Transgenic Technology Can Accelerate Cotton Breeding: Transgenic *ScALDH21* Cotton Significantly Improve Drought Tolerance in Southern and Northern Xinjiang

*Honglan Yang, Dawei Zhang, Tohir A. Bozorov, Abdul Waheed, Jiancheng Wang, Xiaoshuang Li and Zhang Daoyuan*

## **Abstract**

Aldehyde dehydrogenases (ALDHs) contribute to cellular protection against oxidative stress. These enzymes are crucial to organisms' ability to cope with environmental stress. The ALDH21 gene was introduced into upland cotton (*Gossypium hirsutum* L.) from desiccant-tolerant *Syntrichia caninervis* moss, created stable genetic transgenic lines. As a result, drought tolerance is increased and yield penalty is reduced in those transgenic lines. The first study to demonstrate overexpression of *ALDH21* enhances drought tolerance in cotton under multi-location field experiments is presented here. Cotton genotypes containing *ScALDH21* exhibit significant morphological, physiological, and economic benefits. *ScALDH21* functions in the physiology of cotton plants to protect them by scavenging ROS and reducing osmotic stress. The yield of transgenic cotton in northern Xinjiang showed up to 10% improvement under full irrigation and up to 18% improvement in deficit irrigation conditions on fields with purple clay loam soils. Additionally, transgenic cotton can be grown in sandy loam soil in southern Xinjiang with an average yield increase of 40% on different irrigation levels in the desert-oasis ecotone. Using *ScALDH21* as a candidate gene for cotton improvement in arid and semi-arid regions was demonstrated. In addition, we assessed different irrigation protocols and optimized irrigation methods with minimal water requirements for *ScALDH21*-transgenic cotton that could be used in production agriculture.

**Keywords:** transgenic cotton, molecular breeding, *ScALDH21*, drought tolerance, yield improved

## **1. Introduction**

Plants are restricted in their habitat range and productivity by adverse environmental conditions [1]. Drought is the foremost constraint on agricultural production. Cotton (Gossypium spp.) is a major source of textile fibers and oil around the world. More than 32 million ha of cotton are produced in 76 countries [2]. In terms of cotton production, China is ranked among the top two countries in the world [3]. However, cotton production in China, as well as other countries, has recently declined due to increasing drier environments [3, 4]. Chinese agriculture consumes 62% of the country's annual water consumption, and the country is in a moderate water shortage [4]. In agriculture, cotton is the crop with the highest water consumption. In China, cotton is grown mainly in the Xinjiang-Uygur Autonomous Region, an area characterized by very low air humidity and a severe water shortage.

Cotton is the most important crop in China, accounting for around 25% of global fiber production. There is more than one-third of all agricultural land in the Xinjiang-Uyghur Autonomous Region dedicated to cotton plantations [5]. This region has a warm climate with average temperatures of 11.4°C and 49 mm precipitation annually, low groundwater levels, sandy soils, and severe soil salinization [6–9]. In southern Xinjiang, cotton has low germination rates, low survival rates, and low yields [10].

Plants are able to generate significant amounts of reactive aldehydes when faced with a variety of abiotic stresses (such as salinity, desiccation, and cold) [11], which can impair plant growth and crop productivity. Cotton varieties that survive droughts and other adversities must be developed urgently to combat these conditions. In arid lands where freshwater scarcity is a severe constraint on agricultural production, it is necessary to develop more tolerant varieties of plants. It is often difficult to obtain drought-tolerant crops through traditional breeding programs because of the time and labor involved, in addition to the need for large-scale facilities, such as rainout shelters. Interestingly, biotechnological improvements have been attempted since the 1990s, which is inspiring. However, the majority of transgenic cotton is aimed at controlling insect pest damage by expressing a variety of insecticidal proteins from *Bacillus thuringiensis* (i.e., Bt cotton). In transgenic plants, a number of genes have been discovered and studied that have a high potential for improving drought resistance, and some of these genes have shown promise for crop improvement [12–16]. However, only a few of these genes have been successfully deployed in fields of agriculture [17–20].

Plants that are known as bryophytes (mosses, hornworts, liverworts, etc.) are among the oldest species in the world's flora; they are thought to be small, nonvascular, and green plants. Many bryophytes survive even with a total loss of water in their vegetative tissues [21, 22]. The study of drought-tolerant mosses is of particular interest because their genetic engineering properties can be used to increase drought tolerance in arid-zone crops. The desiccation-tolerant moss *Syntrichia caninervis* is distributed in the Gurbantunggut desert in western China and can survive almost complete water loss and recover within 30 seconds after rehydrating [23]. Thus, *S. caninervis* may be a natural gene base for desiccation tolerance (**Figure 1**).

Aldehyde dehydrogenase (ALDH) genes show promise as candidate genes to increase plant resistance, especially *ALDH21* gene from moss *S. caninervis,* which is not found in seed plants. In a desert-oasis ecotone, non-transgenic cotton has an advantage over plants that overexpress *ALDH21* from desiccant tolerant moss, even under different irrigation practices. In addition, we are seeking the best irrigation scheme to reduce the consumption of irrigating water and increase crop production *Transgenic Technology Can Accelerate Cotton Breeding: Transgenic* ScALDH21 *Cotton… DOI: http://dx.doi.org/10.5772/intechopen.103833*

**Figure 1.**

*The natural habitat of* S. caninervis *distributed in the Gurbantunggut desert of western China. Different morphology of moss: dried state and recovered state after applied water.*

in desert areas. Zhu et al. reported that different cotton planting lands gave different yields for some cotton lines [17]. To evaluate the efficacy of transgenes in cotton, irrigation strategy has a crucial role to play, especially in climate-dependent arid regions.

## **2. Cotton drought tolerance breeding with transgene technology**

## **2.1 The overexpression gene types in current drought-tolerant cotton**

It is possible to improve cotton drought tolerance using transgenic technology. The drought tolerance of transgenic cotton has recently been enhanced by using several genes (**Table 1**). As an example, AtLOS5, encoding an aldehyde oxidase cofactor sulfurase; GhAnn1, an annexin gene; isopentenyl transferase (IPT), an enzyme responsible for cytokinin biosynthesis; and 14-3-3 genes involved in plasma membrane H+ -ATPase activity [17, 24–27]. Increased drought tolerance was also observed in transgenic cotton overexpressing the OsSIZ1 gene from *Oryza sativa*, which encodes a SUMO E3 protein [36]. Researchers have used several transcription factor genes as transgenes in cotton to increase drought tolerance, including AtEDT1/ HDG11 (homeodomain-START transcription factor), GhABF2 (bZIP transcription factor), NAC (a transcription factor) in rice (O. sativa L.), and AtRAV (for ABA insensitive3/viviparous1) in cotton [28–32]. It has been demonstrated that the expression of the vacuolar proton-pumping pyrophosphatase gene (AVP1) from Arabidopsis in cotton results in an increase in fiber yield of 20% compared to non-transgenic cotton [33, 34]. Drought tolerance is further improved by co-overexpression of AVP1 and AtNHX1 in cotton [35]. Nonetheless, only AtEDT1/HDG11, transgenic IPT, and

*Cotton*


#### **Table 1.**

*Overexpression of various genes in cotton that reported to enhance drought tolerance.*

transgenic AtAVP1 cotton showed a simultaneous increase in drought tolerance as well as cotton or fiber yield.

## **2.2 Transgenic** *ScALDH21* **cotton significantly improve drought tolerance in southern and northern Xinjiang**

A number of fiber quality parameters and yield were improved with cotton *ScALDH21*. Planting transgenic *ScALDH21* cotton lines on purple clay loam soils in northern Xinjiang, field experiments demonstrated an increase in yields of 10.0% under full irrigation and >18.0% under deficit irrigation conditions (**Figure 2**). Compared to the non-transgenic cotton variety "Xin Nong Mian 1," the transgenic cotton showed an average yield increase of at least 40% grown on sandy loam soil in southern Xinjiang. Compared to the recipient cultivar "Xin Nong Mian 1,"

*Transgenic Technology Can Accelerate Cotton Breeding: Transgenic* ScALDH21 *Cotton… DOI: http://dx.doi.org/10.5772/intechopen.103833*

#### **Figure 2.**

*The experiment sites location and experiment design. (a) Site of Xinjiang in the world map; (b) experiment site in Xinjiang map; (c) experiment design in the north of Xinjiang (44<sup>o</sup> 18*′*13.91*″ *N, 86<sup>o</sup> 13*′*11.03*″ *E, average temperature 18.8°C, average annual rainfall 194.8 mm); (d) experiment design in south of Xinjiang (80o 43*′*45*″ *E, 37o 00*′*57*″ *N, frost-free period 210 days, average temperature 11.9°C, rainfall < 50 mm, evaporation > 2600 mm).*

*ScALDH21*-cotton had substantially improved performance under deficit irrigation, ensuring a more sustainable cotton production in the desert-oasis ecotone [37].

A variety of irrigation protocols were evaluated and optimized to use *ScALDH21* cotton genotypes in production agriculture with minimum water requirements. The following paragraphs describe the characteristics of transgenic *ScALDH21* cotton.

