Other than traditional techniques the Transcription factors (TFs) are emerging as useful resources for genetic engineering to induce drought tolerance in mustard plants, because they act as mjors regulators of various stress-regulatory pathways. Many TFs belonging to families AP2/EREBP, MYB, WRKY, NAC, bZIP have been involved in drought stress tolerance and some TF genes have also been engineered to develop stress resistance in plants. TFs are very important regulators, as they function as terminal transducers and comprehensively regulate the expression of group of downstream genes by combination of specific cis elements in their promoter region [22]. Over-expression of a constitutively active form of AtDREB2A from Arabidopsis has been reported to improve the tolerance to drought and osmotic stresses [23].

#### **2.3 Strategies to develop cold stress tolerance in mustard**

Frost is a sudden crop killer with devastating threat, especially in the north and northeastern parts of India where temperature unexpectedly drops below 0°C. Low-temperature stress not only decreases grain yield but also affects crop grain quality [24]. Shah et al. reported that whole plant death in mustard if frost stress affects the seedling stages. The injury rate of frost stress depends on many important components such as duration and amount of cold stress, different stages of plant growth and moisture content. It has direct effect on the flowering and siliqua development and prevents seed formation, thereby affecting crop productivity, causing considerable yield loss [25].

Cold stress tolerance mechanism in plants is regulated via transcriptional activation or repression. The majority of stress associated proteins such as heat shock, chemical shock and late embryogenesis abundant proteins (HSPs, CSPs and LEA) are accumulate upon extreme temperature stress. They act as molecular chaperones, which are responsible for protecting the cellular machinery in a broad range of cellular processes. Evidence suggests that cold tolerance is linked with the increased expression of genes involved in transcriptional regulation, osmotic adjustment, antioxidant defense and metabolite biosynthesis [26]. Varieties like RGN-48, RK-9001, RH-8816, RGN-13, RH-819, Swaranjyoti, RH-781 have been reported to have good frost tolerance. Mustard crop can be also protected from frost by chemical spray of dimethyl sulphoxide, dithane or 0.15% of H2SO4.

#### **2.4 Strategies to develop heavy metal toxicity stress tolerance in mustard**

Plants largely depend on soil solution to acquire nutrients for their growth and developmental cycle. The recent increase in contamination of arable lands with heavy metals is one of the most important causes of loss in crop productivity [27]. Extensive exposure to heavy metal contamination threatens the sustainability of environmental and agricultural systems. Crops are routinely subjected to metal toxicity due to improper irrigation methods and the addition of excessive quantities of chemical fertilizers, and other synthetic nutrients [28]. Some (potentially toxic) heavy metals, such as Cu, Zn, Ni, Co, Se, and Fe, are also essential elements required for the optimal performance of plants and become toxic when accumulated in excess in soil solution [29, 30]. On the other hand, non-essential elements, such as arsenate (As), cesium (Cs), lead (Pb), and cadmium (Cd), can hamper crop productivity when accumulated in the soil even in trace amounts [31]. Soil contamination with heavy metals causes accumulation of these toxic metals in plant parts, resulting in decreased crop productivity and increased risk to animal and human health [32].

**45**

*Innovative Strategies to Develop Abiotic and Biotic Stress Tolerance in Mustard (Brassicaceae)*

**2.5 Identification of quantitative trait loci (QTL) to develop abiotic stress**

Quantitative trait locus (QTL) is a statistical method developed to analyse and correlate the phenotypic and genotypic data to estimate the genetic variations in complex traits. This technique is less time consuming and gives better mapping resolution by exploring and utilizing each event of recombination that occurs in the evolutionary history. In various crops, QTLs were recognized for a several beneficial agronomic traits, such as enhancing abiotic and biotic stress tolerance, yield and yield contributing factors of the crop, flowering time, root development and uptake of nutrients and nitrogen fixation. Molecular markers linked with various agronomic traits derived from association mapping are reported in crops including soybean [35] and brassica [36, 37]. Lu et al. reported that resequencing of 588 *Brassica napus* accessions from 21 countries has generated 5,294,158 (single nucleotide polymorphisms) SNPsand 1,307,151 indels. The genome-wide association study (GWAS) find 60 loci considerably associated with agronomic traits such as stress tolerance, seed quality etc., which may be proven as a valuable resource for genetic

All these factors such as salt stress, drought and frost decline the crop quality and quantity. These abiotic stresses can be managed by exploring genetic resources material and agronomic factors. Donor lines have been identified for different abiotic stresses and are being used in the breeding programmes for developing tolerant varieties against abiotic stresses. A number of improved cultivars or hybrids have been developed which perform better under different type of stresses resulting in lower reduction upon exposure to stress compared to high yielding varieties. Conventional techniques of plant breeding have not been proved that much successful in addressing abiotic stresses mitigation so far. Therefore reason

The root-associated dark septate endophyte (DSE), *Exophiala pisciphila* isolated from *Zea mays* showed enhanced antioxidant enzyme activity under increased soil Cadmimium (Cd) stress [33]. Three important genes involved in uptake, detoxification and transport of Cd were recognized as downregulation of ZIP, upregulation of PCS and MTP upon inoculation with DSE and exposed to high concentration of Cd. The *Pseudomonas* and *Gigaspora* are reported to alter level of 1-aminocyclopropane-1-carboxylate (ACC) which further increase the tolerance of heavy metals by

*DOI: http://dx.doi.org/10.5772/intechopen.95973*

**tolerance in mustard**

improvement [38].

directly manipulating the ethylene levels in plants [34].

and need for adoption of new molecular approaches.

**3. Biotic stresses in mustard and strategies to develop tolerance**

Besides environmental stresses, biotic stresses are diseases and insect-pests. The fungal diseases are considered as an important biotic constraint in mustard, which leads to significant yield losses of crop world-wide. More than thirty diseases are known to occur on brassica crops in India [39]. However, only few of them are considered as major diseases on the basis of economic yield losses and according to their distribution in the country. Major biotic stresses of rapeseed-mustard in India are Alternaria blight [*Alternaria brassicae* (Berk.) Sacc.], white rust [*Albugo candida* (lev.) Kuntze], Powdery mildew (*Erysiphe cruciferarum*), Downy mildew (Hyaloperonospora parasitica) and Sclerotinia rot (*Sclerotinia sclerotiorum*) which influence the quality and quantity of seed [40–42]. Alternaria blight [*A. brassicae* (Berk.) Sacc.] and White rust [*A. candida* (lev.) Kuntze] have been reported to be

*Innovative Strategies to Develop Abiotic and Biotic Stress Tolerance in Mustard (Brassicaceae) DOI: http://dx.doi.org/10.5772/intechopen.95973*

The root-associated dark septate endophyte (DSE), *Exophiala pisciphila* isolated from *Zea mays* showed enhanced antioxidant enzyme activity under increased soil Cadmimium (Cd) stress [33]. Three important genes involved in uptake, detoxification and transport of Cd were recognized as downregulation of ZIP, upregulation of PCS and MTP upon inoculation with DSE and exposed to high concentration of Cd. The *Pseudomonas* and *Gigaspora* are reported to alter level of 1-aminocyclopropane-1-carboxylate (ACC) which further increase the tolerance of heavy metals by directly manipulating the ethylene levels in plants [34].

#### **2.5 Identification of quantitative trait loci (QTL) to develop abiotic stress tolerance in mustard**

Quantitative trait locus (QTL) is a statistical method developed to analyse and correlate the phenotypic and genotypic data to estimate the genetic variations in complex traits. This technique is less time consuming and gives better mapping resolution by exploring and utilizing each event of recombination that occurs in the evolutionary history. In various crops, QTLs were recognized for a several beneficial agronomic traits, such as enhancing abiotic and biotic stress tolerance, yield and yield contributing factors of the crop, flowering time, root development and uptake of nutrients and nitrogen fixation. Molecular markers linked with various agronomic traits derived from association mapping are reported in crops including soybean [35] and brassica [36, 37]. Lu et al. reported that resequencing of 588 *Brassica napus* accessions from 21 countries has generated 5,294,158 (single nucleotide polymorphisms) SNPsand 1,307,151 indels. The genome-wide association study (GWAS) find 60 loci considerably associated with agronomic traits such as stress tolerance, seed quality etc., which may be proven as a valuable resource for genetic improvement [38].

All these factors such as salt stress, drought and frost decline the crop quality and quantity. These abiotic stresses can be managed by exploring genetic resources material and agronomic factors. Donor lines have been identified for different abiotic stresses and are being used in the breeding programmes for developing tolerant varieties against abiotic stresses. A number of improved cultivars or hybrids have been developed which perform better under different type of stresses resulting in lower reduction upon exposure to stress compared to high yielding varieties. Conventional techniques of plant breeding have not been proved that much successful in addressing abiotic stresses mitigation so far. Therefore reason and need for adoption of new molecular approaches.

#### **3. Biotic stresses in mustard and strategies to develop tolerance**

Besides environmental stresses, biotic stresses are diseases and insect-pests. The fungal diseases are considered as an important biotic constraint in mustard, which leads to significant yield losses of crop world-wide. More than thirty diseases are known to occur on brassica crops in India [39]. However, only few of them are considered as major diseases on the basis of economic yield losses and according to their distribution in the country. Major biotic stresses of rapeseed-mustard in India are Alternaria blight [*Alternaria brassicae* (Berk.) Sacc.], white rust [*Albugo candida* (lev.) Kuntze], Powdery mildew (*Erysiphe cruciferarum*), Downy mildew (Hyaloperonospora parasitica) and Sclerotinia rot (*Sclerotinia sclerotiorum*) which influence the quality and quantity of seed [40–42]. Alternaria blight [*A. brassicae* (Berk.) Sacc.] and White rust [*A. candida* (lev.) Kuntze] have been reported to be

*Brassica Breeding and Biotechnology*

causing considerable yield loss [25].

dimethyl sulphoxide, dithane or 0.15% of H2SO4.

stresses [23].

