*5.5.4 Introgression line (IL) libraries*

IL libraries contain homogenous genetic backgrounds, only differing from one another by the introgressed donor segment. A tomato introgression line population that combines single chromosomal segments introgrossed from the wild, green fruited species *Solanum pennellii* in the background of the domesticated tomato, *Solanum lycopersicum* was used to identify QTL for nutritional and antioxidant contents. Liu *et al.* [12] applied the candidate gene approach to link sequences that have known functional roles in carotenoid biosynthesis to QTLs that are responsible for the variation of the tomato red fruit color.

Marker assisted backcross breeding has been used successfully to incorporate genes or QTL for both qualitative and quantitative traits in a number of crop species especially tomato, cucumber, potato, in some cases leading to the development of improved cultivars. Of late Indian cauliflowers are being introgressed with semidominant mutant *Or* gene to enhance their betacarotene content in an attempt to tackle malnutrition problem by making diverse beta- carotene rich food available to consumers.

Interspecific crosses with wild species transferred the ability to produce small quantities of anthocyanins into the peel of cultivated tomatoes. For example, the dominant gene Anthocyanin fruit (*Aft)*, which induces limited pigmentation upon stimulation by high light intensity, was introgressed into domesticated tomato plants by an interspecific cross with *S. chilense* and the gene Aubergine (*Abg*) from *Solanum lycopersicoides,* Furthermore, the recessive gene atroviolacea (*atv*), derived from the interspecific cross with *Solanum cheesmaniae* that stimulate strong anthocyanin pigmentation in the entire plant, particularly in vegetative tissues. Fruits with either *Aft* and *atv* alleles or *abg* and *atv* alleles have been obtained with higher production of anthocyanins in the peel, ranging in total amount from 1 to 4 mg/g fresh weight of peel. Anthocyanins were found in the skin and flesh of certain cultivars of potato. Total anthocyanin concentrations in Andean potatoes ranged from 14 to 16, 330 μg/g DW [4]. Usually, cultivars high in anthocyanins are low in carotenoids and *vice versa*. The fruit color of red chili is genetically determined by three loci *y*, *cl*, and c*2* Recently the gene for capsanthin-capsorubin synthase (CCs) has been considered as candidate gene for the *y* locus. The relationship between the phytoene synthase and carotenoid content in chili was tested with interval mapping using QTL analysis revealed that they were detected only at the *PSY* locus.

Singh *et al*. [13] observed enormous diversity in pigmentation of European and Asiatic carrot.The Asiatic types are mostly yellow and purple. The Asiatic type collection Local Rewari Black and Local Jaipur Black have higher anthocyanin content. Few molecular markers linked to major genes or QTL have been developed for carotene [14] and the *Y*2 gene and the *Rs* sugar type gene. To date, seven monogenic traits have been mapped for carrot: *yel, cola, Rs, Mj-1, Y,Y*2 and P1.QTL have been mapped for carrot total carotenoids and five component carotenoids: phytoene, x-carotene, ß-carotene, zeta-carotene, and lycopene [14] and the majority of the structural genes of the carotenoid pathway are now placed into this map. Anthocyanin accumulation in the carrot phloem is conditioned by the P1 locus, with purple (P1) dominant to non purple (p1). From the inheritance studies of Eastern carrot germplasm, it is concluded that the *P*1 and *Y*2 loci are unlinked.

The common cucumbers always develop white fruit with lower carotenoid, 22 48 μg/100 g fresh weight. While Xishuangbanna gourd (*C sativus var*.*xishuangbannanesis*) develops orange fruit rich in carotenoid, approximately 700 μg/100 g flesh weight, which makes this germplasm attractive to plant improvement programme. QTL associated with orange color fruit flesh showed two genetic

linkage maps with the markers of RAPD, SCAR, SSR, EST, SNP, AFLP and SSAP, which defined a common collinear region containing four molecular markers on linkage group LG6 inMAP1 and LG 3 in Map 2.

SCAR markers linked to the *Or* gene were identified based on random amplified polymorphic DNA (RAPID) and amplified fragment length polymorphism (ALPH) by performing a bulked segregant analysis (BSA) using a double haploid (DH) population derived from the F1 cross between 91 and 112 (white head leaves) and T12–19 (orange head leaves) *via* microspore culture. On the basis of linkage analysis, the *Or* gene was mapped in a region conversing a total interval of 4.6 cM between two SSR markers derived from BAC clones AC172873 and AC189246 at the end of linkage group 9, which matches with chromosome I of A genome in Chinese cabbage. A genetic map of the 'or' locus was constructed by using five SSR markers and two morphological markers. Three SSR markers were tightly linked to o*r* and two of them, *sau* (C) 586 and *syau* 19, were located on the same side at distances of 1,6 and 1,3 cM, respectively. The other marker, *syau* 15, was located on the other side at a distance of 3.3 cM. Cervantes-Flores et al. [15] have recently reported QTL for dry matter, starch content and ß-carotene content, opening up the possibility of genetic manipulation and further enhancement of sweet potato. Ripley, V.L. and Roslinsky, V [9] identified ISSR marker for 2- propenly glucosinolate content in Brassica. Efforts are also being made to use the genetic and molecular approaches for increasing the leaves of tocopherols in potato tubers through metabolic engineering tools and techniques.

