Keywords

9.1 Introduction

The plant undergoes different kinds of stresses in nature that contribute to adverse growth and compromised plant productivity. Any kind of stress induces a series of adaptive responsive in plant-like alteration in gene expression and induction of defensive cell metabolism. Plant stress could be divided into two major categories termed as biotic (microbial pathogens, nematode, insects, and weed) and abiotic caused through various environmental cues (heat, cold). Both kinds of stressors contribute to both pre- and post-harvest losses. Furthermore, it is also noticeable that with increasing global warming and changing climate situations, agricultural crops significantly encounter an increased event incidence of both abiotic and biotic stresses compromising plant yield (Mahalingam 2015; Ramegowda and Senthil-Kumar 2015; Kumar and Srivastava 2016, 2020a, 2020b; Srivastava et al. 2016; Parmar et al. 2017; Gambhir et al. 2020; Kumar et al. 2018a, 2018b, 2018c). Both kinds of stresses concurrently cause more destruction when compared to either of the stress occurring solely. For instance, abiotic stress directly influences the occurrence, survival, and dissemination of the different pathogens, insects, and weed growth. Additionally, these stress situations also influence plant physiology and host-defense responses to promote plant–pest interactions. However, this effect is not always additive as the outcome could be influenced by the nature of the interaction between these stress factors (Pandey et al. 2015; Ramu et al. 2016).

Tackling any kind of stress is a bigger problem for gradually increasing world population, estimated to reach ten billion by 2050 (Bengtsson et al. 2006; Secretariat UNIS for DR 2015). All this needs a significant increase in crop production by minimizing crop losses in any kind of stressful situation. The improvement could either be achieved by a traditional breeding method like wide-cross hybridization, mutation breeding or by modern technology including transgenics. Despite several attempts, the conventional plant breeding methods have failed in combating this issue. Current studies propose that the tolerance to any kind of stress is multigenic and quantitative QTL (quantitative trait locus) in nature (Collins et al. 2008), which could be a possible reason for this failure. Modern transgenics serve as an alternative to tackle this issue as this includes the introduction of exogenous genes into the host or an alteration in the expression of a host’s gene that can help in improving stress tolerance (Roy and Basu 2009). Besides, modern transgenics require comparably less time than conventional plant breeding methods with a benefit of transfer of only desired gene(s) to the host plant, it always has an upper hand over the conventional crop breeding methods (Yamaguchi and Blumwald 2005). Due to ease in the use of transgenics for crop improvement, the technology has been used extensively worldwide (Wani et al. 2016a). However, the employment of genetic engineering needs the identification of the key genes underlying these processes of plant stress tolerance.

It is well known that any kind of stress induces a series of adaptive responses in the cells contributing to changes in the physiology and molecular makeup of the plants that could be dangerous to the cellular machinery. If we talk at the molecular level, any kind of stress leads to different molecular responses like the production of reactive oxygen species causing damages to cellular molecules like protein, nucleic acids, and lipids by the process of oxidation or peroxidation. In severe conditions, these stress situations may also lead to programmed cell death and thus cause the death of the entire plant (Sharma and Dubey 2007). Hence, exploring the underlying molecular mechanisms and key molecules contributing to stress signaling could be an alternative strategy for crop engineering for stress-tolerant properties, which could be done by application of the modern transgenic approaches like sequencing and functional genomics (Heidarvand and Amiri 2010). Usually, a cell signaling cascade is triggered upon any kind of stress upregulating the different transcription factors inducing various stress-responsive genes coding for proteins helping in stress-tolerant phenotypes. As stress-sensitive plants are compromised in synthesizing these proteins, they are susceptible to all these stressors impeding their overall growth. Several genes have been identified that code for stress-protective compounds and proteins in the different organisms (plants, animals, or microbes). The ideal genes for this kind of targeted insertion have been classified into three major categories; (a) associated with osmolyte synthesis like mannitol, glycine betaine, proline, and heat shock proteins, (b) associated with the uptake of ion and water, and (C) associated to transcriptional control and different signaling pathways. Progress in understanding the underlying mechanism of these stress responses has undergone major development in recent years (Jaspers and Kangasjärvi 2010). The transgenics have been successfully employed to produce plants with different attributes like an increase in grain yield, increase tolerance to high salt and drought stress in rice, barley, maize, etc. (Wani et al. 2016b). Following is a brief introduction of the different kinds of stressors contributing to significant crop losses and the genetic developments created to combat these losses associated with stressful situations. As losses due to both kinds of stressors cause severe crop losses worldwide, it is conceivable that the development of the plant with improved traits for abiotic and biotic stress requires identification and improvement of stress adaptive traits in these plants. This book chapter is a brief introduction about these stresses (biotic and abiotic) and the genetic improvement approaches that have been implicated for improving stress-responsive properties in plants.

9.2 Biotic Stresses

9.2.1 Insect Pest and Nematodes Resistance

The control of insect pests in commercially grown agricultural crops is predominantly based on the indiscriminate use of synthetic insecticides which is deleterious to human health and the environment. Transgenic crops have revolutionized modern agriculture and have become a major tool of integrated pest management leading to a reduction in insecticide use, protecting the environment and human health. Insect-resistant plants were also developed about two decades ago in other crops (Ansari et al. 2015). The search for novel useful genes encoding insecticidal proteins is still in progress and information deriving from the prolonged cultivation of commercialized GM products confirms the efficacy of this biotechnological tool. The new research area includes the use of novel transgenes and improved transformation protocols especially for the development of insect-resistant cultivars in economically grown agricultural crops.

The soil bacterium (B. Thuringiensis, Bt) produces a wide range of proteins (δ-endotoxins) that are included in crystals formed during sporulation and characterized by distinct insecticidal spectra (de Maagd et al. 2001; Sharma and Srivastava 2013). Bt spores contain high levels of δ-endotoxins harmful to specific insects of Lepidopteran, Dipteran, and Coleopteran orders which are the major pests of agricultural crops and perennial tree species. The Bt spores and the crystal (Cry) proteins are ingested by the insect and solubilized within the alkaline midgut. The protoxins are then activated by proteinases and finally, the active Bt toxin binds to specific molecular receptors causing the irreversible damage of the midgut epithelium by colloid osmotic lysis. B. thuringiensis has been used as a commercial insecticide for more than 50 years and to date, an extensive number of reports have demonstrated that Bt proteins have negligible potential adverse effects against humans, animals, and non-target invertebrates. More than 130 Bt genes encoding different δ-endotoxins have been isolated and, among this extremely large gene array, those coding for the CryIA(a) and CryIA(c) proteins have been used to develop transgenic crops resistant to Lepidoptera. Also, the CryIIIA(a) protein has been chosen by different research groups to specifically target Coleopteran pests. Insect resistance was firstly reported in tomato using Bt. gene in 1987. Cotton was the first commercially successful crop in which cry genes were incorporated to provide resistance against lepidopteron insect pest (Perlak et al. 1991). After the success of transgenic cotton, cry genes have been incorporated in many crops, viz., potato rice, canola, soybean, maize, chickpea, alfalfa, and tomato. Insect-resistant transgenic crops have the second largest area under cultivation which is 23.3 million hectares in 2017 (ISAAA 2017), 304 events have been approved in different crops worldwide for commercial cultivation. Out of these events, 208 events comprising various insect resistance (IR) genes in maize have been approved for cultivation. The commercialized crops having various IR genes are cotton (49 events), potato (30 events), soybean (6), rice (3), sugarcane (3), poplar (2), brinjal (1), and tomato (1).