Other than traditional techniques the Transcription factors (TFs) are emerging as useful resources for genetic engineering to induce drought tolerance in mustard plants, because they act as mjors regulators of various stress-regulatory pathways. Many TFs belonging to families AP2/EREBP, MYB, WRKY, NAC, bZIP have been involved in drought stress tolerance and some TF genes have also been engineered to develop stress resistance in plants. TFs are very important regulators, as they function as terminal transducers and comprehensively regulate the expression of group of downstream genes by combination of specific cis elements in their promoter region [22]. Over-expression of a constitutively active form of AtDREB2A from Arabidopsis has been reported to improve the tolerance to drought and osmotic

Frost is a sudden crop killer with devastating threat, especially in the north and northeastern parts of India where temperature unexpectedly drops below 0°C. Low-temperature stress not only decreases grain yield but also affects crop grain quality [24]. Shah et al. reported that whole plant death in mustard if frost stress affects the seedling stages. The injury rate of frost stress depends on many important components such as duration and amount of cold stress, different stages of plant growth and moisture content. It has direct effect on the flowering and siliqua development and prevents seed formation, thereby affecting crop productivity,

Cold stress tolerance mechanism in plants is regulated via transcriptional activation or repression. The majority of stress associated proteins such as heat shock, chemical shock and late embryogenesis abundant proteins (HSPs, CSPs and LEA) are accumulate upon extreme temperature stress. They act as molecular chaperones, which are responsible for protecting the cellular machinery in a broad range of cellular processes. Evidence suggests that cold tolerance is linked with the increased expression of genes involved in transcriptional regulation, osmotic adjustment, antioxidant defense and metabolite biosynthesis [26]. Varieties like RGN-48, RK-9001, RH-8816, RGN-13, RH-819, Swaranjyoti, RH-781 have been reported to have good frost tolerance. Mustard crop can be also protected from frost by chemical spray of

**2.4 Strategies to develop heavy metal toxicity stress tolerance in mustard**

Plants largely depend on soil solution to acquire nutrients for their growth and developmental cycle. The recent increase in contamination of arable lands with heavy metals is one of the most important causes of loss in crop productivity [27]. Extensive exposure to heavy metal contamination threatens the sustainability of environmental and agricultural systems. Crops are routinely subjected to metal toxicity due to improper irrigation methods and the addition of excessive quantities of chemical fertilizers, and other synthetic nutrients [28]. Some (potentially toxic) heavy metals, such as Cu, Zn, Ni, Co, Se, and Fe, are also essential elements required for the optimal performance of plants and become toxic when accumulated in excess in soil solution [29, 30]. On the other hand, non-essential elements, such as arsenate (As), cesium (Cs), lead (Pb), and cadmium (Cd), can hamper crop productivity when accumulated in the soil even in trace amounts [31]. Soil contamination with heavy metals causes accumulation of these toxic metals in plant parts, resulting in decreased crop productivity and increased risk to animal and human

**2.3 Strategies to develop cold stress tolerance in mustard**

**44**

health [32].

most wide spread and destructive fungus diseases of mustard all over the world [43]. The details of these diseases and their causal organism which are affecting rapeseed-mustard crop in India are mentioned in **Table 1** below.

#### **3.1 Conventional approaches for disease management**

The following strategies are helpful for disease management.

Application of more use of pesticides leads to the development of resistance in the target pests, and has negative impacts on biodiversity. More importantly, plants will have increased susceptibility to pests due to the implications of changes of climate. Under such conditions, gaining better knowledge on physiology of plants could lead to sustainable control of biotic stresses. Several plant breeding techniques has been used in extensively to develop biotic stress tolerance in mustard. The stress responses in plants is showed high levels of complexity and redundancy at the sensitivity, response and expression levels with interconnection between stress pathways and over lapping functions between stress metabolites and stress proteins in different stresses. In the case of stress proteins, there are limits on genes of known function that are available but perhaps more importantly the issue of whether single or multiple gene transformations will confer stable resistance. Regular upgradation of technology is required to develop better solution of biotic stresses. Now-a-days, several molecular techniques are considered for a better crop disease management. Lack of disease resistance sources of plant breeding is serious problem and difficult challenge for crop improvement and this problem can be solved through using biotechnology approaches. This is one of the best option and

**47**

*Innovative Strategies to Develop Abiotic and Biotic Stress Tolerance in Mustard (Brassicaceae)*

opportunity to develop strategies for biotic stress tolerance in crop [44]. Several methods has been employed to develop biotic stress tolerance are discussed below.

The conventional breeding techniques are used for the incorporation of genes of interest from inter-crossing species into the crop for the development of biotic stress tolerance; however, these methods proved less effective with undesirable results [45]. Moreover, biotechnological techniques can be effective for the development of stress-tolerant plants. The genetic transformation involves the transfer of stress tolerance gene from gene pools in various plant species for establishment of stress tolerant crops. Genetic engineering could be most effective for the improvement of crop varieties; however, the major trouble associated with this approach is the low transformation competence, silencing of transgene and low gene expression [46]. Recently, tissue culture has proved to be an appropriate and less costly technique for development of stress-tolerant plants. The tissue culture plants are grown in controlled lab conditions requires limited time and space with potential to develop of stress-tolerant plants and leads to the better understanding of biochemical and metabolic pathways of plants growing in harsh environmental conditions [47]. Using partially purified culture filtrates, *B. napus* showing resistance to *Alternaria brassicicola* has been obtained [48] and *B. napus* showing resistance to

*DOI: http://dx.doi.org/10.5772/intechopen.95973*

**3.2 Molecular approaches for disease management**

*3.2.1 In-vitro tissue culture to develop biotic stress tolerance*

*Phoma lingam* has been obtained through embryonic culture [49].

mustard though tissue culture and suited for nontraditional areas.

*3.2.2 RNAi-mediated plant defence to develop biotic stress tolerance*

seed as well as it also have major role in yield and maturity traits.

*3.2.3 Use of microorganisms in biotic stress tolerance*

In vitro selection through enhanced expression of pathogenesis-related (PR) proteins, antifungal peptides or biosynthesis of phytoalexins is an important tool for desirable plant selection [50, 51]. This technology is having an upper hand over transgenic approach for developing improved disease-tolerant crops [52]. Developing pathogen resistance through in vitro selection can be carried out using organogenic or embryogenic calli, shoots, somatic embryos or cell suspensions. By exposing these cultures to different toxins produced by various plant pathogens, tolerant plants can be raised [51]. "Pusa Jaikisan" is the first high yielding variety of

RNA interference (RNAi), is a powerful technology for discovering the functional genetic sequences and harness the down regulation of expression of gene(s) specifically. To accomplish the modified gene expression for a particular trait, gene silencing viz. cosuppression, post transcriptional gene silencing, virus-induced gene silencing etc. can be used. This molecular phenomenon has become a focal point of modern plant biology research across the globe. Thus it has been remarkably used in crop improvement likewise has become a valuable tool for functional genomics in Brassica (Brassica sp.). The rapid adoption of RNAi has replaced previous antisense technology. RNAi has aided in identification of different functions and biological roles of various mustard genes, which are involved in fertility and somatic embryogenesis, resistance to biotic and abiotic stresses and qualitative improvements in oil

The symbiotic interactions between plant and microorganisms may result in several outcomes as defined by fitness benefits by each of the partners [53].

#### **Table 1.**

*Economically important biotic stresses (diseases) of mustard and losses caused by them.*

opportunity to develop strategies for biotic stress tolerance in crop [44]. Several methods has been employed to develop biotic stress tolerance are discussed below.

#### **3.2 Molecular approaches for disease management**

*Brassica Breeding and Biotechnology*

most wide spread and destructive fungus diseases of mustard all over the world [43]. The details of these diseases and their causal organism which are affecting

• Optimization of sowing time is very important as it significantly affects the

• Use of certified seeds, chemicals, disease resistant and tolerant varieties.

• Seed treatment with various biocontrol agents viz., *T. viride*, *G. virens, Pseudomonas* or botanicals like *Allium sativum* etc. Use of bio-control agents (BCA) is advantageous as they are often effective against a wide range of soil-borne pathogens. Moreover, they are ecofriendly, cost effective and their use avoids the risk of development of resistance in the pathogen towards the

• Use recommended doses of N, P and K fertilizers, maintaining optimum plant

Application of more use of pesticides leads to the development of resistance in the target pests, and has negative impacts on biodiversity. More importantly, plants will have increased susceptibility to pests due to the implications of changes of climate. Under such conditions, gaining better knowledge on physiology of plants could lead to sustainable control of biotic stresses. Several plant breeding techniques has been used in extensively to develop biotic stress tolerance in mustard. The stress responses in plants is showed high levels of complexity and redundancy at the sensitivity, response and expression levels with interconnection between stress pathways and over lapping functions between stress metabolites and stress proteins in different stresses. In the case of stress proteins, there are limits on genes of known function that are available but perhaps more importantly the issue of whether single or multiple gene transformations will confer stable resistance. Regular upgradation of technology is required to develop better solution of biotic stresses. Now-a-days, several molecular techniques are considered for a better crop disease management. Lack of disease resistance sources of plant breeding is serious problem and difficult challenge for crop improvement and this problem can be solved through using biotechnology approaches. This is one of the best option and

**Disease Causal organism Yield losses** Alternaria blight *Alternaria. brassicae* (Berk.) Sacc. 10–70 White rust *Albugo candida* (lev.) Kuntze Upto 47 Sclerotinia rot *Sclerotinia sclerotiorum* Upto 35 Downy mildew *Hyaloperonospora parasitica* 17—37 Powdery mildew *Erysiphe cruciferarum* Upto 18

rapeseed-mustard crop in India are mentioned in **Table 1** below.

The following strategies are helpful for disease management.

**3.1 Conventional approaches for disease management**

population with recommended crop spacing.

*Source: Directorate of Rapeseed-Mustard Research (DRMR).*

*Economically important biotic stresses (diseases) of mustard and losses caused by them.*

disease incidence and severity.

control agent.

**46**

**Table 1.**

#### *3.2.1 In-vitro tissue culture to develop biotic stress tolerance*

The conventional breeding techniques are used for the incorporation of genes of interest from inter-crossing species into the crop for the development of biotic stress tolerance; however, these methods proved less effective with undesirable results [45]. Moreover, biotechnological techniques can be effective for the development of stress-tolerant plants. The genetic transformation involves the transfer of stress tolerance gene from gene pools in various plant species for establishment of stress tolerant crops. Genetic engineering could be most effective for the improvement of crop varieties; however, the major trouble associated with this approach is the low transformation competence, silencing of transgene and low gene expression [46]. Recently, tissue culture has proved to be an appropriate and less costly technique for development of stress-tolerant plants. The tissue culture plants are grown in controlled lab conditions requires limited time and space with potential to develop of stress-tolerant plants and leads to the better understanding of biochemical and metabolic pathways of plants growing in harsh environmental conditions [47]. Using partially purified culture filtrates, *B. napus* showing resistance to *Alternaria brassicicola* has been obtained [48] and *B. napus* showing resistance to *Phoma lingam* has been obtained through embryonic culture [49].

In vitro selection through enhanced expression of pathogenesis-related (PR) proteins, antifungal peptides or biosynthesis of phytoalexins is an important tool for desirable plant selection [50, 51]. This technology is having an upper hand over transgenic approach for developing improved disease-tolerant crops [52]. Developing pathogen resistance through in vitro selection can be carried out using organogenic or embryogenic calli, shoots, somatic embryos or cell suspensions. By exposing these cultures to different toxins produced by various plant pathogens, tolerant plants can be raised [51]. "Pusa Jaikisan" is the first high yielding variety of mustard though tissue culture and suited for nontraditional areas.

#### *3.2.2 RNAi-mediated plant defence to develop biotic stress tolerance*

RNA interference (RNAi), is a powerful technology for discovering the functional genetic sequences and harness the down regulation of expression of gene(s) specifically. To accomplish the modified gene expression for a particular trait, gene silencing viz. cosuppression, post transcriptional gene silencing, virus-induced gene silencing etc. can be used. This molecular phenomenon has become a focal point of modern plant biology research across the globe. Thus it has been remarkably used in crop improvement likewise has become a valuable tool for functional genomics in Brassica (Brassica sp.). The rapid adoption of RNAi has replaced previous antisense technology. RNAi has aided in identification of different functions and biological roles of various mustard genes, which are involved in fertility and somatic embryogenesis, resistance to biotic and abiotic stresses and qualitative improvements in oil seed as well as it also have major role in yield and maturity traits.

#### *3.2.3 Use of microorganisms in biotic stress tolerance*

The symbiotic interactions between plant and microorganisms may result in several outcomes as defined by fitness benefits by each of the partners [53]. Interaction to host plants can be positive, neutral or negative. Variations in the outside environment put the plant metabolism out of homeostasis, which creates necessity for the plant to harbour some advanced genetic and metabolic mechanisms within its cellular system [54]. The priming of host response against pathogen is termed as induced systemic resistance, in this case the host response is activated by nonpathogenic plant-associated microorganisms. The ISR induce plant defense mechanisms and protects unexposed part of plants against a future attack by pathogenic microbes and insect pests. Plant hormones ethylene and jasmonic acid plays a regulatory function in the network of interrelated signaling pathways involved in ISR induction [55].

