Science Fair Project on Tomatoes | India

Do you want to create an amazing science fair project on tomatoes ? You are in the right place. Read the below given article to get a complete idea about: 1. Origin of Tomato 2. Botany of Tomato 3. Distribution 4. Genetic Resources 5. Qualitative Genes 6. Breeding Goals 7. Important Donors for Desirable Traits 8. Breeding Methods 9. Fruit Characteristics that Determine Quality 10. Varieties and Others.

Contents:

1. Science Fair Project on the Origin of Tomato
2. Science Fair Project on the Botany of Tomato
3. Science Fair Project on the Distribution of Tomato
4. Science Fair Project on the Genetic Resources of Tomato
5. Science Fair Project on the Qualitative Genes of Tomato
6. Science Fair Project on the Breeding Goals of Tomato
7. Science Fair Project on the Important Donors for Desirable Traits in Tomato
8. Science Fair Project on the Breeding Methods Applicable to Tomato
9. Science Fair Project on the Fruit Characteristics that Determine Quality of Tomato
10. Science Fair Project on the Varieties of Tomato
11. Science Fair Project on the Cultivar Identification of Tomato through Isozymes
12. Science Fair Project on the Heterosis and Its Exploitation in Tomato
13. Science Fair Project on the Hybrid Seed Production in Tomato
14. Science Fair Project on the Cultivar Description of Tomato for Seed Production
15. Science Fair Project on the Main Seed-Borne Tomato Pathogens

Science Fair Project # 1. Origin of Tomato:

Tomato has its origin in Peru, Ecuador and Bolivia on the basis of availability of numerous wild and cultivated relatives of the tomato in this area. From its center of origin, the tomato first moved to Mexico for domestication and cultivation. From Mexico, it arrived in Europe by somewhere 1554. The most likely ancestor of tomato is the wild cherry tomato formerly Lycoperisicon esculentum var. cerasiforme (Dun). Gray.

This species is spontaneous throughout tropical and subtropical America and has spread throughout the tropics of Old World. Most of the evidences support Central American domestication, with Mexico as the primary region of domestication.

One of the most important consequences of the domestication of tomato has been the change from exerted to inserted stigma leading to change from partial allogamy to complete autogamy (self-pollinated). There has been increase in fruit size also. The first recorded mention of the tomato in America was made in 1710. By 1830, the tomato could become popular in the USA.

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Science Fair Project # 2. Botany of Tomato:

Tomato belongs to family Solanaceae and the genus Lycopersicon now Solanum. At present nine species are recognised within this genus (Table 11.1).

SP = Self-pollinated,

CP = Cross-pollinated

SF = Self-fertile,

SI = Self-incompatible.

Interspecific rosses between L. esculentum and L. pimpinellifolium are easily made. Embryo abortion may occur between L. esculentum x L. peruvianum. However, this can be overcome through embryo rescue technique. The tomato flower is normally, perfect. There are four to eight flowers in each compound inflorescence. There is a light protective another cone surrounding the stigma leading to self- pollination.

Emasculation is usually done in afternoon one day prior to anthesis/flower opening. At this stage, the sepals have started to separate and the anthers and corolla are beginning to change from light to dark yellow. The stigma is fully receptive at this stage allowing for pollination even immediately after emasculation.

After a little practice the removal of the anthers should become a simple matter although first attempt will almost inevitably result in breakage of the style. Anthers are removed as a group with or without the surrounding corolla, by inserting forceps between the sepals to grip the base of the anthers and/or petals which are then removed by a firm but steady pull.

If anthers seem reluctant to part company from flower receptacle as a group, it is advisable to remove a single one first by careful manipulation of the forceps. Following this the remaining four may be gripped firmly without any fear of damaging the style.

Pollen next day forenoon is best applied in experimental crosses by slitting the inside of the anthers of mature flowers of the male parent with the forceps in such a way that a small amount of pollen is collected at the tip of the forceps.

This can then be lightly applied to the stigmatic surface and should be visible as a white covering. Forceps should be sterilised by dipping in alcohol or methylated spirit after each pollination. Pollen may be collected in large amounts by inverting the mature flower and tapping pollen into the thumbnail. Protection of pollinated flowers by wrapping with cotton or small pollination bags is essential.

Of the 12 chromosome pairs, chromosome 1 is the largest chromosome, chromosome 2 is a nucleolus organising or satellited chromosome, chromosome 3 has a long achromatic zone present on short arm, chromosome 4 has a short chromatic zone on the short arm, chromosome 5 has a median centromere, chromosome 6 has three large chromatic lumps, chromosome 7 has chromomeres, 8, 9 and 10 have a long chromatic region on the long arm, chromosome 11 has a sub-median centromere and chromosome 12 is the shortest as reviewed by Kalloo (1993). Haploids, tetraploids, trisomics and monosomics have also been produced in the tomato.

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Science Fair Project # 3. Distribution of Tomato:

Tomato (Lycoperiscon esculentum Miller, 2n = 2x = 24) renamed by A. Child (1990) and I.E. Peralta and D.M. Spooner (2006) as Solanum lycopersicum is one of the most important vegetable crops grown throughout the world. The leading tomato growing countries in the world are the USA, several European countries, Japan and China.

Worldwide production of tomatoes reached 122 million tons in 2005. Asia is by far the continent with the greatest production (50%) with Europe ranking as the second (17.5%) followed by Central and North America (12.3%), Africa (11.7%), Latin America and Caribbean (7.8%) and Oceania (0.05%).

China is the major producer of tomato followed by US, Turkey, India and Italy. It is a rich source of vitamins and minerals. In India tomato is grown in about 6.0 lakh hectares. Tomatoes are consumed as fresh and in processed form. World acreage of tomato is more than 3 million hectares.