#### *2.2.1 The aldehyde dehydrogenase (ALDH) enzyme superfamily and its functions*

As ROS are generated, oxidative stress is induced, lipid membranes are destroyed, and 200 types of aldehydes are accumulated, many of which are highly reactive and toxic. Aldehydes must be effectively removed and detoxified in arid environments to improve plant productivity. Plants have developed many enzymatic and non-enzymatic mechanisms to scavenge these toxic compounds [24]. Aldehyde dehydrogenase (ALDH) superfamily proteins may also play a role in scavenging ROS enzymatically [43]. Aldehyde dehydrogenases (ALDHs) have been found to play a central role in plants exposed to stressful conditions in the detoxification of aldehyde [44]. This superfamily of enzymes metabolizes endogenous and exogenous aldehydes to their carboxylic acids by using the coenzyme NAD(P)<sup>+</sup> , producing NAD(P)H and thereby reducing oxidative/electrophilic stress [45]. ALDHs belong to a group of NAD(P)<sup>+</sup>  dependent enzymes. Based on sequence similarity, the ALDH gene superfamily has been classified into 24 protein families by the ALDH Gene Nomenclature Committee (AGNC) [46]. There are 14 ALDH enzyme families in plants. Two of them (ALDH21 and ALDH23) are unique to bryophytes, and the rest (ALDH10, ALDH11, ALDH12, ALDH19, ALDH21, ALDH22, ALDH23, and ALDH24) are unique to higher plants [11, 47, 48]. ALDH21A1 plays a crucial role in the detoxification of aldehydes generated by desiccation stress, and it is proposed that ALDH21A1 expression is a unique stress resistance mechanism. Two classes of resistance pathways have been linked

to the ALDHs superfamily as abiotic or biotic resistance genes. An ALDH acts as an 'aldehyde scavenger' [49]. The increased activity of the Arabidopsis aldehyde dehydrogenase Ath-ALDH3 and soybean ALDH7 was reported to act as a detoxification mechanism that limits the accumulation of aldehyde and oxidative stress in Arabidopsis [1, 50]. In addition, metabolized ALDH products are directly involved in maintaining cellular osmotic homeostasis by catalyzing the synthesis of osmolytes [51, 52]. POD activity was the primary reason for the reduced peroxide levels in transgenic BADH tomatoes compared with SOD, APX, and CAT [52]. ALDH21 confers tolerance to osmotic and oxidative stress in cotton, according to our data. Under deficit stress, *ScALDH21*-cotton showed lower MDA production, increased POD activity, and higher proline and soluble sugar levels than non-transgenic cotton. This indicates that the *ScALDH21* gene may play a significant role in drought tolerance. Several ALDHs participate in drought-tolerant pathways in plants [53–55]. The transcriptional level is believed to be the mechanism by which ALDHs mediate environmental stress. An et al. [56] reported that the treatment of maize plants with NaCl and mannitol increased levels of *ZmALDH7B6* mRNA transcripts. Results of quantitative real-time PCR revealed that osmotic and H2O2 stress increased the expression of the *SiALDH7B1*, *SiALDH12A1,* and *SiALDH18B2* genes of *foxtail millet* (*Setaria italica*) [57]. ALDH overexpression has been shown to positively mitigate environmental stress. In contrast to non-transgenic plants, transgenic Arabidopsis *AtALDH2B8*, *AtALDH3I1*, *AtALDH7B4,* and *SpBADH* were able to survive on media containing high levels of H2O2. Moreover, ROS content in detached leaves of ALDH plants was significantly lower than that of WT [58, 59]. Plants overexpressing *ZmALDH22A1* show increased stress tolerance [60]. *SpALDH10* encodes the drought-inducible betaine aldehyde dehydrogenase (BADH) that catalyzes the oxidation of betaine aldehyde to the compatible solute glycine betaine, resulting in enhanced drought and salinity tolerance in potato plants [61]. ALDHs appear to play an important role in cell metabolism and stress physiology, according to these results. Recently, cotton transformed with *ALDH* genes has been reported to be tolerant to drought and salinity [62]. Transgenic cotton harboring the betA gene (part of the ALDH10 family) improved salt tolerance and cotton yield [62]. However, in only a few cases has a member of *ALDH* been reported as performing a specific function. *ALDH21A1* has previously been identified as a novel eukaryotic aldehyde dehydrogenase that is transcriptionally activated by abiotic stress [11]. Recombinant *Escherichia coli* expressing *ScALDH21* showed higher drought tolerance than control *E. coli* [23]. Compared with the control, tobacco overexpressing *ScALDH21* was more droughttolerant [63]. Abiotic stress tolerance has been demonstrated in transgenic cotton using *ScALDH21* [37, 38, 64].

### *2.2.2 The genetic background of transgenic ScALDH21 cotton*

#### *2.2.2.1 The plant expression vector and the plant transformation*

To make *ScALDH21* expression under CaMV 35S promoter, the open reading frame of *ScALDH21* cDNA (GQ245973) was amplified and cloned into the *Sal* I and *Kpn* I sites of the pCAMBIA2300. A recombinant vector containing selective neomycin phosphotransferase (NPTII) gene was transformed into *Agrobacterium tumefaciens* strain EHA105. Xin Nong Mian 1 (*Gossypium hirsutum*) has been transformed through Agrobacterium-mediated transformation. The Economic Crop Research Institute,

Xinjiang Academy of Agricultural Sciences, China, developed this cotton variety for specific arid zones in Xinjian. It displays good agronomic traits and economic characteristics.

## *2.2.2.2 PCR, RT-PCR detection, and Southern blot analysis*

Using the cetyltrimethylammonium bromide method, genomic DNA was isolated from cotton seedlings at the five-leaf stage. PCR was used to detect the *ScALDH21* transgene in cotton plants using gene-specific primers [38]. The PCR amplification conditions were as follows—initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 s, primer annealing at 60°C for 30 s, and elongation at 72°C for 90 s, with a final elongation at 72°C for 5 min. Electrophoresis of 1% (w/v) agarose gels was used to visualize the PCR products. DNA extracted from cotton seedlings of the T5 generation was digested, run through gel electrophoresis, and transferred to a positively charged nylon membrane (Amersham, USA). Hybridization and chemiluminescence detection were performed according to the manufacturer's protocol (Roche, Germany) using digoxygenin dUTP-labeled probes of the *ScALDH21* gene product. Total RNA was extracted from young cotton leaves to analyze transgene expression. The genomic DNA was removed from the total RNA using DNase I (TaKaRa, Dalian, China). DNA was codified with the cDNA synthesis kit (TaKaRa, Dalian, China) according to the manufacturer's instructions. *ScALDH21* cDNA was used as a positive control for qPCR detection [38]. As an internal control, the *UBQ7* gene (GenBank accession no. DQ116441) was amplified with specific primers [24].

### *2.2.2.3 The transcriptome background of transgenic ScALDH21 cotton*

We collected root samples from cotton seedlings after 1month of growth. Three biological replicates of each treatment were carried out. The total RNA was extracted using the RNAprep Pure Plant Kit (Tiangen, Beijing, China) following the manufacturer's instructions. In accordance with the manufacturer's instructions, sequencing libraries were prepared using the NEB Next Ultra RNA Library Prep Kit for Illumina (NEB, Beverly, CA, USA). Illumina HiSeq 4000 platform was used for sequencing with 150 bp paired-end reads. Based on the length of the gene and the number of reads mapped to the gene (Novogene company, China), the expected fragments per kilobase of genes per million mapped reads (FPKM) of each gene were calculated. Differentially expressed genes were defined using the DESeq R package with an adjusted P-value (q-value) of 0.05. We used the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp/kegg/) to test the statistical enrichment of DEGs in KEGG pathways. Five hundred and seventy-eight co-expressed genes were detected in the two *ScALDH21* transgenic lines, which were differentially expressed from NT and indicate that the target gene *ScALDH21* affected gene expression (**Figure 3a**). In **Figure 3b**, transcription expression patterns for those genes are shown. On the basis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG), 578 genes were identified as overlapping across two transgenic lines compared to NT (**Figure 3c** and **d**). GO shows that ADP binding, O-methyltransferase activity, sulfotransferase activity, and transferase activity are significantly different from those of NT (**Figure 3c**). KEGG annotation shows no significant differences compared with that of NT (**Figure 3d**). Photosynthesis-antenna proteins, phenylpropanoid biosynthesis, and plant-pathogen interactions are the top

#### **Figure 3.**

*The analysis of the difference between two ScALDH21 transgenic lines and non-transgenic cotton based on transcriptomic data. (a) Show the different expression gene numbers in two transgenic lines compared with that in NT in Venn graphs (log2 foldchange > 2, padj < 0.05). (b) Gene Ontology (GO) annotation of the 578 overlap genes in two transgenic lines after compared with NT separately. (c) Kyoto Encyclopedia of Genes and Genomes (KEGG) of annotation the 578 overlap genes in two transgenic lines after being compared with NT separately. NT, non-transgenic cotton; LI and LII are the two ScALDH21 transgenic lines.*

three different pathways. These differences may have contributed to the different biofunctions and phenotypes.