Many studies have been dedicated to the induced systemic resistance SR mediated by free-living rhizobacterial strains [56] the resistance in Mustard (*Brassica juncea*) Induced against Alternaria Black Spot using a virulent *Alternaria brassicae* Isolate-D whereas the strains of *Alternaria alternata* failed to induce resistance against *Alternaria brassicae* [57]. Subsequently attention was drawn to ISR mediated by several other species of genus *Pseudomonas* and the effect was characterized in different plant–pathogen systems. *Pseudomonas* sp. Strain-1 was shown to suppress Sclerotinia stem rot incited by *Sclerotinia sclerotiorum* on stem of mustard. Configuring the functions of endophytes there role in stress tolerance increases immensely. Endophytic microbes improves the plant health by deterring herbivory and pathogenesis while also facilitating plant growth through nutrient uptake, water use efficiency and curtailing of environmental stresses. The endophytic bacteria *Pseudomonas syringae* bacterium was able to induce disease resistance via defense priming [58].

#### **4. Conclusion**

Based on the foregoing chapter, there is no doubt biotic and abiotic stresses are the major barriers in enhancing the productivity of rapeseed-mustard crop. Conventional plant breeding techniques has not provided us full proof protection against abiotic and biotic stresses so far. So, there is an urgent need to minimize the adverse effects of these stresses on the brassica crops to enhance the productivity and production to meet the ever-growing demand of oil in the country. The use of genetic and genomic analysis which helps to identify DNA regions tightly linked to agronomical traits in mustard. Molecular markers for the indirect selection of improved crops speeds up the selection process by alleviating time-consuming approaches direct screening under screen house and field conditions. Use of PGPRs in mustard can also be highly effective as these microorganisms are able to reduce abiotic as well as biotic stress in plants by producing beneficial enzymes, proteins, hormones etc. Tissue culture is another advantageous technique in mustard as this crop is highly susceptible stresses at initial stages. Therefore the use of one particular technique or the mix of these techniques has a potential to improve the stress tolerance in rapeseed mustard.

**49**

**Author details**

Bahaderjeet Singh\*, Amanpreet Singh Sran and Gagandeep Singh Sohi

© 2021 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,

Guru Kashi University, Bathinda, Punjab, India

provided the original work is properly cited.

\*Address all correspondence to<EMAIL_ADDRESS>
*Innovative Strategies to Develop Abiotic and Biotic Stress Tolerance in Mustard (Brassicaceae)*

*DOI: http://dx.doi.org/10.5772/intechopen.95973*

*Innovative Strategies to Develop Abiotic and Biotic Stress Tolerance in Mustard (Brassicaceae) DOI: http://dx.doi.org/10.5772/intechopen.95973*

#### **Author details**

*Brassica Breeding and Biotechnology*

involved in ISR induction [55].

defense priming [58].

ance in rapeseed mustard.

**4. Conclusion**

Interaction to host plants can be positive, neutral or negative. Variations in the outside environment put the plant metabolism out of homeostasis, which creates necessity for the plant to harbour some advanced genetic and metabolic mechanisms within its cellular system [54]. The priming of host response against pathogen is termed as induced systemic resistance, in this case the host response is activated by nonpathogenic plant-associated microorganisms. The ISR induce plant defense mechanisms and protects unexposed part of plants against a future attack by pathogenic microbes and insect pests. Plant hormones ethylene and jasmonic acid plays a regulatory function in the network of interrelated signaling pathways

Many studies have been dedicated to the induced systemic resistance SR mediated by free-living rhizobacterial strains [56] the resistance in Mustard (*Brassica juncea*) Induced against Alternaria Black Spot using a virulent *Alternaria brassicae* Isolate-D whereas the strains of *Alternaria alternata* failed to induce resistance against *Alternaria brassicae* [57]. Subsequently attention was drawn to ISR mediated by several other species of genus *Pseudomonas* and the effect was characterized in different plant–pathogen systems. *Pseudomonas* sp. Strain-1 was shown to suppress Sclerotinia stem rot incited by *Sclerotinia sclerotiorum* on stem of mustard. Configuring the functions of endophytes there role in stress tolerance increases immensely. Endophytic microbes improves the plant health by deterring herbivory and pathogenesis while also facilitating plant growth through nutrient uptake, water use efficiency and curtailing of environmental stresses. The endophytic bacteria *Pseudomonas syringae* bacterium was able to induce disease resistance via

Based on the foregoing chapter, there is no doubt biotic and abiotic stresses are the major barriers in enhancing the productivity of rapeseed-mustard crop. Conventional plant breeding techniques has not provided us full proof protection against abiotic and biotic stresses so far. So, there is an urgent need to minimize the adverse effects of these stresses on the brassica crops to enhance the productivity and production to meet the ever-growing demand of oil in the country. The use of genetic and genomic analysis which helps to identify DNA regions tightly linked to agronomical traits in mustard. Molecular markers for the indirect selection of improved crops speeds up the selection process by alleviating time-consuming approaches direct screening under screen house and field conditions. Use of PGPRs in mustard can also be highly effective as these microorganisms are able to reduce abiotic as well as biotic stress in plants by producing beneficial enzymes, proteins, hormones etc. Tissue culture is another advantageous technique in mustard as this crop is highly susceptible stresses at initial stages. Therefore the use of one particular technique or the mix of these techniques has a potential to improve the stress toler-

**48**

Bahaderjeet Singh\*, Amanpreet Singh Sran and Gagandeep Singh Sohi Guru Kashi University, Bathinda, Punjab, India

\*Address all correspondence to<EMAIL_ADDRESS>
© 2021 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.

#### **References**

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[3] Prasad L, Vishnuvat K. Assessment of yield loss in cauliflower seed crop due to Alternaria blight. Indian Phytopathology. 2006;59:185-189.

[4] Howe GA, Jander G. Plant immunity to insect herbivores. Annual Reviews in Plant Biology. 2008;59:41-66. DOI: 10.1146/annurev. arplant.59.032607.092825

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[7] Vital SA, Fowler RW, Virgen A, Gossett DR, Banks SW, Rodriguez J. Opposing roles for superoxide and nitric oxide in the NaCl stress induce regulation of antioxidant enzyme activity in cotton callus tissue. Environmental and Experimental Botany. 2008;62:60-68.

[8] Shinwari ZK, Nakashima K, Miura S, Kasuga M, Seki M, Yamaguchi-Shinozaki K, Shinozaki K. An Arabidopsis gene family encoding DRE/CRT binding proteins involved in low-temperature-responsive gene expression. Biochemical and Biophysical Research Communications. 1998;250(1):161-170.

[9] Zamani Z, Nezami MT, Habibi D, Khorshidi MB. Effect of quantitative and qualitative performance of four canola cultivars (*Brassica napus* L.) to salinity conditions. Advances in Environmental Biology. 2010;4(3):422-427.

[10] Sharma P, Kannu P, Sardana V, Choudhary OP, Banga SS. Physiological and biochemical basis of salinity tolerance in Indian mustard (*B. juncea*). Abstract number: 171. 15th International Rapeseed Congress held at Berlin on June 16-19, 2019.

[11] Srivastava, A. K., Ramaswamy, N. K., Mukopadhyaya, R., Jincy, M. G. & D'Souza, S. F. Thiourea modulates the expression and activity profile of mtATPase under salinity stress in seeds of *Brassica juncea*. Annals of Botany. 2009;103: 403-410. doi: 10.1093/aob/ mcn229 (2009).

[12] Srivastava AK, Nathawat NS, Ramaswamy NK, Sahu MP, Singh G. Evidence for thiol-induced enhanced in situ translocation of 14C-sucrose from source to sink in *Brassica juncea*. Environmental and Experimental Botany. 2008; 64: 250-255.

[13] Paiva ALS, Passaia G, Lobo AKM, Jardim-Messeder D, Silveira JAG, Margis-Pinheiro M. Mitochondrial glutathione peroxidase (OsGPX3) has a crucial role in rice protection against salt stress. Environmental and Experimental Botany. 2019;158:12-21.

[14] Srivastava AK, Ramaswamy NK, Mukopadhyaya R, Jincy MG, D'Souza SF. Thiourea modulates the expression and activity profile of mtATPase under salinity stress in seeds of *Brassica juncea*. Annals of Botany. 2009;103: 403-410, doi: 10.1093/aob/ mcn229

**51**

*Innovative Strategies to Develop Abiotic and Biotic Stress Tolerance in Mustard (Brassicaceae)*

networks in cellular responses and tolerance Of dehydration and cold stresses. Annual Reviews in Plant

[23] Sakuma Y, Maruyama K, Osakabe Y, Qin F, Seki M, Shinozaki K. Functional analysis of an Arabidopsis transcription factor, DREB2A, involved in droughtresponsive gene expression. Plant Cell.

[24] Dreccer MF, Fainges J, Whish J, Ogbonnaya FC, Sadras VO. Comparison of sensitive stages of wheat, barley, canola, chickpea and field pea to temperature and water stress across Australia. Agricultural Meteorology.

[25] Shah SH, Ali S, Hussain Z, Jan SA, Din JU, Ali GM. Genetic improvement of tomato (*Solanum lycopersicum*) with AtDREB1A gene for cold stress tolerance

using optimized Agrobacteriummediated transformation system. International Journal of Agriculture and

[26] Patade VY, Khatri D, Manoj K, Kumari M, Ahmed Z. Cold tolerance in thiourea primed capsicum seedlings is associated with transcript regulation of stress responsive genes. Molecular Biology Reports. 2012;39:10603-10613.

Biology. 2016;18:471-482.

10.1007/s11033-012-1948-6.

[27] Proshad R, Kormoker T,

ISBN 978-981-10-5744-1.

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83-88.

Mursheed N, Islam MM, Bhuyan MI, Islam MS, Mithu TN. Heavy metal toxicity in agricultural soil due to rapid industrialization in Bangladesh: a review. International Journal of Advanced Geosciences. 2018;6 (1),

[28] Minhas P, Rane J, Pasala RK (Eds.). Abiotic Stress Management for Resilient Agriculture. (Singapore: Springer;),

2018;248: 275-294. 10.1016/j. agrformet.2017.10.006

Biology. 2006;57:781-803.

2006;18:1292-1309.

*DOI: http://dx.doi.org/10.5772/intechopen.95973*

[16] Isayenkov S, Maathuis FJM. Plant salinity stress; many unanswered questions remain. Frontiers in Plant Science. 2019;10:1-11. 10.3389/

[17] Rodriguez RJ, Henson J, Van Volkenburgh E, Hoy M, Wright L, Beckwith F, Kim YO, Redman RS. Stress

tolerance in plants via habitat-

[18] Shekari F, Soltaniband V, Javanmard A, Abbasi A. The impact of drought stress at different stages of development on water relations, stomatal density and quality changes of rapeseed(*Brassica napus* L.). Iran Agricultural Research.

[19] Hasanuzzaman M, Nahar K, Hossain MS, Mahmud JA, Rahman A, Inafuku M, Fujita M. Coordinated actions of glyoxalase and antioxidant defense systems in conferring abiotic stress tolerance in plants. International

Journal of Molecular Sciences.