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Science Fair Project # 4. Genetic Resources of Tomato:

Plant genetic resources are the foundation of any breeding programme. The first source of exploitable variability comes from intraspecific variability. Genes such as sp (self-pruning) gene which conditions the determinate growth habit and the nor (non-ripening) and rin (ripening inhibitor) genes which regulate ripening process in tomato have been located in S. lycopersicum (the cultivated tomato).

These genes have led to dramatic advances in tomato breeding. However, tomato breeders have depended heavily on wild tomato relatives to locate genes conferring resistance to diseases, insects, abiotic stresses and nutritional quality as given in Table 1 1.2.

Interspecific hybridization has facilitated the exploitation of genetic resources in tomato breeding. The incompatibility barriers between some wild species and the tomato have been overcome by the rescue of immature embryos and their in vitro cultivation.

Important germplasm collections of tomato and wild relatives exist in many countries. The C.M. Rick Tomato Genetics Resource Center (TGRC), located at the University of California at Davis, is one of the best collections, and includes wild relatives, monogenic mutants and miscellaneous genetic stocks of tomato. Table 11.3 shows the most important collections of tomato germplasm, which hold more than 900 accessions each from all over the world. AVRDC, Taiwan has the largest collection of tomato accessions.

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Science Fair Project # 5. Qualitative Genes of Tomato:

An exhaustive list of simply inherited characters (characters governed by qualitative genes) has been compiled by Rick (1974). Numerous simply inherited characteristics are part of the architecture of modern-day tomato cultivars including several dominant gene(s) governing resistance to diseases which have been easily exploited by developing F₁ hybrids with resistance to diseases.

For example, the dominant gene I controlling resistance to fursarium wilt in L. pimpinellifolium has been used to evolve first resistant cultivar, Pan American. The genes that have been useful for tomato improvement are given in Table 11.4. A recent compilation on genes for resistance to important diseases of tomato is given in Table 11.5.

The Tomato Genetics Cooperative, initiated in 1951, is a group of researchers who share an interest in tomato genetics, and who have organised informally, for the purpose of exchanging information, germplasm, and genetic stocks. The Report of the Tomato Genetics Cooperative is published annually and contains report of work in progress by members, announcements and updates on linkage maps and materials available.

With C.M. Rick’s passing, the editorship of the TGC is with J. W. Scott, University of Florida, Gulf Coast Research and Education Centre, 5007- 60th Street East, Bradenton, Florida, 34203, USA. For updates on qualitative genes of tomato, relevant reports of TGC should be referred.

Pillen (1996) have listed tomato genes of known function or phenotype that have been mapped onto the molecular map of tomato/potato. A few important genes from this list are given in Table 11.6.

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Science Fair Project # 6. Breeding Goals of Tomato:

1. Earliness

2. Increased fruit yield

3. Fruit quality:

(i) Large round fruit with adequate firmness and shelf life, uniform fruit size-shape, red colour and freedom from external blemishes or abnormalities for fresh market

(ii) Large fruit size, high fruit quality and continuous production for home garden tomatoes

(iii) Deep, uniformly red coloured tomatoes, pH below 4.4, high total soluble solids (4.5-7%) and high alcohol insoluble solids (AIS) in processing tomatoes

4. Indeterminate cultivars for greenhouse production

5. Resistance to diseases:

(i) Fusarium wilt (Fusarium oxysporum f.sp. lycopersici (Sacc.)

(ii) Verticillium wilt (Verticillium albo-atrum Reinke & Berth)

(iii) Late blight (Phytophthora infestans (Mont.) DBy.

(iv) Early blight (Alternaria solani (EII. & G.Martin) Sor.

(v) Septoria leaf spot (Septoria lycopersici Speg.)

(vi) Anthracnose (Colletotrichum phomoides (Sacc.) Chester.) C. coccodes (Wallr.) Hughes

(vii) Bacterial wilt (Pseudomonas solanacearum (E.F.Sm.)

(viii) Bacterial canker (Corynebacterium michiganense (E.M.Sm.) H.L Jens.

(ix) Tomato Yellow leaf curl virus/tomato leaf curl virus

(x) Root knot nematode (Meloidogyne incognita)

6. Resistance to insects:

(i) Fruit borer (Heliothis armigera)

(ii) Whitefly (Bemisia tabaci)

7. Resistance to abiotic stresses:

(i) Cold set varieties

(ii) Hot set varieties

(iii) Drought tolerance

(iv) Salt tolerance

(v) Low temperature germination and growth

(vi) Chilling injury tolerance

(vii) Herbicide tolerance

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Science Fair Project # 7. Important Donors for Desirable Traits in Tomato:

The relevant information is summarised in Table 11.7.

Breeding procedures applicable to self-pollinated crops, viz. introduction, pure line selection, pedigree method, bulk method, single seed descent method and backcross method are applicable to tomato. In introduction, seeds of improved varieties are introduced from one ecological area into another and evaluated. These introductions arc either utilized directly as varieties or utilised into the crosses.

In India, Sioux, HS 110, Roma, Pant T 3, Arka Vikas, Arka Saurabh, S 12, Pant Bahar, Narendra Tomato 2, Narendra Tomato 1 and Margloble are promising tomato introductions.

Backcross method is commonly utilised in wide crosses/interspecific gene transfers for resistance to diseases, etc. Gielen et al. (1996) have mentioned that cucumber mosaic virus (CMV) infections rank among the most devastating diseases in the commercial culture of tomato, for which suitable sources of natural resistance are not available.

The concept of pathogen-derived resistance, however, offers an alternate approach to combat plant viral diseases by transformation of crops with nucleotide sequences derived from the viral genome. The report of these authors demonstrates the successful application of such a pathogen derived gene comprising the CMV coat protein (CP) gene, to generate protection to CMV infection in cultivated tomato.