Apart from cry genes, other insecticidal genes such as Proteinase inhibitors (PIs) are also used to develop insect-resistant GM crops. PIs are natural compounds abundantly found in seeds and storage organs of a wide range of plant species and contributing to the plant defense system against insect pests and pathogens (Schuler et al. 1998). Proteinase inhibitor families are specific for each of the four classes of proteolytic enzymes (cysteine, serine, aspartic, and metalloproteinases). Serpins and Cysteine are the most explored plant PIs against insect pests. Green and Ryan proposed this concept in a pioneer study reporting rapid accumulation of protease inhibitors in potato and tomato leaves attacked by Colorado potato beetles, both locally as well as systemically (Green and Ryan 1972). A few years later, a seminal study by Hilder et al. (1987) reported PI-expressing transgenic tobacco lines by utilizing the potentials of plant genetic transformation. With the advancement of gene transfer technology, this agronomically useful gene was introduced in rice cultivars that enhanced protection to stem borers (Xu et al. 1996) and wheat (Altpeter et al. 1999) to protect from leaf-feeding and storage pest. Since then, various research groups have reported numerous studies of enhanced herbivore protection in multiple plants involving bioassays, PI-expressing transgenic plants, and insect feeding assays. The inhibiting activity of PIs is due to the ability to form stable complexes with proteinases, blocking, altering, or preventing the access to the substrate-binding region of their catalytic site. To develop effective strategies for plant protection against insect pests based on PIs transgenesis, it is imperative to know the class of proteolytic enzymes present in the insect guts, which ultimately results in the extended developmental period, reduce fecundity and increase mortality due to amino acid deficiencies. Different proteinases predominate in different insects. Most of the Lepidopteran species have serine proteinases as the major digestive enzymes. Coleoptera has a wider range of dominant gut proteinases (Schuler et al. 1998). Broadway and Duffey (1986) suggested that the PI mediated inhibition of proteinases is responsible for hyperproduction of digestive enzymes, enhanced loss of essential amino acids, and finally inhibition of insect growth rates. However, some insect species seem to be able to modify dynamically the spectra of their digestive enzymes by the production of insensitive proteinases. Besides several reports of successful PIs-transformed plants enhanced protection to insect pests, this promising strategy of crop protection could not be successfully commercialized.

9.2.2 Nematodes Resistance

Plant-nematode parasitism is one of the most damaging uncontrollable biotic stresses on crops, and the cumulative effect on agriculture is severe. The majority of these losses are inflicted by relatively few species. The most damaging are root-knot nematodes (Meloidogyne species) and cyst nematodes (Heterodera and Globodera species), with root-knot nematodes the major contributors to yield losses (Koenning et al. 1999). Management of nematode parasitism is therefore imperative. Integrated use of chemicals, resistant varieties, and cultural and biological practices provide the most successful management strategy. These approaches are, however, becoming increasingly unsatisfactory. Although conventional intensive farming methods rely largely on the use of nematicides, dependence on this approach must diminish as environmental and health concerns around these toxic chemicals increase. Crop rotation as a strategy to limit nematode infestation has limited utility against those species with cosmopolitan host ranges, such as M. incognita, which may potentially parasitize up to 3000 plant species (Abad et al. 2003). Resistance in plants is therefore an attractive approach for controlling nematode populations. This may be either naturally occurring or transferred to crop cultivars from wild relatives or breeding lines through conventional breeding methods or engineered through molecular techniques. Biotechnology offers several benefits for nematode control in integrated management strategies such as reducing risks to the environment and human health, accessibility for food producers in the developing world, and the possibility of achieving durable, broad-spectrum nematode resistance (Thomas et al. 2006).

Several nematode resistance (Nem-R) genes have been isolated from plants which confer resistance against sedentary endoparasites. The first nematode resistance gene to be cloned was Hs1pro-1 from sugar beet, which confers resistance against the sugar beet cyst nematode (Cai et al. 1997). The encoded protein does not have obvious similarities to known plant genes. However, other cloned Nem-R genes closely resemble known plant R-genes in their domain structure. Four of these genes, Mi-1, Hero A, Gpa2, and Gro1–4, all cloned from tomato or potato relatives, fall into the NBS-LRR class of R-genes. The tomato genes Mi-1 and Hero A confer broad-spectrum resistance against several root-knot nematode species (Milligan et al. 1998) and against several pathotypes of two potato cyst nematode species (Ernst et al. 2002), respectively. By contrast, the potato genes Gpa2 and Gro1–4 confer resistance to a narrow range of pathotypes of a single potato cyst nematode species. Mi-1, Gpa2, and Hero A are members of the NBS-LRR class of plant R-genes that does not contain an N-terminal toll-interleukin receptor-like (TIR) domain. The Hero A gene product is 32% identical to Mi-1 and w22% identical to Gpa2 at the amino acid level (Williamson and Kumar 2006).

9.2.3 Antifungal Resistance

Antifungal proteins like chitinase, glucanase, defensin, thaumatin-like proteins, osmotin-like protein, phytoalexins, RIPs, etc. are produced by different flowering plants (Vigers et al. 1991), fungi (Guo et al. 2008), invertebrates and vertebrates (Raj and Dentino 2002) to combat the fungal pathogens. Some of this plant protein comes under the pathogenesis-related protein group (PR). Genes of these antifungal proteins were transferred to different plants to increase fungal resistance against fungal pathogens. Below is a summarized different antifungal protein which was used to produce transgenic plant against fungi.

