**Author details**

Gopikrishna Madanan1 \*† and Deepti Das Krishna2†

1 Department of Physics, Government Victoria College, Palakkad, Kerala, India

2 Department of Electronics, Cochin University of Science and Technology, Cochin, Kerala, India

\*Address all correspondence to<EMAIL_ADDRESS>
† These authors contributed equally.

© 2020 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.


represents *p* < 0.05 compared with control group; a represents this data was cited in our last paper (Tian et al., 2021) [15].

#### *2.5. Histopathology*

There were some histopathological changes in three group animals, including: slight interstitial inflammation in the lungs; slight stomach glandular dilatation; slight inflammatory cell infiltration and slight vacuolation in livers; slight interstitial inflammatory infiltrate, hyaline casts and calcium deposition in kidneys; testis atrophy and sperm loss in epididymis. Details are shown in Table 5 and Figure 6. Histopathological results in all tissues did not reveal any significant difference between the Gm and Jimai22 groups.

**Table 5.** Histopathological results of three groups at week 13.

"-": Not applicable.

**Figure 6.** *Cont*.

**Figure 6.** Non-specific histopathological changes in rats at week 13. (**A**): slight interstitial inflammation of lung in Jimai22 group; (**B**): Slight glandular dilatation of stomach in Jimai22 group; (**C**): slight inflammatory cells infiltration of liver in Jimai22 group; (**D**,**E**): slight hepatic vacuolation in Jimai22 and Gm group; (**F**–**H**): slight renal interstitial inflammatory cell infiltration in Control, Jimai22 and Gm group, respectively; (**I**,**J**): Slight renal hyaline casts in Jimai22 group and Gm group; (**K**–**M**): slight renal calcium deposition in Control, Jimai22 and Gm group, respectively; (**N**–**P**): testicular atrophy in Control, Jimai22 and Gm group, respectively; (**Q**,**R**): epididymal sperm loss in Control, Jimai22 group.

#### **3. Discussion**

Dehydration responsive element-binding proteins (DREBs) are members of a larger family of transcription factors (TFs), which are involved in the resistance to abiotic stressinduced oxidative damages by regulating the expression of genes in the stress defense pathway [17,18]. In addition, DREBs participate in the induction of salinity tolerance by acting on the downstream (auxin and ethylene signaling pathways) and upstream (ABA-independent signaling pathway) [19]. DREBs are also involved in immunity against several biotic stresses by modulating the expression of several downstream genes of the defense signaling cascade [20]. Therefore, DREB genes were used widely to genetically engineer plants for resistance to abiotic factors. The Gm wheat used in this study is a new variety produced by genetic engineering technology to introduce the GmDREB3 gene to the original wheat.

Previous nutritional analysis showed that this wheat was nutritionally equivalent to its non-Gm counterpart [21]; preceding studies also showed that this Gm wheat was safe in immunotoxicity studies [15,22], and in two-generation reproduction studies [14,15]. According to the OECD Test Guidelines and the Guidelines for Safety Assessment of Genetically Modified Plants of China, 90-day subchronic toxicity experiments in rodents are generally considered necessary for safety evaluation of all genetically modified foods [23–25], but routine 90-day subchronic toxicity assessment in transgenic foods were always conducted in single generation animals. Some studies suggested that long-term and multi-generational assessments are necessary in some cases to further assure the safety of Gm food/feed [26,27]. So far, no 90-day subchronic toxicity study has been performed to address the potential toxicological effects in animals exposed to GM feed over three generations. Consequently, a 13-week subchronic toxicity study was conducted in Wistar rats whose parental animals were pre-exposed to the Gm wheat for two generations.

In the present study, the Jimai22 group was used to test whether this Gm wheat would create any toxic effects due to the insertion of the GmDREB3 gene, while the AIN-93 basic diet was used as a nutritional control to analyze whether the wheat in the diet would affect the nutritional status of the rats. During the period of the 13-week study, compared to Jimai22 group, the body weights of the Gm female group were slightly higher in weeks 2, 3, 4 and 6, which was considered a normal body weight fluctuation. In addition, no remarkable difference was detected in the weekly food consumption, total body weight gains, total food consumption, or total food utilization rate, which also confirmed that the above-mentioned differences were of no toxic concern.

In hematology and serum biochemistry, compared with the Jimai22 group Gm male rats had lower HCT% and Hb and a higher CHOL, Gm females had a lower BUN and a higher CHOL. When compared with the control group: Gm females had lower HCT%, BUN, ALT and AST, higher Glu, CHOL, TP and Ca2+; Gm males had lower HCT%, AST, Na+, Cl− and a higher Glu. All of the above-mentioned differences were considered to have no toxicological significance since these values were similar to those of the control group and the Jimai22 group; in addition, these observed differences were within the normal reference ranges for rats of this age in our laboratory. Serum total T4, T3 and TSH should be measured on samples obtained from each animal according to OECD 408 (2018 revised version), but these indexes was absent in this study because the investigation was performed in 2016. We acknowledge it was a limitation of the study. We will strictly refer to OECD guideline 408 (2018 version) in the future study of 90-day subchronic toxicity.

With respect to organ weights, significant increases were presented in absolute and relative weights of livers and adrenal glands in female rats of the Gm group, as compared with the Jimai22 group, which were considered to be not treatment-related since the respective findings were not supported by the biochemical or histopathological data generated in our study. Compared with the control group, Gm female rats had higher absolute weights of heart, liver, spleen, kidneys and thymus, and lower relative weights of brain and ovaries; also, Gm male rats had lower relative weights of heart and brain, which were attributed to increased body weights (females and males in Gm group) at the time of necropsy and were considered to have no toxicological significance.

In the histopathological analysis, some kinds of lesions were observed in the Gm group, but the severity and frequency of these lesions were comparable between the Gm and Jimai 22 group. Furthermore, the incidences of these findings were all within the normal reference range for such data established in our laboratory. Thus, all of the abovementioned histopathological alterations in the Gm group were interpreted as incidental and were considered to be of no significance related to toxicity.

#### **4. Materials and Methods**

#### *4.1. Plant and Diet*

The Gm and Jimai22 wheats were provided by the Chinese Academy of Agricultural Sciences. Both seeds were processed into flour, then the two kind of wheat flour were formulated into rodent diets; both were at a percentage of 69.55% as previous diets of two generations, and meanwhile, the AIN-93 diet was set as a nutritional control [28]. The target genes in Gm, Jimai22 and control diets were detected using the PCR method [14].

#### *4.2. Experiment Animals and Breeding Condition*

In total, 120 specific pathogen-free (SPF) Wistar rats (60 males and 60 females) were obtained from previous two-generation reproductive experiments. The rats were housed in a good experimental environment and could eat and drink ad libitum. All experimental processes were approved and supervised by the Hubei Provincial Animal Management and Use Committee (NO. 2015022).

#### *4.3. Study Design and Administration*

The previous two generations of rats (F0 and F1 generation) were fed Gm diet, Jimai22 diet and AIN-93 control diet for their lifetime. A total of 20 males and 20 females of the third generation rats (F2 generation) were randomly selected from each group and were given the same diet as their parents for 13 weeks, to investigate the subchronic toxicity of this Gm wheat.

#### *4.4. Clinical Observations, Body Weight and Food Consumption*

All animals were observed daily for signs of toxicity and the availability of food. Animal body weights and food consumption of each group were recorded per week; total body weight gains, total food consumption and total food utilization rate for weeks 1–13 were calculated.

#### *4.5. Urinalysis*

Urine samples of all rats were collected by metabolism cages one day prior to necropsy. During the process of urine collection water was available to all rats freely. After observing and recording the urine appearance and volume, the urine indices, including BIL, UBG, KET, PRO, NIT, GLU, LEU, BLD, SG and PH, were determined using an automatic urine analyzer (Dirui; Changchun; China).

#### *4.6. Hematology and Serum Biochemistry*

At the end of week 13, all animals of the three groups were fasted overnight for 16 h and were given drinking water as before, then the blood samples were collected from abdominal aorta under anesthesia. RBC, WBC, PLT, HCT, Hb, NEUT%, LYMPH%, MONO%, EO% and BASO% were determined using an automatic blood analyzer XT2000iv (Sysmex Corporation; Godo; Japan). PT and APTT were measured using a coagulation function analyzer CA510 (Sysmex Corporation; Godo; Japan). GLU, BUN, CREA, CHOL, TG, ALT, AST, TP, ALB, ALB/GLO, Na+, K+, Cl− and Ca2+ were measured using an automatic analyzer AU680 (Beckman Coulter, Inc.; California; America).

#### *4.7. Histopathology*

All animals were sacrificed and subsequently submitted to detailed necropsy and histopathology examination. Brain, heart, liver, spleen, kidneys, adrenal glands, thymus, ovaries (female), uterus (female), testes (male), epididymides (male) and prostate (male) were weighed, and the relative organ weights were calculated. The following organs were examined under a microscope: brain, pituitary, spinal cord, sciatic nerve, thyroid, parathyroid, salivary gland, sternum, thymus, oesophagus, trachea, heart, lung, stomach, spleen, liver, duodenum, jejunum, ileum, colon, rectum, pancreas, adrenals, kidneys, aorta, ovaries, uterus, mammary gland, testes, epididymides, prostate, urinary, bladder skeletal muscle, skin, eyes, and lymph node. In the histopathological analysis, the incidence and semi-quantitative score system used was recommended by Shackelford et al. [29]. The degree of lesions in each item was graded from one to five depending on severity: 1 = minimal (<1%); 2 = slight (1–25%); 3 = moderate (26–50%); 4 = moderate/severe (51–75%); 5 = severe/high (76–100%).

#### *4.8. Statistical Analysis*

Quantitative data in this study including animal body weights, food consumption, organ weight, hematology and biochemistry data were analyzed using one-way analysis of variance (ANOVA), then followed by Student's *t*-test when the variance of three group data was homogeneous. Urine data were analyzed using the Kruskal–Wallist test, and histopathological changes were analyzed using Chi-square test and Fisher's exact test. Statistically significant differences were considered if the *p*-value between the groups was less than 0.05.