Transformation of an inbred tomato line with the CMV-CP gene isolated from subgroup I strain, engendered high levels of protection to various CMV strains, including a virulent strain causing lethal necrosis and a typical subgroup II strain.

Moreover, when challenged by natural infection through aphid vectors in open field, levels of protection were largely maintained in hemizygous hybrids. In all, these results demonstrate that synthetic resistance genes based on the CMV-CP gene make excellent sources of broad spectrum resistance to CMV infections for introgression into tomato breeding programmes.

Pedigree method has been the most common breeding method in tomato. In this method single plant selection is initiated in F2 and is continued through successive generations till pure-lines are obtained (generally till F6).

In early generations, the selection emphasis is on highly heritable characters like growth habit, resistance to diseases, fruit characteristics, etc., whereas characters with low heritability like fruit yield and quality, etc., are taken into account in the later generations.

In single seed descent method conceptualized by Brim (1966), also referred to as ‘modified pedigree’ method, F2 population is planted and one seed is obtained from each plant to grow the next generation till F6 in which individual superior plants are selected for individual plant progeny planting and selection of superior progeny rows in the next generation.

This method is now encouraged to be used by tomato breeders as generations can be advanced in the off-seasons and this allows maintenance of broad genetic base in advanced generations.

Interspecific hybridization has been used extensively in tomato due to its narrow genetic basis. Embryo rescue has been used to produce interspecific hybrids due to the sexual incompatibility that exists between tomato and the wild Solarium peruvianum.

Some accessions of S. peruvianum have been shown to be more compatible with S. lycopersicum and can be used as a genetic bridge in transferring characteristics of interest from other accessions of S. peruvianum to S. lycopersicum.

Backcrosses to S. chilense obtained from interspecific hybrids between S. lycopersicum and S. chilense have also been employed as a bridge for the introgression of genes of interest from incompatible accessions of S. peruvianum into S. lycopersicum. S. lycopersicum is not as incompatible with S. chilense as with S. peruvianum.

In this case, the use of the pollen mixture method made obtaining interspecific hybrids possible. Other more distant species, such as S. lycopersicoides, which possess resistance or tolerance to several diseases affecting tomato as well as to chilling have also been crossed to S. lycopersicum but the introgression of characteristics of interest has not been possible due to the male sterility of the hybrids and their unilateral incompatibility when used as the pistillate parent.

The method used for introgressing characters controlled by a single major gene, both from wild species and from a different tomato cultivar, has generally been the backcross. This is the case of the R genes of resistance, whose control is usually monogenic. More recently, backcross has also been used for the development of substitution lines, advanced backcross and backcross inbred lines.

These populations signify advantages for the mapping and introduction of quantitative trait loci (QTLs) from exotic germplasm, including those related to yield and fruit quality or resistance to diseases as reviewed by Diez and Nuez, 2008.

Doubled haploid technology is a powerful alternative to classic plant breeding strategies, mostly due to the significant time and resource savings that can be achieved through its application.

At present, doubled haploids have been obtained in many crops of agricultural interest (see http://www(dot)scri(dot)sari(dot)ac(dot)uk/assoc/COST851/Default(dot)htm for a detailed list), and this set of techniques is currently being routinely applied to breeding programmes in rapeseed, maize, barley and solanaceae crops such as pepper and eggplant.

However, this technology is still poorly developed in tomato, as mostly due to the extreme recalcitrancy of this crop. In recent years, it has been possible to obtain haploid and doubled haploid plants from in vitro anther cultures, but at a very low frequency. However, this method poses several drawbacks which prevent it from being efficiently applied on a routine basis.

Among these, the most important is the unavoidable presence of somatic diploids coming from the anther walls. This critical issue can be overcome by an in vitro culture of isolated microspores. Very recently, it has been demonstrated that microspore derived haploid embryogenesis can be achieved in tomato but this technique is still in its infancy and much work must still be devoted to this topic.

Fruit size, shape, external colour, smoothness, uniformity and freedom from external defects are important characteristics for fresh use of tomato. For this purpose, usually large sized, round fruits with uniform red colour and firm texture are preferred. However, external appearance of the fruits may be of less importance if tomatoes are to be used for crushing and processing purpose.

Red colour is the most preferred fruit colour. In practical tomato breeding programme, the crimson (ogc) and high-pigment (hp) have been utilised to enhance fruit colour. The ogc gene increases lycopene at the expense of beta carotene and reduces vitamin A content.

The hp gene, on the other hand, increases total fruit carotenoids resulting in excellent colour and increased vitamin A level. Several undesirable effects associated with hp gene (slow germination, slow- growth, premature defoliation) have limited its use in a tomato improvement programme.

Soft and juicy tomatoes are usually preferred for fresh consumption. However, firm-fruit cultivars are preferred for long distance transportation and mechanical harvesting.

Variations in sugar and acid content are the most important identifiable contributors to difference in flavour (sensation of smell and taste). Cultivar differences in sugar and acid content not only result in differences in sweetness and sourness but also are key to variation in overall flavour intensity. Differences in acid levels among cultivars make a much greater contribution to flavour-intensity variation than do differences in sugar.

Genotypic variations in levels of citric and malic acid have greater potential to affect flavour than the known variation in fructose and glucose. Volatile compounds, maturity and ripeness at picking, differences in the composition of locular and pericarp tissue also affect flavour in various ways. Therefore, in breeding programmes to improve flavour, compromises will be necessary between a greater locular portion and fruit characteristics such as size, pericarp thickness, and firmness.

Nutritional value of tomato is primarily determined in terms of levels of vitamins A and C. On an average tomato contains 900 IU /100 g vitamin A and 23 mg/100 g vitamin C. Differences in pro-vitamin A beta carotene content of more than 100 fold have been reported in progeny from crosses between L. esculentum and L. hirsutum.