9.3 Chitinase and Glucanase

Most of the fungi contain chitin and glucan in their cell wall as major components. Chitinase and glucanases are hydrolytic enzymes that can degrade chitin and glucan, thus leads to the degradation of the cell wall of fungi. Chitinase comes under the glycosyl hydrolase family and it hydrolyzes glycosidic bond in chitin. On the basis of isoelectric pH, the sequence at N-terminal, enzyme localization, and signal peptides chitinase enzyme was found to belong to 18 and 19 families of glycosyl hydrolases. Classes III and V of chitinase come under glycosyl hydrolase 18 and I, II, and IV under family 19. These enzymes are PR-3 group members (PR-Pathogen-related proteins) are first described in the orchid bulb by Bernard as an antifungal factor (Sharma et al. 2011). These are probably the most frequently studied and described PR proteins. These hydrolytic enzymes can cause lysis of fungal hyphae, inhibition of fungal growth, and exhibit in vitro antifungal activity (Boller 1993; Neuhaus 1999). Chitinase was reported to be present in plants, animals, microbes, and human beings. But the most preferred one is microbial chitinase because they can be easily produced in bulk and also available is more. But the first group of PR families is plant chitinases which are abundantly present in the plant kingdom and found to be effective against many phytopathogenic fungi like Ascomycota and Basidiomycota phyla (Punja 2004). It was also reported that these hydrolytic enzymes can also be produced in plants in response to abiotic stress as well as growth conditions. The number of chitinase gene isolated from different sources has been studied, sequenced, cloned as well as transformed into plants to develop fungal resistance. It was found out that when glucanase and chitinase genes were expressed in transgenic plants, then it results in more resistance to fungal pathogens (Nishizawa et al. 1999). But the expression of these genes in transgenic plants at a low level is a key issue. Lee and Raikel (1995) reported that in rice and tobacco, the expression of chitinase genes increased the plant’s resistance to phytopathogenic fungi. Jabeen et al. (2015) studied for the first time that transgenic tomato plants showed resistance to two major fungal pathogens, i.e. Fusarium oxysporum f. sp. lycopersici (Fol) causing fusarium wilt and Alternaria solani causing early blight (EB) when rice chitinase (RCG3) was expressed in tomato. Agrobacterium-mediated transformation of cotyledonary petioles with an endochitinase gene (chit33-cDNA) isolated from Trichoderma atroviride under CaMV35S constitutive promoter showed increased resistance against Sclerotinia sclerotiorum in canola (R line Hyola 308) (Solgi et al. 2015). EuCHIT2, a new chitinase gene that was isolated from Eucommia ulmoides Oliver was overexpressed in tobacco plants showed resistance to Erysiphe cichoracearum DC (Dong et al. 2017). Khan et al. (2017) also developed transgenic potatoes using the Agrobacterium-mediated method that overexpressed endochitinase gene and showed resistance against Alternaria solani. Novel chitinase gene LOC_Os11g47510 from indica rice Tetep provides enhanced resistance against sheath blight pathogen Rhizoctonia solani in rice (Kamboj et al. 2017). Chitinase enzyme gained attention towards biocontrol of fungal pathogen, but glucanase enzymes are less studied as compared to chitinase. Only a few reports are available on glucanase gene transformation in plants, but some transgenic plants overexpressing glucanase gene was successfully produced. Glucanase gene of tobacco was overexpressed in groundnut which showed tolerance to Cercospora arachidicola and Aspergillus flavus (Sundaresha et al. 2010). It was found out that transgenic groundnut was not only resistant to fungi but also produced less aflatoxin. In another report, grapevine, b-1,3-glucanase (VvGHF17) gene was overexpressed in Arabidopsis plants, showed resistance to Colletotrichum higginsianum and Botrytis cinerea (Fujimori et al. 2016). However, the synergistic action of these hydrolytic enzymes with each other as well as with other antifungal proteins has resulted in excellent in vitro and in vivo antifungal action (Melchers and Stuiver 2000). For example, when chitinase (chi11) and osmotin (ap 24) antifungal proteins encoding genes isolated from rice and tobacco, respectively, were expressed in tobacco, then this synergistic action can cause enhancement of sheath blight tolerance in transgenic rice (Sripriya et al. 2017).

9.4 Defensin

Defensin is a small antimicrobial cationic peptide that is present in various living organisms such as plants, microbes, and mammals. It contains about 45–54 amino acid residues which form a highly conserved structure scaffold with cysteine amino acid to forms αβ conformation. In plants, γ-thionin of wheat and barley was renamed as defensin based on structure and function similarity with insect defensin. The tertiary structure of plant defensin is formed of 3 antiparallel strands and 1 α- helix strand stabilized with disulfide bridges is highly conserved to form CSα/β (Cysteine stabilized α-helix β sheet motif) (Zhu et al. 2005). 3D Structures of different plant defensins are almost similar instead of low-level amino acid identity. Plant defensin is secreted in extracellular space in plant cells except for some floral defensin which is targeted to the vacuole. Plant defensin is naturally synthesized and present in every organ of plants. In plants defensing, major role is in the inhibition of phytopathogenic fungal growth at a very less concentration (Lay and Anderson 2005). Main mechanism of how defensin prevents fungal growth is not clear but it was found out that defensin bound to fungal cell membrane because of some electrostatic or hydrophobic interactions and at a very high concentration it causes membrane permeabilization which leads to the death of fungi (Sagaram et al. 2011; Thevissen et al. 2003; Valente et al. 2013; Hayes et al. 2013). The transgenic expression of defensins has enhanced plant resistance to phytopathogenic fungi. However, so far, there are no reports of enhanced resistance through the transgenic overexpression of defensin genes in those plants from which it was initially originated. Defensin gene isolated from plants was overexpressed in many plant species. For example, a defensin gene Rs-AFP2 was isolated from Raphanus sativus and overexpressed in transgenic rice showed antifungal ability against Rhizoctonia solani and Magnaporthe oryzae (Jha and Chattoo 2010). This gene causes direct inhibition of theses pathogen (Lacerda et al. 2016). Spore germination and growth of obligate biotrophic fungi Fusarium tucumaniae and Colletotrichum gossypii var. cephalosporioides was inhibited in transgenic Pichia pastoris expressing rDrr230a defensin protein gene. This gene also showed inhibition of the Asian soybean rust pathogen Phakopsora pachyrhizi and was used against cotton and soybean fungal diseases. A defensin gene J1–1 was overexpressed in transgenic pepper showed increased resistance against Colletotrichum gloeosporioides fungi which is the causal agent of fruit-specific anthracnose fungus (Seo et al. 2014). Transgenic poplar plant expressing a putative defensin gene showed enhanced resistance against Septotis populiperda (Wei et al. 2020).

9.5 Thaumatin-like Proteins

Thaumatin-like proteins are present in plants such as Kalemfe which is a tropical flowering plant. It is a very sweet tasting protein that is almost about 100,000 times sweeter than sucrose. These proteins also come under the PR-5 Proteins family. These are low molecular weight proteins of about 20–24 kDa with 200 residues and 16 conserved cysteine which are involved in 8 disulfide bond formation which gives stability to this protein (Fierens et al. 2009). TLPs are present in different kingdoms such as plants (angiosperms, gymnosperms), animals, and fungi also (Liu et al. 2010). These proteins also expressed in plants against biotic and abiotic stress (Muoki et al. 2012; Singh et al. 2013). TLPs are also found to be antifungal proteins when overexpressed in transgenic plants (Singh et al. 2013; Wang et al. 2011a, b; Liu et al. 2012; Mahdavi et al. 2012; Acharya et al. 2013). It’s unclear how these thaumatin-like antifungal proteins interact with the fungal pathogen, and more research is needed. Thaumatin-like proteins possess the antifungal activity and overexpression of these proteins showed tolerance to fungal pathogens (Wang et al. 2011a, b; Liu et al. 2012; Mahdavi et al. 2012; Acharya et al. 2013; Singh et al. 2013). Thirty-three putative TLPs gene of grape was studied for grape disease resistance and it was found that overexpression of TLP29 in Arabidopsis thaliana causes powdery mildew resistance (Yan et al. 2017). ObTLP1 which is an ocimum thaumatin-like protein was found to be an antifungal protein and was reported to inhibit the growth of Ceratonia sclerotiorum and Botrytis cinerea. When this gene was overexpressed in transgenic Arabidopsis, then it led to resistance against these fungi and also against dehydration and salt stress; thus suggesting their role in abiotic stress also (Misra et al. 2016). Agrobacterium-mediated transformation of Ostlp, a thaumatin-like protein in cassava inhibits Colletotrichum gloeosporioides f. sp. Manihotis growth (Ojola et al. 2018).