#### **5. Conclusions**

The results of the 13-week subchronic toxicity study of the Gm wheat did not exhibit any toxic effects in Wistar rats, whose ancestors had been pre-exposed to the same wheat for two generations. This suggests that Gm wheat is as safe for food as its parental wheat in our current study.

**Author Contributions:** Writing—original draft: J.T.; investigation: J.T., X.-H.K., Y.Y., W.-X.Y., X.-Q.T., L.-J.P. and J.F.; methodology: B.-L.F., J.T., X.-G.Y. and Q.Z.; supervision: B.-L.F. and J.-F.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by Ministry of Agriculture of the People's Republic of China (2016ZX08011005) and Hubei Provincial Public Health Academic Leader Program (No. 2021-73).

**Institutional Review Board Statement:** All experimental procedures were approved and supervised by the Hubei Provincial Animal Management and Use Committee (No. 2015022).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are availability on request from the corresponding author. The data are not publicly available due to privacy.

**Acknowledgments:** We thank Ming Chen for kindly providing the samples of Gm wheat and Jimai22 control wheat.

**Conflicts of Interest:** The authors declare that they have no conflict of interest.

#### **References**


**4. Salt tolerance approaches**

Considering the adverse effects of salt stress in wheat plant, biologists are trying to find out the salt-tolerant strategies in plants by different approaches. Many researchers found positive effect in using exogenous phytoprotectants in alleviating salt-induced damages in wheat. In this section, some of the evidences are discussed.

#### **4.1. Use of osmoprotectants**

To prevent the adverse effects of various environmental stresses including salt stress, plants demonstrate a variety of adaptive mechanisms both at the cellular and organismal levels. Under salt stress conditions, to cope with the salt-induced osmotic, ionic as well as oxidative stresses, plant synthesizes and accumulates organic compatible solutes or osmolytes [48, 65, 66]. Accumulation of these compatible solutes is one of the most important physiological strategies employed by plants under salt stress conditions. Osmoprotectants or osmolytes are small, highly soluble, uncharged, and nontoxic organic molecules which help to survive organisms in extreme osmotic stresses. Osmoprotectants comprise of (i) α-amino acids such as proline (Pro) and ectoine; (ii) ammonium compounds such as glycine betaine (GB), β-alanine betaine, dimethylsulfoniopropionate (DMSP), and choline; and (iii) polyols, sugars, and sugar alcohols such as trehalose (Tre), sorbitol, and mannitol, etc. These osmoprotectants perform vital functions in osmotic adjustment, stabilizing proteins and membranes. Thus, enhanced salt stress tolerance is observed in plants overexpressing the osmoprotectants biosynthetic and metabolic genes. Enhanced salt exposure causes increased biosynthesis of osmoprotectants (Pro, GB, Tre, ecotine, and sorbitol, etc.) which provides enhanced osmotic stress tolerance generated from salt stresses [67, 68] (**Table 3**). For mitigating salt-induced damages, in recent decades, the use of exogenous osmoprotectants has been found effective [12, 69]. Several research findings demonstrated that the use of osmoprotectants provided significant protection against adverse effects of salt stresses in *T. aestivum* seedlings (**Table 3**). At the same time, several research studies proved Pro as a potent protectant against the adverse effects of salt. Proline acts not only in osmotic adjustment as a compatible solute, but also in scavenging ROS, chelating metal, activating detoxification pathways, balancing cells redox status, buffering cytosolic pH, storing energy (carbon and nitrogen), stabilizing subcellular membranes and structures including photosystem II (PS II), and as signaling molecule [70–74]. Raza et al. [75] demonstrated the effect of exogenous GB (50 mM and 100 mM) in moderately salt-sensitive (MH-97) and salt-tolerant (S-24) wheat cultivars grown under salt stress (15 dS m−1 NaCl). Glycine betaine treatment ameliorated the salt-induced photosynthetic reduction as well as increased the photosynthetic capacity, water use efficiency, and osmotic adjustment where salt-tolerant (S-24) cultivar showed better performance against salt stress compared to moderately salt-sensitive (MH-97) cultivar. Later, with the same experimental procedure, they again suggested that the exogenous GB modulated the activities of antioxidant enzymes such as SOD, CAT, and POD which contributed significantly to salt stress tolerance in *T. aestivum* [76]. It has been reported that accumulation of Pro protects *T. aestivum* from the salt-induced damages by maintaining a higher K+ /Na+ ratio and reducing ionic toxicity [38], increasing the major antioxidant enzymes (CAT, APX, SOD, and POD) activities [77]. In *T. aestivum*, GB (10 mM and 30 mM) supplementation with salt stress (150 mM NaCl) increased the germination percentage, shoot Ca content, total chl content, and thus confer salt stress tolerance [78]. Khan et al. [79] reported that increased grain yield in *T. aestivum* associated with the increased Pro, chl content and K+ /Na+ ratio. Overexpression of GB in transgenic *T. aestivum* lines T1, T4, T6, and Shi 4185 (wild type line) caused enhanced salt stress (200 mM NaCl) tolerance by enhancing ROS scavenging, osmotic adjustment and regulating ion homeostasis [80]. Salt stresses (10 dS m−1 NaCl) were imposed in two wheat cultivars (cv. Seher and Lasani). In both wheat cultivars, salt stresses caused significant reduction in the germination percentage, chl contents and growth. Exogenous Pro (50 and 100 mM) application alleviated the adverse effects of salt stress by improving the germination percentage, seedling growth and chl contents of wheat plants but 100 mM Pro was found more effective compared to 50 mM Pro [81]. Mahboob et al. [82] reported that the supplementation of Pro (50 and 100 mM) ameliorated the salt (60 and 120 mM NaCl) induced reduction of plant growth, photosynthetic pigments and ionic balance by increasing shoot and root length, chl *a*, *b* contents*,* FW and DW of seedlings and endogenous Pro, GB, and K+ /Na+ ratio in *T. aestivum* seedlings. Exogenous Pro (60 ppm) upregulated the endogenous hormones (gibberelic acid (GA3 ) and indole acetic acid (IAA)), ammonium compounds (GB and choline) and downregulated the MDA content and growth inhibitor abscisic acid (ABA) in salt stressed *T. aestivum* [83]. Salt (50, 150, and 300 mM) induced disruption of photosynthetic pigments and protein polypeptide synthesis in *T. aestivum* were prevented by the exogenously applied Pro (50 ppm) and at the same time by protecting the turnover machinery of proteins [84]. Besides osmotic adjustment, GB is also involved in ROS scavenging, stabilizing macromolecules (nucleic acids, proteins, and lipids) and various components of photosynthetic machinery such as PS II complexes and RuBisCO and acts as reservoir of carbon and nitrogen sources [85–87]. Upon salt exposure (150 mM NaCl), reduced lipid peroxidation, increased glutathione (GSH) and GB concentrations, enhanced plasma membrane protection, increased cell solute potential and improved ion homeostasis were observed when caryopsis of *T. aestivum* were primed with GB (25, 50, 100 mM) [88]. Increasing the K+ /Na+ and Ca+ /Na+ ratios, reducing MDA content, protecting photosynthetic apparatus, improving plasma membrane integrity and stabilizing macromolecules (proteins, PS II and transporters) GB (20 mM) imparted in salt stress tolerance in *T. aestivum* [83]. Exogenous GB (5 mM) application improved chl *a*, total chl and K+ content of roots, increased root length, plant height, FW and DW of *T. aestivum* under salt stresses (100 and 200 mM NaCl) [89]. Rao et al. [57] suggested that the enhanced production of Pro and GB in six salt-tolerant cultivars (*T. aestivum* cv. AUQAB-2000, PUNJAB-85, PIRSABAK-05, BAKHAR-2002, FARKHARE-SARHAD and KAGHAN-94) alleviated the damaging effects of salt stress by activating their antioxidant enzymes. Endogenous Pro and GB mediated salt stress (8 EC, 16 EC) mitigation in fifteen *T. aestivum* cultivars were further reported by Rao et al. [57]. They suggested that the five cultivars of wheat (SEHAR-2006, LU26-CTR, NARC-2009, BARS-2009, PIRSABAK-09) showed obvious salt stress tolerance by increasing the production of Pro and GB. Yan and Zheng [90] demonstrated that pretreatment with Tre (2, 20, and 40 mM) alleviated the adverse effects of salt stress (3 g L−1 NaCl) in *T. aestivum* cv. Yangmai-19. Various beneficial effects were observed in different physiological parameters. Increased relative growth rate, relative chl content, N content, DW and biomass plant-1 were observed with Trehalose supplementation. Trehalose application also improved Pro accumulation, K+ accumulation and K+ /Na+ ratio. In addition,

**4. Salt tolerance approaches**

160 Wheat Improvement, Management and Utilization

**4.1. Use of osmoprotectants**

ages by maintaining a higher K+

/Na+

antioxidant enzymes (CAT, APX, SOD, and POD) activities [77]. In *T. aestivum*, GB (10 mM and

ratio and reducing ionic toxicity [38], increasing the major

this section, some of the evidences are discussed.