By backcrossing PI 126445 (L. hirsutum) to Indian Baltimore and Rutgers, a variety Caro-Red has been developed. Caro-Red has 10 times more beta-carotene than the recurrent parent. Caro-Red is similar to Rutgers for most characters except for its distinctive red-orange colour due to higher beta carotene level. Another variety in this category released at Purdue is Caro-Rich. It may be mentioned, however, that still preference is for normal cultivars with red colour.

The hp and ogc genes have also been reported to affect vitamin A content. The ogc gene results in about a 40% decrease in beta- carotene content, whereas, hp gene results in about 40% increase in beta-carotene. Some breeders have avoided using ogc alone because of its negative effect on vitamin A, but processing cultivars have been developed (e.g., Vermillion) which contain this gene.

Large variations in vitamin C content among tomato varieties (13-44 mg/100 g) and among species, cultivars and strains (8—119 mg/100 g) have been reported. Cultivars with about 50 mg/100 g vitamin C have been developed in USA (e.g., Double Rich) but these have not got commercial importance. This is primarily due to negative relationship between vitamin C levels and fruit yield. Further, there appears to be a negative correlation between vitamin C content and fruit size.

1. Fruits to be uniform in size and shape to allow mechanical harvesting

2. Uniform, intense red colour

3. Lack of fruit cracking

4. Small pedicel scar and flexible skin to permit easy peeling

5. Firm pericarp to reduce losses during harvesting and transport

6. Firm and thick central pericarp column

7. Lack of pufllness in locules and with an optimum number of three

8. Rich in intense red colour due to high lycopene

9. Brix in the range of 4 to 6

10. pH less than 4.4

Processing quality in tomato is determined by five distinct parameters.

Fruit colour determined by colorimeter is a measure of fruit maturity and also influences grade and standards of the processed commodity.

Fruit pH affects the heating time required to achieve sterilisation of the processed commodity. Longer times are required as the product pH increases. Values above pH 4.5 are generally unacceptable for such purposes. For cultivar and breeding line comparisons, pH is measured directly with fresh juice obtained from a uniform sample of fully ripe fruits.

Over-mature fruits should be avoided. Fruit pH is the most significant component of tomato acidity contributing to heat inactivation of Bacillus coagulans Hammer, a thermophilic organism. There has been fear that home processing of low-acid, fresh market cultivars could result in an unsafe product.

Titratable acidity provides a measure of organic acids (total acidity) present in a fruit sample, which in turn estimates tartness. To determine titratable acidity, 10 ml of fresh raw juice is diluted to 50 ml with distilled water. The volume of 0.1 N NaOH required for titration to pH 8.1 is multiplied by a correction factor (0.064) to determine titratable acid as percentage of citric acid.

There is frequently a highly significant — ve correlation between pH and TA, although some authors have noted poor correlations. Stevens (1986) concluded that the pH in tomato fruits can best be reduced by increasing citric or malic acids and by reducing the phosphate levels. In most cultivars, citrate makes a greater contribution to sourness than malate because it occurs at higher concentrations.

As per published reports, inheritance of acidity is largely quantitative, but there is evidence of a single major gene conditioning high acidity also. Major component of genetic variance affecting acidity has been found to be additive with heritability estimates of 0.38 for pH and 0.64 for TA. It has been possible to transfer high acidity from small fruited, high- acid lines to advanced breeding lines.

10. Total Soluble Solids (TSS):

The total solids are composed of all fruit components except water and those volatilized during drying. The most common means of estimating solid content is by refractive index, the value obtained is often called total soluble solids (TSS) or natural tomato soluble solids (NTSS) which is reported as Brix and is highly correlated with TSS. The major constituents of TSS are glucose, fructose, and sucrose.

High soluble-solid cultivars give more cases of finished product per ton of raw fruit and thus require less energy in concentration. Consequently, this parameter of quality has been of major interest to the processing industries that manufacture concentrated tomato products.

Tomato fruit solid content is influenced by both environmental and genetic factors. High light intensity, long photoperiods and dry weather at harvest favour high fruit solids. Similarly, there is a negative correlation between yield and solid contents. As a consequence, selection for high yield or compact growth habit frequently results in sacrifices in solid content.

It has been usually observed than indeterminate forms have more soluble solids than the determinate forms. Wild Lycopersicon species could be used as a genetic source for increasing TSS of tomato cultivars with a concentrated fruit set, but a compromise in total yield, fruit size and earliness seems to be unavoidable.

There is a negative correlation not only between total yield and TSS, but also between fruit-size and TSS. This is explainable on the ground that tomato fruits grow by cell expansion and not by increase in number of cells. Cell expansion is accompanied by increase in water content leading to dilution of TSS. A 408, A 276 and Red Cherry are good parents for high TSS breeding programmes.

Viscosity is an important parameter of established grades and standards. Quality of juice, sauces, soup and paste are influenced by consistency. The acid efflux method is commonly used to evaluate raw fruit sample. About 2 kg of fully ripe fruit is blended in 30 ml of concentrated HCl to inactivate pectic enzymes.

This blend is passed through an extractor fitted with a 400 mesh screen to remove skin and seeds. The juice is deaerated for 3 minutes and passed through a standard viscometer where viscosity is expressed as the time required for 100 ml to flow through the viscometer column.

The insoluble solids also provide a measure of fruit viscosity potential. The insoluble solids are called water insoluble solids (WIS) if water is used to remove soluble components, or alcohol insoluble solids (AIS) if alcohol (usually 80% ethanol) is used to remove soluble component.

Usually, AIS is slightly greater than WIS, because the less complex carbohydrate polymers are more soluble in water than the alcohol. A very high correlation has been reported between AIS content of tomato fruits and viscosity of juice.