9.6 Osmotin-like Proteins

Osmotin or osmotin like proteins is a multifunctional protein that comes under the PR-5 protein family because they are homologous to thaumatin. Osmotin structure shows three motifs with similar folding as thaumatin and other PR-5 proteins. It consists of three domains. Singh et al. (1987) characterized osmotin from salt adapted cultures tobacco (Nicotiana tobaccum) cells. Osmotin is a multifunctional stress-responsive protein that enhances biotic and abiotic stress resistance in plants (Anu et al. 2015; Le et al. 2018; Su et al. 2017). Osmotin gene expression and protein formation is induced by any biotic stress such as fungal attack and its overexpression in transgenic plants leads to less disease symptoms (LaRosa et al. 1992; Liu et al. 1994; Zhu et al. 1996). Osmotin protein attacks specifically the plasma membrane of the pathogen which leads to signaling for cell death. Cell wall composition also determines osmotin toxicity because it governs osmotin protein access to the plasma membrane (Ibeas et al. 2000, 2001; Narasimhan et al. 2001, 2005). Oryza sativa, Glycine max, Capsicum chinense, Vitis vinifera, and Sesamum indicum are examples of plants in which osmotin gene was being transformed and showed increased resistance to fungal growth (Kim et al. 2004; Elvira et al. 2008; Weber et al. 2014; Katam et al. 2015; Chowdhury et al. 2017). ObTLP1, which showed similarity to stress-responsive osmotin protein as well as to thaumatin-like protein, was isolated from Nicotiana tabacum and showed resistance to Botrytis cinerea, Sclerotinia sclerotiorum, and to salt stress and dehydration when expressed in Arabidopsis (Misra et al. 2016). Chowdhury et al. (2017) reported that SindOLP when overexpressed in sesame showed resistance against biotic as well as abiotic stresses. Transgenic lines of potato cultivar “Kufri Chipsona 1” were developed containing OsmWS osmotin gene isolated from Withania somnifera. These transgenic lines showed 22 fold expression of this gene within 3 days and inhibit Alternaria solani growth (Kaur et al. 2020).

9.7 Plant Ribosome-Inactivating Proteins

RIPs are RNA N-glycosidase which causes depurination of the highly conserved region, i.e. α-sarcin loop of 28s rRNA and thus inactivates ribosome by inhibiting the eF-Ia to bind with the ribosome. This inhibition leads to blockage of translation on the ribosome (de Virgilio et al. 2010). RIPs are widespread in nature and are distributed among different plant genera within different tissues. A number of RIPs are found to possess different antimicrobial activities in nature such as antifungal, antitumoural, antibacterial, and antiviral activities (Stirpe 2004; Puri et al. 2009; Bian et al. 2010). In agriculture, it is demonstrated in vitro and in transgenic plants that RIPs have been connected to defense by antifungal, antibacterial, antiviral, and insecticidal activities (Akkouh et al. 2015). For example, transgenic tobacco plant containing maize proRIP antifungal protein showed increased resistance against R. solani (Maddaloni et al. 1997). Yuan et al. (2002) reported that blast disease in transgenic rice was found to be inhibited by a type I RIP TCS (Yuan et al. 2002). Curcin-2 isolated from Jatropha curcas leaves was expressed in tobacco plants showed antifungal activity against R. solani, this protein was found to exhibit activity against different other stresses also (Huang et al. 2008). Agrobacterium-mediated transformation of the potato cultivar “Desirée” with Ribosome-Inactivating Protein (rip30) gene of barley produced a transgenic which showed enhanced resistance to Rhizoctonia solani in greenhouse condition (M’Hamdi et al. 2013). Plant RIPs also showed enhanced resistance when co-expressed with other antifungal proteins, e.g. when rice basic chitinase (RCH10) and modified maize RIP (MOD1) were co-expressed in rice, it showed good resistance against R. solani (Kim et al. 2003). Transgenic plants of blackgram co-expressing chitinase gene from barley and RIP showed Corynespora leaf spot fungal growth inhibition (Chopra and Saini 2014). Transformation of RIP α-MMC gene into rice showed increased resistance to blast fungus (Qian et al. 2014). A transgenic potato lines expressing PhRIP I gene of Phytolacca heterotepala coding for a ribosome-inactivating protein was found to possess more resistance to Botrytis cinerea and Rhizoctonia solani fungal pathogens (Gonzales-Salazar et al. 2017).

9.8 Phytoalexin

Phytoalexin term was originally coined by Müller (1958) and they come under low molecular weight plant antibiotic group. These are naturally produced secondary metabolites that possess antimicrobial activity. These are produced naturally in plant cells as normal growth metabolites or can be induced in the presence of pathogen attack or other stress. They can inhibit bacteria, fungi, insects, nematodes, toxic against animals or plants itself. About 350 phytoalexins have been identified and characterized from 30 plant families, Leguminosae plant family produces maximum 130 phytoalexins. These phytoalexins are well diversified in the plant kingdom and are characterized among different classes of chemical compounds such as coumarins, diterpenes, flavonoids, alkaloids, phenolic compounds, luteolinidin, apigenidin, and apigeninidin. Pisatin was the first phytoalexin isolated and characterized from garden pea, Pisum sativum (Cruickshank and Perrin 1960). The molecules that signal plants to begin the process of phytoalexin synthesis are called elicitors. Elicitors of biotic origin may be involved in the interaction of plants and potential pathogens, whereas abiotic elicitors are not involved in normal host–pathogen interactions. Phytoalexins only showed resistance in a sufficient concentration which will be produced by one or more phytoalexins along with another component. Phytoalexins were biosynthesized by phenylpropanoid pathways mainly around resistant tissue and also in necrotic lesions. Also, the acetate-mevalonate and shikimate pathways are involved in flavonoid biosynthesis. These all pathways are interconnected and are involved in the synthesis of some important enzymes which play a crucial role in resistance such as chalcone isomerase (CHI), chalcone synthase (CHS), phenyl-alanine ammonia lyase (PAL) CoA ligase, and stilbene synthase. Overexpression of these potential enzymes shows resistance against different diseases. Stark-Lorenzen et al. (1997) expressed the stilbene synthase gene of grapevine in rice and found that disease resistance was increased. Resveratrol synthase and isoflavone methyltransferase gene was also expressed in alfalfa plants to increase disease resistance. Similarly, isoflavone reductase (GmIFR) isolated from soybean enhanced resistance against Phytophthora sojae in Soybean (Cheng et al. 2015).