Brassica. 2015;6(2):265-272.

[22] Yamaguchi-Shinozaki K,

[20] Kaur S, Sharma P. Biochemical characterization of Indian mustard (*Brassica juncea* L.) genotypes in response to moisture stress and irrigation modules. Journal of Oilseed

[21] Cheema, M.A.;Wahid, M.A.; Sattar, A.; Rasul, F.; Saleem, M.F. Influence of different levels of potassium on growth, yield and quality of canola (*Brassica napus* L.) cultivars. Pakistan Journal of Agriculture Science. 2012; 49: 163-168.

Shinozaki K. Transcriptional regulatory

adapted symbiosis. The ISME Journal.

Suprasanna P. Thiourea priming enhances salt tolerance through co-ordinated regulation of microRNAs and hormones in Brassica juncea. Scientific Reports. 2017;7, 45490. doi:

10.1038/srep45490.

fpls.2019.00080

2008;2:404-416.

2015;34(2):81-90.

2017;18(1):200-228.

[15] Srivastava AK, Sablok G, Hackenberg M, Deshpande U, *Innovative Strategies to Develop Abiotic and Biotic Stress Tolerance in Mustard (Brassicaceae) DOI: http://dx.doi.org/10.5772/intechopen.95973*

Suprasanna P. Thiourea priming enhances salt tolerance through co-ordinated regulation of microRNAs and hormones in Brassica juncea. Scientific Reports. 2017;7, 45490. doi: 10.1038/srep45490.

[16] Isayenkov S, Maathuis FJM. Plant salinity stress; many unanswered questions remain. Frontiers in Plant Science. 2019;10:1-11. 10.3389/ fpls.2019.00080

[17] Rodriguez RJ, Henson J, Van Volkenburgh E, Hoy M, Wright L, Beckwith F, Kim YO, Redman RS. Stress tolerance in plants via habitatadapted symbiosis. The ISME Journal. 2008;2:404-416.

[18] Shekari F, Soltaniband V, Javanmard A, Abbasi A. The impact of drought stress at different stages of development on water relations, stomatal density and quality changes of rapeseed(*Brassica napus* L.). Iran Agricultural Research. 2015;34(2):81-90.

[19] Hasanuzzaman M, Nahar K, Hossain MS, Mahmud JA, Rahman A, Inafuku M, Fujita M. Coordinated actions of glyoxalase and antioxidant defense systems in conferring abiotic stress tolerance in plants. International Journal of Molecular Sciences. 2017;18(1):200-228.

[20] Kaur S, Sharma P. Biochemical characterization of Indian mustard (*Brassica juncea* L.) genotypes in response to moisture stress and irrigation modules. Journal of Oilseed Brassica. 2015;6(2):265-272.

[21] Cheema, M.A.;Wahid, M.A.; Sattar, A.; Rasul, F.; Saleem, M.F. Influence of different levels of potassium on growth, yield and quality of canola (*Brassica napus* L.) cultivars. Pakistan Journal of Agriculture Science. 2012; 49: 163-168.

[22] Yamaguchi-Shinozaki K, Shinozaki K. Transcriptional regulatory networks in cellular responses and tolerance Of dehydration and cold stresses. Annual Reviews in Plant Biology. 2006;57:781-803.

[23] Sakuma Y, Maruyama K, Osakabe Y, Qin F, Seki M, Shinozaki K. Functional analysis of an Arabidopsis transcription factor, DREB2A, involved in droughtresponsive gene expression. Plant Cell. 2006;18:1292-1309.

[24] Dreccer MF, Fainges J, Whish J, Ogbonnaya FC, Sadras VO. Comparison of sensitive stages of wheat, barley, canola, chickpea and field pea to temperature and water stress across Australia. Agricultural Meteorology. 2018;248: 275-294. 10.1016/j. agrformet.2017.10.006

[25] Shah SH, Ali S, Hussain Z, Jan SA, Din JU, Ali GM. Genetic improvement of tomato (*Solanum lycopersicum*) with AtDREB1A gene for cold stress tolerance using optimized Agrobacteriummediated transformation system. International Journal of Agriculture and Biology. 2016;18:471-482.

[26] Patade VY, Khatri D, Manoj K, Kumari M, Ahmed Z. Cold tolerance in thiourea primed capsicum seedlings is associated with transcript regulation of stress responsive genes. Molecular Biology Reports. 2012;39:10603-10613. 10.1007/s11033-012-1948-6.

[27] Proshad R, Kormoker T, Mursheed N, Islam MM, Bhuyan MI, Islam MS, Mithu TN. Heavy metal toxicity in agricultural soil due to rapid industrialization in Bangladesh: a review. International Journal of Advanced Geosciences. 2018;6 (1), 83-88.

[28] Minhas P, Rane J, Pasala RK (Eds.). Abiotic Stress Management for Resilient Agriculture. (Singapore: Springer;), ISBN 978-981-10-5744-1.

[29] Khan ZI, Ugulu I, Sahira S, Ahmad K, Ashfaq A, Mehmood N.

**50**

*Brassica Breeding and Biotechnology*

Stat\_OS 2018.pdf.

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[31] Khalid S, Shahid M, Bibi I, Sarwar T, Shah A, Niazi N. A review of environmental contamination and health risk assessment of wastewater use for crop irrigation with a focus on low and high-income countries. International Journal of Environmental Research. 2018;15:895. 10.3390/ ijerph15050895.

[32] Couto R, Comin JJ, Souza M, Ricachenevsky FK, Lana M, Gatiboni L. Should heavy metals be monitored in foods derived from soils fertilized with animal waste? Frontiers in Plant Science. 2018;9: 732. 10.3389/ fpls.2018.00732.

[33] Wang JL, Li T, Liu GY, Smith JM, Zhao ZW. Unravelling the role of dark septate endophyte (DSE) colonizing maize (*Zea mays*) under cadmium stress: physiological, cytological and genic aspects. Scientific Reports. 2016; 6: 22028

[34] Friesen ML, Porter SS, Stark SC, Von Wettberg EJ, Sachs JL, Martinez-Romero E. Microbially mediated plant functional traits. Annual Review of Ecology, Evolution, and Systematics. 2011;42: 23-46.

[35] Hu Z, Zhang D, Zhang G, Kan G, Hong D, Yu D. Association mapping of yield-related traits and SSR markers in wild soybean (*Glycine soja*

Sieb. and Zucc.). Breeding Science. 2014;63:441-449

[36] Qu C, Jia L, Fu F, Zhao H. Genome-wide association mapping and Identification of candidate genes for fatty acid composition in *Brassica napus* L. using SNP markers. BMC Genomics. 2017;18:232.

[37] Zhu Q, King GJ, Liu X, Shan N, Borpatragohain P, Baten A, Wang P, Luo S, Zhou Q. Identification of SNP loci and candidate genes related to four important fatty acid composition in *Brassica napus* using genome wide association study. PLoS One. 2019;14:e0221578.

[38] Lu K, Wei L, Li X, Wang Y, Wu J. Whole-genome resequencing reveals *Brassica napus* origin and genetic loci involved in its improvement. Nature Communications. 2019;10:1154

[39] Saharan GS, Mehta N and Sangwan MS. Diseases of oilseed crops. Indus Publication Co., New Delhi. 2005. ISBN 10:<PHONE_NUMBER> / ISBN 13:<PHONE_NUMBER>764

[40] Saha LR and Singh HB. Diseases of rapeseed and mustard and their management. Review of Tropical Plant Pathology. 1988;5:47-77

[41] Kolte SJ. Tackling fungal diseases of oil seed Brassicas in India. Brassica. 2005;7 (1/2): 7-13.

[42] Mehta N, Sangwan MS, Saharan GS. Fungal diseases of rapeseed mustard. Diseases of Oil Seed Crops. 2005;15-86.

[43] Kotle SJ. Diseases of Annual Edible Oilseed Crop. Rapeseed mustard and sesame diseases. Vol.II. C.R.C. Press, Boca Raton, Florida, U.S.A. 1985;320.I SBN:<PHONE_NUMBER>437

**53**

*Innovative Strategies to Develop Abiotic and Biotic Stress Tolerance in Mustard (Brassicaceae)*

Plant Cell, Tissue and Organ Culture.

[52] Jayashankar S, Li Z, Gray DJ. In vitro selection of *Vitis vinifera* Chardonnay with Elsinoe ampelina culture filtrate is accompanied by fungal resistance and enhanced secretion of chitinase. Planta

2008; 93:85-96.

2000;211:200-208

2010;48: 909-930.

2009;164:493-513.

29: 96-99.

1985;29-39.

[53] Lewis DH. Symbiosis and mutualism: crisp concepts and soggy semantics. The Biology of Mutualism: Ecology and Evolution, ed. Boucher.

[54] Gill SS, Tuteja N. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiology and Biochemistry.

Berendsen RL, Weller DM, Van Wees SC, Bakker PA. Induced systemic resistance by beneficial microbes. Annual Reviews in Phytopathology. 2014;52:347-375.

Interactions of *Bacillus* spp. and plants with special reference to induced systemic resistance (ISR). Microbiological Research.

[57] Vishwanath, Kolte SJ. Methods of inoculation for resistance to Alternaria blight of rapeseed and mustard. Journal of Mycology and Plant Pathology. 1999;

[58] Ardanov P, Ovcharenko L, Zaets I, Kozyrovska N, Pirttila AM. Endophytic bacteria enhancing growth and disease resistance of potato (*Solanum tuberosum* L.). Biological Control. 2011;56:43-49

[55] Pieterse CM, Zamioudis C,

[56] Choudhary DK, Johri BN.

*DOI: http://dx.doi.org/10.5772/intechopen.95973*

biotechnological interventions for trait improvement: status and prospects. Planta. 2020;252 (54):3-18. https://doi. org/10.1007/s00425-020-03465-4

[45] Manoj K, Rai MK, Kalia RK, Rohtas RS, Dhawan A. Developing stress tolerant plants through in vitro selection—An overview of the recent progress. Environmental and Experimental Botany. 71(1):89-98. DOI:

10.1016/j.envexpbot.2010.10.021

[47] Pérez-Clemente RM,

Newsletter. 1985;10:97-100.

Gómez-Cadenas A. 2012; In vitro tissue culture, a tool for the study and breeding of plants subjected to abiotic stress conditions. Book chapter In Techopen. 2012 DOI: 10.5772/50671

[48] MacDonald MV, Ingram DS. In vitro selection for resistance to *Alternaria brassicicola* in *Brassica napus* spp. oleifera (winter oilseed rape) using partially purified culture filtrates. Cruciferae

[49] Sacristan MD. Resistance response to *Phoma lingum* of plant regenerated from selected cells and embryogenic cultures of haploid Brassica napus. The or Appl Genet. 1982;61:193-200.

[50] Ganesan M, Jayabalan N. Isolation of disease-tolerant cotton (*Gossypium hirsutum* L. cv. SVPR 2) plants by screening somatic embryos with fungal culture filtrate. Plant Cell, Tissue and Organ Culture. 2006; 87:273-284.

[51] Kumar JV, Ranjitha Kumari BD, Sujatha G, Castano E. Production of plants resistant to *Alternaria carthami* via organogenesis and somatic

embryogenesis of safflower cv. NARI-6 treated with fungal culture filtrates.

[46] Rai MK, Rajwant KK, Rohtas S, Manu PG, Dhawan AK. Developing stress tolerant plants through in vitro selection – an overview of the recent progress. Environmental and Experimental Botany. 2011;71:89-98.

