The grape genotypes which are resistance under this temperature range can be screened as good for powdery mildew resistance. Therefore, QTLs influencing these traits are identified in the chromosome position before introgression to the susceptible varieties for resistance improvement(Topfer et al., 2011). Introgression of QTLs of interest should be subject to yield as the important parameter expected from the consumers and farmers. Run 1 gene originated from mascadine grapes(V. rotundifolia) is resistant to powdery mildew1. It is found in the chromosome number 12. It has transferred resistance to the genetic engineered grape cultivars. However, recent investigation has detected sporulation of powdery mildew pathogen on introgressed grape lines with RUN1 locus which is contributed by breakdown of resistance of the pathogen(Cadle-Davidson et al., 2011).

Through marker assisted selection, new seedless grape varieties have been introduced by crossing seed versus seedless. By crossing seed and seedless grape genotypes produce 1:1 that provides opportunity of reducing the cost for evaluation. For instance, Sultanina is one of the seedless grapes in the grape breeding possessing QTL SD1 which controls the seedless(Karaagac et al., 2012). In addition, marker VMC7F2 accounts for decreasing of progeny size enhancing seedless screening. Although marker assisted selection identifies QTLs influencing the traits, QTLs are influenced by the environmental condition and geographical locations. Therefore, using large population size in the screening of QTLs and genes associated with traits of interest simplify identification. Grapevine seedlings take 3-6 years to produce fruits, thus, the use of molecular tools may shorten the breeding cycle.

The challenge to achieve this, it needs large space of vineyard establishment where management may not be efficient by plant breeders. In this regard, marker assisted breeding approaches become supportive. Previous studies have reported some of the useful QTLs for mapping berries in grape(Doligez et al., 2010). Through their effort, molecular markers linked to major loci for traits of interest in grape genotypes have been available for breeding enhancement. They have boosted conventional breeding method including establishment of seedless grape species which is influenced by SD1 QTLs(Karaagac et al., 2012). The optimization of protocol for different markers to increase efficiency is suggested(Riaz et al., 2009). Furthermore, marker assisted selection is very useful during pyramiding in breeding since it involves manipulation of many genes for disease resistance. Thus, it contributes to yield improvement. Fig.1 shows the application of marker assisted selection in grape improvement(Topfer et al., 2011).

Functional Genomics in Grape: Grapevine cultivars include many species adapted in different climate. The study of functional genomics in these species is part of updating data in the web data source of the family Vitaceae(Doddapaneni et al., 2008). The database for annotated Expressed Sequence Tags(EST) and gene expression data involved in both V. vinifera and outside the V. vinifera species are available online(Doddapaneni et al., 2008). The database is controlled online for easily accessibility by every user globally. This enhances data availability in the world. Grape species information has been stored in the ExpDB database for referencing of above 320,000 EST sequences with high proportion of V. vinifera than other species. It includes grape species of both local and hybrid varieties.

The online database for annotating grape species involves in the retrieval of putative homologous of the EST in various species with a long message of possession nucleotide identifies polygenic comparison and useful applicable primers(Doddapaneni et al., 2008). Functional genomics contribute storing of functional genes of grape species for gene exploitation. EST use bioinformatics techniques to widen its efficient application for the specialization of genomic, marker design and gene annotation(Dong et al., 2005). Functional genomics can regulate effect of excessive cold and chilling temperature affecting grape growth in most temperate countries. Coldness increases dormancy in grape, though, by maintaining gene sequences can assist in reduction of dormancy by screening method. It is possible to exhibit genetic variation of dormancy adaptation using genetic model. These mechanisms can be described by functional genomics approach using molecular markers.

This approach uses molecular markers for mapping biochemical and physiological processes producing both induced short photoperiod and minimum temperature. This has provided plant breeders with grape species which are resistant(Fennell, 2014). They are regulating dormancy so that can be used on the required time. Studies have been conducted to exploit traits from seedless grape species(Sultani Cekirdeksiz syn. Sultanina) for developing new seedless varieties. This is achieved by sequencing for easy exploitation of the novel strategy for further studies(Di Genova et al., 2014). Other studies have concentrated deep in the heterozygosity genome and functional genomics analysis(Di Genova et al., 2014). To accomplish this strategy, SNP markers are used for identification of seedless trait position in the chromosome. SNP catalogue is available for grape species. It is one of the powerful tools of functional genomics analysis and very helpful for updating genome information in grapes(Di Genova et al., 2014).

