**13. Biotechnological approaches**

The Bt-brinjal has been developed by inserting cry1Ac gene from a soil bacterium called *Bacillus thuringiensis* through *Agrobacterium tumefaciens* mediated method. Bt brinjal contains three genes, cry1Ac gene, which encodes an insecticidal protein Cry1Ac, is derived from a common soil bacterium and is driven by CaMV 35S promoter (cauliflower mosaic virus 35S). It also has nptII gene (neomycin phosphor transferase-II) which contains an antibiotic resistance marker and another marker gene "aad "for amino glycoside adenyl transferase. The cry1AC protein formed in Bt brinjal is analogous in structure and function to that found in nature. The resistance against fruit and shoot borer of brinjal is provided by cry1Ac genes and it minimizes damages and facilitate for the reduction in pesticide sprays and thus it is eco-friendly. *Bacillus thuringiensis* and *B. t* var. *Kenyae* (B.t.k) microbial formulations have been found to be highly specific to target insect pests, and do not have deleterious effects on non-target organisms such as beneficial insects, birds, fish, and mammals including human beings. The confirmation by ELISA revealed the presence of the Cry1Ac protein. The quantitative estimates established significant levels of Cry1Ac protein (2.46–4.33 ng ml−1) in the leaf extract of the transformed plants. The expression of this insecticidal protein in high levels resulted in significant amount of mortality of larvae and also stunted the growth of any surviving larva on transformed plant tissue.

Incongruity occurs in inter-specific crosses as a result of a lack of genetic information in one partner to complete pre- and post-pollination processes in the other. After fertilization the growth of the embryo is restricted due to some post-fertilization barriers. For the developmental process an equilibrium has to be established between embryo and endosperm for sharing the nutrients. When this equilibrium in the development of the zygote is disturbed, first division is delayed and abortion of the young embryo or disintegration of endosperm happens. This abortion may occur in any stages of development of the young seed. Based on the stage of embryo abortion *in vitro* methods have been developed to overcome post-fertilization barriers in a number of plant species. Embryo rescue techniques are the oldest and most successful *in vitro* procedures.

In many instances, progeny from wild crosses of inter-varietal and inter-specific is difficult to produce owing to several barriers like pre zygotic and post-zygotic barriers. The development of young zygote may be arrested by hybrid breakdown, hybrid sterility and hybrid non viability.Post-zygotic barriers such as endosperm abortion and, at later stages, embryo degeneration are of common occurrence, leading to low fertility but these have been overcome through the use of embryo rescue.

Embryo culture is one of the earliest forms of *in vitro* culture applied to practical problems that has proven of greatest value to breeders. Among the very important strategies hybrid embryo rescue, and related applications like ovule/ovary/placental cultures through sequential embryo cultureis especially useful in vegetable crops and culture of embryos has also been demonstrated in tomato, brinjal, capsicum, hot pepper, onion, potato, tomato including cucurbits for rescuing useful hybrids.

Ovule and ovary culture are more suitable than embryo culture for small seeded species or very young embryos. When abortion occurs in a very young stage and maternal tissue has no negative influence on the development of seeds, ovary culture can be applied. Ovary culture has been applied in many *Brassica* species. The ovule culture is applied in *Lycopersicon*. Depending on the genotypic combination of the inter-specific crossing, the percentage of seedlings obtained from ovule culture varied from 0.5–22.5 per cent, whereas in the *in vivo* situation on the plant no seeds could be harvested.

Embryo rescue technique was attempted in an interspecific hybridisation of tomato variety MT-3 and Kashi Amrit with wild relative *S. peruvianum* (WIR-3957)*.* The optimum time for rescuing the embryos was standardized as twenty five days after pollination. The most effective media for germination of the immature putative hybrid embryos was Murashige and Skoog's medium supplemented with 1 mg/L GA3, 0.1 mg/L NAA and 0.5 mg/L BAP. The confirmation of hybridity of this inter-specific crosses was done using RAPD markers. Verba et al. [43] made inter-specific reciprocal crossings between *Solanum melongena, S. aethiopicum.*

**Confirmation of resistance**: The confirmation of resistance in the developed varieties/hybrids are usually undertaken by artificial screening studies.

**Artificial Screening***:* This is artificial epidemics created by inoculation of pathogen onto the plant population. The most common methods used to inoculate *Colletotrichum* on chili plants either involve using the fruit puncture method or the spraying method in the laboratory or in the field [44]. The injection of very small amount of conidia suspension into the fruit pericarp is known as microinjection or fruit puncture method. The resistance can be known by the lesions shown at the injection or inoculation area [20, 21, 45]. Secondly, spraying method in which the conidia suspension is sprayed on plants at flowering and fruiting. But this method is not safe and risky. So the puncture of detached fruits in the laboratory has paid an outstanding development of anthracnose resistance evaluation in chili.

Different artificial screening methods are employed based on the mode of spread of the diseases as given below


### **14. Conclusion**

In vegetables the biotic stresses are pests, diseases and nematodes. The damages induced by these factors affect the production, productivity and quality. Breeding of resistant varieties offer the cheapest means of pest/disease/nematode management. Resistant varieties obviate the use of chemicals, thus reduce environmental pollution. Effectiveness of resistant varieties depend upon the stability of their performance in various environmental conditions and changing climatic scenario.

*Breeding Approaches for Biotic Stress Resistance in Vegetables DOI: http://dx.doi.org/10.5772/intechopen.94983*

However like any other concept, resistant breeding also has its own limitations. That is breakdown of resistance. However, horizontal resistance being durable but difficulty relates to an accurate & reliable assessment of the level of resistance. A variety resistant to a stress may be susceptible for other. So future planning should require far greater effort for introgression of genes from different resistant sources and develop multiple resistant varieties against several biotic stresses than that required for single.