Considering the adverse effects of salt stress in wheat plant, biologists are trying to find out the salt-tolerant strategies in plants by different approaches. Many researchers found positive effect in using exogenous phytoprotectants in alleviating salt-induced damages in wheat. In

To prevent the adverse effects of various environmental stresses including salt stress, plants demonstrate a variety of adaptive mechanisms both at the cellular and organismal levels. Under salt stress conditions, to cope with the salt-induced osmotic, ionic as well as oxidative stresses, plant synthesizes and accumulates organic compatible solutes or osmolytes [48, 65, 66]. Accumulation of these compatible solutes is one of the most important physiological strategies employed by plants under salt stress conditions. Osmoprotectants or osmolytes are small, highly soluble, uncharged, and nontoxic organic molecules which help to survive organisms in extreme osmotic stresses. Osmoprotectants comprise of (i) α-amino acids such as proline (Pro) and ectoine; (ii) ammonium compounds such as glycine betaine (GB), β-alanine betaine, dimethylsulfoniopropionate (DMSP), and choline; and (iii) polyols, sugars, and sugar alcohols such as trehalose (Tre), sorbitol, and mannitol, etc. These osmoprotectants perform vital functions in osmotic adjustment, stabilizing proteins and membranes. Thus, enhanced salt stress tolerance is observed in plants overexpressing the osmoprotectants biosynthetic and metabolic genes. Enhanced salt exposure causes increased biosynthesis of osmoprotectants (Pro, GB, Tre, ecotine, and sorbitol, etc.) which provides enhanced osmotic stress tolerance generated from salt stresses [67, 68] (**Table 3**). For mitigating salt-induced damages, in recent decades, the use of exogenous osmoprotectants has been found effective [12, 69]. Several research findings demonstrated that the use of osmoprotectants provided significant protection against adverse effects of salt stresses in *T. aestivum* seedlings (**Table 3**). At the same time, several research studies proved Pro as a potent protectant against the adverse effects of salt. Proline acts not only in osmotic adjustment as a compatible solute, but also in scavenging ROS, chelating metal, activating detoxification pathways, balancing cells redox status, buffering cytosolic pH, storing energy (carbon and nitrogen), stabilizing subcellular membranes and structures including photosystem II (PS II), and as signaling molecule [70–74]. Raza et al. [75] demonstrated the effect of exogenous GB (50 mM and 100 mM) in moderately salt-sensitive (MH-97) and salt-tolerant (S-24) wheat cultivars grown under salt stress (15 dS m−1 NaCl). Glycine betaine treatment ameliorated the salt-induced photosynthetic reduction as well as increased the photosynthetic capacity, water use efficiency, and osmotic adjustment where salt-tolerant (S-24) cultivar showed better performance against salt stress compared to moderately salt-sensitive (MH-97) cultivar. Later, with the same experimental procedure, they again suggested that the exogenous GB modulated the activities of antioxidant enzymes such as SOD, CAT, and POD which contributed significantly to salt stress tolerance in *T. aestivum* [76]. It has been reported that accumulation of Pro protects *T. aestivum* from the salt-induced damTre has functions in stabilizing the biomolecules and structures like membrane lipids, proteins under salt stress [91–93]. Salt-sensitive wheat cultivar (*T. aestivum* cv. Kızıltan-91) under salt stress (100 mM NaCl) showed physiological alteration. However, pretreatment with exogenous mannitol (100 mM) reversed the deleterious salt effects by increasing antioxidant enzymes (such as SOD, POD, CAT, APX, and GR) activities, appearance of SOD and POD isozyme activity bands and reducing lipid peroxidation [59].

romolecules (proteins, PS II, and transporters)

**Table 3.** Protective effects of various exogenously applied osmoprotectants under salt stress in *T. aestivum*.

Priming of *T. aestivum* seeds with choline (5 and 10 mM) reduced the damaging effects of NaCl (150 mM) by increasing the K+ , Ca2+, GB accumulation, improved ion homeostasis and decreased Na+ and Cl<sup>−</sup> in both shoot and root, mitigated PM permeability and reduced lipid peroxidation of leaf [94]. Expression of *mtlD* gene encoding mannitol-1-phosphate dehydrogenase resulted in enhanced salt stress tolerance in *T. aestivum* due to defensive roles of mannitol against salt stress [95]. The *mtlD* gene encoding mannitol-1-phosphate dehydrogenase transformation in *T. aestivum* cv. Giza 163 conferred salt stress tolerance by inducing mannitol and reducing sugars in tissues of plant [96]. Kerepesi et al. [97] demonstrated that increased fructan contents in salt resistant (Sa) and moderately salt-tolerant (Ch) varieties of *T. aestivum* showed increased tolerance against salt stress (200 mM NaCl). Sharbatkhari et al. [98] investigated the role of fructan in salt-tolerant (Bam) and salt-sensitive (Ghods) cultivars of *T. aestivum*. They found higher fructan accumulation and remobilization in salt-tolerant Bam cultivar, which contributed to the higher salt stress tolerance by increasing the photosynthetic capacity and decreasing the salt induced severe yield loss.

#### **4.2. Plant hormone**

Tre has functions in stabilizing the biomolecules and structures like membrane lipids, proteins under salt stress [91–93]. Salt-sensitive wheat cultivar (*T. aestivum* cv. Kızıltan-91) under salt stress (100 mM NaCl) showed physiological alteration. However, pretreatment with exogenous mannitol (100 mM) reversed the deleterious salt effects by increasing antioxidant enzymes (such as SOD, POD, CAT, APX, and GR) activities, appearance of SOD and POD

> • Increased shoot and root length • Increased FW and DW of seedlings

• Increased chl *a*, *b* contents • Improved Pro, GB, K+

substomatal CO2

CO2

tion rate)

• Improved WUE

and GR

damage

**Protective effects References**

contents, and K+

concentration, and transpira-

• Improved gaseous exchange parameters (net

• Increased chl *a*, *b*, and total chl contents

• Increase activities of SOD, POD, CAT, APX,

• Reduced lipid peroxidation and membrane

• Increased the GSH and GB concentrations • Enhanced plasma membrane (PM) protection

• Increased endogenous hormones (GA and IAA)

and Ca+

• Improved PM integrity and stabilization of macromolecules (proteins, PS II, and transporters)

/Na+ ratios

• Increased photosynthetic capacity • Increased stomatal conductance

• Reduced lipid peroxidation

• Increased the cell solute potential • Improved ion homeostasis

• Increased chl *a* and *b*

• Increased K+

• Increased GB and choline

• Reduced MDA content

• Decreased MDA content and ABA

/Na+

• Improved K, Ca, and Zn contents

• Protected photosynthetic apparatus

assimilation rate, stomatal conductance,

/Na+ ratio Mahboob et al. [82]

Talat et al. [81]

Raza et al. [75]

Seckin et al. [59]

Salama et al. [88]

Hendawey et al. [83]

Hendawey et al. [83]

isozyme activity bands and reducing lipid peroxidation [59].

**Doses of osmolytes**

50 and 100 mM Pro, foliar spray

50 mM and 100 mM Pro, foliar spray

50 mM and 100 mM GB, foliar spray

100 mM mannitol, pretreatment, 24 h

25, 50, and 100 mM GB, caryopsis priming, 24 h

60 ppm Pro, foliar spray

20 mM GB, foliar spray

**Cultivars Salinity doses**

ESW-9525 and kherman

Seher and Lasani

MH-97 and S-24

Kızıltan-91 100 mM

Gomeza 7 150 mM

Sakha 93 and Gimmeza7

Sakha 93 and Gimmeza7

**and duration**

162 Wheat Improvement, Management and Utilization

60 and 120 mM NaCl, 7 d

10 dS m−1 NaCl, 6 d

15 dS m−1 NaCl

NaCl, 5 d

NaCl, 38 d

10.04 dS m−1 (soil), 35–65 d 7.33 dS m−1 (irrigation water), 35–65 d

10.04 dS m−1 (soil) 7.33 dS m−1 (irrigation water), 35 to 65 d

Plant hormones are chemicals produced within the plants at low concentration involved in regulation of plant development and tolerance towards various stresses including salinity [99]. Now-a-days, various kinds of plant hormones such as ABA, auxin, cytokinins (CK),

**Table 4.** Protective effects of various exogenously applied phytohormones under salt stress in *T. aestivum*.

#### **4.3. Plant nutrient**

brassinosteroids and GA3

164 Wheat Improvement, Management and Utilization

**Cultivars Salinity dose and duration**

(15 ds m−1)

150 mM NaCl, entire growth period

150 mM NaCl, entire growth period

50, 100, 150, and 200 mM NaCl),entire

15 dS m−1, entire life

life cycle

cycle

Auxin (Tryptopan) 4.89 × 10−4 mM, 12 h seed priming

4.89 × 10−1 mM auxin (tryptophan), 12 h seed

priming

spray

15 dS m−1, 8 d 100, 150 and 200 mg L−1

150 mg L−1 GA3

cytokinins (kinetin and BAP), 12 h seed priming

100, 150 and 200 mg L–1 cytokinins (kinetin and BAP), 12 h seed priming

, foliar

MH-97 (salt intolerant), Inqlab-91 (salt tolerant)

MH-97 (salt intolerant), Inqlab-91 (salt tolerant)

Sohag 3 (sensitive), Giza 168 (tolerant)

MH-97, Inqlab-91

MH-97, Inqlab-91

GA3

GA3

are externally used for alleviating various kinds of abiotic stresses

**Dose of phytohormones Protective effects References**

• Increased net assimilation rate

• Increased productive tiller and

assimilation rate

Iqbal and Ashraf [44]

Iqbal and Ashraf [100]

Shaddad et al. [102]

Iqbal et al. [105]

Iqbal and Ashraf [104]

• Increased CO2

• Increased growth

• Increased germination percentage

• Improved ion homeostasis • Increased shoot DW

• Improved leaf area, photosynthetic pigment, carobohydrate, protein, amino acid and Pro content, grain weight

• Increased germination rate • Increased early seedlings growth such as shoot DW and

• Increased plant height, shoot

1000-grain weight, grain yield

root DW

dry biomass • Increased fertile tiller,

grain yield

including salinity (**Table 4**). The plant growth hormone auxin increased the germination percentage, shoots DW and maintained ion homeostasis under salt stress condition [100]. Iqbal and Ashraf [101] reported that seed priming with different auxins alleviated salt stress (15 dS m–1) by maintaining hormonal balance and assimilation rate and improved growth and yield of both tolerant and sensitive cultivars under salt stress condition. Seed priming with

 alleviates the drastic effect of salinity and increases grain weight and grain quality by improving photosynthetic pigments, leaf area and plant growth [102]. Foliar application of

 also confers salt stress tolerance by increasing germination percentage, plant growth and upregulating antioxidant enzyme [103]. Seed priming with cytokinin such as kinetin and benzylaminopurine (BAP) increase germination percentage and grain yield by increasing plant growth, productive tiller and 1000-grain weight under salt stress condition [104, 105]. Gurmani et al. [106] noted that, seed priming with ABA improved salt stress tolerance by increasing net assimilation rate, chl content and decreasing Na uptake. It is also evident that phytohormone brassinosteroid plays role in alleviating salt stress. Ali et al. [107] reported that brassinosteroid increased grain yield by improving photosynthetic attribute, assimilation rate and transpiration rate under salt stress condition (150 mM NaCl). Eleiwa et al. [22] also showed brassinosteroid-induced positive response in wheat seedlings under salt stress conditions (**Table 4**).