#### *2.2.3 The phenotype of ScALDH21-transgenic cotton*

Following the identification of *ScALDH21* transgenic cotton, the growth performance of non-transgenic cotton (NT) and transgenic cotton (TC) under drought stress was examined and compared in northern Xinjiang from 2011 to 2014 and in southern Xinjiang from 2016 to 2018. Plants of all three independently transformed *ScALDH21* transgenic lines grew significantly taller than NT recipient plants in both full irrigation (26% higher) and deficit irrigation (23% higher). Transgenic and non-transgenic lines did not differ in leaf shape (length/width ratio) in either condition. However, the *ScALDH21* cotton had a greater leaf area compared to the NT plants in both full irrigation (79% increased) and deficit irrigation (51% increased). A significant difference was not observed between transgenic and NT plants in the full irrigated group. However, 24% more branches and 32% more bolls were observed in the deficit stress group. In general, the results showed that transgenic plants outperformed NT in morphological features like plant height, leaf area, leaf number, stem diameter, and root length. Similarly, morpho-physiological traits like fresh weight and dry weight of transgenic plants were greater than those of recipient plants. In transgenic plants, drought stress triggered lateral roots and increased leaf area significantly [38, 64]. Under drought stress conditions, *ScALDH21* overexpression appeared to

### *Transgenic Technology Can Accelerate Cotton Breeding: Transgenic* ScALDH21 *Cotton… DOI: http://dx.doi.org/10.5772/intechopen.103833*

enhance plant growth in TC. Thus, overexpression of *ScALDH21* in cotton significantly increased the number of lateral roots, which consequently accelerated leaf growth following drought stress compared to NT. The height, leaf area, and leaf color of the transgenic *ScALDH21* cotton were all enhanced under normal and stress conditions in addition to the root system. The phenotypic results were consistent with the performance of other transgenic cotton [25, 31]. Under normal and drought conditions, overexpression of the rice NAC gene improves the root system [31]. In cotton, overexpressing a vacuolar pyrophosphatase gene increased root length and lateral root number, which improved the plant's water-absorbing abilities [33, 65]. After a 90-day water deficit, expression of the isopentenyl transferase gene IPT in cotton led to increased cotton height and roots. Overexpression of the Arabidopsis 14-3-3 protein gene GF14 in cotton results in a "stay-green" phenotype [27]. A potato sucrose synthase gene ectopically expressed in cotton accelerates leaf expansion and vegetative growth [66]. Additionally to demonstrating drought-tolerant phenotypic characteristics in *ScALDH21* transgenic cotton, our data also explain cotton's stress memory in terms of phenotype and physiology with two continuous water retention experiments that showed double water deficiency was worse than single water deficiency [38].

### *2.2.4 The physiological character of ScALDH21-transgenic cotton*

Various biotic and abiotic stresses trigger ROS accumulation in plant cells, which leads to oxidative stress with lipid peroxidation, which also causes free radical reactions involving membrane polyunsaturated fatty acids [67]. ROS are produced when toxic aldehydes accumulate from lipid peroxidation [68]. Aldehyde-detoxifying enzymes ALDH3I1 and ALDH7B4 are both significant ROS scavengers and proteins that inhibit lipid peroxidation in Arabidopsis. In Arabidopsis, overexpression of these genes reduced lipid peroxidation under drought and salt stress [60].

It remains to be seen whether overexpression of *ScALDH21* improves overall plant health and performance under deficit irrigation. As a result of the toxicity of ROS produced by environmental stress, plants experience reduced growth, delayed development, and decreased yield. The *ScALDH21* cotton lines in this study responded to deficit irrigation with an increase in POD activity. This is consistent with previous reports [69], and therefore, it is reasonable to assume that they are better equipped to negate drought-induced ROS production. The transgenic cotton plants also showed decreased levels of MDA, an indicator of peroxidation [70] that indicates an improved ability to combat oxidative stress. Additionally, the *ScALDH21* cotton lines exhibit a more pronounced proline accumulation response to deficit irrigation, which is a well-distributed, multifunctional osmolyte that aids osmotic stress tolerance. *ScALDH21* overexpression reduced ROS-induced membrane peroxidation (lower MDA), increased ROS protection (elevated POD activity), and increased proline levels. It is likely that *ScALDH21* maintains a more intact cell system to counteract the negative effects of water deficiency. The level of proline and soluble sugars in transgenic cotton was increased by overexpression of *ScALDH21*. After one or two continuous water-withholding treatments, we measured the levels of free proline and soluble sugar in the leaves of seedlings and flowers [38]. It was found that proline accumulation increased after two continuous water withholdings, compared with other treatments. As a result of all drought treatments, NT and TC accumulate more soluble sugar, but TC accumulates more.

*ScALDH21* cotton lines are able to maintain photosynthetic homeostasis and chlorophyll levels despite drought-induced oxidative stress. Southern Xinjiang

experienced an increase in SPAD values in either 2017 or 2018 [37]. Furthermore, the photosynthetic characteristics of plants under drought stress conditions were investigated at different stages of development (germination and flowering). As a result of full irrigation, the TC plants had a greater photosynthetic rate than the NT plants. The net photosynthetic rate of both TC and NT cotton was significantly reduced under drought stress, however, the TC cotton still maintained a significantly higher rate than NT cotton. The TC showed higher stomatal conductance and transpiration rates than the NT cotton [38]. Those can provide insight into the possible reason for the increase in biomass in *ScALDH21*-cotton plants.

#### *2.2.5 The yield and fiber performance of ScALDH21-transgenic cotton*

From 2013 to 2018, transgenic lines of cotton were grown in northern and southern Xinjiang to determine whether the *ScALDH21*-cotton was effective in the field.

TC lines in northern Xinjiang have been found to be better in growth and development than NT lines to some extent, after applying six different water retention treatments at different growth stages. Water deficit stress during the bud stage will cause the plant stalk length and boll number to decrease. Cotton yields were significantly decreased if twice deficit stresses were met during the bud or flower stage. Cotton growth and yield are critically dependent on water availability during the bud stage. Fiber parameters such as fiber strength, ginning out-turn of the fibers, fiber length, and length uniformity of the *ScALDH21*-cotton lines were not significantly different from NT plants. Comparatively to NT plants, TC lines produced stronger, more uniform, and longer fibers. TC had a micronaire value similar to or slightly lower than NT [38].

During harvesting season, boll weight, seed index, cotton yield, and fiber yield were measured in managed treatment plots under full and deficit irrigation conditions in northern Xinjiang in 2014 and 2016. Under drought stress, both *ScALDH21* transgenic cotton lines and NT lines had greatly reduced boll weight. Compared with NT plants, the seed index was higher in transgenic lines under both full and deficit irrigation conditions, and it reached up to 22% under stress. In both conditions, cotton yield per hectare and fiber yield did not differ significantly [64]. Under both full and deficit irrigation conditions in both managed treatment plots and production fields, the fiber length, uniformity, strength, elongation, and micronaire value of *ScALDH21* transgenic lines were improved or significantly improved compared with NT.

To determine the performance of the *ScALDH21* transgenic cotton under oasis field conditions in southern Xinjiang, three kinds of irrigation schedules were used (root zone model-simulated forecast irrigation (F), soil moisture sensor-based irrigation (S), and flood irrigation based on experience estimates (E) and two full (FI) and deficit (DI) irrigation conditions were employed [37]. Under all deficit irrigation conditions, the number of bolls and cotton yield of TC plants decreased compared to full irrigation, however, they were higher than those of NT plants. Over 3 years of experiments, TC plants showed a significant increase in cotton yields of up to 58.7% compared to NT plants [37]. Furthermore, in soil moisture sensor-based deficit irrigation (SDI) treatment, cotton yields are the lowest. In years, the cotton yield of *ScALDH21*-cotton lines grown under forecasted full irrigation (FFI) increased from 37–73% compared to NT plants. NT and Forecasted deficit irrigation (FDI), Soil moisture sensor-based full irrigation (SFI), SDI, Experience-based full irrigation (EFI), and Experience-based deficit irrigation (EDI) differed for all 3 years.

#### *Transgenic Technology Can Accelerate Cotton Breeding: Transgenic* ScALDH21 *Cotton… DOI: http://dx.doi.org/10.5772/intechopen.103833*

Furthermore, yield increases of transgenic lines were highest in SDI (from 67.5% to 92.3% in 3 years), compared to NT plants. Average data for 3 years in the SDI showed a large increase in cotton yield with a smaller deviation (83.5%).

In addition to treatments, cotton yields vary by year. As with the boll number per plant, the cotton yield per hectare, fiber yield per hectare, and cotton yield per plant were significantly higher in the transgenic lines than in the NT lines. The average seed yield for all treatments was 68% (variable from 14–128%) and 41% (variable from 10–102%) in 2017 and 2018, respectively [37]. Fiber elongation was increased in transgenic lines. Fiber strength also increased in transgenics after irrigation. There were no significant differences in fiber uniformity and micronaires between genotypes.