9.9 Antibacterial Proteins

These are small-sized lytic peptides which are having amphipathic α-helical structure. These proteins produce pores in the bacterial cell membrane causing lysis of bacterial cells (Boman 1991). These antimicrobial proteins are produced by the different living organisms from bacteria to animals as defense proteins. Some of the antibacterial proteins which are transferred to plants to increase resistance against bacteria are summarized below;

9.9.1 Cecropins

These are positively charged antimicrobial peptides which are isolated from giant silk moth (Hyalophora cecropia) hemolymph. Cecropin term was given because of its source of isolation. They are proteinaceous in nature consisting of 31–39 amino acid residues and synthesized as lipid bodies in cells. Cecropin mainly lyses the cell membrane of bacteria, on interaction with bacterial membrane, it forms an α-helical structure interaction which causes ion channel formation. It also inhibits proline uptake and leads to leaky membranes. It acts as the main constituent of insects immune system of bacteria and at low concentration (0.1–5μM) inhibits many gram-positive bacteria as well as some gram-negative bacteria (Chen et al. 1997). Antibacterial activity of cecropins isolated from Antheraea pernyi, Hyalophora cecropia, and Bombyx mori has been demonstrated towards different genes (Jaynes et al. 1993; Sharma et al. 2000). Agrobacterium-mediated transformation of antimicrobial peptide cecropin P1 (cecP1) in rapeseed (Brassica napus L.) was done and it has shown that these transgenic plants showed resistance to the bacterial and fungal pathogens Erwinia carotovora and Fusarium sporotrichioides (Zakharchenkoa et al. 2020). Cecropin B isolated from Chinese tasar moth (Antheraea pernyi) has been expressed in transgenic citrus to eliminate the effect of Huanglongbing (HLB), associated with Candidatus liberibacter asiaticus bacteria (Zou et al. 2017).

9.9.2 Attacins

Attacins are also another type of antibacterial proteins that are much larger than cecropins, i.e. about 180–190 aminoacids. There are about six different types of attacins (A-F) that have been isolated from a moth, i.e. H. cecropia. These proteins, i.e. A-F attacins differ from each other because of the processing step during synthesis, protein from A-D constitute a basic group whereas E and F are acidic. Attacins are found to attack gram-negative bacteria but these proteins do not cause lysis but disrupt outer membrane structure. Attacin proteins are not broad spectrum like cecropins but it can inhibit the growth of some bacteria like E. coli, Acinetobacter calcoaceticus, and Pseudomonas maltophilia (Hultmark et al. 1983). Apples were transformed using cDNAs coding for attachin E which were coupled to plant promoters (Norelli et al. 1994). Transformed plants of a susceptible apple rootstock N.26 possessed increased resistance to the fire blight pathogen Erwinia amylovora compared to the untransformed control, but were still more susceptible than the naturally resistant rootstock Liberty. Attacin expressed in transgenic potato enhanced its resistance to bacterial infection by E. carotovora subsp. atroseptica (Arce et al. 1999). Transgenic pear and apple expressing attacin genes have significantly enhanced resistance to E. amylovora in in vitro and growth chamber tests (Ko et al. 2000). Transgenic apple expressing attacin targeted to the intercellular space, where E. amylovora multiplies before infection, has significantly reduced fire blight, even in apple plants with low attacin production levels (Ko et al. 2000). Attacin A gene was transferred to citrus under the control of a phloem-specific promoter to control Huanglongbing disease (Tavano et al. 2019).

9.9.3 Lysozyme

This is a low molecular weight self-defense enzyme that was discovered in 1922 by Alexander Fleming. These enzymes come under antimicrobial proteins because they are hydrolytic in nature and attack the peptidoglycan layer of bacteria. It specifically cleaves between N-acetylmuramic acid and N-acetylglucosamine of cell wall peptidoglycan (Wohlkönig et al. 2010). The number of reports suggested that lysozyme can kill gram +ve and gram –ve bacteria. T4 lysozyme (T4L), human lysozyme, and Hen egg-white lysozyme (HEWL) are some of the classes of this antimicrobial protein gene, which have been cloned and transformed to different plants. Transgenic tobacco plants expressing these lysozyme genes were found to be more resistant against plant pathogenic bacteria (Trudel et al. 1995; Kato et al. 1998). E. carotovora causes soft rot disease in potato, resistance against this disease has been conferred in potato by expressing T4L gene from T4-bacteriophage (Düring et al. 1993). Fungal and bacterial growth was inhibited in transgenic tobacco expressing human lysozyme gene suggesting its potential use for controlling plant disease (Nakajima et al. 1997). A plant lysozyme was isolated from Momordica charantia L., which can be used further to increase bacterial resistance in plants. Resistance to these diseases could also be achieved by engineering potato with lysozyme gene (chly) from chicken (Serrano et al. 2000), and complete resistance was achieved by expression of the phage T4 lysozyme (Ahrenholtz et al. 2000).

9.10 Herbicide Resistance

Weeds are a major constraint to crop production because they compete for nutrients and other resources with the main crops, posing a serious threat to crops (Fartyal et al. 2018). Herbicide resistance is the most predominant trait that has been adopted for cultivating GM crops. In the early 1990s, GM crops resistant to broad-spectrum herbicides such as glyphosate and glufosinate have first been cultivated commercially. These GM crops are highly valued worldwide and have shown economic, social, and ecological benefits (Green and Castle 2010; Heap and Duke 2018). The herbicide glyphosate is known to inhibit 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS), a key enzyme in the shikimate and phenylpropanoid pathway that is responsible for the biosynthesis of aromatic amino acids and several secondary metabolites. In 1996, the first glyphosate-tolerant soybean (“Roundup Ready”) harboring cp4epsps gene was commercialized and many other commercialized crops harbor this gene. Glyphosate-resistant maize was introduced in the year 1998. Till now, maximum events, i.e. 210 have been approved for maize for herbicide resistance (ISAAA database 2020). Another important herbicide glufosinate also known as phosphinothricin inhibits glutamine synthetase (GS) enzyme that catalyzes the assimilation of ammonia with glutamate to form glutamine. The inhibition of glutamine synthetase leads to the assimilation of ammonia that inhibits photosystem I and II reactions and indirectly inhibits photosynthesis (Wang et al. 2018). Two glufosinate resistance genes bar and pat, were isolated from soil bacteria. Out of these two, pat (phosphinothricin N-acetyltransferase) gene was isolated from Streptomyces viridochromeogenes and bar gene from S. hygroscopicus. These genes encode resistance to phosphinothricin (PAT) and bialaphos (L-Alanyl-L-alanyl-phosphinothricin; Bar). Thus, researchers primarily focused on these two genes in basic research for the development of herbicide-resistant genetically engineered crops. In the past few years, the glyphosate and glufosinate resistant genes (EPSPS and bar) have been introduced into pepper, soybean, maize, millet, potato, and other crops (Zhao et al. 2020). Besides these two above-mentioned herbicides, transgenic crops specific to other herbicide groups such as 2,4-D (aad-1 and aad-12 genes), dicamba (dmo gene), isoxaflutole, mesotrione, oxynil, and sulfonylurea, have been commercialized recently. Currently, different multinational companies such as Monsanto, Dow, Bayer, Syngenta, and BASF are developing new herbicide-resistant traits in different crops. Agrobacterium-mediated gene transfer technology has been used to introduce the EPSPS and bar genes, which confers resistance to glyphosate, and glufosinate into castor (Zhao et al. 2020). Maize, soybean, cotton, and canola are among the most widely grown commercial crops that confer herbicide resistance (Brookes and Barfoot 2015).