[44] Singh RK Prasad A, Muthamilarasan M, Parida SK, Prasad M. Breeding and

*Innovative Strategies to Develop Abiotic and Biotic Stress Tolerance in Mustard (Brassicaceae) DOI: http://dx.doi.org/10.5772/intechopen.95973*

biotechnological interventions for trait improvement: status and prospects. Planta. 2020;252 (54):3-18. https://doi. org/10.1007/s00425-020-03465-4

*Brassica Breeding and Biotechnology*

Determination of toxic metals in fruits of *Abelmoschus esculentus* grown in contaminated soils with different irrigation sources by spectroscopic method. International Journal of Environmental Research. 2018;12:503- 511. 10.1007/s41742-018-0110-2.

Sieb. and Zucc.). Breeding Science.

Genome-wide association mapping and Identification of candidate genes for fatty acid composition in *Brassica napus* L. using SNP markers. BMC Genomics.

[37] Zhu Q, King GJ, Liu X, Shan N, Borpatragohain P, Baten A, Wang P, Luo S, Zhou Q. Identification of SNP loci and candidate genes related to four important fatty acid composition in *Brassica napus* using genome wide association study. PLoS One.

[38] Lu K, Wei L, Li X, Wang Y, Wu J. Whole-genome resequencing reveals *Brassica napus* origin and genetic loci involved in its improvement. Nature Communications. 2019;10:1154

Sangwan MS. Diseases of oilseed crops. Indus Publication Co., New Delhi. 2005. ISBN 10:<PHONE_NUMBER> / ISBN 13:

[40] Saha LR and Singh HB. Diseases of rapeseed and mustard and their management. Review of Tropical Plant

[41] Kolte SJ. Tackling fungal diseases of oil seed Brassicas in India. Brassica.

[42] Mehta N, Sangwan MS, Saharan GS. Fungal diseases of rapeseed mustard. Diseases of Oil Seed Crops. 2005;15-86.

[43] Kotle SJ. Diseases of Annual Edible Oilseed Crop. Rapeseed mustard and sesame diseases. Vol.II. C.R.C. Press, Boca Raton, Florida, U.S.A. 1985;320.I

[39] Saharan GS, Mehta N and

[36] Qu C, Jia L, Fu F, Zhao H.

2014;63:441-449

2017;18:232.

2019;14:e0221578.

9788173871764

Pathology. 1988;5:47-77

2005;7 (1/2): 7-13.

SBN:<PHONE_NUMBER>437

[44] Singh RK Prasad A, Muthamilarasan M, Parida SK, Prasad M. Breeding and

[30] Narendrula-Kotha R, Theriault G, Mehes-Smith M, Kalubi K, Nkongolo K. Metal toxicity and resistance in plants and microorganisms in terrestrial ecosystems. Reviews of Environmental Contamination and Toxicology. 2020;249, 1-27. 10.1007/398\_2018\_22.

[31] Khalid S, Shahid M, Bibi I, Sarwar T, Shah A, Niazi N. A review of environmental contamination and health risk assessment of wastewater use for crop irrigation with a focus on low and high-income countries. International Journal of Environmental

Research. 2018;15:895. 10.3390/

[32] Couto R, Comin JJ, Souza M, Ricachenevsky FK, Lana M, Gatiboni L. Should heavy metals be monitored in foods derived from soils fertilized with animal waste? Frontiers in Plant Science. 2018;9: 732. 10.3389/

[33] Wang JL, Li T, Liu GY, Smith JM, Zhao ZW. Unravelling the role of dark septate endophyte (DSE) colonizing maize (*Zea mays*) under cadmium stress: physiological, cytological and genic aspects. Scientific Reports. 2016; 6:

[34] Friesen ML, Porter SS, Stark SC, Von Wettberg EJ, Sachs JL, Martinez-Romero E. Microbially mediated plant functional traits. Annual Review of Ecology, Evolution, and Systematics.

[35] Hu Z, Zhang D, Zhang G, Kan G, Hong D, Yu D. Association mapping of yield-related traits and SSR markers in wild soybean (*Glycine soja*

ijerph15050895.

fpls.2018.00732.

22028

2011;42: 23-46.

**52**

[45] Manoj K, Rai MK, Kalia RK, Rohtas RS, Dhawan A. Developing stress tolerant plants through in vitro selection—An overview of the recent progress. Environmental and Experimental Botany. 71(1):89-98. DOI: 10.1016/j.envexpbot.2010.10.021

[46] Rai MK, Rajwant KK, Rohtas S, Manu PG, Dhawan AK. Developing stress tolerant plants through in vitro selection – an overview of the recent progress. Environmental and Experimental Botany. 2011;71:89-98.

[47] Pérez-Clemente RM, Gómez-Cadenas A. 2012; In vitro tissue culture, a tool for the study and breeding of plants subjected to abiotic stress conditions. Book chapter In Techopen. 2012 DOI: 10.5772/50671

[48] MacDonald MV, Ingram DS. In vitro selection for resistance to *Alternaria brassicicola* in *Brassica napus* spp. oleifera (winter oilseed rape) using partially purified culture filtrates. Cruciferae Newsletter. 1985;10:97-100.

[49] Sacristan MD. Resistance response to *Phoma lingum* of plant regenerated from selected cells and embryogenic cultures of haploid Brassica napus. The or Appl Genet. 1982;61:193-200.

[50] Ganesan M, Jayabalan N. Isolation of disease-tolerant cotton (*Gossypium hirsutum* L. cv. SVPR 2) plants by screening somatic embryos with fungal culture filtrate. Plant Cell, Tissue and Organ Culture. 2006; 87:273-284.

[51] Kumar JV, Ranjitha Kumari BD, Sujatha G, Castano E. Production of plants resistant to *Alternaria carthami* via organogenesis and somatic embryogenesis of safflower cv. NARI-6 treated with fungal culture filtrates.

Plant Cell, Tissue and Organ Culture. 2008; 93:85-96.

[52] Jayashankar S, Li Z, Gray DJ. In vitro selection of *Vitis vinifera* Chardonnay with Elsinoe ampelina culture filtrate is accompanied by fungal resistance and enhanced secretion of chitinase. Planta 2000;211:200-208

[53] Lewis DH. Symbiosis and mutualism: crisp concepts and soggy semantics. The Biology of Mutualism: Ecology and Evolution, ed. Boucher. 1985;29-39.

[54] Gill SS, Tuteja N. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiology and Biochemistry. 2010;48: 909-930.

[55] Pieterse CM, Zamioudis C, Berendsen RL, Weller DM, Van Wees SC, Bakker PA. Induced systemic resistance by beneficial microbes. Annual Reviews in Phytopathology. 2014;52:347-375.

[56] Choudhary DK, Johri BN. Interactions of *Bacillus* spp. and plants with special reference to induced systemic resistance (ISR). Microbiological Research. 2009;164:493-513.

[57] Vishwanath, Kolte SJ. Methods of inoculation for resistance to Alternaria blight of rapeseed and mustard. Journal of Mycology and Plant Pathology. 1999; 29: 96-99.

[58] Ardanov P, Ovcharenko L, Zaets I, Kozyrovska N, Pirttila AM. Endophytic bacteria enhancing growth and disease resistance of potato (*Solanum tuberosum* L.). Biological Control. 2011;56:43-49

**55**

**Chapter 4**

**Abstract**

**1. Introduction**

Embryo Culture and Embryo

Somatic embryogenesis is the best demonstration of totipotency in higher plants

in which somatic cell produce whole plant like zygotic embryo. It is also demonstrated that immature, weak, hybrid or sometimes inviable embryos can be saved through *in vitro* culture to prevents its degradation. It may help to cross the reproductive barriers when interspecific hybrids developed. *Brasssica* is an economically valuable oil yielding and vegetable crop and India is the largest producer of oil seed rape in the world. Various factors affect the embryo rescue in *Brassica* like growth stage of the embryos, types and composition of the rescue medium etc. The embryo regeneration potential can improve through the modification of culture conditions in both zygotic as well as somatic embryo. Except the embryo culture other parts like ovule, ovary culture can also be done to developed interspecific hybrids. This chapter is focused on the embryo rescue techniques in the genus *Brassica* and

**Keywords:** *Brassica*, embryo rescue, hybrids, embryo culture, somatic embryos

producer of the oilseed rape in the world. The genus belongs to the family Brassicaceae having about 38 different species [1]. Crossing and hybridization in these species was done to developed new cultivars worldwide for improving traits and yields. The naturally occurring genetic variation is the basis of the improvements in *Brassica* to produce new morphotypes in interspecific hybridization program [2]. The interspecific hybridization is a difficult process due to pre and post-fertilization barriers and abortion of the hybrid embryo. The embryo degeneration after some hybridization experiments takes place very early [3]. This may be due to the poor endosperm development or sometimes endosperm may not be developed [4]. But in non-endospermic embryos the post fertilization barrier cannot crossed and embryos are defective, disformed and aborted early. The hybrid embryos can be regenerated through various technique that's comes under the embryo rescue. The embryo rescue by culturing on nutrient medium that would support and orderly development of embryos. If the embryos are disformed, secondary embryogenesis may be induced by the manipulation of medium and growth regulators combination [5]. New biotechnological tool like transgenic technology would be better in improving the varieties of *Brassica* crop. Large number of transgenics, both biotic and abiotic stress resistant plants were developed [6, 7]. It is desired to produce transformed plant through somatic embryogenesis. It is preferred because of the

*Brassica* is an important vegetable and oilseed crop of India and it is the largest

summarizes possible ways of improving the technique used.

Rescue in *Brassica*

*Mohammad Akmal*

#### **Chapter 4**

## Embryo Culture and Embryo Rescue in *Brassica*

*Mohammad Akmal*

#### **Abstract**

Somatic embryogenesis is the best demonstration of totipotency in higher plants in which somatic cell produce whole plant like zygotic embryo. It is also demonstrated that immature, weak, hybrid or sometimes inviable embryos can be saved through *in vitro* culture to prevents its degradation. It may help to cross the reproductive barriers when interspecific hybrids developed. *Brasssica* is an economically valuable oil yielding and vegetable crop and India is the largest producer of oil seed rape in the world. Various factors affect the embryo rescue in *Brassica* like growth stage of the embryos, types and composition of the rescue medium etc. The embryo regeneration potential can improve through the modification of culture conditions in both zygotic as well as somatic embryo. Except the embryo culture other parts like ovule, ovary culture can also be done to developed interspecific hybrids. This chapter is focused on the embryo rescue techniques in the genus *Brassica* and summarizes possible ways of improving the technique used.