## **6. Transgenic approach**

Three genes, encoding phytoene synthase (*CrtB*), phytonene desaturase (*Crt1*) and lycopene beta – cylase (*CrtY)* from *Erwinia* have been introduced in potato to produce beta carotene. Romer *et al*. [16] developed transgenic tomato to enhance the carotenoid content with the bacterial carotenoid gene (*crtl*) encoding the enzyme phytoene desaturase, which converts phytoene into lycopene. Lu et al. [17] suggested that transgenic cauliflower with *Or* transgene is associated with a cellular process that triggers the differentiation of proplastids into chromoplasts for carotenoid accumulation and *Or* can be used as a novel genetic tool to induce carotenoid accumulation in a major staple food crops.

One of the most obvious benefits of enhancing carotenoid levels is the increase in pigmentation, which can lead to more deeply colored vegetables that are often preferred by consumers. Thus, increasing levels of carotenoid is doubly beneficial, both in terms of nutrition and esthetics. There are a range of other approaches to enhance the carotenoid levels in potatoes and other root vegetables. Diretto *et al.* [18] have silenced the first step in the ß-epsilon branch of carotenoid biosynthesis, lycopene epsilon cyclase (LCY-e) in potato which is low in carotenoids. This antisense tuber-specific silencing of the gene results in significant increases in carotenoid levels, with up to 14-fold more ß-carotene.

**Enhancing anthocyanins:** Potato does not normally produce anthocyanin, but germplasm expressing anthocyanin pigment has been developed and is attracting interest from consumers. One of these genes which encode a novel single -domain MYB transcription factor has the potential to influence anthocyanin pigment production in potato. The resulting purple potato might offer both novelty and health functionality to consumers, who can also benefit from native Andean potatoes that do not always show desired tuber shapes for both table and processing industry.

**97**

**7. Conclusion**

*Breeding Vegetables for Nutritional Security DOI: http://dx.doi.org/10.5772/intechopen.95349*

traits through crossbreeding of transgenic tomato plants.

can gain a protective benefit from *B. rapa* [20].

**Folates rich tomato:** Diaz de la Garza et al. [19] developed transgenic tomatoes by engineering fruit specific over expression of GTP cyclohydrolase I that catalyzes the first step of pteridine synthesis, and amino deoxychorismate synthase that catalyzes the first step of PABA synthesis. Vine ripened fruits contained on average 25 fold more folate than controls by combinig PABA and pteridine overproduction

The extent to which vegetable brassicas protect against cancer probably depend on genotype of the consumer, in particular the allele present at the GSTM 1 locus. This gene codes for the enzyme glutathione transferase, which catalyzes the conjugation of glutathione with isothiocyanates. Approximately, 50% of humans carry a deletion on the GSTM1 gene which reduces their ability to conjugate, process and excrete isothiocyanates. Individuals with two null alleles for GSTM1 might gain less protection from these cultivars of vegetable. The most commonly consumed *Brassica* vegetable in Asia is *Brassica rapa*. *B. rapa* contains different isothiocyanates to *B. oleracea* and recent evidence suggests that individuals who are null for GSTM1

A 10 fold increase in the level of 4-methylsulphinylbutyl glucosinolate was obtained by crossing broccoli cultivars with selected wild taxa of the *Brassica oleracea* (chromosome number, n = 9) complex. Tissue from these hybrids exhibited 100 fold increase in the ability to induce quinone reductase in Hepa1cIc7 cells over broccoli cutlivars, due to an increase in 4 – methylsulphinylbutyl glucosinolate content. Vegetables of the *Allium* genus such as onion, garlic, leek and chive are among the oldest crops associated with health-related properties. Three sets of transgenic onion plants containing antisense alliinase gene constructs (a CaMV 35S-driven antisense root alliinase gene, a CaMV 35S-driven antisense bulb alliinase, and a bulb alliinase promoter-driven antisense bulb alliinase) have been recently produced [21]. Transgenic hybrid onion seed from these transgenic lines has been developed by crossing a nontransgeneic open- pollinated parental line with a transgenic