Several authors have concluded that tomato cell walls and the juice component are essential to high viscosity and that cellulose is the cell wall component closely related to viscosity. Further, major constituents of AIS are poly-galacturonoides and poly-sacharides. The carbohydrates found in the AIS are arabinose, ribose, xylose, mannose, galactose, and glucose.

Inheritance studies on viscosity involving low viscosity genotype VF 145 B-7879 and high viscosity genotype VF 109 and 9039 M have indicated the involvement of less than 3 genes, with high heritability estimates (0.68 and 0.75).

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Science Fair Project # 10. Varieties of Tomato:

A large number of tomato varieties have been developed in India. In initial stages, most lines were straight selections from introduced germplasm lines and in the later stages the emphasis shifted on pedigree method of breeding.

A summary description of some popular varieties is given in Table 11.12 based on information given by Gill et al. (1988), Kalloo and Bhutani (1993) and other additional sources. Prominent tomato hybrids in India are NS 2535, Abhinav, Mahalaxami, Himsohna, US 618, Navin 2000, JK Desi, Laxami, Shaktiman, KVTH 143, KVTH 245, KVTH 246 and KVTH 278, etc.

Science Fair Project # 11. Cultivar Identification of Tomato through Isozymes:

A precise description of a newly bred cultivar is necessary to distinguish it from other cultivars of the same kind to protect the rights of plant breeders and producers. Traditionally, morphological characters are taken into account to detect seed contamination and establish cultivar identity.

These methods need large field areas or greenhouse space, are labour intensive and require an expert to distinguish between morphologically similar cultivars of a crop such as tomatoes. Biochemical methods, e.g. isozyme analysis and restriction fragment length polymorphism (RFLPs) would be of great value for cultivar identification.

Isozyme analyses are not only quicker and less labour intensive than traditional methods, but, are also more reliable since the expression of isozyme loci are co-dominant and not altered by environmental factors. RFLP-analysis would be the preferred method.

Isozymes of cultivated and wild tomatoes have been studied mostly by starch gel electrophoresis. These and other studies have suggested that polymorphism in cultivated tomatoes is finite due to popular breeding methods by which the genetic uniformity is promoted.

This lack of polymorphism may be the reason why the identification of tomato cultivars, using isozyme analysis, has usually not been reported. Henn et al. (1992) used isoelectric focusing to determine if the resolving power of vertical isoelectric focusing could be used to distinguish between different tomato cultivars.

The isozymes assayed were:

Alcohol dehydrogenase (ADH)

Acid phosphatase (APS)

Esterase (EST)

Phosphoglucoisomerase (PGI)

Phosphoglucomutase (PGM)

Phosphogluconate dehydrogenase (PGDH)

Seventeen cullivars were chosen for this study. The extraction buffer contained 100 mM Tris (hydroxymethyl — amino methane, pH 8.5), 10 mM beta mercaptoethanol, 15% (v/v) glycerol, 10 mM sodium tetra-borate, 2 mM ethylenediamine-tetra-acetic acid (EDTA), 0.02% (v/v) Triton X —100 and 5 mg/ml fatty acid free bovine serum albumin (BSA).

70 mg seeds were crushed with a mortar and pestle in liquid nitrogen. The extraction buffer (0.5 ml) was added after the mortar has reached room temperature. The extract was centrifuged at 10000 X g for 5 minutes at 4°C and the supernatant used for electrophoresis.

Gels were prepared by mixing 2 ml 30% (w/v) acrylamide and 0.8% (w/v) N, N’ methylene bisacrylamide, 7 ml distilled water, 2.4 ml 50% glycerol and 0.45 ml ampholytes (pH range, 4-6). The mixture was degassed before addition of 50 µl of 10% (w/v) ammonium per-sulphate and 20 µl of N, N, N’, N’ tetra-methylene diamine (TEMED). Gels were left overnight at room temperature to polymerize.

Twenty five microlitre aliquotes of the supernatant were loaded per well. The cathode solution consisted of 20 mM sodium hydroxide and the anode solution of 20 mM acetic acid, precooled to 4°C. Electrophoresis was performed at room temperature (25°C) for 1.5 hrs at 200 constant volt, where-after it was increased to 400 constant volt for an additional 1.5 hrs. Gels were stained as per Vallejos (1983).

Genetic polymorphisms within two of the six enzymes were sufficient to identify the majority of the cultivars based mainly on qualitative differences. Nine ADH phenotypes were distinguished and together with APS allowed the identification of 12 of 17 cultivars. The other four isozymes investigated showed less polymorphism and did not assist in identification of additional cultivars.

A large number of DNA fingerprinting techniques are now available for varietal characteri­zation and identification. These include AFLP, ALP, AP-PCR, AS-PCR, CAPS, DAF, ISA, RAPD, RFLP, SAP, SCAR, SSCP, SSLP, SSR, STS and SNPs, etc.

Single nucleotide polymorphism (SNP) refers to a specific and defined position at a chromosomal site at which the DNA sequence of the two genotypes differs by a single base. This might be the result of a transition (purine-purine or pyrimide-pyrimidine) or transversion (purine-pyrimidine or vice-versa) event. SNPs are now more commonly used.

Some of the favourable features of SNPs as evident from human genome sequencing programmes are:

1. SNPs are more stable than STMS (sequence tagged microsatellites) and hence, are more reliable.

2. SNPs are much more prevalent than STMS.

3. Frequency of occurrence of SNPs is far greater than that of STMS.

4. SNPs are more non-uniform in distribution.

5. SNP density within genetic sequences is low but may be useful genetic variation for exploitation in breeding programme.

6. Amenable to non-gel based assays and hence very high throughput can be achieved.

DNA sequencing is used to detect polymorphism in single nucleotides at specific and defined position on chromosome. SNPs are becoming markers of choice due to their abundance and amenability to automation and data-basing.