**Keywords:** *Brassica*, embryo rescue, hybrids, embryo culture, somatic embryos

#### **1. Introduction**

*Brassica* is an important vegetable and oilseed crop of India and it is the largest producer of the oilseed rape in the world. The genus belongs to the family Brassicaceae having about 38 different species [1]. Crossing and hybridization in these species was done to developed new cultivars worldwide for improving traits and yields. The naturally occurring genetic variation is the basis of the improvements in *Brassica* to produce new morphotypes in interspecific hybridization program [2]. The interspecific hybridization is a difficult process due to pre and post-fertilization barriers and abortion of the hybrid embryo. The embryo degeneration after some hybridization experiments takes place very early [3]. This may be due to the poor endosperm development or sometimes endosperm may not be developed [4]. But in non-endospermic embryos the post fertilization barrier cannot crossed and embryos are defective, disformed and aborted early. The hybrid embryos can be regenerated through various technique that's comes under the embryo rescue. The embryo rescue by culturing on nutrient medium that would support and orderly development of embryos. If the embryos are disformed, secondary embryogenesis may be induced by the manipulation of medium and growth regulators combination [5]. New biotechnological tool like transgenic technology would be better in improving the varieties of *Brassica* crop. Large number of transgenics, both biotic and abiotic stress resistant plants were developed [6, 7]. It is desired to produce transformed plant through somatic embryogenesis. It is preferred because of the

genetic stability during culture and very low rate of genetic variations which is otherwise results into somatic variations or somaclones [8]. There are however, some exceptions as in wheat (*Triticum durum* Desf.) somaclonal variation had been reported for several agronomic and phenotypic traits, such as plant height, leaf size, pollen fertility, tolerance to aluminum toxicity, albinism and leaf malformations [9]. *Cymbopogon winterianus* Jowitt showed somaclonal variations when regenerated via somatic embryogenesis [10]. But these variations are restricted to only few plant genera and it is found that the somatic variation produced frequently during tissue culture but generally not in somatic embryo or secondary embryos culture.

The embryogenesis in higher plants is controlled by many genes and related embryos specific proteins. Loss of function mutants were used to identified the genes involved in There are about 220 *EMB* genes in *Arabidopsis* required for normal embryo development. These were identified through duplication of alleles or molecular complementation [11]. Some proteins also required for the somatic embryogenesis and in embryo rescue in Carrot (*Dacus carrota*), these are mainly glycoproteins secreted into the culture medium such as endichitinase and arabinigalactan proteins (AGPs) are required for somatic embryogenesis. These AGPs contains glucosamine and *N*-acetyl-*D*-glucosaminyl [12]. *Rhizobium leguminosarum nod* gene metabolic product contains *N*-acetylglucosamine lipooligosaccharides that promotes carrot embryo rescue [13].

#### **2. Zygotic embryos, genetic embryos and somatic embryos**

In higher plants, formation and development of embryo are the two distinct phenomena that can takes place inside the ovule. An ovule has different parts like chalaza, nucellus, micropyle, integuments, and most important embryo sac. The embryo sac develops after the reduction division of the megaspore mother cell. Only one haploid megaspore cell give rise to the birth of embryo sac that's contains many cells like synergid cells, antipodal cells and one egg cell. Instead of all these cells there is a central nucleus (So called because it is not surrounded by distinct cell wall). The egg cell is polar and contain a distinct nucleus on cytoplasm-rich chalazal pole while there are vacuoles at micropylar end [14]. Egg cell when fertilized called the zygote and gives rise to the embryo. The two important process alternate in each generation, the meiosis (reduction division resulting two haploid cells called the gametophyte) and the fertilization (fusion of the two-haploid nucleus called the sporophyte). The three process are distinct with one another. Formation of zygote after fertilization, formation of the embryo and development of the embryo. Failure of any process disturbed the embryogenesis and cease the formation and development of embryo. After the formation of complete embryo successfully the mature embryo enters in desiccated and metabolically quiescent state [15] or it undergo the period of dormancy that's complete the process. The dormancy is the process of adaptation to withstand unfavorable conditions. The zygotic embryo in the angiosperm after maturation develops into seeds and it is composed of several tissues, including the embryo, the endosperm, and the testa. The mature embryo has the bipolar axis, on which root and shoot meristem are present and gives rise to the root and shoot during plant development. In contrast to the zygotic embryo, the genetic embryo is formed without fertilization through the process termed as apomixis. Apomixis refers to the formation of embryo in the ovule from the somatic cells. These somatic cells when diploid i.e. nucellus or integument directly gives rise to the embryo and termed as sporophytic apomixis. However, when embryo sac originates either from megaspore mother cells by mitosis or incomplete meiosis in diplospory is termed as gametophytic apomixis.

**57**

*Embryo Culture and Embryo Rescue in* Brassica *DOI: http://dx.doi.org/10.5772/intechopen.96058*

**3. Embryo culture**

of the somatic embryos resemble with the zygotic embryos.

storage of the food material in cotyledon and leaf explants.

Embryo rescue is an *in vitro*-culture technique that is used to save weak, immature and hybrid or sometimes inviable embryos to prevents its degradation. The procedure involves excising weak, immature plant embryos and culture them on specially devised culture medium. It plays an important role in plant breeding of important crop plants. In *Brassica*, the interspecific and intergeneric hybridization was done from very earlier because the crop yield losses due to disease, biotic and abiotic stresses is very high. Interspecific, intergeneric and intervarietal hybrids have been generated in mango, banana, seedless grape, papaya and seedless citrus using embryo rescue [28]. It is not only an important oilseed crop but used as vegetable, and fodder for the farm animals. The earlier attempts were made in Chinese cabbage varieties i.e. *B. oleracea* and *B. campestris* [3]*.* The hybrid embryos were carefully removed and

**4. Embryo rescue in** *Brassica*

cultured on the nutrient medium *in vitro*.

Totipotency is the most spectacular demonstration of potencies in the cells of higher plants. The somatic embryogenesis is the generation of bipolar structure from any somatic cell that have distinct root and shoot pole. It is very rare phenomenon present in nature and restricted to some plants like *Kalenchoe*, *Bryophyllum* etc. But it may be introduced into other plants artificially through tissue culture technique. How somatic cell triggered to gives rise to the embryo? It is somewhat unclear but it is suggested that the irregular distribution of auxin stimulates the establishment of embryonic structure [16]. However, the stages of the development

In *Brassica*, somatic embryogenesis can be induced using various auxins like 2,4-D and NAA at higher concentrations separately or in combinations with the cytokinin [17]. 2,4-D alone proved best hormone that used to induced polarity in the somatic cells as compared to the combination with the cytokinin like kinetin as it may ceased the further proliferation of somatic embryos [18]. Various culture medium was used with the auxins like SH Medium [19], B5 medium [20], MS medium [21], Kao's medium [22] etc., but rapid propagation of somatic embryos in *Brassica* proved to be the best in MS medium [17]. MS basal medium and low pH (3.5–5) was also used to induced somatic embryos in *Brassica napus* using immature seeds 14 to 28 days after pollination [23, 24]. Not only in *B. napus* but in *B. oleracea* varieties, cabbage and cauliflower immature zygotic embryos gives rise to the somatic embryos with high frequency [5], confirming that the stress condition either due to the PGR (mainly auxin) reprogrammed the zygotic immature cell to induced embryogenesis in *Brassica*. There is not only induction and establishment of polarity but also maintenance of the root and shoot meristem. It is also noticed that the zygotic embryos when immature have higher embryogenic potential than the mature embryo [23, 25]. The low pH value i.e., 3.5–5 in *Brassica napus* increase the exchange of ions and ionic nutrients that's accumulate inside the embryogenic tissue or callus. The stored food material mainly lipid bodies in the cells of *Brassica* indicates the good exchange of nutrient under auxin induced stress condition. The leaf explant is best for the induction of somatic embryos as compared to the other explants like stem and hypocotyl sections (**Figure 1A-D** and **F**) [17, 26, 27]. This is because the large number of vacuolated protoplasts that's provide the space for the

*Embryo Culture and Embryo Rescue in* Brassica *DOI: http://dx.doi.org/10.5772/intechopen.96058*

Totipotency is the most spectacular demonstration of potencies in the cells of higher plants. The somatic embryogenesis is the generation of bipolar structure from any somatic cell that have distinct root and shoot pole. It is very rare phenomenon present in nature and restricted to some plants like *Kalenchoe*, *Bryophyllum* etc. But it may be introduced into other plants artificially through tissue culture technique. How somatic cell triggered to gives rise to the embryo? It is somewhat unclear but it is suggested that the irregular distribution of auxin stimulates the establishment of embryonic structure [16]. However, the stages of the development of the somatic embryos resemble with the zygotic embryos.

#### **3. Embryo culture**

*Brassica Breeding and Biotechnology*

promotes carrot embryo rescue [13].

genetic stability during culture and very low rate of genetic variations which is otherwise results into somatic variations or somaclones [8]. There are however, some exceptions as in wheat (*Triticum durum* Desf.) somaclonal variation had been reported for several agronomic and phenotypic traits, such as plant height, leaf size, pollen fertility, tolerance to aluminum toxicity, albinism and leaf malformations [9]. *Cymbopogon winterianus* Jowitt showed somaclonal variations when regenerated via somatic embryogenesis [10]. But these variations are restricted to only few plant genera and it is found that the somatic variation produced frequently during tissue

culture but generally not in somatic embryo or secondary embryos culture.

**2. Zygotic embryos, genetic embryos and somatic embryos**

The embryogenesis in higher plants is controlled by many genes and related embryos specific proteins. Loss of function mutants were used to identified the genes involved in There are about 220 *EMB* genes in *Arabidopsis* required for normal embryo development. These were identified through duplication of alleles or molecular complementation [11]. Some proteins also required for the somatic embryogenesis and in embryo rescue in Carrot (*Dacus carrota*), these are mainly glycoproteins secreted into the culture medium such as endichitinase and arabinigalactan proteins (AGPs) are required for somatic embryogenesis. These AGPs contains glucosamine and *N*-acetyl-*D*-glucosaminyl [12]. *Rhizobium leguminosarum nod* gene metabolic product contains *N*-acetylglucosamine lipooligosaccharides that

In higher plants, formation and development of embryo are the two distinct phenomena that can takes place inside the ovule. An ovule has different parts like chalaza, nucellus, micropyle, integuments, and most important embryo sac. The embryo sac develops after the reduction division of the megaspore mother cell. Only one haploid megaspore cell give rise to the birth of embryo sac that's contains many cells like synergid cells, antipodal cells and one egg cell. Instead of all these cells there is a central nucleus (So called because it is not surrounded by distinct cell wall). The egg cell is polar and contain a distinct nucleus on cytoplasm-rich chalazal pole while there are vacuoles at micropylar end [14]. Egg cell when fertilized called the zygote and gives rise to the embryo. The two important process alternate in each generation, the meiosis (reduction division resulting two haploid cells called the gametophyte) and the fertilization (fusion of the two-haploid nucleus called the sporophyte). The three process are distinct with one another. Formation of zygote after fertilization, formation of the embryo and development of the embryo. Failure of any process disturbed the embryogenesis and cease the formation and development of embryo. After the formation of complete embryo successfully the mature embryo enters in desiccated and metabolically quiescent state [15] or it undergo the period of dormancy that's complete the process. The dormancy is the process of adaptation to withstand unfavorable conditions. The zygotic embryo in the angiosperm after maturation develops into seeds and it is composed of several tissues, including the embryo, the endosperm, and the testa. The mature embryo has the bipolar axis, on which root and shoot meristem are present and gives rise to the root and shoot during plant development. In contrast to the zygotic embryo, the genetic embryo is formed without fertilization through the process termed as apomixis. Apomixis refers to the formation of embryo in the ovule from the somatic cells. These somatic cells when diploid i.e. nucellus or integument directly gives rise to the embryo and termed as sporophytic apomixis. However, when embryo sac originates either from megaspore mother cells by mitosis or incomplete meiosis in diplospory

**56**

is termed as gametophytic apomixis.