> Along with other physiological and biochemical functions, plant nutrients play positive roles in alleviating damage effects of abiotic stresses including salinity (**Table 5**). Exogenous application of K enhanced salt stress tolerance in wheat seedlings by improving photosynthetic pigments, antioxidant enzyme activity, K uptake and decreasing Na uptake [109, 110]. Foliar application of phosphorus (P) also alleviated salt-induced damage by increasing plant biomass, leaf area and decreasing Na uptake [111]. Application of CaSO4 increased plant growth, water status and K and Ca uptake under salt stress condition [112]. Later on, Tian et al. [113] noted that application of Ca(NO3 )2 reduced saltinduced oxidative damage by decreasing lipid peroxidation and electrolyte leakage in wheat seedlings.

**Table 5.** Protective effects of plant nutrients under salt stress in *T. aestivum*.

#### **4.4. Antioxidant**

Antioxidants are important for plants to maintain the ROS level lower. Plant possesses various non-enzymatic antioxidants in their cellular components to protect themselves from oxidative stress. The major antioxidant includes AsA, GSH, tocopherol and some phenolic compounds. Some of these antioxidants showed advanced protection against salt-induced oxidative stress when they were applied exogenously (**Table 6**). However, these are mostly dose dependent. A number of studies have been reported the positive effects of AsA in mitigating salt stress in wheat. Athar et al. [20] studied the effect of AsA on wheat plants subjected to salt stress. Salt stress (150 mM NaCl) caused reduction in growth and photosynthesis which were associated with decrease in tissue K+ /Na+ ratio in both sensitive and moderately tolerant varieties. However, root applied AsA (100 mg L−1) counteracted the adverse effects of salt stress on the growth of tolerant variety which was due to the enhanced endogenous AsA level and CAT activity, and higher photosynthetic capacity, and accumulation of K+ and Ca2+ in the leaves. Their study supports the notion that exogenous AsA counteracts the adverse effects of salt stress on growth of wheat by improving photosynthetic capacity of wheat plants against salt-induced oxidative stress and maintaining ion homeostasis, however, these effects were cultivar specific [20]. Ascobin (compound composed of ascorbic acid and citric acid) was found to be effective in mitigating salt-induced damages in wheat as reported by Elhamid et al. [114]. Salt stress markedly increased the lipid peroxidation while the activities of antioxidant enzymes (SOD, CAT, POD, APX and GR) dramatically increased. However, foliar treatment of wheat cultivars with ascobin could partially alleviate the harmful effect of salinity especially at the lower levels of salinity imposed in the two cultivars of wheat at most of the studied parameters [114]. Apart from the dose, mode of application is also a factor to initiate the protective effect by exogenous AsA. In their study Athar et al. [115] found differential effects when AsA was applied through the rooting medium, or as seed soaking or as foliar spray to salt stressed (120 mM NaCl) wheat plants. Exogenous AsA mitigated the adverse effect, e.g. improved leaf ascorbic acid, activities of CAT, POD, and SOD. Root applied AsA caused more enhancements in photosynthetic capacity and more reduction in leaf sodium (Na+ ) compared with AsA applied as seed soaking or foliar spray. However, the effects were also cultivar specific [115]. In a hydroponic experiment Khan et al. [116] showed that foliar applied AsA (50 and 100 mg L−1) could not alleviate the adverse effects of salt stress on plants, but it improved the growth of nonstressed plants. Since AsA failed to enhance the antioxidant defense, it enhanced the Na+ accumulation in the leaves but did not change the K+ accumulation in the salt-stressed plants. Azzedine et al. [21] observed that the exogenous AsA improved the plant growth under salt stress condition which was partly due to the increased leaf area, improved chl and carotenoid contents, enhanced Pro accumulation, and decreased H2 O2 content. Melatonin (*N*-acetyl-5-methoxytryptamine) is also considered a potential antioxidant in plants which is distributed in many parts of the plant. Due to its universal hydrophilic and hydrophobic nature and solubility in both water and lipid, it can cross cell membranes easily and enter subcellular compartments and hence, considered as an antioxidant and a modulator in multiple plant developmental processes and various stress responses [117]. In their pot experiment, Sadak et al. [117] observed that wheat seeds presoaked with melatonin (100 and 500 μM) provided better growth, photosynthetic pigments, yield, and quality in wheat under salinity (3.85 and 7.69 dS m−1). Melatonin treatments at different levels caused significant increase in yield and yield attributes, carbohydrate, protein, N, P, K, flavonoids, phenolic contents, and antioxidant activity either in nonstressed and salinity-stressed plants relative to their corresponding controls. Importantly, 500 μM melatonin was more effective than 100 μM. Farouk [118] reported that both AsA and α-tocopherol minimized salt-induced senescence of flag leaves of wheat. This was due to enhanced activities of antioxidant enzymes which led to the lower lipid peroxidation and H2 O2 accumulation. Exogenous antioxidants also decreased membrane permeability, Na and Cl content. These higher levels of antioxidants and lower level of H2 O2 in flag leaf might be the prerequisite for delayed leaf senescence in antioxidants-sprayed plants [118].

**4.4. Antioxidant**

**Cultivars Salinity dose and duration**

> 150 mM NaCl, 113 d

166 Wheat Improvement, Management and Utilization

40, 80, and 120 mM NaCl, 90 d

PUNJAB-85 50 mM NaCl, 34 d 3 and 6 mM

Jimai 22 100 mM NaCl, 15 d 17.5 mM

50, 100, 150 and 200 mM K2

25 and 150 mg

O kg–1 soil,

P L–1, foliar application

CaSO4

Ca(NO3

**Table 5.** Protective effects of plant nutrients under salt stress in *T. aestivum*.

)2, 15 d

106 d

K2

150 mM NaCl 400 and 800 mg

110 d

SO4 ,

NAYAB-11 and MILLAT-11

Gemiza 9, Sakha 93

Antioxidants are important for plants to maintain the ROS level lower. Plant possesses various non-enzymatic antioxidants in their cellular components to protect themselves from oxidative stress. The major antioxidant includes AsA, GSH, tocopherol and some phenolic compounds. Some of these antioxidants showed advanced protection against salt-induced oxidative stress when they were applied exogenously (**Table 6**). However, these are mostly dose dependent. A number of studies have been reported the positive effects of AsA in mitigating salt stress in wheat. Athar et al. [20] studied the effect of AsA on wheat plants subjected to salt stress. Salt stress (150 mM NaCl) caused reduction in growth and pho-

**Plant nutrients Protective effects References**

• Increased root length and biomass • Increased plant height and biomass

• Increased plant height and biomass • Increased chl *a*, chl *b* and carotenoid

• Increased SOD and POD activity

• Increased plant height, root length, root and shoot biomass

• Increased leaf number, leaf area and

• Decreased Na uptake and increase

• Increased root and shoot biomass • Increased root and leaf RWC • Increase K and Ca uptake

> •– and H2 O2 contents

• Decreased lipid peroxidation, electro-

• Increased SOD, POD, and CAT

uptake and decreased

Kausar and Gull

El-Lethy et al. [109]

Khan et al. [111]

Zaman et al. [112]

Tian et al. [113]

[110]

• Increased K+

content

chl content

K uptake

• Decreased O2

lyte leakage

activities

Na+ uptake

and moderately tolerant varieties. However, root applied AsA (100 mg L−1) counteracted the adverse effects of salt stress on the growth of tolerant variety which was due to the

/Na+

ratio in both sensitive

tosynthesis which were associated with decrease in tissue K+

**Table 6.** Protective effects of various exogenously applied antioxidants under salt stress in *T. aestivum*.

#### **4.5. Signaling molecules**

**Cultivars Dose and duration of stress**

168 Wheat Improvement, Management and Utilization

Sids 1 and Giza

168

S-24 and MH-97 150 mM NaCl, 58 d 50, or 150 mg L−1

3000 and 6000 mg L−1 NaCl,

throughout the growth duration

75 d

S-24 and MH-97 120 mM NaCl,

S-24 and MH-97 150 mM NaCl,

Waha 150 mM NaCl,

Giza 168 0.23, 3.85, and 7.69

Giza 168 0.8, 7.5, and 11.5 dS

75 d

4 weeks

2 weeks

dS m−1 salinity,

m−1 salinity, 65 d

Huaimai 17 300 mM NaCl, 7 d 100 μM SNP

AsA

200-600 mg L−1 ascorbin (ascorbic acid and citric acid 2:1)

50 and 100 mg L−1

100 mg L−1 AsA or α-tocopherol

(sodium nitroprusside, a nitric oxide/NO donor)

AsA

**Antioxidants Major effects References**

• Improved photosynthesis • Increased AsA content and CAT activities • Improved growth

• Decreased MDA content • Decreased activities of antioxi-

dant enzymes

POD, and SOD

• Decreased Na+

machineries

contents

• Decreased H2

500 μM melatonin • Improved shoot height, number

of shoot

pigments

dant activity

activities • Reduced H2

• Decreased Na+

activities

• Improved germination • Deceased Na content and increased K content • Enhanced CAT and SOD

• Lower Na+

0.7 mM AsA • Increased leaf area

• Improved photosynthesis

• Protection of photosynthesis

• Improved chl and carotenoid

• Enhanced Pro accumulation

• Increased photosynthetic

• Increased carbohydrate, protein, N, P, K, flavonoids, phenolic contents, and antioxi-

• Enhanced antioxidant enzymes

O2

lipid peroxidation, and membrane permeability

accumulation,

contents

and Cl<sup>−</sup>

O2 content

of leaves per plant, FW and DW

content

accumulation

100 mg L−1 AsA • Increased activities of CAT,

content, and

Athar et al. [20]