Miraculin rich vegetables: For reduction of bitterness in lettuce, the gene for sweetness and taste modifying protein miraculin, from the pulp of berries of West African shrub *Richadella dulcifica* was cloned [22]. This gene, with the CaMV 35S promoter, was introduced into the lettue cultivar "Kaiser" using *A. tumefaciens* GV2260. Expression of this gene in transgenic plants led to the accumulation of

Protein rich potato: The genetically modified potato developed at CPRI in collaboration with NIPGAR "Protato" contains 60% enhanced protein content. This has been achieved by introducing *AmA1* gene (*Amaranth Albumin 1*) from edible amaranth plant into seven commercial varieties of potatoes. The GM potato plants were tested in India and the results demonstrated greater harvest and moderate increase in tuber yield. Safety evaluation indicated that the transgenic potatoes are suitable for commercial cultivation and have no negative effects on animal health. In addition, the concentration of several essential amino acids increased significantly in transgenic tubers which are otherwise limited in potato. This resulted in a significant increase in yield and enhanced nutrition. The *AmA1* gene has been reported to have potential for the nutritional improvement of other food crops as well [23].

Vegetables are nutritional powerhouses, key sources of micronutrients needed for good health. They add diversity, flavor and nutritional quality to diets.

parental plant carrying a single transgene in the hemizygous state.

significant concentrations of the sweet enhancing protein.

### *Breeding Vegetables for Nutritional Security DOI: http://dx.doi.org/10.5772/intechopen.95349*

**Folates rich tomato:** Diaz de la Garza et al. [19] developed transgenic tomatoes by engineering fruit specific over expression of GTP cyclohydrolase I that catalyzes the first step of pteridine synthesis, and amino deoxychorismate synthase that catalyzes the first step of PABA synthesis. Vine ripened fruits contained on average 25 fold more folate than controls by combinig PABA and pteridine overproduction traits through crossbreeding of transgenic tomato plants.

The extent to which vegetable brassicas protect against cancer probably depend on genotype of the consumer, in particular the allele present at the GSTM 1 locus. This gene codes for the enzyme glutathione transferase, which catalyzes the conjugation of glutathione with isothiocyanates. Approximately, 50% of humans carry a deletion on the GSTM1 gene which reduces their ability to conjugate, process and excrete isothiocyanates. Individuals with two null alleles for GSTM1 might gain less protection from these cultivars of vegetable. The most commonly consumed *Brassica* vegetable in Asia is *Brassica rapa*. *B. rapa* contains different isothiocyanates to *B. oleracea* and recent evidence suggests that individuals who are null for GSTM1 can gain a protective benefit from *B. rapa* [20].

A 10 fold increase in the level of 4-methylsulphinylbutyl glucosinolate was obtained by crossing broccoli cultivars with selected wild taxa of the *Brassica oleracea* (chromosome number, n = 9) complex. Tissue from these hybrids exhibited 100 fold increase in the ability to induce quinone reductase in Hepa1cIc7 cells over broccoli cutlivars, due to an increase in 4 – methylsulphinylbutyl glucosinolate content.

Vegetables of the *Allium* genus such as onion, garlic, leek and chive are among the oldest crops associated with health-related properties. Three sets of transgenic onion plants containing antisense alliinase gene constructs (a CaMV 35S-driven antisense root alliinase gene, a CaMV 35S-driven antisense bulb alliinase, and a bulb alliinase promoter-driven antisense bulb alliinase) have been recently produced [21]. Transgenic hybrid onion seed from these transgenic lines has been developed by crossing a nontransgeneic open- pollinated parental line with a transgenic parental plant carrying a single transgene in the hemizygous state.

Miraculin rich vegetables: For reduction of bitterness in lettuce, the gene for sweetness and taste modifying protein miraculin, from the pulp of berries of West African shrub *Richadella dulcifica* was cloned [22]. This gene, with the CaMV 35S promoter, was introduced into the lettue cultivar "Kaiser" using *A. tumefaciens* GV2260. Expression of this gene in transgenic plants led to the accumulation of significant concentrations of the sweet enhancing protein.

Protein rich potato: The genetically modified potato developed at CPRI in collaboration with NIPGAR "Protato" contains 60% enhanced protein content. This has been achieved by introducing *AmA1* gene (*Amaranth Albumin 1*) from edible amaranth plant into seven commercial varieties of potatoes. The GM potato plants were tested in India and the results demonstrated greater harvest and moderate increase in tuber yield. Safety evaluation indicated that the transgenic potatoes are suitable for commercial cultivation and have no negative effects on animal health. In addition, the concentration of several essential amino acids increased significantly in transgenic tubers which are otherwise limited in potato. This resulted in a significant increase in yield and enhanced nutrition. The *AmA1* gene has been reported to have potential for the nutritional improvement of other food crops as well [23].