The DNA markers provide a very effective means to be used in IPR issues. Intellectual property rights are defined as the rights granted by a state authority for certain products of intellectual efforts and ingenuity. The Protection of Plant Varieties and Farmers’ Rights Act-2001 of GOI provides for plant breeder’s rights.

The Biological Diversity Act-2002 of GOI prohibits access to biological resources without the approval of designated and competent authority established as per law. For registration of plant varieties and consequent IP protection, a candidate variety must meet the criteria of distinctness, uniformity and stability (DUS).

Though morphological data provide the basis for DUS testing, these do have some drawbacks. Morphological expression is influenced by environmental factors leading to error in scoring. On the other hand. DNA fingerprinting has the advantages of having high degree of non-tissue specific polymorphism, minimal influence of environment and simple inheritance system.

However, for the moment, DNA fingerprint as a proof of unique identity of a plant variety is not acceptable in India by the International Union for Protection of New Varieties of Plants (UPOV). Nonetheless, plant breeders may seek to strengthen their claim for protection of new varieties by including molecular profile as supplementary information to establish the distinctness of their varieties.

Molecular profiles may be particularly relevant in cases of biotechnologically developed varieties where only small apparent phenotypic differences exist between new and extant variety. Bhat and Karihaloo (2006) were able to distinguish certain varieties in a collection of 27 tomato genotypes using RAPD in India.

Science Fair Project # 12. Heterosis and Its Exploitation in Tomato:

The information under this section is primarily based on Yordanov (1983). It is almost unanimously accepted that heterosis manifestation in tomato is in the form of the greater vigour, faster growth and development, greater earliness, and productivity, higher resistance to disease and unfavourable environmental conditions.

The first step towards a large scale extension of hybrid tomato cultivars in the practice and proper organisation of hybrid seed production took place in Bulgaria. After the second world war tomato heterosis breeding developed quickly in the Netherlands, England, France, USA, Japan and other countries. In India, tomato hybrid cultivar on commercial scale was introduced in 1973 by Indo American Hybrid Seed Company.

In addition to advantages of hybrid tomato cultivars as given above, heterosis breeding makes possible the attainment of a given breeding task in the shortest, more precise and surest way, by combining the valuable dominant genes of both the parents.

Such is the case when number of disease resistances are fixed together. If, for example, the female parent is resistant to the tobacco mosaic virus and to the leaf mold and the male parent is Verticillium and Fusarium resistant, the F₁ hybrid will combine resistance to all four diseases.

Not less than 10 generations will be necessary for development of a similar true breeding cultivar by use of the other classical methods of breeding. However, F₁ hybrid cultivars are preferred by plant breeders and seed producing companies for commercial consideration also. They see in heterosis sure way to preserve their originators’ rights on the cultivars developed by them.

Elucidating why hybrid organisms show greater vigour than their inbred parents (heterosis) has intrigued biologists since Darwin first sought to explain why out-crossing is prevalent in nature. Although heterosis is used extensively in agriculture, a lack of knowledge of the mechanism(s) responsible for heterosis has precluded boosting heterotic effects beyond present values.

Three classical models have been proposed to for dissecting heterosis into genetic and molecular components. The dominance model assumes that distinct sets of deleterious recessive alleles undergo genome-wide complementation in hybrids.

The over-dominance model states that intra- locus allelic interactions at one or more heterozygous genes leads to increased vigor. Genes showing over-dominance are the most noteworthy and sought after from both a fundamental and applied perspective, because with over-dominance theoretically only a single heterozygous gene is needed to achieve heterosis.

Although many over-dominant quantitative trait loci (QTLs) have been identified through genetic mapping experiments, further studies have revealed several examples supporting the third model, pseudo-over-dominance which is dominance that mimics over- dominance because the mutations involved are linked.

Consequently, there is little support for single gene over-dominance. Nevertheless, notable classical reports in non-crop plants and animals have suggested, amid controversy, that heterozygosity for single gene mutation can cause over- dominance (see Krieger et al., 2010 for details of cross references).

Working under the hypothesis that mutations have the potential to be over-dominant, Krieger et al., (2010) searched for genes that cause heterosis in tomato. To identify over-dominant mutations for yield, they crossed 33 diverse fertile mutants with the matching non-mutagenized parent known as M 82 to create isogenic mutant heterozygotes and compared their yield.

They reported that heterozygosity for tomato loss-of-functional allele of SINGLE FLOWER TRUSS (SFT) which is the genetic originator of the flowering hormone florigen , increases yield by up to 60%. Yield over-dominance from SFT heterozygosity was robust, occurring in distinct genetic backgrounds and environments.

These authors showed that several traits integrate pleiotropically to drive heterosis in a multiplicative manner and these effects derive from a suppression of growth termination mediated by SELF PRUNING (SP), an antagonist of SFT. These findings provide the first example of a single over-dominant gene for yield and suggest that single heterozygous mutation may improve productivity in other agricultural organisms.

Tomatoes are a self-pollinating inbred crop with perfect flowers. Although genetic sterility is available in tomato, hand emasculation and hand pollination are preferred for making hybrids. Crossing is performed in countries where labour costs are low. Seed number per pollination is high.

Tomatoes are a high value crop, grown for fresh market or for processing, and seeding rates are very low compared with the value of the commercial crops. In the USA, 100% of fresh market and 80% of processing tomatoes are F₁ hybrids.

Although tomato hybrids can exhibit heterosis for yield, the amount of yield increase in absence of stress is small or even nonexistent. The unique utility and attraction of hybrid tomatoes is that they allow breeders to assemble, in one cultivar, complementary genes for disease-resistance as well as for traits affecting product quality such as shelf life.

Breeders of hybrid tomatoes do not need to place all desirable resistance genes in one inbred cultivar, which accentuates problems with linkage drag; they instead can hybridize two complementary inbred lines to produce a hybrid with the desired full set of resistance genes.