#### *2.2.6 The irrigation strategy of ScALDH21-transgenic cotton*

Cotton productivity and yield are largely influenced by a variety of factors, including genetics and irrigation methods. In this study, the TC lines performed better than the NT lines, despite soil, air humidity, and temperature affecting plant yield [71–73]. This study evaluated the drought tolerance ability of *ScALDH21*-cotton lines at field stations located in southern Xinjiang, China, a region classified as a desert-oasis ecotone with sandy loam soils, as well as at Manas Experimental Station, northern Xinjiang (**Figure 2**). The results indicated that our transgenic cotton had improved performance and could adapt to a wide range of cotton culture environments.

The lack of rainfall makes irrigation vital for agricultural production in arid and semi-arid lands. In arid zones, for example, normal irrigation above 600 mm during the vegetation period is sufficient for stable cotton harvesting [5]. Our desert oasis drought experiments in southern Xinjiang with sandy loam soils designed the 75% deficit irrigation and less than 600 mm different irrigation strategies to conserve more irrigation water and keep cotton yield constant. We used three irrigation schedules: DSSIS (Decision Support System for Irrigation Scheduling) forecasts (F), soil moisture sensors (S), and experience irrigation (E). Full irrigation (FI) and deficit irrigation (DI, 75% of full) were applied from 2016 to 2018 (**Figure 2**). Different irrigation protocols and water consumption affected the growth and yield of cotton, and the "Smart Irrigation" irrigation scheme based on the Root zone water quality model (RZWQM2) was found to be the best irrigation scheme for sustainable cotton production in an arid land. The results indicated that deficit irrigation schemes can be utilized in the desert-oasis ecotone, and in conjunction with the use of *ScALDH21*-cotton lines, the yields are sufficient for viable and sustainable agriculture.

Moreover, through mixed model analysis, we found that the cotton line always has a significant effect on plant phenotype, physiology, and yield components in southern Xinjiang, and cotton line and irrigation scheduling both have significant effects on cotton growth and development separately. In addition, irrigation scheduling and irrigation levels have a significant interaction effect. The relationship between yield and crop water use was calculated as overall water use efficiency (WUE). The EI schedule consumed more water (EFI, 547 mm, and EDI, 409 mm) than either the FI (FFI, 385 mm, and FDI, 288 mm) or SI (SFI, 254 mm, and SDI, 186 mm) schedules. Compared to NT plants, WUE was higher in *ScALDH21*-cotton lines each year. A high WUE was observed in the forecast, with drip irrigation leading to the highest WUE, and flood irrigation leading to the lowest WUE. In all treatments, the WUE value of *ScALDH21*-cotton lines increased by 59.6% compared to NT.

The individual irrigation level and timing significantly affected vegetative growth parameters, plant height, and leaf area, but the differences did not differ substantially between years despite differing precipitation levels. In each irrigation treatment, the TC, and especially the L16, grew significantly higher than the NT controls from 2016 to 2018. There was a dramatic reduction in the leaf area of NT under SDI in all years, but there was no difference in the leaf area of TC [37]. Therefore, the irrigation treatments can be ranked as forecast irrigation > flood irrigation > soil moisture irrigation based on their ability to maintain high instantaneous water use efficiency (IWUE).

We also used managed treatment plot experiments and field-scale in purple clay loam soil sites at Manas Experimental Station, northern Xinjiang. The two experiments differed in terms of growth space and water consumption. In the managed plot experiment, 50% less of full irrigation significantly reduced cotton vegetative growth and cotton yield (\*50% loss of cottonseed and lint yield compared with full irrigation), whereas, in the field, 30% less of full irrigation did not affect cotton vegetative growth or yield. The reason for this can be explained by the amount of water used, which was 675 L of water m−2 with full irrigation and 472 L m−2 with deficient irrigation in the field, 566 L m−2 (control), and 283 L m−2 (stress) in the managed treatment areas.

The study also provides guidelines for optimal irrigation protocol and minimum water requirements for the use of *ScALDH21*-transgenic cotton lines in arid regions.

## **3. Conclusions and perspectives**

*ScALDH21*-transgenic cotton exhibits improved plant growth and developmental phenotype through sustained net photosynthetic rate, greater tolerance to osmotic and oxidative stress, and improved cotton yield and fiber quality. Transgenic cotton can also be grown in sandy loam soils in southern Xinjiang and purple clay loam soils in northern Xinjiang that are more productive than non-transgenic recipients. This transgenic variation of *ScALDH21* is significantly better than recipient cultivars that can be commercially exploited when irrigation is scarce, enabling a more sustainable cotton production in the desert oasis ecotone. For *ScALDH21* transgenic cotton that can be used in agricultural production, we evaluated various irrigation protocols and optimized irrigation regimes with minimal water requirements. Using multiple growing seasons and multiple studies, ectopic expression of the moss *ScALDH21* gene in cotton improves drought tolerance and reduces yield penalties. *ScALDH21* overexpression enhances drought tolerance in cotton, suggesting that *ScALDH21* could be a candidate gene for improving cotton in arid-arid regions. To ensure the safety of transgenic lines, they must be tested with a GMO undergone a biosafety protocol. Currently, biosafety and risk assessment based on GMO requirements are being tested. To breed the next generation of crop varieties, updated germplasm, knowledge, and breeding techniques will be needed [74]. There are numerous reports of genes that confer stress tolerance, most of which are from Arabidopsis, very few of which have been successfully tested in crop plants due to potential side-effects of candidate genes on growth and morphology. It is therefore urgent to develop many drought-tolerant gene resources from drought-resistant plants. Reports indicate that introducing foreign genes from xerophytic plants or overexpressing certain cotton genes improve its performance under drought conditions [35, 36, 38]. Therefore, it is reasonable to assume that an in-depth comparative study of the expression and function of members of the same gene families in extreme xerophytes will eventually aid in breeding drought-tolerant crop plants.

## *Transgenic Technology Can Accelerate Cotton Breeding: Transgenic* ScALDH21 *Cotton… DOI: http://dx.doi.org/10.5772/intechopen.103833*

It has been widely used in plant biotechnology to improve crop traits with CRISPR/Cas9 technology [75, 76] and it will be applied to crop breeding in the near future [77], especially for gene knock out in crops. To overcome complex and changing adversity, crop breeding must be multi-resistant because climate change leads to an increase in both biotic and abiotic stresses. The pursuit of homologous genes from extreme xerophytes plants, but with a low degree of identity to crop, will significantly increase drought tolerance. *ScALDH21* orthologs were not found in cotton genomes, but transgenic lines exhibited better growth and development, as well as greater photosynthesis ability in a water-scarce environment. Furthermore, this gene was also discovered to be salt-tolerant as well as resistant to *Verticillium*, which indicates that moss as the first landing plant may be an excellent resistant gene resource library, especially the extreme drought-tolerant moss.

## **Acknowledgements**

This work was supported by grants from the Tianshan Youth Program (Grant No. 2019Q035), the National Natural Science Foundation of China (Grant No. 31700289), The third comprehensive scientific investigation in Xinjiang (2021xjkk0502), and the West Light Talents Cultivation Program of Chinese Academy of Sciences (2016-QNXZ-B-20). We are grateful to Xin Wei and Professor Jianhui Xu from the Research Institute of Economic Crops, Xinjiang Academy of Agricultural Sciences, China, for their support in the fieldwork. We thank Fanjiang Zeng and Xiangyi Li from Cele National Station of Observation and Research for Desert-Grassland Ecosystem, Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences for providing experimental conditions. We are also thankful to the anonymous reviewers who put a great deal of effort into reviewing and editing this chapter.

*Cotton*

## **Author details**

Honglan Yang1,2, Dawei Zhang3 , Tohir A. Bozorov4 , Abdul Waheed1,2, Jiancheng Wang1,2, Xiaoshuang Li1,2 and Zhang Daoyuan1,2\*

1 Xinjiang Key Laboratory of Conservation and Utilization of Plant Gene Resources, Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences, Urumqi, China

2 Turpan Eremophytes Botanical Garden, Chinese Academy of Sciences, Turpan, China

3 Research Institute of Economic Crops, Xinjiang Academy of Agricultural Sciences, Urumqi, China

4 Institute of Genetics and Plants Experimental Biology, Uzbek Academy of Sciences, Kibray, Tashkent Region, Uzbekistan

\*Address all correspondence to: zhangdy@ms.xjb.ac.cn

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Transgenic Technology Can Accelerate Cotton Breeding: Transgenic* ScALDH21 *Cotton… DOI: http://dx.doi.org/10.5772/intechopen.103833*

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## **Chapter 6**

## Cotton Breeding in the View of Abiotic and Biotic Stresses: Challenges and Perspectives

*Venera Kamburova, Ilkhom Salakhutdinov and Ibrokhim Y. Abdurakhmonov*

## **Abstract**

Global climate change manifested in average annual temperature rise and imbalance of most natural factors, such as changes in annual mean rainfall, air humidity, average temperature of cold and warm months, soil quality, etc., lead to climatic zones displacement. All these have a significant impact on agricultural production in total, including cotton growing. Cotton is one of the most important technical crops in the world. However, it is very sensitive to environmental changes. The influence of abiotic stresses (high temperature, changes in the mean rainfall and soil salinity) causes a dramatic decrease yield of this crop. Moreover, temperature anomalies and climatic zones displacement cause a change in the area of pathogens and pests distribution, which also reduces the cotton yield. One of the possible ways to increase the cotton yield under the influence of abiotic and biotic stresses is the development of new resistant varieties, using both classical breeding methods and genetic engineering achievements.