Elhamid et al. [114]

Athar et al. [115]

Khan et al. [116]

Azzedine et al. [21]

Sadak et al. [117]

Farouk [118]

Zheng et al. [119]

and Ca2+ content

• Decreased Na+

increased K+

Although there are specific signaling roles of phytohormones and antioxidants present in plants, which have been discussed in previous sections, this part will discuss the role of exogenously applied signaling molecules. Among the signaling molecules, nitric oxide (NO) has been widely studied in recent decades, due to its diverse role in tolerance to several abiotic stresses including salinity. Nitric oxide exerts its signaling role through various pathways and through interaction with other molecules (**Figure 3**) [26]. In the last decade, exogenous application of NO through different donors was found to enhance crop growth and productivity under stressful conditions [26]. Zheng et al. [119] observed great improvement in seed germination of wheat under high salinity (300 mM NaCl). Wheat seeds soaked in SNP solution provided better germination under salinity which was associated with decreased Na<sup>+</sup> concentration and increased K+ concentration in the seeds. Exogenous SNP also helped in increasing starch and amylase content in seeds which increased the weights of coleoptile and radical. Moreover, exogenous NO enhanced the activities of SOD and CAT which decreased the oxidative damages evident with lower level of lipid peroxidation, O2 •−, and H2 O2 [119]. Kausar and Shahbaz [120] found the positive effect of foliar applied NO in mitigating salt stress in wheat. Wheat seedlings grown under 100 mM NaCl exhibited reduced growth and photosynthetic rate. However, NO spray ameliorated the effect by enhancing FW of plants, leaf area, stomatal conductance, and internal CO2 concentration. However, NO could not take part role in enhancing PS II activity [120]. In our laboratory, we examined the effect of exogenous NO in conferring salt stress tolerance in wheat [24]. Wheat plant exposed to any level of salt (150 and 300 mM NaCl) caused significant increase in oxidative stress (as indicated by MDA and H2 O2 content). Salt stress-induced oxidative stress was due to the disruption of antioxidant defense. However, the seedlings which were pretreated with NO donor (1 mM SNP) showed enhanced tolerance which was due to increased nonenzymatic antioxidants (AsA and GSH pool) and the activities of monodehydroascorbate reductase (MDHAR), DHAR, GR, glutathione *S*-transferase (GST), GPX, glyoxalase (Gly) I, and Gly II. Therefore, we concluded that both antioxidant defense and glyoxalase systems worked together in enhancing salt stress tolerance as induced by NO [24]. As shown in **Figure 3** Arg is one of the precursors of NO production. Few studies have indicated the role of exogenous Arg in salt stress tolerance in wheat. Qados et al. [121] observed that Arg could alleviate the salt-induced adverse effects in wheat. When wheat plants were exposed to different levels of salinity (2000–8000 ppm NaCl), plant mass, relative water content, yield components (spike length, spike weight, and spikelets per spike), grain yield, straw yield, biological yield, and harvest index decreased in dose dependent manners. Salt stress also deteriorated the chemical constituents of the grains. However, when the grains were presoaked with Arg, they provided better growth, yield components, yield as well as the quality aspects (nutrient content) at harvest [121]. Polyamines are often considered as signaling molecules which interact with NO and also exert direct beneficial effects [124–126]. Saeidnejad et al. [122] found the positive effect of spermine (Spm) in mitigating salt stress (100 and 200 mM NaCl) effect in wheat. In general, although seed priming with Spm showed a slight effect on germination process on both susceptible and tolerant cultivars, Spm application was an effective approach in salinity tolerance induction of wheat cultivars mostly through the activation of enzymatic antioxidants and increasing osmolytes production [122]. H2 O2 , which was previously thought to be a toxic substance and a major ROS recently been considered as signaling molecules. The double role of H2 O2 is now an interesting topic of research of many plant scientists. However, as exogenous application, most of the experiments were conducted using H2 O2 as priming agents or pretreatments rather than using as cotreatment. Signaling cross talk of H2 O2 with NO is also well established since last two decades [127]. Exogenous H2 O2 protected wheat plants from salt-induced damages by enhancing antioxidant defense as reported by Li et al. [123]. The seedlings supplemented with H2 O2 (0.05 μM) decreased the levels of MDA and O2 •−, which was associated with the increased activities of SOD, POD, CAT and APX and the concentration of GSH and carotenoid under salt stress (150 mM NaCl). Exogenous H2 O2 also increased plant height, shoot length, root length, and biomass under saline condition. The results were reversed when H2 O2 scavenger was used that indicated a clear role of H2 O2 in initiating its signaling role when applied at lower concentration [123].

 **Figure 3.** Interaction with PA, H2 O2 , and Arg during NO biosynthesis.

#### **4.6. Seed priming**

tration and increased K+

170 Wheat Improvement, Management and Utilization

H2 O2

using H2

H2 O2 O2

the levels of MDA and O2

O2

O2

cross talk of H2

Exogenous H2

clear role of H2

stomatal conductance, and internal CO2

signaling molecules. The double role of H2

O2

concentration in the seeds. Exogenous SNP also helped in increasing

•−, and H2

concentration. However, NO could not take part role

O2

[119]. Kausar

O2

(0.05 μM) decreased

is now an interesting topic of research of many

O2

scavenger was used that indicated a

, which

starch and amylase content in seeds which increased the weights of coleoptile and radical. Moreover, exogenous NO enhanced the activities of SOD and CAT which decreased the oxi-

and Shahbaz [120] found the positive effect of foliar applied NO in mitigating salt stress in wheat. Wheat seedlings grown under 100 mM NaCl exhibited reduced growth and photosynthetic rate. However, NO spray ameliorated the effect by enhancing FW of plants, leaf area,

in enhancing PS II activity [120]. In our laboratory, we examined the effect of exogenous NO in conferring salt stress tolerance in wheat [24]. Wheat plant exposed to any level of salt (150 and 300 mM NaCl) caused significant increase in oxidative stress (as indicated by MDA and

activation of enzymatic antioxidants and increasing osmolytes production [122]. H2

as reported by Li et al. [123]. The seedlings supplemented with H2

saline condition. The results were reversed when H2

was previously thought to be a toxic substance and a major ROS recently been considered as

plant scientists. However, as exogenous application, most of the experiments were conducted

CAT and APX and the concentration of GSH and carotenoid under salt stress (150 mM NaCl).

protected wheat plants from salt-induced damages by enhancing antioxidant defense

as priming agents or pretreatments rather than using as cotreatment. Signaling

with NO is also well established since last two decades [127]. Exogenous

also increased plant height, shoot length, root length, and biomass under

O2

in initiating its signaling role when applied at lower concentration [123].

•−, which was associated with the increased activities of SOD, POD,

O2

 content). Salt stress-induced oxidative stress was due to the disruption of antioxidant defense. However, the seedlings which were pretreated with NO donor (1 mM SNP) showed enhanced tolerance which was due to increased nonenzymatic antioxidants (AsA and GSH pool) and the activities of monodehydroascorbate reductase (MDHAR), DHAR, GR, glutathione *S*-transferase (GST), GPX, glyoxalase (Gly) I, and Gly II. Therefore, we concluded that both antioxidant defense and glyoxalase systems worked together in enhancing salt stress tolerance as induced by NO [24]. As shown in **Figure 3** Arg is one of the precursors of NO production. Few studies have indicated the role of exogenous Arg in salt stress tolerance in wheat. Qados et al. [121] observed that Arg could alleviate the salt-induced adverse effects in wheat. When wheat plants were exposed to different levels of salinity (2000–8000 ppm NaCl), plant mass, relative water content, yield components (spike length, spike weight, and spikelets per spike), grain yield, straw yield, biological yield, and harvest index decreased in dose dependent manners. Salt stress also deteriorated the chemical constituents of the grains. However, when the grains were presoaked with Arg, they provided better growth, yield components, yield as well as the quality aspects (nutrient content) at harvest [121]. Polyamines are often considered as signaling molecules which interact with NO and also exert direct beneficial effects [124–126]. Saeidnejad et al. [122] found the positive effect of spermine (Spm) in mitigating salt stress (100 and 200 mM NaCl) effect in wheat. In general, although seed priming with Spm showed a slight effect on germination process on both susceptible and tolerant cultivars, Spm application was an effective approach in salinity tolerance induction of wheat cultivars mostly through the

dative damages evident with lower level of lipid peroxidation, O2

Seed priming is one of the easiest and cheapest techniques for successful crop production under various abiotic stress conditions including salinity [128, 129]. Seed priming is a presowing, controlled hydration technique that regulates and increases pregermination metabolic activity during early germination stage, but before radical projection [130, 131]. Seed priming has been effectively affirmed to improve germination percentage and seedling establishment in many crops such as wheat, rice, maize, soybean, canola, sunflower, sugarbeet, etc. [29, 132, 133]. Positive effects of seed priming might originate from de novo synthesis of certain germination-promoting substances, enhancing pregermination metabolites [131], early DNA replication, greater ATP availability, enzyme activation, osmotic adjustments [134], and membrane reorganization through restoring their original structures and reducing leakage of metabolites. Along with synchronous and fast emergence, primed seeds show reduced photo and thermodormancy, a wider range of germination temperatures and better capacity to compete with weeds and pathogens [135, 136]. Seed priming can be an easy solution for crops to overcome adverse environmental situations; it is reliable, simple, low cost, and also low risk technique [128, 137]. Various priming techniques such as hydropriming (soaking seed in water), osmopriming (soaking seed in nutrient, hormone, or chemicals), and halopriming (soaking seed in salt solution) have been developed to increase speed of germination, uniform seedling establishment, and crop production [138].