Functional genomics, which is current tool in grape will enhance plant molecular breeding and therefore improving the production. Table 1 shows sources of Vitis species and the corresponding number of expressed sequence tags available in grape(Doddapaneni et al., 2008). It is time for plant breeders and molecular biologist to apply this tool for plant and animal genome storage in the database which have contribution for yield improvement.

Bioinformatics Tool: Bioinformatics is the branch of biology which deals with the study of the application of computing methods during analysis of biological information which are related to biomolecules on the large scale(Knowles et al., 2013). It provides a coverage of the targeted topic like the study of genomic and gene expression. It compares biological data of different plants and animals. This tool is used in the gene sequence prediction on the gene protein and the gene sequence of different plant genome(Luscombe et al., 2001). It works with the data stored in the gene bank repository of nucleotide sequence and database responsible protein arrangement. Because of high demand by researchers, there has been rapid increase of information for storage(Benson et al., 2000). Grape regulates part of sequencing reads using FASTA or FASTQ format techniques which works in high efficiency using bioinformatics tool.

Grape can run the set with high quality control producer followed by aligning readings to the genome. During the alignment, part of it is removed from read order. Grape can approximate the gene and the efficiency of transcript expression findings the exon and new transcripts identification. Grape genome can be run in computer with mapping specification and identification tools. But, there must be modular design for guiding. There are tools which play a major role in controlling data exchange formats which can be integrated(Knowles et al., 2013).

Genetic Engineering

Methodologies of Genetic Engineering in Grape: Genetic engineering in grapes is the transformation of genes from one plant species to another purposely for modification. One of the recommendations in genetic engineering is that the foreign gene should not negatively affect the genes in the recipient plant. The methodologies used in this tool for transformation is achieved through the application of Agrobacterium tumefaciens whereas the gene of interest is transferred from one species to Agrobacterium for multiplication. Thereafter, they are transferred to the target new plant species through Transfer DNA followed by DNA replication and gene expression. The second approach is biolistic bombardment which directly inserts the gene of interest from a donor to a grapevine genotype.

It needs large number of DNA copies because of low multiplication compared to Agrobacterium mediated transformation. Grape industries utilize these technologies for maximization of production through diseases control in the vineyards(Vivier and Pretorius, 2000). V. vinifera was the first species to be transformed in grape(Mullins et al., 1990). The number of explants of different grape species has been successfully transformed followed by regeneration for gene expression of optimized protocol through somatic embyogenesis(Kim et al., 2013). The variety Chancellor was developed through introgression of tfdA gene which is tolerant to 2, 4-D herbicides(Mulwa et al., 2007). Testing the combination of technologies like functional genomics, QTLs and their position in chromosome, marker assisted selection; cloning and genetic engineering enhance tackling of problems in crops(Vivier and Pretorius, 2000).

Conventional breeding through introgression of genes in crops like grape, potato, banana, apple and strawberry is tedious. Therefore, Cisgenes are transferred to another plant species with no linkage drag as in conventional method(Holme et al., 2013).

Transgenic Grape Cultivars: A number of grape varieties have been developed through genetic engineering to address various problems in grape production(Costantini et al., 2007). Transgenic grape cultivars Chardonnay(Vouillamo et al. 2006), Thompson Seedless, Silcora(Mezzetti et al. 2002), Chancellor and Koshusanjaku(Perl and Eshdat 1998) have been developed through genetic engineering. Fig. 2 illustrates the phenotypic differences between transgenic and non-transgenic grape varieties(Costantini et al., 2007).