**Keywords:** cotton, global climate change, abiotic and biotic stresses, cotton breeding, genetic engineering

## **1. Introduction**

Cotton (*Gossypium* ssp. L.) is the major source of quality natural fiber and widely contributed to textile and seed oil industry [1]. Currently, the annual global cotton fiber production is about 25 million metric tons; the market value estimated is \$ 12 billion [2].

Because cotton is a subtropical plant, it is well adapted to survive with dry and hot environment [3]. Despite this, cotton nevertheless reacts to an environmental change such as temperature and rainfall in instance. Long-term exposure of negative factors such as a drought, salinity, and temperature stress causes a significant decrease of yield and fiber quality [4, 5]. Such negative effect on cotton is due to the fact that drought, salinity, and temperature stress cause osmotic imbalance, membrane disorganization, growth decrease, inhibition of cell fission and reproduction; this

also leads to decline of photosynthesis level and hyperproduction of reactive oxygen species (ROS) [6, 7].

In addition to abiotic stresses, the cotton production is greatly influenced by biotic factors, such as pests and diseases that also cause a significant (up to 10–30%) reduction in yield [8]. At the same time, global climate changes responsible for temperature factors and climatic zone displacement also affect their development, geographical distribution, pathogenicity or injuriousness [4].

In this regard to these threats to cotton production, breeders are facing the important task of new cotton varieties resistant to abiotic and biotic stresses. However, this problem-solving by the classical genetics methods has become complicated due to the resistance traits generally having multigenic nature with a complex type of inheritance [9]. Additionally, the breeding oriented on resistance is further complicated by the "bottle-neck" effect such as narrow genetic basis typical for cultivated cotton [10]. Nevertheless, these disadvantages may be successfully overcome by the use of genetic engineering methods: transgenesis, RNA interference, and genome editing approaches.

In this chapter, we would like to analyze and summarize information about increase of cotton resistance to abiotic and biotic factors using genetic engineering approaches.

## **2. Increasing resistance to abiotic stresses**

Abiotic stresses are a direct consequence of climate change. The world increase of temperature is primarily caused by carbon dioxide effect, i.e., its content in the atmosphere. The increase in the average annual temperature is the cause of increase of water evaporation from the soil, which directly leads to osmotic (by drought) and salt stress. One of the features of abiotic stresses is their simultaneous exposure. In other words, they have usually a similar effect on plants and defense mechanisms appearance in plants [11].

Abiotic stresses may affect cotton upon all development stages and lead to significant decrease in both yield and quality of cotton fiber [12, 13]. Thus, an increase of temperature at 2–3°C from the optimum can decrease biomass and yield, as well as increase fiber micronaire [13]. Drought and salinity also cause a decrease in the yield and quality of cotton fiber [6, 14].

In this regard, increase of a cotton resistance to abiotic stresses will reduce a negative effect and can raise the yield and quality of fiber. In this chapter, we consider the impact of abiotic stresses on the morphological and physiological parameters, as well as the mechanisms of resistance development and methods for increasing the adaptive potential of cotton to negative environmental factors.

#### **2.1 Influence of abiotic stresses on morphophysiological parameters in cotton**

Influence of abiotic stresses on cotton plants manifested in various forms of morphophysiological and biochemical changes, which reduce yield and fiber quality of cotton [6, 13, 14]. They negatively affect both morphological (seed germination, plant height and architecture, length and area of root system, leaf area, shoot and root biomass, boll development) and physiological parameters (chlorophyll content, photosynthetic efficiency, transpiration rate, stomatal conductance) [6, 13, 14].

*Cotton Breeding in the View of Abiotic and Biotic Stresses: Challenges and Perspectives DOI: http://dx.doi.org/10.5772/intechopen.104761*

In addition, prolonged exposure to abiotic stresses leads to a decrease in yield and fiber quality. Yield reduction manifested by both a decrease in the number and weight of bolls and fiber yield [6, 13, 14]. At the same time, this negative effect on yield is explained by a decrease in the activity of catabolic processes, including photosynthesis [6, 13, 14]. The fiber quality reduction manifested in a decrease in fiber length and an increase in micronaire. Such influence of abiotic factors on one of the most important agronomic traits of cotton is caused both by reduction of carbohydrate synthesis due to reduction of photosynthesis activity and by disruption of elongation process due to changes of membrane permeability and organization of microtubules and cytoskeleton [13, 14].

Disruption of photosynthesis under abiotic stresses is associated with an increase in ion permeability of chloroplast thylakoids and a decrease in chlorophyll levels, as well as inhibition of the activity of the key enzyme of carbohydrate synthesis 1,5-bisphosphate carboxylase [13–16].

#### **2.2 Mechanisms of resistance to abiotic stresses**

To reduce the negative impact of abiotic stresses in plants, including cotton, they have developed some adaptations on physiological and molecular level.

Physiological adaptations include accumulation of soluble substances in vacuoles to maintain cell turgor and decrease of stomatal conductance to reduce transpiration [11, 13, 14].

Molecular defense mechanisms against abiotic stresses include accumulation of osmolytes (proline, betaine, and soluble sugars), changes in activity of antioxidant system reducing level of ROS, regulation of cell ion balance and hormonal activity, as well as changes in activity of heat stress proteins [6, 7, 13, 14, 17]. Let us consider each mechanism separately.

*Antioxidant system.* One of the aftereffects of abiotic stresses on cotton is an increased level of ROS due to disruption of cell respiration and photosynthesis [6, 7, 13, 14, 17–20]. An increased ROS level leads to oxidative damage to proteins, DNA and lipids, destabilization of membranes, and increase of their permeability [19, 21]. Neutralization of ROS in plants is carried out by antioxidant system that includes nonenzymatic antioxidants (flavonoids, carotenoids, tocopherols, glutathione, etc.) and various antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase, peroxidase, ascorbate peroxidase, and glutathione reductase) [21–24]. In most crops, including cotton, the increased activity level of antioxidant enzymes is associated with resistance to abiotic stresses: higher activity of antioxidant enzymes had been seen in more resistance varieties [17, 19, 25].

*Ion balance regulation in cell.* Ion imbalance and toxicity accompanied by Na+ accumulation are the main consequences of salt and osmotic stress [6, 7, 11, 14, 18, 26]. To reduce ion toxicity and restore ion balance, plant cells use the Ca2+-dependent salt supersensitive (SOS) regulatory pathway, which regulates ion homeostasis by modulating Na+ /H+ antiporter activity during salt stress [7, 14, 26, 27]. The SOS pathway consists of plasma membrane Na+ /H+ -antiporter (SOS1), protein kinase (SOS2), and two calcium sensors— SOS3 and SCaBP8 (SOS3-like calcium-binding protein 8) [26, 28].

The excessive accumulation of Na+ in the cytoplasm also results in the accumulation of Ca2+, which interacts with SOS3/SCaBP8, activating the serine/threonine protein kinase SOS2. Then, SOS2 phosphorylates SOS1, which increases Na+ /H+ -antiporter activity, restoring the ion balance in the cell and enhancing salt tolerance [7, 20, 26]. SOS3/SCaBP8-SOS2 also regulates the activity of other transporters involved in ion

homeostasis: K+ - and Na+ -transporters, vacuolar Na+ /H+ -exchanger (NHX), vacuolar H+ -ATPases, and pyrophosphatases (PPase) [18, 20, 26].

*Accumulation of osmolytes.* Most abiotic stresses lead to water imbalance and, as a consequence, the induction of osmotic stress, which reduces cell turgor and the activity of many enzymatic systems [7, 11, 14, 18, 20]. To reduce the osmotic stress affects, plant cells accumulated the following osmoprotectors such as proline, betaine, soluble sugars, etc. [7, 11, 14, 18]. These agents protect membrane lipids and proteins from oxidative damage, increase the photosynthesis rate, and restore the osmotic potential of the cell [7, 20, 26, 29, 30].

*Hormonal regulation.* Abscisic acid (ABA), ethylene, salicylic acid (SA), jasmonic acid (JA), and brassinosteroids (BR) are the main plant stress hormones [7, 26, 31]. ABA is considered a major stress hormone whose activity increases during drought and salinity [7, 31]. ABA promotes the accumulation of K<sup>+</sup> , Ca2+, and osmolytes, reducing the inhibitory effect of abiotic stresses [7, 26, 31]. SA and BR are also involved in plant responses to abiotic stress [7, 26, 31]. An increase in BR under salt stress contributes to the maintenance of ion and osmotic homeostasis, increasing the stress tolerance of plants [7, 26, 31]. In addition, the BR signaling cascade intersects with the SOS pathway. BR leads to calcium accumulation in the cytosol, which activates the SOS pathway through SOS3/SCaBP8 [7, 26, 31]. The protective effect of SA and JA under stress is due to the activation of plant antioxidant system [7, 26, 31].