9.11 Virus Resistance

Plant viruses have been a major threat to agricultural production and with the advent of transgenic technology, there has been a revolutionary enhancement in the production of GM crops resistant to various plant viruses through various strategies. Significant resistance to a variety of plant viral diseases has been accomplished by gene silencing techniques. Bonfim and co-workers in 2007 explored the concept of using an RNA interference construct to silence the sequence region of the AC1 viral gene and generated highly resistant transgenic common bean plants (Bonfim et al. 2007). This method has also been adopted for the engineering of plum pox virus resistance (Scorza et al. 2013). Exceptional progress has been perceived for transgenic alfalfa, melon, potato, rice, tomato, and tobacco against a broad spectrum of plant viruses, including alfalfa mosaic virus, cucumber mosaic virus, potato virus X (PVX), potato virus Y (PVY), and potato leaf roll virus (Parray et al. 2019). In recent years, non-coding RNAs (ncRNAs) have gained unprecedented attention for regulating cellular processes and engineering plant virus resistance (Taliansky et al. 2021).

9.12 Abiotic Stress Tolerance

Variable photoperiod, nutrient deprivation, starvation, drought, high salt conditions, temperature, and osmotic changes are some of the situations that contribute to abiotic stress (Hirayama & Shinozaki 2010; Zhu 2016). These are of critical importance as studies have estimated the loss caused to reach around 50% by 2050 by this kind of stress situation (Bengtsson et al. 2006; Ahuja et al. 2010; Thakur et al. 2010; Lobell and Gourdji 2012). Furthermore, it is also estimated that the frequency of drought, salinity, and heat will increase in the coming years (Easterling et al. 2000; Bernstein et al. 2008), standing as a challenge to agriculture production. Plant encounters various kinds of abiotic stresses that contribute to a significant crop loss worldwide. All the abiotic stresses are interconnected and include the following.

9.12.1 Salinity

High salt situations in the soil and drought are two main abiotic stresses affecting around 20% of the crop losses in irrigated fields (Qadir et al. 2014). The ions that contribute to high salinity situations are Na+, Ca2+, Mg2+, K+, Cl−, SO42−, HCO3−, CO32−, and NO3 (Flowers and Flowers 2005). The accumulation of either a single type of ions or more than one type of ion may result in a condition that is termed salination. High saline conditions can be caused due to various reasons like mineral weathering, precipitation followed by the movement of salt towards land surfaces are some primary causes of the salinity. High salt has a variable effect on crop plants, though the primary effect includes osmotic stress and toxicity. These primary effects may lead to another secondary effect on the cell-like compromised cell and membrane expansion thereby a compromised metabolism. It is quite difficult to assess which factor contributes to salinity tolerance in crops. It is conceivable that the proper correlation studies between a trait and salinity tolerance could help in easing out the direct relationship between the effects of any salt on crop productivity. In past, many studies have been proposed to characterize the response of any biological molecule, e.g. transcription factors and biomolecules (Negrão et al. 2017) towards any kind of salt stress, however, these studies are still very limited. We propose that engineering the biomolecules to produce stress-resistant crops could be a promising approach because they all play a significant role in the plant’s adaptation to variable salt conditions. Some of the development in major food crops has been described in the coming sections.

9.12.2 Heat

Global warming has led to a significant increase in temperature. It has been estimated that average temperature rise by 3–4 °C has estimated to increase the crop losses to 15–35% in tropical regions like Africa and Asia and by 25–35% in the Middle East (Ortiz et al. 2008). Despite a significant increase in global food production, a food deficit still survives as global cereal production is declining because of increasing temperature (Fischer and Edmeades 2010). The most common effect of high temperature on any crop is an effect on their overall reproductive development due to pollen infertility (Zinn et al. 2010). Heat stress is a major abiotic stress that plant gets exposed in nature and affects every stage of plants life, however, the variability could be seen in the patter they affect the plant that differs from species to species (Sakata and Higashitani 2008). The noticeable effects of the increase of temperature on plants are leaf and stem scorching, abscission, and senescence, inhibition of root and shoot growth, finally causing the fruit damage and results in a decrease in plant productivity (Vollenweider and Günthardt-Goerg 2005). In certain cases, high temperature may also result in changes in plant architecture like elongated hypocotyls and petioles (Hua 2009). Like any other stress, in response to heat stress, plants undergo a series of cellular and metabolic responses that are necessary to survive in these high-temperature situations. This includes changes in a cellular organization like changes in cytoskeleton and membrane functions. These structural changes are also accompanied by production transcription faction producing biomolecules like heat shock proteins (HSPs) (Bray et al. 2000), and the production of biomolecules like phytohormones (abscisic acid; ABA) and antioxidants (Maestri et al. 2002).

9.12.3 Drought

Drought is a situation where the plant does not have a sufficient amount of water which is necessary for the optimum growth of the plant. Various reasons responsible for this kind of stress are low rainfall and compromised irrigation conditions. Drought has a variety of effects on plant growth. The first is a lack of germination and seedling establishment in various crops (Ashraf and Harris 2004; Kaya et al. 2006). It has been reported that in rice, drought during a vegetative state severely compromises overall plant growth (Manickavelu et al. 2006). Drought situations are often associated with compromised photosynthetic activity and finally wilting, thereby compromising plant yield which could be followed by plant death.

9.12.4 Cold

Sensitivity to low temperature is another important abiotic stress that is of critical importance contributing towards the significant yield loss in crops. Cold affects plant life in different aspects, however, an ability to tackle this kind of stress may lead to cell death. Plant encounters temperature fluctuations in the natural environment and thus need a different mechanism to different responses to minimize cellular damages. Response to cold may involve an initiation of a signaling cascade to cause metabolic changes significantly adding to the increased tolerance to chilling temperatures (Chinnusamy et al. 2003). These changes are quite beneficial to plant because these changes ease a plant to cope up with cold stress. These changes/processes induce upon stress are collectively termed as “low temperature-induced signal transduction (LTST).” LTST leads to the upregulation of certain genes that produces specific proteins that help is an adaptation to freezing temperatures. From the above sections, it is clear that any kind of abiotic stress on plants induces a series of adaptive responses in the plant that are quite common in every kind of stress.