Mutation Breeding

Conventional Mutagenesis in Grapes: Conventional mutagenesis plays an important role in grape production. It involves diversification of grape through spontaneous mutation which occurs naturally(Hensz, 1981). Spontaneous mutation in grape increases genetic diversity however, it takes long time to occur. Application of grape conventional mutagenesis can lead to production of seeded or seedless grape. However, spontaneous mutagenesis has been useful for development of seedless grapevine which is formed by parthenocarpy or stenospermocarpy(Vardi et al., 2008). Conventional breeding in grape has little genetic base which do not allow sufficient genetic diversity materials for screening due to long duration of spontaneous mutation and long life cycle(Hensz, 1981; Ulukapi and Nasircilar, 2015). Application of spontaneous mutation facilitates creation of variability for introduction of new seeded or seedless grape cultivars(Pathirana, 2011).

Cultivars at this stage may need further screening to obtain the best genotypes for future improvement. Nevertheless, for seedless grape, induced mutation is among the methods which supplement spontaneous mutation to fasten genetic diversity to enhance production in grapes(Ahloowalia and Maluszynski, 2001). The promising mutants originated by spontaneous mutation are screened and treated with induced mutagens like gama-rays, x-rays and thermal neutrons which create new mutants.

In vitro Mutagenesis and Selection: Mutation induction technology is one of the tools used in grape improvement. Application of in vitro-induced mutagenesis such as chemical, x-rays, gama-rays fasten improvement of grape by producing mutants which may respond well in response to yield, disease resistance, quality wine and nutrient contents(Khawale et al., 2007). Improvement is done through screening the best traits and growing in the controlled tissue culture laboratories depending on the objective of the study(Khawale et al., 2007). This helps production of potential varieties. In vitro mutagenesis in plant is achieved by various methods like somatic embryogenesis, embryo rescue, and micropropagation. These tools increase the genetic variation. The in vitro mutagenesis in combination with tissue culture can enhance variability in grape breeding(Sinski et al., 2014).

This is done by multiplying large number of explants, which later are managed in the screen house for acclimatization before planting in the field. This technology has simplified the availability of seed in grape production programs. Effort has increased breeding for new seedless grape because of tremendous increase of demand. This has widened the market for these species(Bergamini et al., 2013). The challenge is small size of the fruit which is influenced by damage of embryo stenospermocarpic grape species that affect signalling performance(Sweetman et al., 2012).

Hybridization

Conventional Hybridization of Grape: Conventional hybridization is the method of plant breeding which involves screening the best genotypes for improvement(Hansen, 2000). It is useful for generation of new grape genotypes with quality wine, raising, juices and table grapes. Conventional hybridization is achieved by transferring the pollinizer pollens to the stigma of the recurrent maternal parents to develop new varieties. The progenies created after crossing contrasting traits are screened to obtain the best for further evaluation(Hansen, 2000). Only small portion of the target trait is transferred to the genotype which is lacking. The best populations are selfed or backcrossed to the recurrent for trait enhancement. Selfing allows segregation of progenies which helps breeders to select the promising population by phenotyping method. Population that remain stable until final stage of evaluation are released the new varieties for adoption.

Though, practically it is difficult to achieve conventional hybridization through conventional approach(Aazami, 2010).

In vitro Embryo Rescue: In vitro embryo rescue is a tissue culture technology used to promote weak immature ovule embryo which fails to develop into full plants after fertilization(Li et al., 2015). It is grown in vitro tissue culture using media that contain nutrient concentrations specific for particular plant variety. Embryo rescue is commonly applied in weak embryo, interspecific hybrid plant species and breeding seedless grapes of various Vitis species where the embryo fails to develop after fertilization. Therefore, it needs boosting to grow into complete plant(Li et al., 2015). Uses of seedless grape varieties as female parent in grape breeding produce less than 15% proportion of viable seed due to abortion of fertilized ovule embryo(Yang et al., 2007; Zhang et al., 2013). Interestingly, uses of in vitro embryo rescue technique helps recovery of aborted ovule embryo during early growth stage which allows growth into complete plant(Guo et al., 2015).

In ovulo embryo rescue technique is applicable under in vitro culture condition with controlled environment. It involves aseptic excision of weak ovule embryo allowing ovule culture in the culture medium. Depending on the varieties some embryo may be in vitro cultured without excising(Sharma et al., 1996). This facilitates recovery of embryo which grows into normal complete plants same as in conventional approach(Valdez, 2005). In vitro embryo rescue promotes the development of the aborted ovule embryo from diploid and tetraploid cross varieties. Only few seed amount is produced depending on the genotypes crossed, medium concentration and growth regulators(Zhao and Guo, 2004). Fig. 3 shows embryo rescue and plant regeneration of seedless grape from ovule embryo by in vitro culture using recommended media concentration in comparison with the controls(Guo et al., 2015).