*Heat shock proteins (HSPs)*. HSPs are molecular chaperones and play an important role in plant resistance to temperature stress [13, 32]. Depending on the molecular weight, the following HSP groups are distinguished: small HSP (sHSP), HSP60, HSP70, HSP90, and HSP100 [13, 32]. sHSP and HSP100 bind to proteins, prevent their denaturation and aggregation, promote their refolding with the participation of ATP-dependent chaperones (ClpB/DnaK) [13, 32]. HSP60 (mitochondrial chaperone or chaperonin 60) contributes to the maintenance of normal mitochondrial and chloroplast functioning under heat stress by keeping the native state of the inner mitochondrial membrane proteins and chloroplast thylakoids [13, 32]. HSP70 is involved in protein folding and prevention of protein aggregation [13, 32]. In addition, inhibition of HSP70 gene expression in cotton leads to oxidative stress by increasing H2O2 levels, which suggests the involvement of this chaperone in the regulation of several antioxidant enzymes activity [13, 32]. HSP90 together with HSP70 regulates protein folding by participating in signal transduction through signaling kinases and hormone receptors [13, 32].

Thus, plants have numerous mechanisms to promote abiotic stresses resistance. The genes mediating these defense mechanisms may be potential objects of interest for enhancing the adaptive potential of plants to environmental stress conditions.

#### **2.3 Improving the adaptive potential of cotton to abiotic stresses**

A significant decrease in the yield and fiber quality under the influence of abiotic stresses assigns a task for breeders to create cotton varieties resistant to these stresses. To solve this problem, it can use the methods of classical breeding, methods of molecular selection, and genetic engineering. Let us consider the application, advantages, and disadvantages of these methods.

*Classical breeding.* Inheritance of abiotic stress tolerance traits in cotton is multigenic with complex intergenic interaction including additive and nonadditive, dominant, and epistatic effects [9, 14]. The complex mechanism of trait inheritance *Cotton Breeding in the View of Abiotic and Biotic Stresses: Challenges and Perspectives DOI: http://dx.doi.org/10.5772/intechopen.104761*

and "bottle-neck" effect make it difficult to use classical breeding methods to obtain cotton varieties resistant to abiotic stress. Moreover, these methods require a lot of time to develop new varieties.

*Marker-associated selection (MAS).* The use of molecular markers and Quantitative Trait Loci (QTL) mapping made it possible to overcome the disadvantages of classical breeding in developing cotton varieties resistant to abiotic stress [13, 14, 33–35]. Simple sequence repeat (SSR) and single-nucleotide polymorphisms (SNPs) are most commonly used to identify QTL [13, 14, 33–35]. Thus, using SSR markers, 11 QTLs localized on eight chromosomes (c9, c11, c15, c16, c21, c23, c24, c26) associated with salt tolerance traits were identified in the test population from *G. tomentosum* and *G. hirsutum* cross [36]. In the same population, QTLs associated with drought tolerance were also localized on chromosomes c5, c8, c9, and c16 as well as some QTL clusters for same trait on chromosomes c2, c3, c5, c6, c9, c14, c15, c16, and c21 [37]. Additionally, 165 QTLs have been identified in an introgressed population of *G. hirsutum* under abiotic stress conditions using 481 SNPs and 523 SSR markers covered of most cotton chromosomes. In total, 15 of them have been common QTLs of tolerance to abiotic stresses localized in 12 chromosomes: c1, c2, c5, c6, c8, c9, c10, c12, c20, c23, c25, and c26 [14].

Presently, various strategies, including genotyping by sequencing (GBS), SNP arrays, and genome-wide association study (GWAS), as well as populations of recombinant inbred lines (RIL) and backcross inbred lines (BIL), are used to improve the efficiency of QTL mapping [14]. Thus, 95 loci that associated with salt tolerance in *G. hirsutum* were found using GWAS in combination with SSR markers [38]. GWAS in combination with polymorphic SNPs of the CottonSNP63 K array applied to determine resistance of upland cotton has revealed a drought tolerance QTLs on chromosomes c8, c15, c21, c24, c25, and c26 and salt tolerance QTLs on chromosomes c1, c9, c11, c12, c13, c14, c18, c21, and c24 [39]. These data have confirmed using GWAS in combination with SNPs for MAGIC population of *G. hirsutum* including of 550 RILs. It has found that 11 QTLs associated both drought and salt tolerance [40].

In addition, the use of meta-analysis allows improving the accuracy of QTL mapping associated with abiotic stresses. For example, this approach has identified 23 stress tolerance QTL clusters on 15 different cotton chromosomes: c3, c4, c5, c6, c7, c11, c14, c15, c16, c19, c20, c23, c24, c25, and c26 [41].

Summarizing the above, the use of molecular markers and associative mapping data can significantly reduce the time to breed resistant cotton varieties.

*Transgenic approaches.* These approaches are widely used to increase cotton resistance to abiotic stresses. Thus, overexpression of *AVP1* and *OsSIZ1* genes in cotton enhances its resistance under both drought and heat shock stresses [42]. Overexpression of *HSP101* gene also increases resistance of cotton to temperature stress [13]. Further, transformation of cotton by *AsHSP70* gene from *Agave sisalana* resulted in improvement of a number of physiological parameters under heat stress [43].

Application of transgenic approaches also allows increasing cotton resistance to drought and salinity. Many transcription factors, regulating the activity of functional genes, can influence drought and salt tolerance in cotton [13]. Thus, overexpression of transcription factor *GhABF2* increases both drought and salt tolerance in cotton through regulation of ABA cascade genes [44]. Overexpression of other transcription factor genes (*AtRAV1/2*, *AtABI5,* and *SNAC1*) also increases cotton resistance to drought and salinity [13].

Increase in defense capacity of cotton due to increase level of osmoprotectants and activity of antioxidant enzymes and ion antiporters also enhance the adaptive resistance of the crop to abiotic stresses [13]. Overexpression in cotton of *AtEDT1/HDG11* gene from *A. thaliana* led to the increase of proline level and activity of antioxidant enzymes, increasing the resistance to salt and osmotic stress [45]. Moreover, transformation of cotton by the H+ -phosphatase gene (*TsVP*) from *Thellungiella halophile* allowed to reduce the negative effect of salt stress on photosynthetic activity [13]. Individual and coexpression of H+ -pyrophosphatase (*AVP1*) and vacuolar Na+ /H+ antiporter (*AtNHX1*) genes from *A. thaliana* led to the increase of cotton salt tolerance due to more efficient regulation of ion balance [13].

Regulation of hormonal status by overexpression of their biosynthesis genes can also increase the adaptive potential of cotton resistance to salt and osmotic stress. Thus, overexpression of isopentenyltransferase (*IPT*) gene, one of cytokinin biosynthesis genes, increased cotton resistance to drought and salinity [13, 46]. Furthermore, cotton transformation with AtLOS5 gene (involved in ABA biosynthesis) from *A. thaliana* increased drought tolerance of the crop [13].

In this way, the application of transgenic methods makes it possible to effectively increase cotton resistance to abiotic stresses. However, those approaches are limited by the legislative regulation of GMO in many countries, according to this all transgenic crops obliged to undergo a full cycle of biosafety assessment [47].

*Modern methods of genetic engineering.* To overcome the biosafety constraints of transgenic cotton, researchers use modern genetic engineering methods including RNA interference (RNAi) and genome editing (GE) approaches.

RNAi is one of promising approaches both for studying of resistance genes and developing new cotton varieties resistant to abiotic stresses [10, 48]. For example, the use of VIGS-mediated RNAi revealed that R2R3-type *GbMYB5* transcription factor increases cotton resistance to abiotic stresses due to proline accumulation and increase antioxidant enzymes activity [10]. It has been also found that the expression levels of several miRNAs in leaves (miR156, miR157, miR162, miR172, miR397, miR398, miR399) and roots (miR172, miR397, miR398, miR399) change under salt and osmotic stress [49]. In addition, RNAi of phytochrome A1 gene increased the resistance of cotton to salt stress by activation of antioxidant enzymes [17].

Application of GE approaches to increase the adaptive potential of cotton in accordance to abiotic stresses is currently quite limited. However, there are successful applications of GE in cotton. For example, the target editing of *GhRDL1* and *GhPIN1–3* genes by the use of CRISPR/Cas9 system has allowed to obtain droughtresistant cotton lines [50].

Summarizing the above, it should be noted that presently, marker-associated selection and transgenic methods have the greatest importance in breeding of cotton resistant to abiotic stresses.

## **3. Improving resistance to biotic factors**

Biotic factors (insect pests and pathogens) are among the most important factors that reduce cotton productivity [4, 8, 51]. For example, losses of cotton yield from pests may be up to 84% [51] and due to pathogens up to 30% [8]. As in the case of abiotic factors, global climate change leads to a shift of climatic zone, affecting the growth, development, and spread of insect pests and pathogens [4]. As results, this leads to the emergence of new pests and pathogens in these areas.