Seed priming has been effectively shown to increase germination and emergence of seeds of many crops in the tropical and subtropical areas, especially under salt stress conditions [139]. Increased germination rates and better seedling establishment resulted in higher levels of salt stress tolerance and crop yields when seeds were primed. Seed priming has recently been applied to overcome the salt stress problem on agricultural land [137]. Several research findings evidenced the role of seed priming to improve salt stress tolerance in wheat (**Table 7**). Hydropriming for 12 h on six Indian wheat cultivars showed 50% reduction of mean germination time under saline condition [140]. Effect of hydropriming was studied in salt-sensitive (MH-97) and salt-tolerant (AUQAB-2000) cultivars of wheat under salt stress (15 dS m−1) condition [16]. It is well documented that seed osmopriming helps to improve salt stress tolerance in wheat seedlings. Seed osmopriming with PEG-8000 solution showed increased germination percentage, germination index, root and shoot length, and seedling FW and DW than salt-affected wheat seedlings at different salinity levels (4, 8, 12, and 16 dS m−1). It has been reported that seed osmopriming with AsA helped to increase the endogenous AsA content and CAT activity which increased the salt stress tolerances in wheat [141]. Increased germination percentage, early seedling establishment, accumulation of ABA and Pro, and plant growth were featured due to seed osmopriming with 0.05 mM SA in wheat under salt stress condition [142]. Seed halopriming improves plant salt-tolerance by maintaining ion homeostasis mechanism. Salt stress increases the accumulation of Na+ concentrations in the roots and shoots of wheat plants and decreases the uptake of beneficiary nutrients. However, seed halopriming helps to maintain the ion homeostasis by decreasing Na+ concentration and increasing K+ , Ca2+ concentration, and K+ /Na+ ratio in roots and shoots. Increasing K+ and Ca2+ absorption, K+ /Na+ ratio due to seed halopriming under salt stress was connected with vigorous seedling growth and crop production, increased photosynthetic activity, and reduced electrolyte leakage. Seed halopriming with CaCl2 helps in the maintenance of ionic balance by reducing the Na+ and increasing the K+ absorption consequently improves salt stress tolerances [143]. Salt stress also induced oxidative damage by producing ROS. Seed halopriming detoxifies the ROS by increasing the activity of enzymatic antioxidant such as SOD and CAT [43]. Iqbal and Ashraf [100] demonstrated that halopriming with 100 mM KCl, NaCl, and CaCl2 reduced the salt stress affect on growth and grain production of two wheat cultivars. Priming with phytohormone increased germination with better seedling establishment and tolerance to various stresses including salinity. Seed priming of wheat with IAA increased germination percentage by improving amylase activity [144] and mitigated the growth inhibitory effect of salinity [16]. Seed priming of three wheat cultivars with auxin (0, 1, and 2 mg L−1) increased germination percentage, root and shoot length, seedling FW and DW, and yield under salt stress condition [18]. Priming with SA (100 mg L−1) solution for 24 h enhanced growth, photosynthetic pigments such as chl *a*, chl *b* and also increased total soluble and reducing sugar for maintaining osmotic adjustment during salt stress [145]. Iqbal and Ashraf [101] reported that seed priming with GA (150 mg L−1) played a potential role in alleviating salt stress damages by reducing Na+ and Cl<sup>−</sup> concentrations, Na+ / K+ ratio, and increasing K+ and Ca2+ contents. Moreover, seed priming with GA increased germination percentage, seedling growth and yield contributing components under salt stress condition.

Seed priming has been effectively shown to increase germination and emergence of seeds of many crops in the tropical and subtropical areas, especially under salt stress conditions [139]. Increased germination rates and better seedling establishment resulted in higher levels of salt stress tolerance and crop yields when seeds were primed. Seed priming has recently been applied to overcome the salt stress problem on agricultural land [137]. Several research findings evidenced the role of seed priming to improve salt stress tolerance in wheat (**Table 7**). Hydropriming for 12 h on six Indian wheat cultivars showed 50% reduction of mean germination time under saline condition [140]. Effect of hydropriming was studied in salt-sensitive (MH-97) and salt-tolerant (AUQAB-2000) cultivars of wheat under salt stress (15 dS m−1) condition [16]. It is well documented that seed osmopriming helps to improve salt stress tolerance in wheat seedlings. Seed osmopriming with PEG-8000 solution showed increased germination percentage, germination index, root and shoot length, and seedling FW and DW than salt-affected wheat seedlings at different salinity levels (4, 8, 12, and 16 dS m−1). It has been reported that seed osmopriming with AsA helped to increase the endogenous AsA content and CAT activity which increased the salt stress tolerances in wheat [141]. Increased germination percentage, early seedling establishment, accumulation of ABA and Pro, and plant growth were featured due to seed osmopriming with 0.05 mM SA in wheat under salt stress condition [142]. Seed halopriming improves plant salt-tolerance by maintaining ion homeostasis mechanism. Salt stress increases the

concentrations in the roots and shoots of wheat plants and decreases

helps in the maintenance of ionic balance by reducing the Na+

absorption consequently improves salt stress tolerances [143]. Salt stress

and Ca2+ absorption, K+

and Cl<sup>−</sup>

and Ca2+ contents. Moreover, seed priming with GA increased

, Ca2+ concentration, and

concentrations, Na+

ratio due to

and

reduced

/

/Na+

the uptake of beneficiary nutrients. However, seed halopriming helps to maintain the ion

seed halopriming under salt stress was connected with vigorous seedling growth and crop production, increased photosynthetic activity, and reduced electrolyte leakage. Seed

also induced oxidative damage by producing ROS. Seed halopriming detoxifies the ROS by increasing the activity of enzymatic antioxidant such as SOD and CAT [43]. Iqbal and

the salt stress affect on growth and grain production of two wheat cultivars. Priming with phytohormone increased germination with better seedling establishment and tolerance to various stresses including salinity. Seed priming of wheat with IAA increased germination percentage by improving amylase activity [144] and mitigated the growth inhibitory effect of salinity [16]. Seed priming of three wheat cultivars with auxin (0, 1, and 2 mg L−1) increased germination percentage, root and shoot length, seedling FW and DW, and yield under salt stress condition [18]. Priming with SA (100 mg L−1) solution for 24 h enhanced growth, photosynthetic pigments such as chl *a*, chl *b* and also increased total soluble and reducing sugar for maintaining osmotic adjustment during salt stress [145]. Iqbal and Ashraf [101] reported that seed priming with GA (150 mg L−1) played a poten-

germination percentage, seedling growth and yield contributing components under salt

Ashraf [100] demonstrated that halopriming with 100 mM KCl, NaCl, and CaCl2

concentration and increasing K+

accumulation of Na+

halopriming with CaCl2

ratio, and increasing K+

stress condition.

increasing the K+

K+ /Na+

K+

homeostasis by decreasing Na+

172 Wheat Improvement, Management and Utilization

ratio in roots and shoots. Increasing K+

tial role in alleviating salt stress damages by reducing Na+

• Increased SOD, CAT, APX activities

**Table 7.** Beneficial effects of seed priming in improving salt stress tolerance in *T. aestivum*.


**Abstract**

Current advances in microelectronics depend on novel approaches based on the synergistic use of charge and spin dynamics of electrons in multi-functional materials. Such new concepts have already found practical applications in magnetoelectronics or spintronics (e.g., spin valves or nonvolatile memory components). For efficient spintronic devices, it is desirable to have an enhanced spin polarization, that, to work with nearly 100% spin-polarized currents. Since half metallic materials have electrons of unique spin polarization around the Fermi level (finite density of states in only one spin channel), they are promising candidates for use as spin injectors in spintronic devices. Although the Heusler compounds reported in the literature presenting half-metallic ferro/ferrimagnetism are numerous, only a few contain elements with low toxicity, as for example zirconium, being also susceptible of convenient preparation and processing. Therefore, in future, zirconium-based compounds could become a much suitable alternative to the presently known cobalt, iron, chromium, titanium, manganese, or scandium-based half-metallic Heusler compounds, being of interest especially in biomedical spintronic related applications involved in corrosive/active environment.

**Keywords:** Heusler compounds, spintronics, half metallic, spin gapless semiconductors


**7. Concluding remarks**

Inspired by Wacquant's theoretical work connected to advanced marginality, which in symbolic form seems to emerge through territorial stigmatization processes, this chapter has focused on children and young people who grow up in socially

deprived housing areas in Denmark. Children and young people, all of whom have an ethnic minority background, and who live their everyday lives in these housing areas characterized by stigmatization and marginalization. The chapter's analyses show how specific urban and housing areas in Denmark stand out as housing areas that, on the one hand, are identified as holding specific difficulties, for example, with crime, people without a connection to the labor market as well as stories about so-called parallel societies, and on the other hand, is woven into welfare state policies. Welfare state policies, which include both social housing and pedagogical efforts and massively present in the physical space, as in no other urban and housing areas in Denmark and in all aspects of the lives of the people who live in these housing areas.

Wacquant [19, 20, 34, 35] emphasizes that processes of territorial stigmatization can neither be viewed as a static condition nor a neutral process, but something that highly functions through housing policy decisions—in the Nordic context, particularly connected to the public housing sector, and delimited to special housing areas designated as having special difficulties with, for example, unemployment, lack of education, and crime, as turning factors.

Thus, this chapter is based on three housing areas in Denmark, where welfare state policies have designated these housing areas as deprived, and with a specific view to the upbringing and everyday life of children and young people in these housing areas, as well as the work of pedagogues with the children and the young people in the locally rooted leisure and club facilities.

There is nowhere in the data material where the children and young people identify the housing areas they grow up in as places they do not want to live, although both the young boys and men refer to the risk of crime as an omnipresent condition to be dealt with. Strategies for dealing with local stigma seem to highlight how the housing area provides a community for the children and young people, across the three housing areas. The community is both about living close to one's "best friends" and living "close to one's family." At the same time, there seems to be experienced attention to how "easy" involvement in crime is in this housing area, or perhaps rather that it requires something special from the young boys to stay out of it. As actively acting young boys, the movements into the leisure club seem to provide a form of protection, which at the same time provides opportunities to participate in leisure activities, such as sports, computer games, and music with friends and pedagogical staff. For the older young men, who are on their way into adulthood, their experiences, however, include stories about how difficult it can be to create a secure future for yourself, through school, education, and work. The precarious existence, or the contours of it, seems to be connected to lack of schooling, lack of upper secondary education, several interrupted educational courses, the risk of or actual movements into crime, and experiences of having difficulties in finding a spare time job.