In *Brassica*, somatic embryogenesis can be induced using various auxins like 2,4-D and NAA at higher concentrations separately or in combinations with the cytokinin [17]. 2,4-D alone proved best hormone that used to induced polarity in the somatic cells as compared to the combination with the cytokinin like kinetin as it may ceased the further proliferation of somatic embryos [18]. Various culture medium was used with the auxins like SH Medium [19], B5 medium [20], MS medium [21], Kao's medium [22] etc., but rapid propagation of somatic embryos in *Brassica* proved to be the best in MS medium [17]. MS basal medium and low pH (3.5–5) was also used to induced somatic embryos in *Brassica napus* using immature seeds 14 to 28 days after pollination [23, 24]. Not only in *B. napus* but in *B. oleracea* varieties, cabbage and cauliflower immature zygotic embryos gives rise to the somatic embryos with high frequency [5], confirming that the stress condition either due to the PGR (mainly auxin) reprogrammed the zygotic immature cell to induced embryogenesis in *Brassica*. There is not only induction and establishment of polarity but also maintenance of the root and shoot meristem. It is also noticed that the zygotic embryos when immature have higher embryogenic potential than the mature embryo [23, 25]. The low pH value i.e., 3.5–5 in *Brassica napus* increase the exchange of ions and ionic nutrients that's accumulate inside the embryogenic tissue or callus. The stored food material mainly lipid bodies in the cells of *Brassica* indicates the good exchange of nutrient under auxin induced stress condition. The leaf explant is best for the induction of somatic embryos as compared to the other explants like stem and hypocotyl sections (**Figure 1A-D** and **F**) [17, 26, 27]. This is because the large number of vacuolated protoplasts that's provide the space for the storage of the food material in cotyledon and leaf explants.

#### **4. Embryo rescue in** *Brassica*

Embryo rescue is an *in vitro*-culture technique that is used to save weak, immature and hybrid or sometimes inviable embryos to prevents its degradation. The procedure involves excising weak, immature plant embryos and culture them on specially devised culture medium. It plays an important role in plant breeding of important crop plants. In *Brassica*, the interspecific and intergeneric hybridization was done from very earlier because the crop yield losses due to disease, biotic and abiotic stresses is very high. Interspecific, intergeneric and intervarietal hybrids have been generated in mango, banana, seedless grape, papaya and seedless citrus using embryo rescue [28]. It is not only an important oilseed crop but used as vegetable, and fodder for the farm animals. The earlier attempts were made in Chinese cabbage varieties i.e. *B. oleracea* and *B. campestris* [3]*.* The hybrid embryos were carefully removed and cultured on the nutrient medium *in vitro*.

#### **Figure 1.**

*Somatic embryos of* Brassica juncea *L. A, B, C, and D, The cotyledon derived somatic Embryos, E and F, hypocotyl derived somatic embryos.*

The survival rate of the embryos can be increased when rescued because wide hybridization crosses fail to complete normal sexual reproduction cycle. In *Brassica* the best adopted methods for the embryo rescue are the direct ovule culture, siliqua culture and immature embryos culture. Sometimes embryo-nurse endosperm for embryo transplant was also adopted [29]. Very young embryo is difficult to culture on artificial culture medium. Due to this young fertilized pistil were cultured. Therefore, the chances of embryo abortion can be minimized. For siliqua culture young fertilized pistils excised 4 to 6 days after pollination and cultured on the MS medium. It absorbed the medium start to grow gradually but regular subculturing is required. The swollen pistil again excised to dissect out developing ovule. Ovules can also be culture in a similar manner and selection day after pollination may varies from plant to plant and to identify the fertilized pistil, pollinated pistil fixed in 70% ethanol 24 to 48 hour after pollination and stained in aniline blue, the pollen germination and pollen tube growth may be observed under the microscope [30]. Ovary culture in *Brassica* can be done 4 to 14 days after pollination. The stalk

**59**

*Embryo Culture and Embryo Rescue in* Brassica *DOI: http://dx.doi.org/10.5772/intechopen.96058*

embryoids derived from isolated microspore culture [34].

**5. Factors affecting embryo rescue**

secondary embryogenesis should be avoided.

**5.1 Nurse tissue**

and inconspicuous.

**5.2 Culture medium**

of the ovary cut from the base and cultured on the nutrient medium [31]. For embryos culture, the siliqua was collected after 10, 15, 20, 25, and 30 days after pollination and after the sterilization the ovules are dissect out from siliquae and the young embryos cultured on the MS medium [21]. All these methods of embryo rescue depend on the type and the condition of the hybrid embryos and the type of hybridization experiments [32]. Considerable progress in embryo rescue has been takes place but the rescue of hybrid embryos seems to be difficult when abortion occurs at very early. Embryo implantation technique was adopted to overcome this problem which is known as embryo-nurse endosperm transplant where excised hybrid embryo inserted into a cellular endosperm dissected from third species or one of the parents. The nurse endosperm with the transplanted embryo cultured on an artificial medium [33]. Recently double haploids were developed through

Various factors affect the embryo rescue in *Brassica* and other plants. It depends on the age of the embryo, intactness of the suspensor [35], excision procedure, sterilization, culture medium supplementation, temperature and light requirements etc. Highly immature embryo rescue is very difficult and requires special medium requirements. Excision procedure is designed in that way, no or minimum injury should be there in embryo proper specially when embryo excised at very early stage of development. The aim should be rescue for primary embryos

The early stage embryo culture during rescue is very difficult to culture on artificial medium. The nurse tissue provides a natural condition and nutrition so that the chances of embryo abortion reduces. The best nurse tissue for the embryo culture is endosperm and if it is some days old it efficiently increases the chances of survival of the young embryo. The somatic embryos can be used as nurse tissue for culturing the hybrid zygotic embryos as done in *Pinus* [36]. The ovule is another nurse tissue which is used to generate very young stage embryos. It is not necessary to excise young embryo from the ovule but excision of ovule is easy. Sometime ovary culture can also be used and this was done when the embryos are very small

There are various nutrients formulations used to culture the embryos during rescue. Murashige and Skoog formulation [21] and Gamborg's B-5 [20] are most frequently used in *Brassica* [37]. Sometimes both hormones were used as in

*B. oleracea* [5]. However, there are examples in which B5 vitamins are used with MS salts in embryo rescue of *R. sativus* [38]. Other mediums used for the embryo rescue in other plants are Knop's medium; Heller's medium [39], Monnier's medium [40] etc. The early heterotrophic immature embryos take its nutrition form the endosperms and surrounding tissue but when embryos mature it is partly autotrophic and it requires only basic mineral salts and sucrose. In *Brassica*, the embryos become autotrophic very late after globular stages [41]. The early globular stage of proembryo culture was achieved earlier using double-layer culture system and in embryo culture medium which contains mineral salts, sugars, amino acids, organic acids and *Embryo Culture and Embryo Rescue in* Brassica *DOI: http://dx.doi.org/10.5772/intechopen.96058*

*Brassica Breeding and Biotechnology*

The survival rate of the embryos can be increased when rescued because wide hybridization crosses fail to complete normal sexual reproduction cycle. In *Brassica* the best adopted methods for the embryo rescue are the direct ovule culture, siliqua culture and immature embryos culture. Sometimes embryo-nurse endosperm for embryo transplant was also adopted [29]. Very young embryo is difficult to culture on artificial culture medium. Due to this young fertilized pistil were cultured. Therefore, the chances of embryo abortion can be minimized. For siliqua culture young fertilized pistils excised 4 to 6 days after pollination and cultured on the MS medium. It absorbed the medium start to grow gradually but regular subculturing is required. The swollen pistil again excised to dissect out developing ovule. Ovules can also be culture in a similar manner and selection day after pollination may varies from plant to plant and to identify the fertilized pistil, pollinated pistil fixed in 70% ethanol 24 to 48 hour after pollination and stained in aniline blue, the pollen germination and pollen tube growth may be observed under the microscope [30]. Ovary culture in *Brassica* can be done 4 to 14 days after pollination. The stalk

*Somatic embryos of* Brassica juncea *L. A, B, C, and D, The cotyledon derived somatic Embryos, E and F,*

**58**

**Figure 1.**

*hypocotyl derived somatic embryos.*

of the ovary cut from the base and cultured on the nutrient medium [31]. For embryos culture, the siliqua was collected after 10, 15, 20, 25, and 30 days after pollination and after the sterilization the ovules are dissect out from siliquae and the young embryos cultured on the MS medium [21]. All these methods of embryo rescue depend on the type and the condition of the hybrid embryos and the type of hybridization experiments [32]. Considerable progress in embryo rescue has been takes place but the rescue of hybrid embryos seems to be difficult when abortion occurs at very early. Embryo implantation technique was adopted to overcome this problem which is known as embryo-nurse endosperm transplant where excised hybrid embryo inserted into a cellular endosperm dissected from third species or one of the parents. The nurse endosperm with the transplanted embryo cultured on an artificial medium [33]. Recently double haploids were developed through embryoids derived from isolated microspore culture [34].

#### **5. Factors affecting embryo rescue**

Various factors affect the embryo rescue in *Brassica* and other plants. It depends on the age of the embryo, intactness of the suspensor [35], excision procedure, sterilization, culture medium supplementation, temperature and light requirements etc. Highly immature embryo rescue is very difficult and requires special medium requirements. Excision procedure is designed in that way, no or minimum injury should be there in embryo proper specially when embryo excised at very early stage of development. The aim should be rescue for primary embryos secondary embryogenesis should be avoided.

#### **5.1 Nurse tissue**

The early stage embryo culture during rescue is very difficult to culture on artificial medium. The nurse tissue provides a natural condition and nutrition so that the chances of embryo abortion reduces. The best nurse tissue for the embryo culture is endosperm and if it is some days old it efficiently increases the chances of survival of the young embryo. The somatic embryos can be used as nurse tissue for culturing the hybrid zygotic embryos as done in *Pinus* [36]. The ovule is another nurse tissue which is used to generate very young stage embryos. It is not necessary to excise young embryo from the ovule but excision of ovule is easy. Sometime ovary culture can also be used and this was done when the embryos are very small and inconspicuous.

#### **5.2 Culture medium**

There are various nutrients formulations used to culture the embryos during rescue. Murashige and Skoog formulation [21] and Gamborg's B-5 [20] are most frequently used in *Brassica* [37]. Sometimes both hormones were used as in *B. oleracea* [5]. However, there are examples in which B5 vitamins are used with MS salts in embryo rescue of *R. sativus* [38]. Other mediums used for the embryo rescue in other plants are Knop's medium; Heller's medium [39], Monnier's medium [40] etc. The early heterotrophic immature embryos take its nutrition form the endosperms and surrounding tissue but when embryos mature it is partly autotrophic and it requires only basic mineral salts and sucrose. In *Brassica*, the embryos become autotrophic very late after globular stages [41]. The early globular stage of proembryo culture was achieved earlier using double-layer culture system and in embryo culture medium which contains mineral salts, sugars, amino acids, organic acids and coconut water [42]. The coconut water induces cell division in plant and necessary for the development of very young embryo [43]. The other additive components that used during embryo culture are tomato juice, banana pulp, different fruit juices, fish emulsion, leaf extract, potato extract etc. [44]. Sucrose is a very important constituent of the culture medium as an energy source and it maintains the osmotic potential of culture medium. High sucrose contents trigger the formation of embryos because it mimics the high osmotic potential of the embryo sac [29], and it help for the induction of embryos from embryogenic calli [17].

#### **5.3 Silver nitrate**

The silver nitrate is another very important ethylene antagonistic component that's play important role in *Brassica* embryo culture. It is added into the medium at a concentration that varies between (1–10 mgl−1). It significantly increases the regeneration potential in various *Brassica* species through both embryogenesis and organogenesis. During somatic embryogenesis the presence of AgNO3 increase the no of embryos significantly specially in *B. oleracea* and *B. rapa* [45].