Somatic Cell Hybridization: Somatic embryogenesis is a propagation method for improving the genetic variation through the use of cellular techniques(Mezzetti et al., 2002). It forms non-zygotic embryos from the single somatic cell or group of somatic cells with enhancing developmental pathways which helps asexual grape embryo to develop into complete plant. Somatic hybrid can be tested from a number of genotypes using in vitro tissue culture. This technique involves use of media composed Murashige and Skoog medium containing 2, 4-dichlorophenoxyacetic acid(2, 4-D) and 6-benzylaminopurine in addition of casein hydrolysate(Prado et al., 2010). The somatic hybrid grape varieties show callus development in the medium while those without somatic cells will not be expressed(Ola'h et al., 2009).

The source of plant tissue somatic embryo callus development is anther; nevertheless, other tissue parts of young grape plants like immature leaf, nucellar cells, vegetative cells of mature plants, and embryonic tissues are recommended(Perl and Eshdat, 1998). It is one of the methods of crop breeding achieved by interspecific and intragenic hybrid. It is achieved by fusing protoplasts of two different cultivars and screen the targeted hybrid cells and thus, generating into full hybrid plant(Grosser and Gmitter Jr, 2011). Protoplasts are produced from embryonic grape callus or suspension cultures of one cultivars and another protoplast from next cultivar. Protoplasts are fused, however in in vitro culture, at least some protoplasts must be embryonic to enhance regeneration of the new cultivars of fusing protoplast cultivars(Grosser and Gmitter Jr, 2011). Protoplast fusion is used for genetic transfers of traits of interest from one plant species to another thus, enhancing breeding in crops.

The explants of grapes such as shoots, embryo, leaves, petioles and flowers produce somatic embryos(Mezzetti et al., 2002). To make the cultivar stable, explants are tested from the top reserve part of the cultivars. Some explants become active within a short time and are generated easily in culture medium with high generation of somatic embryogenesis(Perl and Eshdat, 1998). They maintain genetic stability of cultivar which enhances somatic embryogenesis for propagation and avoids rapid genetic variability to maintain genotype for a long time.

Table 1. Sources of Vitis and the ESTs sequences available in the database.

Sr.###Vitis species/Hybrids###Number of ESTs

No.

1###V. vinifera(wine grape)###303,054

2###V. shuttleworthii###10,704

3###V. hybrid cultivar###6,533

4###V. rupestris x V. arizonica###5421

5###V. aestivalis###2,101

6###V. riparia###1,910

7###V. pseudoreticulata###122

8###V. cinerea x V. rupestris###61

9###V. cinerea x V. riparia###58

Conclusion: Conventional and molecular grape breeding methods play a significant role to address biotic and abiotic stresses for grape improvement. Germplasm collection ensures availability of breeding materials. The gene banks for conservation of different germplasm need to be established, repaired and maintained for future use. Breeding broadens varieties which may adapt into multi-environments. Breeding should involve phenotyping, conventional method and molecular breeding tools such as molecular markers, marker assisted selection, genomic, genome wide association, genetic engineering and tissue culture to shorten duration for release of new varieties. The breeding should involve wild grape species which carry important traits for instance, drought tolerance, and disease resistance, quality of wine and high content of medicinal substances for preventing the disease.

Introgression of QTLs associated with traits of tolerance like, abiotic stress(salinity, drought, temperature and deficiency of soil fertility) and biotic stress(diseases and pests) to susceptible grape cultivars may enhance production.

Conflicts of Interest: The authors declare that there is no conflict of interest regarding the publication of this paper.

Funding Statement: This review has not been funded by any organization.

Acknowledgments: TARI-Makutupora Centre is acknowledged for providing conducive environment during preparation of the manuscript. Prof. Jameel M. Al-Khayri is acknowledged for reviewing and commenting on this review.

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