At the same time, the analyses show how the pedagogical staff takes on the local stigma in their work in the leisure and youth clubs. In particular, the emphasis on supporting and helping children and young people to move out of the risk of not completing primary school, not obtaining leisure jobs, and upper secondary education, as well as crime prevention efforts, fills the pedagogical strategies aimed at helping children and young people.

With the concept of precariat, this chapter's analyses show how young men with an ethnic minority background experience processes of exclusion from school, education, and the labor market. And at the same time points to the importance of recognizing the connection between the individual subjective experienced point of view and perspective in contexts with wider and deeper societal structures of social and economic inequality.

*Processes of Precarious Living Conditions: Young Men of Ethnic Minority Background Growing… DOI: http://dx.doi.org/10.5772/intechopen.110646*


Figure 1 visualizes the conceptual model of the present study. The conceptualization is based on what determining factors have been covered by previous advertising disclosure research in connection with children (i.e., advertising disclosures factors and individual factors). It further includes factors retrieved from persuasion knowledge development literature that might also be responsible for advertising disclosure effectiveness in children (i.e., environmental and situational factors). Their potential role for a more understandable disclosure for children will be discussed in the following chapter (RQ2b). In addition, other potential factors that could be important in this context but have not received any scientific attention at this time are discussed. This offers opportunities for future research.

#### **5. Discussion**

Based on the results of the literature review, a great part of previous empirical research on advertising disclosures and children focused on persuasion outcomes and thus direct effects. This also includes the activation of persuasion knowledge in terms of the brand to which children are exposed to. It follows that it remains unclear what factors might be especially important in this context and potential moderators.

# *5.1 Advertising Disclosures Factors*

Previous findings for advertising disclosures factors in connection with children appear to be mixed and insufficient. Half of these studies revealed no effects of advertising disclosure factors on children's persuasion knowledge. Other studies showed that specific advertising disclosures factors can affect children's knowledge of persuasion, including disclosure type and content, modality, timing, and repetition. These factors thus seem to play important roles for that advertising disclosures can effectively communicate their message and merit more attention of scholars in the future (De Jans et al. 2019).

While a great part of advertising disclosure factors examined were theoretically derived from disclosure studies with adults (van Reijmersdal et al. 2017), other factors such as the repetition of advertising disclosures were found to be especially relevant in connection with children (Spielvogel, Naderer, and Matthes 2019; Uribe and Fuentes-García 2019). We therefore need to specify other advertising disclosures factors that might be relevant for young consumers. Against this background, the authors de Jans et al. (2018) aimed to create an advertising disclosure more suitable for children. By doing so, they paid special attention to the visual appearance of the disclosure. Based on cocreation workshops with children, an eye-tracking study tested various disclosure designs which differed in their *visual appearance* (e.g. color, font size) *and format* (e.g. symbols, exclamation marks). Future research is highly encouraged to test additional visual appearances more suitable for children. Furthermore, it is recommendable to test how the visual appearance of various advertising disclosures not only affects children's visual attention but also children's disclosure awareness, disclosure understanding, and in the last instance their knowledge of persuasion. Special focus should be given to *symbols*, especially when it comes to young children whose reading skills are still developing.

# *5.2 Individual Factors*

In terms of individual factors, it gets apparent that previous advertising disclosure studies in connection with children build on persuasion knowledge development research. In other words, it is assumed that individual factors such as age do not only directly improve children's knowledge of persuasion but also moderate advertising disclosures effects on persuasion knowledge activation. However, in most of the included studies the effect of advertising disclosures on children's knowledge of persuasion was not contingent on young consumers' developmental stages.

Despite this fact, other individual factors than children's age have received little to no attention by scholars in this research field. Age as a crucial factor for persuasion knowledge development might be less important than previously theorized (Lapierre 2017), especially in the case of contemporary advertising targeted at children (Nairn and Fine 2008). Therefore, other determining individual factors than children's age might be very relevant for the success of an advertising disclosure in the sense of being both more understandable and effective for children.

One advice for future research is to focus more on 'mutable' supporting constructs than on 'immutable' supporting constructs such as age. This advice builds on literature on persuasion knowledge development. It is criticized that scholars predominantly use age as a proxy although persuasion knowledge has been framed as a cognitive skill (Lapierre 2015). When it comes to the question which individual factors support or reduce the effectiveness of advertising disclosures in children, other factors should thus be examined. For instance, to my knowledge no study so far considered the moderating role of children's *reading skills* when testing disclosure effectiveness. While linguistic competence might be an important factor to consider for the development of persuasion knowledge in children (Chernin 2007), reading ability might be even more important for disclosure effectiveness. While consumer's reading ability is not a necessity for the recognition of branded content, the wording of advertising disclosures currently used in practice often comprises full sentences (e.g. "PP – This program contains product placement") or only signal words (e.g. "PP – product placement"; see Spielvogel, Naderer, and Matthes 2020). This highlights that various advertising disclosures call for full developed reading skills in consumers and in some cases symbol comprehension when only using an abbreviation (e.g. "PP").

Inspired by the authors (Boerman, Van Reijmersdal, and Neijens 2015a), another potential influential factor might be children's *disclosure familiarity*. In their study, adult consumers who were familiar with the use of advertising disclosures also increased the chance that the disclosure was recognized. To my knowledge, this potential moderating influence has not yet been studied with children. However, if children are frequently exposed to advertising disclosures and are thus familiar with these cues, this might also increase the chance that children are more aware of disclosures.

#### *5.3 Environmental and Situational Factors*

Compared to the listed factors above, determining factors that take the environment and situation into account constitute a dearth of research in this research field. However, especially when it comes to children, environmental factors such as their family environment are viewed as relevant influencing factors for both advertising effects (Hudders and Cauberghe 2018) and the development of children's advertising skills (Nelson et al. 2017; Terlutter and Spielvogel 2010). This might also be the case for advertising disclosure effects. Furthermore, situational factors such as the amount of visual attention that consumers pay toward the respective area of interest might not only be relevant for advertising effects but also for advertising disclosure effects. Eye-tracking studies in this context already showed that children's amount of visual attention serve as important mediators for the effect of advertising disclosures on children's conceptual persuasion knowledge (Spielvogel, Naderer, and Matthes 2019; van Reijmersdal et al. 2020). However, moderating situational influences have not been subject of research so far.

This circumstance could, however, neglect significant influencing factors for advertising disclosure effects. Another potential influential factor that takes situational aspects into account is consumer's *involvement with the media content*. There is empirical evidence for that adult viewers who are highly involved with the media content are also less likely to remember the disclosure (Boerman, Van Reijmersdal, and Neijens 2015a). The authors conclude that the more viewers engage with a plot, the less cognitive resources remain for disclosure processing. Future research could also examine this assumption with children, for example by manipulating involvement (Matthes, Schemer, and Wirth 2007). When it comes to more contemporary advertising formats such as advergames, children's *experience of game flow* might not only be positively associated with persuasion knowledge (Vanwesenbeeck, Ponnet, and Walrave 2016) but also explain advertising disclosure effects.

#### *5.4* **The Important Role of Disclosure Awareness**

Finally, it has to be pointed out that the results of two sampled studies indicated that advertising disclosures affect several components of conceptual persuasion knowledge *only* among children who correctly remembered seeing the disclosure (Boerman and van Reijmersdal 2020; van Reijmersdal et al. 2017). Furthermore, disclosure recognition appears to serve as an mediator for the effect of advertising disclosures on children's persuasion knowledge (van Reijmersdal et al. 2020). These results highlight that *disclosure awareness* constitute a precondition so that an advertising disclosure can have its intended effect on the activation of persuasion knowledge. Studies with adults also emphasized the necessity of disclosure awareness for advertising disclosure effects (Boerman, van Reijmersdal, and Neijens 2012; Boerman, Van Reijmersdal, and Neijens 2015a; 2015b). Future research should therefore consider disclosure awareness more intensively.

**Fig. 2.:** Overview of Potential Determining Factors for Advertising Disclosures Effectiveness in Children.

Figure 2 summarizes the findings and gives an overview of potential determining factors for advertising disclosures effectiveness in children (assessed by conceptual and attitudinal persuasion knowledge activation). Based on the findings of the literature review of advertising disclosure studies in connection with children, there is a scientific focus on direct effects (grey arrow). Furthermore, Figure 2 visualizes all determining factors that have been covered by previous research in this field (marked in bold). Furthermore, it contains additional identified factors that might be important in this context (both marked in bold and shown in italics). Moreover, it is assumed that some factors do interact with each other (double-headed arrows). Against this background, several studies paid attention to interaction effects of advertising disclosures (factors) and age on children's knowledge of persuasion (e.g. Hoek et al. 2020). Interestingly, one study investigated the interaction between advertising type and the use of disclosures on children's persuasion knowledge (Daems et al. 2019). Although the authors did not examine specific advertising disclosure characteristics, they paid attention to the interplay of disclosures and environmental factors and how this interaction affects children's knowledge of persuasion. Moreover, situational factors such as the amount of visual attention children pay to the commercial are viewed as important mediators for the effects of disclosures on children's persuasion knowledge (Spielvogel, Naderer, and Matthes 2019). However, situational factors may not only constitute mediators but also may moderate the effect of advertising disclosures (factors) on children's persuasion knowledge. In this light, the authors Vanwesenbeeck and colleagues (2016) for instance showed that children's experience of game flow was positively associated with children's knowledge of persuasion.

# **6. Conclusion**

The present study delivers several scientific implications. In sum, the findings of the literature review indicate that advertising disclosures only partly foster components of children's conceptual persuasion knowledge. That is, some studies revealed positive effects while other found no effects at all. These derived effects, however, are only limited to specific components of persuasion knowledge (i.e., mostly the first component of conceptual persuasion knowledge, namely recognition of advertising). When considering the findings for both conceptual and attitudinal persuasion knowledge, it also gets apparent that determining factors might be responsible for the emergence of advertising disclosure effects.