9.13 Genetic Engineering for Stress-Tolerant Properties

It is conceivable that the introduction of these alien/novel stress-responsive genes into plants to bring a stress-tolerant property is a promising approach to tackle these stress-sensitive phenotypes (Mittler 2002; Mittler and Blumwald 2010). Therefore, an extensive part of the current genetic research has been dedicated to producing stress-resistant plants by this kind of gene introduction. With the advancement of genetic engineering technology, the cloning and overexpression of stress-resistant genes has become an easy task. The conventional Agrobacterium-mediated gene introduction is the common method for gene introduction into the host plant. Besides, the Agrobacterium-mediated transformation other non-agrobacterium species that have been identified for genetic transformation in plants are Rhizobium sp. NGR234, Sinorhizobium meliloti, and Mesorhizobium loti. Herein, we describe some important genes/proteins that have been identified and modified in plants for their stress-responsive roles. Depending upon the type of response they belong to; these genes can be divided into two major groups; ones involved in cellular protection (osmoprotectants, membrane stabilization, detoxification), transcription factors, and signaling molecules (Vendruscolo et al. 2007).

9.14 Abscisic Acid (ABA) Response Genes

Abscisic acid is one of the most important plant hormones that serves variable functions in the plant. ABA is an important messenger that is involved in different adaptive responses like regulation of accumulation of osmolytes, LEA (Late Embryogenesis Abundant) protein synthesis, and antioxidant enzymes (Chaves et al. 2003; Verslues et al. 2006). ABA levels in plants increase in response to different stressors and result in stomatal closure to minimize the water loss occurring due to the process of transpiration from leaves. Different cellular responses are largely dependent on the ABA levels (Sreenivasulu et al. 2012), thus conceivable that engineering this trait for crop improvement could be a promising approach. ERA1 is one such gene that has been identified in Arabidopsis, and ß-subunit of a farnesyltransferase. It has been shown that plants that do not have this gene are shown to have increased drought tolerance. This has been also shown that downregulation of ERA1 by expressing under a drought inducible promoter through antisense expression of ERA1 in Arabidopsis and canola (Jalakas et al. 2017). Similarly, the mutants for ABA receptors like pyrabactin resistance 1-like 1 (pyl1), pyl4, and pyl6 in rice have been shown to improve plant improvement for drought resistance (Miao et al. 2018). Following a similar strategy, a Canadian company is developing new transgenic plants under the name Yield Protection Technology™ and it has developed transgenic plants for maize, soybean, and cotton since 2011.

9.15 Gene Encoding Compatible Solute

Stress-induced biosynthesis and accumulation of various organic metabolites is the common and most effective defense mechanism plant display in response to any kind of stress. Osmoprotective adaptation to a stressful situation is a widespread response that is conserved in all kinds of living organisms (Saxena et al. 2013). These solutes also act as scavengers for free radicals and stabilize the plant proteins during stress (Nahar et al. 2016). These compounds have low molecular weight and do not inhibit normal cellular functions and are termed as compatible osmolytes. These chemicals are fundamental to all organisms from bacteria to plants and can be characterized into different categories depending on their biochemical nature (Khan et al. 2009; Jewell et al. 2010) (Table 9.1). The majority of these proteins are hydrophilic and uncharged in nature and function in a vast variety of functions including scavenging of the ROS (reactive oxygen species), as osmoprotectants, pH stabilizers, proteins, enzymes, and membrane. The first report of this type of gene to plant lies in the early 90s, where the introduction of these osmolytes has shown to confer the cold and salt resistance properties to the host plants. Glycine betaine, β-alanine, proline, and mannitol are some common compatible solutes that are conventionally used for metabolic engineering for stress-resistant properties. However, glycine betaine is regularly used for this purpose in different crops. Various transgenic plants that have been produced by this kind of gene introduction are listed in Table 9.2.

Table 9.1 Categories of plant protectants based upon their biochemical nature
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Table 9.2 Recent examples of the plant development through gene modification in agriculturally important crops
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9.16 Free Radicle Scavengers

Exposure to any kind of stress situation finally leads to the production of reactive oxygen species, production of which negatively affects different processes like enzyme and biochemical activities and thus affects the biosynthesis of DNA, protein and carbohydrates, thus exceeding the oxidative stress in a cell. The exposure of plants to these environmental stresses leads to reactive oxygen species (ROS) (Verslues et al. 2006; Jewell et al. 2010). Production of the free radicle upon stress situation is another phenomenon of the stress response that is seen in plants upon waterlogging, drought, high salinity, and high temperatures. It is evident that ROS influences the expression of different genes that influence growth, cell cycle, response to pathogens, plant development, and even cell death (Gill and Tuteja 2010). Thus, modification and expression of the gene helping in scavenging these reactive oxygen species could be an alternative approach to produce stress-tolerant plants. Some of these genes that have already been shown to be successfully introduced in plants for imparting stress-tolerant properties are; ascorbate peroxidase, superoxide dismutase, and glutathione reductase.

9.17 Genetic Engineering of the LEA Proteins Coding Genes

The late embryogenesis abundant (LEA) protein is a stress-induced protein produced in vegetative tissues of the plants. Although the exact functions of these stress-induced proteins are not known, it has been known that these proteins are water-binding molecules that are dehydration and cold-responsive. These are the proteins that help in protecting the desiccation and protect the seed development during salt stress, dehydration, and cold. These proteins are encoded by different genes in different plant species, for instance, responsive to dehydration (RD), early responsive to dehydration (ERD), inducible to cold (KIN), regulated by cold (COR), and responsive to abscisic acid (RAB). Two members of this class of proteins are HVA1 (protein from barley) and LE25 (protein from tomato). The reports have shown that the introduction of the LEA gene from barley into rice imparts tolerance to salinity and water stress (Xu et al. 1996). A similar observation was made in yeast transformed with LE24 from tomato (Imai et al. 1996). Similarly, expression of the HVA gene in wheat has also been reported to improve the quality of plants to grow in water-deficient situations with an ability to produce high biomass. A recent example of this kind of gene introduction for stress resistance was achieved by expressing Melon Y3SK2-Type LEA gene in tobacco. A recent study has shown that the introduction of this gene increases resistance to drought and salt (Aduse Poku et al. 2020).

9.18 Molecular Chaperones

Heat shock proteins (HSPs) are the to-date known molecular chaperons that help incorrect folding of the proteins. Besides these, conventions protein folding molecules other protein folding molecules that have been extensively studied are known as peptidyl-prolyl-isomerases. It has been reported that HSP is produced in response to rapid heat stress. Besides HSP’s are also known to get produced under different stages of plant development like embryogenesis, seed germination, development of pollen, and fruit maturation (Prasinos et al. 2005). The modification of these molecules for developing stress-tolerant plants lies early in 1987, when the transgenic tobacco expressing HSP17 were produced, though no conclusion was made on the stress-tolerant property as a promoter was incompetent (Schöffl et al. 1987).