In this regard, improving plant resistance to biotic factors allows effectively control of pests and pathogens to reduce yield losses. In this part, we are looking at the characteristics of the main pests and pathogens, as well as a natural defense mechanisms and methods of improving cotton resistance to them.

## **3.1 Characteristics of major pests and pathogens of cotton**

*Insect pests.* Cotton pest insects can be divided into two groups according to the mechanism of plant damage: chewing and piercing-sucking [52]. The first group includes insects that feed the plant biomass: cotton bollworm (*Helicoverpa armigera*), fall armyworm (*Spodoptera frugiperda*), pink moth (*Pectinophora gossypiella*), spotted bollworm (*Earias vittella*), and cotton leafworm (*Alabama argillacea*). The pests of this group of insects are larvae (caterpillars) that feed on immature bolls and leaves [8, 52].

The second group includes sap feeding insects that damage phloem: boll weevil (*Anthonomus grandis*), cotton aphid (*Aphis gossypii*), thrips (*Frankliniella spp*, *Thrips tabaci*, *Neohydatothrips variabilis*, and *Scirtothrips dorsalis*), cotton seed bug (*Oxycarenus hyalinipennis*), tarnished plant bug (*Lygus lineolaris*), cotton fleahopper (*Pseudatomoscelis seriatus*), and two-spotted spider mite (*Tetranychus urticae*) [8, 52]. The pests in this group are adults and/or nymphs [52].

In addition, soil nematodes can also cause a significant cotton yield reduction [8]. Nematodes parasitizing on cotton include the root-knot nematode (*Meloidogyne incognita*), reniform nematode (*Rotylenchulus reniformis*), and sting nematode (*Belonolaimus longicaudatus*) [8].

*Phytopathogens.* Cotton pathogens include viruses, bacteria, and fungi [8, 53]. Fungi of genera *Fusarium*, *Rhizoctonia*, *Pythium,* and *Thielaviopsis* affect cotton seedlings causing seedling root rot [53]. Blackspot causes with fungus of *Alternaria macrospora* Zimm, leading to leaves damage. *Ramularia areola* causes Ramularia blight of cotton [53]. Cotton boll rot is a complex disease caused by several fungal pathogens such as *Fusarium moniliforme*, *Calletotrichum gossypii*, *C. capsici*, *Aspergillus flavus*, *A. niger*, *Rhizopus nigricans*, *Nematospora nagpuri,* and *Botryodiplodia sp*. This disease affects the bolls, spreads to inner tissues, and leads to rotting of the seeds and fibers [53]. The most dangerous form of boll-rot is anthracnose, caused by *Calletotrichum gossypii* Southw. Anthracnose in cotton can occur in all growth stages of the plant, and it can affect all tissues, causing seedling or young plants to wilt and die, as well as a severe reduction in fiber and seed yields [53].

*F. oxysporum* f. sp. *vasinfectum* causes development of cotton Fusarium wilt at seedling stage with cotyledon lesions [53]. Verticillium wilt is caused by *Verticillium dahliae* Kleb, which affects cotton leaves in the budding or immature bolls stages [53].

The viral diseases include cotton leaf curl disease (CLCuD) and (CLCrD) [53]. CLCuD is caused by begomoviruses that lead to leaf injury (swollen veins, leaf curl, enation, and stunting). When affected in the early stages of development, there is a significant reduction in yield [53]. The cotton leaf curl virus (CLCrD) affects the leaves resulting in leaf discoloration and vein hypertrophy, leaf curl, shortening of internodes, and growth stunting. The infestation degree depends on the stage of plant development [53].

Bacterial blight of cotton is one of the most serious diseases causing significant yield losses [8, 53]. Disease results from infection by *Xanthomonas citri* pv. *malvacearum* [8, 53]. Affected plants show the following symptoms such as defoliation, swelling and darkening of stems, bolls detachment. By severely affecting, the fiber quality is decreased due to coloration and the plant death [53].

### **3.2 Mechanisms of resistance to biotic factors**

The long coevolution of cotton and insect pests and pathogens has resulted in mechanisms to reduce the damage from biotic factors. Molecular mechanisms of pathogen resistance include the activation of resistance genes (R-genes) in response to exposure. R-gene activation triggers a large number of intracellular cascades leading to the synthesis of protective substances that reduce the damage by pathogens [54, 55]. Morphological and chemical defense mechanisms have been developed in cotton to reduce the pest influence degree [56, 57]. Let us in more detail consider mechanisms of resistance to insect pests and pathogens correspondingly.

*Resistance to pests*. Morphological defenses and chemicals (secondary metabolites) of cotton directly influence the insect (imago or larvae) affecting important parameters of their life cycle [56]. Trichomes are considered as the major morphological adaptation of cotton that increases its insect resistance. These provide protection by forming a physical barrier or excreting chemical repellents, toxins, or adhesive substance [56].

Terpenoids, flavonoids, tannins, and anthocyanins are among the secondary metabolites providing direct protection of cotton plants from insects [56]. Terpenoids are the most studied protectors of cotton. Terpenoids synthesized in cotton include gossypol, hemigossypol, hemigossypolone, and heliocides H1, H2, H3, and H4 contained in small subepidermal and intracellular pigment glands [56]. Cotton terpenoids have direct toxic effect on insect pests including *H. virescens*, *H. zea*, *H. armigera*, *P. gossypiella*, *Estigmene acrea*, *E. insulana,* and *E. vitella*. In addition, gossypol and gossypol-like compounds are toxic to the gall nematode *Meloidogyne incognita* [56].

It should also be noted that damage of cotton by pests and pathogens causes induction of terpenoid biosynthesis by activation of JA-, SA-, and ethylene-dependent signaling pathways [56, 58]. These pathways activation occurs due to elicitors, which, interacting with specific receptors, lead to an increase in intracellular Ca2+. This in turn activates calcium-dependent proteins, including Ca2+-dependent protein kinases (CDPKs) [58]. CDPK, by phosphorylating proteins and changing gene expression patterns, activates mitogen-activated protein kinases (MAPK), leading to JA and SA formation, on the one hand, and the ethylene pathway, on the other [58].

*Resistance to pathogens.* Plant resistance to pathogens (plant immunity) is controlled by resistance genes (R-genes) [54]. R-genes encode surface (receptor-like kinases—RLK) or intracellular receptors (nucleotide-binding proteins with leucinerich repeats—NLR) activating a various mechanism under interaction with them [54, 59–61]. One consequence of receptor activation is an increase intracellular Ca2+ concentration, leading both to the activation of the Ca2+-dependent signaling cascade and an increase in ROS levels [59–61]. At the same time, ROS play the function of an intracellular signaling molecule contributing to the development of systemic acquired resistance (SAR). It should also be noted that both Ca2+- and ROS-dependent signaling cascades by activation of JA-, SA-, and ethylene-dependent signaling pathways lead to synthesis of phytoalexins (mainly gossypol and gossypol-like terpenes), which play an important role in cotton resistance to pathogens [59–61]. For example, induction of gossypol synthesis in cotton has been proved by infestation with *Verticillium dahlia*, *Fusarium oxysoporum* f.sp. *vasinfectum*, *Rhizoctonia solani*, *Rhizobium rhizogenes,* and *Xanthomonas spp* [56].

Thus, plants have a various mechanisms that provide resistance to pests and pathogens. Genes mediating these defense mechanisms may be potential genes for improving cotton resistance to biotic factors. In addition, the control of genes of the causative agents themselves, playing an important role in their life activity, may also be of potential interest.

#### **3.3 Improving cotton resistance to biotic factors**

Biotic factors (pathogens and pests) are one of the main reasons for significant yield losses (up to 84% due to insects and up to 30% for pathogens) in agriculture [4, 8, 51]. At the same time, strategies to control infestations are an increase in the internal defense mechanisms of plants or introduction of pathogen-targeted constructs into the genome [62, 63]. Methods of classical breeding, molecular breeding, and genetic engineering are used to develop new varieties that are resistant to the impact of biotic factors. Let us consider the application, as well as advantages and disadvantages of each of these methods.

*Classical breeding.* Classical breeding methods increase the internal defense mechanisms of the plants and use the cotton germplasm reserves to produce new resistant varieties. For example, among all cultivated cotton species (*G. hirsutum* L., *G. barbadense* L., *G. arboretum* L., and *G. herbaceum* L.), only *G. barbadense* has sufficient resistance to *Verticillium dahlia*. However, transgenesis of the resistance into upland cotton by classical breeding methods has so far not been successful [59].

Such interspecific crossing for the purpose of transfer wilt resistance genes is complicated by different type of these traits inheritance in *G. hirsutum* and *G. barbadense L*. Studies of interspecific crossing show dominant or partially dominant inheritance of resistance traits, while by intraspecific crossing of *G. hirsutum*, the traits inheritance is more complex [59]. A number of studies report wilt resistance control by a single dominant gene, while others state that resistance is a quantitative trait [59, 64]. An additional difficulty is the fact that varieties with high wilt resistance have low fiber yield and quality, as well as crop yield [59]. In addition, it should be noted that classical breeding methods are time-consuming, which reduces the effectiveness of this approach in breeding pathogen-resistant cotton varieties.