Above all, future research is highly encouraged to pay more attention to the investigation of determining factors than solely testing direct effects. Thereby, scholars can build on and expand the proposed conceptualization of determining factors of the present study. Additionally, it is imperative to provide empirical evidence for the additional factors theoretically derived and proposed by the present study. All of these efforts enable us to develop a more effective and understandable advertising disclosure for children.

#### **References**

# **Sampled studies**


**4. Sustainability, vernacular architecture, and identity**

 In the past three decades, the world witnessed great awareness toward environmental dilemmas such as global warming, resources depletion, energy, air and water pollution, waste, population growth, and globalization. These issues come within the responsibility of arguments on sustainability indicating the necessity for sustainability integration in the ways we live, act, use of resources, and build. In meeting the human needs, sustainable architecture represents the interrelationship between natural, cultural, social, and economic resources to create optimum relationships between people and their environments [18].

The word "sustain" means "to hold up" or "to support from below," a society needs to be supported by its populations in the present and future. Most traditional societies took care of their community through the amalgamation of natural and physical environment with cultural, social, and mystical values. These are the societies where sustainability is evident and livable as daily practices.

When discussing sustainability, we need to go beyond definitions, looking for the essence of the meaning. Sustainability is not merely the integration of environmental, social, and economic issues and improving quality of life [19]. The idea of sustainability is to make sure that our decisions and actions today do not compromise the future generations to meet their own needs. Re-evaluating and analyzing vernacular architecture support the interrelation between policy makers and the society [20]. We have to make sure that our actions go along with our environmental systems not in opposition to them. Sustainable design implies responsibility and it implies a far-reaching respect for natural systems and resources, respect for people and cycle of life [4].

Sustainability is by nature "architecture." If we are to achieve it, we must concretely engage the design issues associated with ambient environment resources. We must address, more fully, the underlying influence(s) of political, economic, and social issues comprising the cultural and spiritual background in which the desire to achieve sustainability in itself reflects such a significant value shift [21]. Sustainable design is a philosophical approach to design that seeks to maximize the quality of the built environment [4].

Vernacular architecture is the result of constrains on resource availability. Historically and without modern means, extraordinary enterprise produced architecture often of the most distinctive character and identity with only limited means available [22]. Vernacular architecture was built on inherently sustainable principles such as resource limitations imposed by economic or natural factors, yet succeeded in offering rational solutions to harsh climates and human need [23]. Elements of sustainable design are integral to every established form of vernacular architecture that are tried and proven solutions and have evolved over long periods of trial and error using local materials and technology emerging from ambient natural and cultural environment.

Vernacular architecture, in the Arab World, is a model for sustainability; it embodies different cultural values, which may be applied in the conceptual design of buildings today. These values are about the way of construction, which is responsive to the climate and suits the style of living, traditions, customs, social values, and culture of people; it is of a great sustainability potential (**Figure 3**).

Sustainability in architecture is an essential part to determine the identity of the architectural regionalism. Hidayatun, Prijotomo, and Rachmawati assert that: "Identity has a permanent nature, permanence is supported by both natural environment and cultural, such as the natural topography, material and cultural includes views and habits. All of this is contained in the sustainable criteria. So the nature of sustainability is an important part in recognizing the identity" [16].

#### **Figure 3.**

*Accommodation to the ambient environment in vernacular architecture in the Arab World: solid walls, small openings, light finishing, courtyards, and wind catchers (Source: Salman, Maha).*

Since our predecessors built sustainability, they did not need to enforce methods to reveal their society or place identity. Identity was evident by being adhered to natural and social environment while society evolution through time. They built naturally in accordance with ambient environment and available resources and building materials; architecture embraced identity as it reflected its place, people, and culture.


**1. Introduction**

Flavanols are the most abundant substances of polyphenols found in cocoa, with a degree of polymerization ranging from monomers to polymeric proanthocyanidins [1]. Therefore, unfermented cocoa bean is composed of 1.3–3.3% methylxanthines [2] and of about 6% condensed flavan-3-ols [3]; as a result, it is has been listed as the 4th richest dietary source of polyphenols [4]. Cocoa is one of the top crops in Colombia in terms of economic impact, with a national production record of 59,665 tons in 2019 according to National Cacao Producers Federation. There is also a government initiative aiming at a switch from cocaine to cocoa and to join forces with the private sector to enhance Colombia's competitiveness at an international level, which has been established in the comprehensive national program for the replacement of illegal crops [5].

Polyphenols have gained increasing attention as supplements and additives in functional foods due to their nutraceutical properties and beneficial health properties. The recovery of polyphenols from cocoa and its by-products through several technologies such as maceration [6], microwave [7], and pressurized liquid extraction [8] has been previously reported. Most of them use powder cocoa beans obtained from processing operations consisting mainly of (a) drying and particle size reduction pretreatment, (b) degreasing, and (c) recovery of secondary metabolites. During the pretreatment stage, low drying temperatures and short times are preferred to avoid oxidation of flavonoids while small particle size is suggested to increase mass transfer.

The total fat content of whole cocoa beans is over 50% (on a dry basis) [9], which constitutes a barrier for the release of polyphenols from the cells; therefore, a degreasing process is commonly employed that can include pressing of the beans, or using a solvent extraction (i.e., hexane, chloroform, petroleum ether or other non-polar solvents) [10,11] to achieve a final fat content lower than<12 wt.% [12]. When defatting using solvents, an additional step for residual solvent removal is thus needed. Supercritical fluid CO2 can be used, which leaves no residue in the final product, but it is more expensive [13].

During the last stage, the extraction yield can vary not only due to the pre-treatment step, but also due to the type of solvent, contact time, temperature, solid to solvent ratio, the structure of the solid matrix and pH [14]. Although several solvents can be used to extract cocoa polyphenols, polar solvents approved by food regulations agencies (i.e., FDA and EFSA) for human consumption (that is, water, ethanol or a mixture of both) are preferred.

Some patented processes can be found in the literature for producing cocoa polyphenol concentrate; these include a series of sequential steps consisting on inhibition of enzymatic browning (blanching with hot water or steam), pressing and supercritical CO2 extraction for fat removal or degreasing with hexane over 6 h [13], reduction of particle size (<500 um) using cryogenic milling (under −5 ◦C), and extraction of polyphenols using hot water [13], 60% 2-propanol [15], acetone/water/acetic acid [16] and aqueous methanol [17].

These are good examples showing that no single universal extraction processes can be employed for the extraction of polyphenols from different plant sources [18]. Therefore, the goal of the present work was to establish the optimal experimental conditions to enhance higher recovery of polyphenols from cocoa beans without degreasing. Thus, a suitable method that was reproducible for large-scale production (0.05–10 L), cheap, used food grade solvents and reliable for food applications was developed based on ultrasound-assisted solid-liquid extraction using aqueous ethanol. The best operational conditions for the pre-treatment process (drying and particle size reduction to avoid the degreasing) and for the recovery of polyphenols were established. In addition, chemical characterization and antioxidant activity of the cocoa polyphenol extract were also studied.

#### **2. Material and Methods**

#### *2.1. Reagents*

All the chemicals used were analytical or reagent grade and were not purified further. Folin-Ciocalteu reagent, gallic acid, sodium carbonate, ascorbic acid, l-cysteine, sodium phosphate monobasic monohydrate (NaH2PO4·H2O), disodium hydrogen phosphate (Na2HPO4), caffeine, and theobromine were obtained from Sigma Aldrich (St. Louis, MO, USA). (+)-Catechin hydrate (≥99%; ASB-000003310), (−)-epicatechin (≥99%; ASB-00005127), procyanidin B2 (≥90%; ASB-00016231) were purchased from ChromaDex Inc. (Irvine, CA, USA). Acetonitrile, methanol, (HPLC-grade), ethanol, *n*-hexane, citric acid and formic acid were acquired from Merck (Merck, Germany). Deionized water (18 MΩ/cm) from an Aqua Solution system (Aqua solution, Inc. Jasper, Georgia, USA) was used for the preparation of all solutions.


**Author details**

Nischal Sharma1 , Rohtash Saini1 , Sreehari K1 , Akash Pathaikara1,2, Pravin Punde1,3 and Raju Attada1 \*

1 Department of Earth and Environmental Sciences, Indian Institute of Science Education and Research Mohali, Punjab, India

2 Division of Environmental Science and Technology, Pohang University of Science and Technology, South Korea

3 Department of Physics and Technology, UiT The Arctic University of Norway Tromsø, Norway

\*Address all correspondence to<EMAIL_ADDRESS>
© 2023 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.


*2.2. Participants and Recruitment*

A total of 10 GA maintenance engineers took part in the study. Participants were recruited using a purposeful sampling approach with ages ranging from 21 to 60 (*M*age = 41, *SD* = 11.37). Nine males and one female took part, broadly representing the gender ratio in GA maintenance engineering. Experience in GA maintenance engineering ranged from 1 to 42 years (*M*exp = 17.8, *SD* = 10.5).


*Edited by Mohammad Manjur Shah and Umar Sharif*

The book discusses the recent advances in basic and applied approaches including research on the genetics of insects, its application in resolving the consequences of world population growth, its impact on agriculture, and control strategies and their implications on the fast-depleting insect resources. The application of insects as a probable nutrient substitute along with the role of sex hormones among insects has been thoroughly discussed. The entire book basically contains five chapters spread over two sections: Section I mainly focuses on diversity, conservation and nutrition, while Section II is concerned with economic importance and up-to-date information on the role of peptides. The book is well illustrated with diagrams, graphical representations and flow charts for easy understanding the important information discussed in the book.

ISBN 978-1-78923-454-1

Insect Science-Diversity, Conservation and Nutrition

Published in London, UK © 2018 IntechOpen © ConstantinCornel / iStock

## Insect Science-Diversity, Conservation and Nutrition

*Edited by Mohammad Manjur Shah and Umar Sharif*

**INSECT SCIENCE-**

**CONSERVATION AND**

Edited by **Mohammad Manjur Shah**

**DIVERSITY,**

**NUTRITION**

and **Umar Sharif**