9.19 Proteins for Ion Homeostasis Across the Membrane

A high salt situation causes osmotic stress in the cell and leads to an increase in the high salt situations in the cytoplasm. To counter this, plant has to develop a mechanism where a plant can utilize these ions to minimize the adverse effects of these ions in the cell. This is regulated by genes that regulate the ion channels like Na+/H+ antiports and stress signaling through calcium- and calmodulin-dependent protein phosphatase calcineurin. The first successful example of this kind of gene introduction was achieved by overexpressing a single endogenous gene (AtNHX1) encoding a vacuolar Na+/H+ antiport protein in Arabidopsis. The transgenic Arabidopsis was found to be thriving well in the high salt situation, i.e. 200 mM sodium chloride, which correlated well with higher levels of the AtNHX1 transcripts and protein and vacuolar Na+/H+ antiport activity. Similarly, expressing catalytic and regulatory subunit of yeast calcineurin in tobacco has also been shown to generate transgenic tobacco with salt-tolerant properties.

9.20 Transcription Factors

A number of genes that are activated upon abiotic stress are controlled by a complex network of transcription factors (Yamaguchi-Shinozaki and Shinozaki 2006). The stress-responsive genes can generally be classified into two groups; regulatory and functional (Shinozaki et al. 2003). Functional proteins include different enzymes, membrane proteins (water channel and transporters), heat shock proteins, all these proteins have a direct role in stress response. Regulatory proteins include different transcription factors, kinases, and phosphatases that regulate various stress responses. Furthermore, a response to any kind of stress on the plant is multigenic in nature, involving the role of two or more genes in a stress response mechanism. Thus, the introduction of only a single gene is not sufficient to induce a series of changes that may be necessary for a specific stress adaptation. Thus, engineering the different transcription factors involved in controlling specific traits together may be a promising alternative approach to achieve a specific stress-tolerant trait. Some successful initial examples of this kind of gene modification have been achieved by the overexpression of the HSPs have known to confer thermotolerance in Arabidopsis (Lee et al. 1995). Similarly, overexpression of the CBF1 (“C-repeat binding factor”), a transcriptional activator has been shown to confer tolerance to freezing in Arabidopsis inducing the expression of four COR (“cold-regulated”) genes (Jaglo-Ottosen 1998). Another transcription factor is the dehydration-responsive Element (DREB1A), this TF is also known to upregulate different stress-responsive genes. Both DRE and CBF factors are known as a cis-acting element that regulates the gene expression in response to variable dehydrating stressors (salt, cold, and drought) (van Rensburg and Krüger 1994). A member of the DRE family, DREB1A, is reported to impart drought resistance in Arabidopsis thaliana via inducing the expression of different stress-responsive genes (Pellegrineschi et al. 2003). Another family of a transcription factor that is involved in stress tolerance is the NAC gene family members. These transcription factors are known to get expressed in different stages of growth and response to the environment. SNAC1 is a member of this family, expression of which is known to improve drought resistance in rice (Hu et al. 2006). Other transcription factors like bHLH, bZIP, NAC, AP2/ERF, MYB, Zinc finger, WRKY, and kinases are associated with increase crop yield in rice (Dubouzet et al. 2003; Hu et al. 2006; Hossain et al. 2010).

9.21 Recent Advances in Plant Improvement for Abiotic Stress Tolerance

In the above section, we have successfully described the traditional transgenic methods to crop improvement. The current section focuses on the recent advancement that has been made in crop development programs to improve plant for the desired traits.

9.22 Development of Abiotic Stress-Tolerant Crops by miRNA

MicroRNA (miRNA) can be described as the single-stranded RNAs that are approximately 21–24 base pairs in length (Zhao et al. 2011). Studies have shown an involvement of these microRNAs in imparting stress resistance to variable stress on plants (Xia et al. 2012). These miRNAs inhibit the expression of target RNA by binding to the 3′ end of the RNA, thus inhibiting the translation (Meng et al. 2010; Li et al. 2011; Ding et al. 2011). These miRNAs are involved in different cellular processes like transcription, protein stability, and degradation (Shen et al. 2010; Ding et al. 2011). Several studies have shown that 11 miRNAs exert tissue-specific expression towards major abiotic stresses in Arabidopsis thaliana. According to the reports in Arabidopsis thaliana, miRNA-169 has been shown to contribute towards drought resistance. Similarly, other miRNA’s like 159, 396, and 393 also showed to contribute to other abiotic stressors (salinity, cold, and heat) (Table 9.3).

Table 9.3 Recent examples of the plant development through miRNA technology
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9.23 Development of Abiotic Stress-Tolerant Crops by CRISPR (Lustered Regularly Interspaced Short Palindromic Repeats)/Cas9

CRISPR-Cas9 is a recent development developed for crop improvement in recent years. This system depends on an RNA-DNA recognition system that employs a double-strand break in the host genome. The technology has an upper hand over the other crop improvement methods as this is a comparably fast, and efficient gene-editing method for crop improvement (Mao et al. 2013). Besides, it is preferred over the other gene-editing tools as this is simple in designing and efficiently introduces mutation with a targeted introduction at desired locations (Ma et al. 2015; Malzahn et al. 2017). The technique is preferred over other genetic improvement techniques as this is comparable to less tricky and avoids the tedious screening of the desired clone. Besides mutation, this technique can also be used to induce or repress the expression of the particular gene using modified CRISPR where an inactive form of Cas9 (dCas9) is fused with a transcriptional activator or a repressor (Bortesi and Fischer 2015). Due to its vast potential in genome improvement, it has the potential to replace other gene improvement methods. While the system is employed for the improvement of animal cells (Gilbert et al. 2013; La Russa and Qi 2015); the system has limited reports of successful use of this method in plant improvement (Piatek et al. 2015). Some recent examples of CRISPR-Cas mediated plant improvement are summarized in Table 9.4.

Table 9.4 Some recent examples of crop improvement via employing CRISPR/Cas9 system
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9.24 Conclusion

Overall, we have summarized the major biotic and abiotic stress mechanisms that can be engineered for imparting stress-resistant properties to different plants. Though, a huge effort has been already made in this area, there is a considerable challenge that remains to be addressed. As the plant undergoes multiple combinations of stress in field conditions, this area needs further addressing. We propose that the plant’s response to multiple different stressors cannot be inferred from assessing the plant’s response to individual stress. Thus, it is essential to test different improved varieties to multiple stressors in field conditions where all stressors occur at once. Another challenge to getting these improved varieties to farmers is their development expenses and the approvals these GM plants require for their field trials. As multiple precautions are already in the guideline to ensure the safety associated with the GM crops, precautions to ensure this safety should not become a barrier for future crop development programs.