Hybrids are essential for expression of the slow ripening trait governed by the gene nor. The homozygous wild type, +/+ , ripens too fast; homozygous nor/nor does not ripen at all, but the heterozygote nor/+ ripens slowly, as desired by the market. Tomato hybrids also exhibit increased yield stability, perhaps because they have a better balance of genes for disease resistance.

The success of hybrid tomatoes shows that hybrids can be commercially successful in an inbred crop. Expensive means of seed production, such as hand pollination, are feasible with tomatoes because of the high value of the commercial crop, the relatively low seed requirement, and the large number of seeds produced per pollination.

This example also points up the fact that heterosis for yield need not be the major factor in determining whether hybrids will be successful. Hybrids can provide many advantages over non-hybrid cultivars, in addition to heterosis for yield.

Bulk of the hybrid seed in tomato is produced by hand emasculation and pollination. The female and the male cultivars are usually planted for convenience on separate neighbouring plots. One pollinator plant is planted for three-four female parent plants. Before emasculation and pollination a thorough examination of lines is done and off types plants are rogued out.

Before beginning of crossing all flowers in bloom in the female plants are removed. Then regular daily emasculation and pollination starts. Emasculation is done in afternoons. The most suitable phase for flower emasculation is when the corolla leaves have just opened and forms an angle up to 45° in respect to flower axis.

At this phase no self-pollination has yet occurred, because the pollen anthers have not burst open. Emasculation of flower buds, when the corolla leaves have a light yellow colour, is also allowed.

All flowers, which have not been emasculated in time and where a certain doubt that self-pollination has occurred exists, should be removed, collected and destroyed. In India currently about 50 tons of hybrid seed of tomato is produced and marketed. Half of this quantity is accounted for by regular oval types followed by TYLCV tolerant acidic types.

Emasculation is generally done by hand is mass hybrid seed production. The left hand holds the flower, while the thumb and the forefinger of the right hand take the corolla leaves near the base and with great attention by only one upward pull pick off the corolla leaves together with all stamens. This manipulation is quickly mastered by the pollinating workers.

Emasculated flowers are pollinated in the next morning. Pollination on the day of emasculation is not recommended because the pistil is not fully mature and fruits with less seeds arc produced. Pollen needed for pollination is collected previously from flowers in full bloom. Labour productivity raises sharply when the pollen is placed in the glass tube.

Each pollinating worker receives a glass tube filled with pollen in the morning before work begins. He inserts the stigma of the emasculated flower into the glass tube and pollen sticks to it abundantly. Before or just after pollination, pollinated flower are marked by cutting off the ends of two neighbouring calyx leaves.

Genetic male sterility, though available in tomato, has not been used on large scale in tomato hybrid seed production, because the female line segregates in 1: 1 ratio for fertile and sterile plants and detection and removal of fertile plants before anthesis is not possible.

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Science Fair Project # 13. Hybrid Seed Production in Tomato:

The procedure described here is based on recommendations of Asian Vegetable Research and Development Centre, Shanhua, Taiwan. It is broadly similar to the procedure in use by private sector seed companies in hybrid seed production of tomato around Ranibennur area in Karnataka in India.

The F₁ hybrids of tomato compared to open-pollinated varieties are superior in yield. They mature early and uniformly; they also have better fruit quality and disease resistance. Therefore, farmers prefer hybrid seeds although hybrid seeds are costlier than open-pollinated seeds.

The high profit margins of F₁ hybrid seeds attract the interest of entrepreneurs and farmers in hybrid seed production. However, farmers need to be familiar with specific skills such as emasculation, pollination, roguing off-type plants and other techniques essential in successful tomato hybrid seed production before they can embark on this venture.

For successful hybrid tomato seed production, the area selected should meet the following requirements (a) favourable environment for flowering, fruiting and seed setting; (b) day temperatures of 21-25°C ; and (c) night temperatures of 15-20°C. Moreover, low atmospheric humidity (relative humidity-60%) at fruit maturity and harvesting will minimise disease and insect problems and increases seed yields.

Hand-pollination is practiced effectively for commercial hybrid seed production in most countries. However, hybrid seed production in developing countries is more economical because of the comparative advantage of cheaper labour cost.

Steps:

1. Plant the female parent seedlings at a spacing of 60 centimeters between rows and 50 centimeters between hills.

2. Plant the male parent seedlings in a separate plot near the female parent. Spacing’s of 150 centimeters between rows and 40 centimeters between plants in single-row raised beds is convenient for crop management operations.

However, to facilitate pollination, planting time of the parents should be adjusted to synchronize flowering in male and female parents. Usually male parent seedlings are planted 10 to 15 days earlier than female parent to obtain adequate pollen from the very beginning of the pollination programme.

The female parent, regardless of growth habit, is staked after the opening of flowers in the first cluster. Staking facilitates plant handling during emasculation and pollination and field management operations. Later, staking also keeps the ripening fruits above the ground and prevents rotting losses. In the male parent, only indeterminate types need to be staked to facilitate crop management operations.

3. Inspect the fields regularly throughout the cropping season to identify and remove off-type and suspected off-type plants. Regular inspection of parental lines and removal of virus-infected and other diseased plants throughout the cropping season is also recommended.

4. Be ready with enough simple operational tools such as fine-pointed forceps, sharp scissors, pollen-collecting cups and hand gloves before emasculation and pollination are started. Flower buds about to open in two to three days are ready for emasculation. The petals are slightly out of the flower bud, but not opened, and the corolla colour is slightly yellow or even paler.

It is important to first sterilize the forceps, scissors and hands by dipping them in alcohol before emasculation is started. If gloves are used, these should also be sterilized to prevent pollen contamination.