*Marker-associated selection (MAS).* MAS and QTL mapping have been widely used in the development of cotton varieties resistant to Verticillium and Fusarium wilt. For example, more than 400 QTL of resistances to both kind of wilt have been identified, which are distributed over all 26 pairs of chromosomes [34, 59, 64]. These data were obtained both using mapping of chromosome-substituted and RIL populations with the help of various markers type and GWAS [59, 64–68].

Furthermore, a meta-analysis of the consensus map of Cotton Marker Database (CMD) based on *G. hirsutum* × *G. barbadense* cross, five mutagenesis "hot spots" of wilt resistance were identified on c16 and c23 chromosomes [69]. Same meta-analysis revealed that 74 QTLs of nematode resistance are localized on all chromosomes. Thus, 71 QTLs of them are associated with resistance to root-knot nematode, and three remains with resistance to reniform nematode. Especially, the greatest number of QTLs for this trait was identified on chromosomes c7 and c11. The mutagenesis hotspot of nematode resistance is also located on chromosome c7 [69, 70]. Additionally, this study shown that two QTLs of resistance to *Xanthomonas campestris* pv. *Malvacearum* are localized on chromosomes c5 and c14 [69].

The obtained data of a QTL mapping can be successfully used in further MAS and genomic breeding programs.

*Transgenic approaches.* These approaches are currently the most effective method for creating cotton varieties resistant to insect pests [8]. According to ISAAA, transgenic cotton occupies about 79% of the total cultivated area of this crop [71]. Despite the existence of various strategies to develop insect-resistant transgenic crops (use of several genes with insecticidal properties such as inhibitors of insect's digestive proteases, α-amylase, lectin, etc.), most transgenic insect-resistant (IR) crops, including cotton, are based on insertion of *cry* genes encoding *Bacillus thuringiensis* (Bt) toxin in the host plant genome [8, 72]. Bt (or Cry) toxins have specific activity against insect from orders such as *Lepidoptera*, *Coleoptera*, *Hymenoptera,* and *Diptera*, as well as for nematodes [8, 72]. The cultivation of Bt-cotton has significantly reduced the use of insecticides in cotton-growing countries [72]. The use of transgenic cotton plants with Bt-gene sets further expands the potential of transgenic cotton and reduces the emergence of resistant insect populations [8, 72].

Thus, vegetative insecticidal proteins (*Vip*s) from *B. thuringiensis* with insecticidal activity against *Gossypium spp*. pests are promising for the creation of transgenic IR-cotton [8]. The proteins *Vip1* and *Vip2* are binary toxins, which are very toxic to some representatives of *Coleoptera* and *Hemiptera*. The action mechanism of *Vip3* is similar to that of Bt-toxins [8].

In order to create varieties resistant to fungal pathogens (*Rhizoctonia solani*, *Alternaria alternata*, *Alternaria macrospora,* and *Fusarium oxysporum*), transformation of cotton with genes encoding chitinases has been used [8]. Glucose oxidase genes were introduced into the cotton genome to improve resistance to *V. dahliae*, while the harpin encoding gene (*hpa1Xoo*) from *Xanthomonas oryzae* pv. *oryzae* is used to provide resistance to various pathogens [8]. Transformation of cotton with the antisense movement protein (*AV2*) and antisense coat protein (*ACP*) genes from CLCuV results in resistance to CLCuD [8].

In accordance with above, the application of transgenic technology is currently the most used and commercially successful for creating pest and pathogen resistant crops. However, the most serious disadvantage of this technology is the need for longterm biosafety assessment of transgenic cotton to minimize risks of human health and the environment [47].

*RNA interference (RNAi).* The host-induced gene silencing (HIGS) approach, in which a construct is introduced into the host genome that induces posttranslational suppression of gene expression in pathogen or pest through dsRNA upon infection, is most commonly used to achieve resistance to biotic factors with RNAi [8, 10, 59]. Thus, introduction of RNAi construct to hygrophobins1 (*VdH1*) gene of *V. dahliae* into cotton genome provides resistance to this pathogen [73]. A similar effect is achieved by HIGS to *V. dahliae VdRGS1* gene mediated by tobacco rattle virus [74].

The use of HIGS to the genes encoding proteins that play an important role in the life maintenance of insect allows the development of cotton IR lines. Thus, silencing of cytochrome P450 gene of insect monooxygenase (*CYP6AE14*) involved in gossypol detoxification leads a significant increase in cotton resistance to cotton bollworm (*Helicoverpa armigera*) [10, 75].

Another approach to improve cotton resistance to biotic stresses is virus-induced gene silencing (VIGS) of the host genome [10]. Thus, VIGS-mediated suppression of *GhNDR1*, *GhMKK2,* and *GbVE1* gene expression in cotton increased its resistance to *V. dahliae* [10].

Summarizing these, RNAi is a promising approach to develop cotton varieties resistant to biotic stresses. However, the application of this approach is limited by high probability of effect on nontarget organisms and complexity of cotton genome, due to tetraploidy [10].

*Genome editing approaches.* GE methods are also promising for developing pathogen and pest-resistant cotton varieties. For example, CRISPR/Cas9-mediated editing *Cotton Breeding in the View of Abiotic and Biotic Stresses: Challenges and Perspectives DOI: http://dx.doi.org/10.5772/intechopen.104761*

of Gh14–3-3d gene, which is a negative regulator of disease resistance, has allowed obtaining cotton lines with high resistance to *V. dahliae* [50, 76]. However, despite the promise of GE methods, their application is limited by the complexity of the genome of cultivated tetraploid cotton species, needing to edit both homologs in A- and D-subgenomes.

Thus, summarizing the data above, transgenic methods are currently the most used and commercially successful strategy for developing of new insect pest and pathogen-resistant varieties.

## **4. Conclusion and future perspectives**

Global climate change has a significant impact on cotton production through the complex impact of abiotic and biotic factors, reducing yields and fiber quality [4, 8, 13, 14, 51]. This poses a task to breeders of developing new cotton varieties that are resistant to abiotic and biotic stresses. To challenge it, breeders use both classical and molecular breeding methods and genetic engineering.

By developing cotton varieties resistant to abiotic stresses, molecular breeding methods are more often used, while genomic transgenomic methods improve resistance to insect pests and pathogens [8, 14]. However, the use of modern genetic engineering approaches, including cis- and intragenesis methods, is limited by the complexity of the genome of cultivated tetraploid cotton species. Therefore, the application of RNAi and GE methods to obtain cotton varieties resistant to abiotic and biotic stresses is currently insignificant [8, 14].

In addition, the insignificance of using molecular breeding methods to create pest and pathogen-resistant cotton varieties should be noted. This is due to the insignificant number of mapped insect and pathogen resistance loci in the cotton genome [59–70].

Fundamental understanding of molecular and genetic mechanisms underlying cotton resistance to abiotic and biotic stresses will allow application of cis- and intragenesis methods as well as RNAi and GE technologies in new resistant varieties development. Thus, the genes encoding DRE-binding protein 1 (*GhDBP1*), Na+ /H+ antiporter (*SOS1*; *GhNHX1*), and H+ -pyrophosphatase (*AVP1*) are promising genes for improving drought and salt tolerance [14]. Overexpression of own heat shock genes can be used to improve resistance to heat stress [13]. RNAi and GE technologies can also be used to reduce the expression level of negative regulators of resistance to abiotic stresses.

Studying the mechanisms of interaction between the host plant and insect pests or pathogens, as well as the molecular and genetic basis of life support functions of causative agents, will allow more successful use of the HIGS, RNAi, and GE technologies to suppress key genes and cisgenesis technologies to enhance the host plant defense mechanisms. Genes encoding Vacuolar-type ATPase (*V-ATPaseE*), tubulin-folding cofactor D (*TBCD*), choline acetyltransferases, receptor for activated C kinases (*RACK*), and zinc finger transcription factor (*HUNCHBACK*) are promising for HIGS approach application. Overexpression of key genes of stress hormone biosynthesis (SA, JA, and ethylene) can be used to enhance the protective properties of cotton. In addition, pyramiding the genes for different resistance traits to develop varieties with combined resistance to stresses is promising.

Thus, modern molecular biology technologies have great potential to reduce the negative effect of global climate changes on cotton yield and fiber quality.

*Cotton*

## **Author details**

Venera Kamburova\*, Ilkhom Salakhutdinov and Ibrokhim Y. Abdurakhmonov Center of Genomics and Bioinformatics, Academy of Science of Republic of Uzbekistan, Tashkent, Uzbekistan

\*Address all correspondence to: venera.k75@gmail.com

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*Cotton Breeding in the View of Abiotic and Biotic Stresses: Challenges and Perspectives DOI: http://dx.doi.org/10.5772/intechopen.104761*

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## **Chapter 7**