5. Use sharp-pointed forceps to force open the selected buds. Then, spilt open the anther cone. Carefully, pull the anther-cone out of the bud, leaving the calyx, corolla and pistil. Sometimes the corolla could also be completely taken out in the same operation. To help identify the hybrid fruits from selfed fruits at the time of harvest, cut the calyx (all or two sepals).

6. Collect flowers from the male parent to extract pollen. The best time for pollen collection is during the early morning hours when anthers have dehisced; most of the pollen sheds off if done later. Remove the anther-cones from the flowers and put them in suitable containers, such as cellophane or paper bags or plastic pans.

Dry the anther containers under a 100-watt lamp (about 30 centimetres above the bag) for 24 hours. Temperature for drying is about 30°C. The pollen can also be sun-dried, but avoid drying at midday when temperature is very high. Put the dried anther-cones in a plastic pan or cup.

7. Cover the cup with a fine mesh screen and then seal it with a similar tight-fitting cup (serving as a lid). Shake the cup so that pollen is collected in the lid cup. Transfer the pollen into a small convenient to handle container for pollination. Fresh pollen is best for good fruit-set. It can be kept for one day at moderate room temperature.

When weather conditions are not suitable for pollination, dried or dehydrated pollen can be stored in a sealed container (capsule or vial) and kept in the freezer for about a week. Without freezing, the pollen can be kept in an ordinary refrigerator for two to three days without any significant loss in viability.

The pollen should be rehydrated before use by keeping the open pollen container in a moist (high humidity) closed jar for about one hour. Avoid wetting the pollen in the process.

Remove naturally pollinated flowers and developing selfed-fruits of the female plant to prevent mixture of the hybrid seed lots and avoid competition with hybrid fruits for plant nutrient utilisation.

The number of hybrid fruits produced per plant depends on the average fruit-size and seeds per fruit of the maternal parent. As a rule of thumb, maintain the following: 20 fruits for large-fruited parent; 30 fruits for medium-fruited parent; and more than 30 fruits for small-fruited parent.

8. Keep the pollinated fruits on the plant until they become fully mature, preferably pink or red ripe. This will enable the seeds to develop normally and fully. Check the cut calyx while harvesting, to ensure that only hybrid fruits are picked. If harvesting is done at mature green or pink fruit stage, keep the fruits in a covered dry place until they become red ripe.

9. For small-scale seed production, seeds are commonly extracted manually. Collect fully ripe fruits in non-metallic containers, such as plastic buckets, crates, tubs, troughs or cemented tanks. Metallic containers may react with acids in the tomato juice and affect seed viability. Hence, they should not be used. Crush the fruits by trampling with feet. For a quick and efficient seed extraction, put the harvested fruits in fine net nylon bags and then crush.

10. Gather the bags containing crushed fruits into big plastic containers and ferment to separate the gel mass embedding the seeds. To hasten the fermentation process, put weights over the bags or keep the fruits submerged in the liquid fruit mass.

The time of fermentation depends upon the ambient room temperature. If temperature is above 25°C, one-day fermentation may be sufficient. If below 25°C, two-to three- day fermentation may be needed. However, fermentation for more than three days may spoil the seeds’ quality.

11. To wash the seeds, pour the fermented pulp into an open plastic container and stir vigorously to let the skin, flesh and gel mass float and the heavier seeds sink. Remove the floating mass. Repeal washing until all the skin and fleshy portions arc removed and only the clean seed is left at the bottom.

12. Spread the washed seed in a thin layer for drying under shade. An electric fan may be used for quick drying. The seeds can be dried under the sun the following day. Seeds must be well dried up to 6-8% moisture for safe storage.

Seeds can be dried in air drier also. Drying continues for two to three days by maintaining a temperature of 28°-30°C and until the seeds are dried to about 6 to 8% moisture content. High temperature at the time of drying may cause seeds to germinate.

13. Pack and deliver the dried seeds according to specifications of the seed company or contract agency.

Isolation Distance:

Breeder/foundation seed – 50 m

Certified seed – 25 m

The isolation distance is primarily to avoid admixture at harvesting time otherwise 3 m distance is sufficient. Planting ratio of male: female -1:4

Roguing Stages:

a. Before flowering — Growth habit, leaf morphology, reaction to pathogen

b. Early flowering and first fruit set — General plant morphology

c. First ripe fruit — Fruit morphology, colour and size

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Science Fair Project # 14. Cultivar Description of Tomato for Seed Production:

There are open-pollinated and F, hybrid cultivars.

The following outline is based on George (1999):

1. Plant:

Growth type: determinate or indeterminate

Foliage:

Attitude: semi-erect, horizontal or drooping

2. Leaf:

Length: short, medium or long

Width: narrow, medium or broad

Division of blades: pinnate or bi-pinnate

3. Peduncle:

Abscission layer: absent or present

4. Fruit:

Size: very small, small, medium, large or very large

Shape in longitudinal section: flattened, slightly flattened, round, rectangular, cylin­drical, heart-shape, obvoid, ovoid, pear-shape or strongly pear-shape

Ribbing at stem end : absent, very weak, weak, medium, strong or very strong

Predominant number of locules : 2, 2 and 3, 3 and 4, more than 4

Green shoulder before maturity: absent or present

Colour at maturity: yellow, orange, pink or red

5. Time of maturity: early, medium or late

6. Resistance to specific pests and/or pathogens

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Science Fair Project # 15. Main Seed-Borne Tomato Pathogens:

These are listed in Table 11.11.

Seed Yield and 1000 Seed Weight:

The seed yield varies considerably depending upon growth habit, number of fruit trusses/ plant, and plant density. In general, in greenhouse production, 1 kg of fruit produces about 4 g of seed (approximately 1200 seeds). In field production, the thumb rule is that the seed weight is about 1% of fruit weight. Generally, seed yield is 250-400 kg/ha. The 1000 seed weight is about 2.5-3.3 g. Seed multiplication ratio is 1000.