#### **5.4 Temperature and light**

Embryo culture during embryo rescue influenced with temperature and light and their requirements are also varies in different plants [46]. The low temperature regime is best for the embryo rescue but in some cases at high temperature regime (26.4 °C/10.4 °C embryo rescue through ovule culture method gives better results than low temperature regime period (19.4 °C/4.3 °C) (1.75%) in *B. oleracea* [47]. According to Mei et al., [48] there is a significant quadric relation between the effective accumulated temperature (EAT) and the efficacy of ovule culture. The hybrid *B. napus* x *B. oleracea* siliqua can be collect for the excision of ovule on the basis of EAT instead of siliqua age.

#### **6. Cytology of cultured embryos**

Several biochemical and molecular changes occurs at cellular level as the embryo formed, grow in length and approaches to the maturity. The developmental studies of the somatic embryos showed that the lipid, protein and polysaccharides produced during at varying degrees when embryos were cultured on ABA containing medium. At first one or two weeks polysaccharides were produced and after that polysaccharides lipid and protein accumulated [49]. The analysis of the cultured embryos cells of oil yielding crop like *Brassica*, under electron microscopy revealed the presence of lipid bodies (spherosomes) associated with the endoplasmic reticulum [50]. The embryos cell contains several other components like some dense granules (ribonucleoprotein), endoplasmic reticulum, mitochondria, amyloplast and some irregular bodies etc. It was noted earlier that when a cell start to convert into an embryo, it start to contains smaller vacuoles in dance cytoplasm, large nucleus (Nu) with numerous organelles and stored bodies [17]. These are the steps towards the development of seed and the stored food material is the reserve for germination. The stored food material in the small somatic embryos is similar to as in the endosperms of zygotic embryos as in *Acromia aculeata*. The endosperm of zygotic embryos showed the accumulation of lipid and proteins which may consumed in the initial stages of germination and plantlet establishment (**Figure 2A-E**). However, their somatic embryos does not showed such types of deposition and this results in low conversion of these

**61**

**Figure 2.**

*Embryo Culture and Embryo Rescue in* Brassica *DOI: http://dx.doi.org/10.5772/intechopen.96058*

embryos into plants [51]. In other plants like orchids some early stage protocorm behave like somatic embryos and contains protein bodies and starch granules and it is supposed to be similar with the zygotic embryo development [52]. The deposition of the polysaccharides starts toward the basal end of the suspensor

*A Light micrographs of* Brassica juncea *somatic embryo sections, cells showed the stored food material in the form of lipid B, C, D and E Light micrographs of different histochemical tests of* Acromia aculeata *endosperm of zygotic embryos showed lipid (l) and protein bodies (pb). Source: Akmal et al [17], Moura et al. [51].*

*Embryo Culture and Embryo Rescue in* Brassica *DOI: http://dx.doi.org/10.5772/intechopen.96058*

*Brassica Breeding and Biotechnology*

**5.3 Silver nitrate**

**5.4 Temperature and light**

EAT instead of siliqua age.

**6. Cytology of cultured embryos**

induction of embryos from embryogenic calli [17].

coconut water [42]. The coconut water induces cell division in plant and necessary for the development of very young embryo [43]. The other additive components that used during embryo culture are tomato juice, banana pulp, different fruit juices, fish emulsion, leaf extract, potato extract etc. [44]. Sucrose is a very important constituent of the culture medium as an energy source and it maintains the osmotic potential of culture medium. High sucrose contents trigger the formation of embryos because it mimics the high osmotic potential of the embryo sac [29], and it help for the

The silver nitrate is another very important ethylene antagonistic component that's play important role in *Brassica* embryo culture. It is added into the medium at a concentration that varies between (1–10 mgl−1). It significantly increases the regeneration potential in various *Brassica* species through both embryogenesis and organogenesis. During somatic embryogenesis the presence of AgNO3 increase the

Embryo culture during embryo rescue influenced with temperature and light and their requirements are also varies in different plants [46]. The low temperature regime is best for the embryo rescue but in some cases at high temperature regime (26.4 °C/10.4 °C embryo rescue through ovule culture method gives better results than low temperature regime period (19.4 °C/4.3 °C) (1.75%) in *B. oleracea* [47]. According to Mei et al., [48] there is a significant quadric relation between the effective accumulated temperature (EAT) and the efficacy of ovule culture. The hybrid *B. napus* x *B. oleracea* siliqua can be collect for the excision of ovule on the basis of

Several biochemical and molecular changes occurs at cellular level as the embryo formed, grow in length and approaches to the maturity. The developmental studies of the somatic embryos showed that the lipid, protein and polysaccharides produced during at varying degrees when embryos were cultured on ABA containing medium. At first one or two weeks polysaccharides were produced and after that polysaccharides lipid and protein accumulated [49]. The analysis of the cultured embryos cells of oil yielding crop like *Brassica*, under electron microscopy revealed the presence of lipid bodies (spherosomes) associated with the endoplasmic reticulum [50]. The embryos cell contains several other components like some dense granules (ribonucleoprotein), endoplasmic reticulum, mitochondria, amyloplast and some irregular bodies etc. It was noted earlier that when a cell start to convert into an embryo, it start to contains smaller vacuoles in dance cytoplasm, large nucleus (Nu) with numerous organelles and stored bodies [17]. These are the steps towards the development of seed and the stored food material is the reserve for germination. The stored food material in the small somatic embryos is similar to as in the endosperms of zygotic embryos as in *Acromia aculeata*. The endosperm of zygotic embryos showed the accumulation of lipid and proteins which may consumed in the initial stages of germination and plantlet establishment (**Figure 2A-E**). However, their somatic embryos does not showed such types of deposition and this results in low conversion of these

no of embryos significantly specially in *B. oleracea* and *B. rapa* [45].

**60**

embryos into plants [51]. In other plants like orchids some early stage protocorm behave like somatic embryos and contains protein bodies and starch granules and it is supposed to be similar with the zygotic embryo development [52]. The deposition of the polysaccharides starts toward the basal end of the suspensor

#### **Figure 2.**

*A Light micrographs of* Brassica juncea *somatic embryo sections, cells showed the stored food material in the form of lipid B, C, D and E Light micrographs of different histochemical tests of* Acromia aculeata *endosperm of zygotic embryos showed lipid (l) and protein bodies (pb). Source: Akmal et al [17], Moura et al. [51].*

and gradually it increases and move inwards in the cortex of developing embryos. The stored lipid bodies in embryo at its peak during the cotyledon development and after that decreases. The most abundant fatty acids in somatic embryos of *Picea abies* are linolic, oleic, palmitic and 5,9-octadecanioc acids [53]. There is a quantitative difference in the composition of the fatty acids in *in vitro* and *in vivo* cultured somatic embryos [54].

#### **7. Genes involved in embryogenesis**

When a somatic cell enters into embryogenic state it starts to modify the gene expression level. There are number of genes involved in somatic embryogenesis, showed increase expression. These can be categorized in various groups [55]. These are hormone responsive genes, house-keeping genes, genes expressed during maturation, genes coding for extracellular proteins, homeotic genes, HSPs,(Heat-Shock Proteins) germins, zygotic mutants and genes for signal transductions, except these there are the genes of transcription factors. The various transcription factors regulate several genes that showed expression in the somatic embryo induction [15]. In *Arabidopsis thaliana* the basic pattern of embryo formation is the polarity of apical-basal axis and other perpendicular to the axis. It consists of shoot meristem, cotyledons, hypocotyl, root, and root meristem along the apical–basal axis and a concentric arrangement of epidermis, subepidermal ground tissue, and central vascular cylinder along the radial axis For the apical-basal axis formation asymmetric *PIN7* and *WOX2* expression is important to establish apical cell identity at the apical and basal cell of the zygote while another *gnom* mutant argues against the possibility that the different sizes of the daughter cells per se are important for apical–basal development [56]. There are some genes that interact with one another and control the pattern formation. The mutant analysis showed that the resulting mutant disturbed the pattern formation related to the primary shoot and root meristems [57]. Some embryogenesis-related genes similar expression pattern in both in vitro and in vivo embryogenesis and these were *LEA* (Late Embryogenesis Abundance) genes, *SERK*, (*SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE*), *AGL15 (AGAMOUS-LIKE15*), *BBM* (BABY BOOM gene*), LEC1*(LEAFY COTYLEDON*)*, *FUS3* (FUSCA3, B3-domin transcription factor) and *AB13 (ABSCICSIC ACID INSENSETIVE3)* [58]. There are about 250 EMB genes required for the normal development of the embryo and for complete seed development additional 219 genes product required [11, 59]. A dataset of about 510 *EMBRYO*-*DEFECTIVE* (*EMB*) genes were identified in Arabidopsis [60]. The TARGET OF RAPAMYCIN (TOR) kinase has been recognize as a key developmental regulator in both plants and animals which integrates environmental and nutrient signals to direct growth and development [61]. The rapamycin kinase receptor gene in *Arabidopsis.*

(AtRaptor1) is responsible for the early development of embryo [62]. The genes also essential for the post-embryonic plant growth because the AtRaptor1A, AtRaptor1B double mutants are defective in meristem driven growth during post embryonic stage [63].

In near future, the techniques of somatic embryos induction, and embryo rescue in *Brassica* can be further improved so that there should be about 99% chances of hybrid embryos survival. This will not only increase their survival rate but number of plantlets regenerated also improved, especially in those intergeneric crosses where the hybrid embryos survival rate is very low. This will give an opportunity to increase the productivity of the *Brassica* crop through various crop improvement programs.

**63**

**Author details**

Mohammad Akmal

College, Balrampur, India

Advanced Plant Physiology Laboratory, Department of Botany, M. L. K. PG.

© 2021 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,

\*Address all correspondence to<EMAIL_ADDRESS>
provided the original work is properly cited.

*Embryo Culture and Embryo Rescue in* Brassica *DOI: http://dx.doi.org/10.5772/intechopen.96058*

In this chapter, the somatic embryos and zygotic embryos with embryo rescue techniques are discussed. The hybrid embryo rescue technique in *Brassica* and other plants provide a useful tool to developed intergeneric and interspecific hybrids. The modification of the culture conditions, use of plant growth regulators and other complex medium components, the immature hybrid embryo can be successfully rescued at an early stage. The hybrid embryo in *Brassica* directly gives rise to new plants or some time it is desired to generate secondary embryos. The cytology of the somatic and zygotic embryos and the genes involved are also briefly discussed.

The author acknowledges Prof. M. Z. Abdin lab, Jamia Hamdard, New Delhi for

carried out research work on somatic embryogenesis in *Brassica juncea* L.

**8. Conclusion**

**Acknowledgements**

**Acronyms and abbreviations**

*BBM* BABY BOOM gene *SERK, LEC* Leafy cotyledon

*AGL15* Agamous-like15 *WUS* WUSCHEL

*LEA* Late Embryogenesis Abundance

*SERC* Somatic embryogenesis receptor-like kinase

*AB13* Abscicsic acid insensetive3

### **8. Conclusion**

*Brassica Breeding and Biotechnology*

cultured somatic embryos [54].

**7. Genes involved in embryogenesis**

and gradually it increases and move inwards in the cortex of developing embryos. The stored lipid bodies in embryo at its peak during the cotyledon development and after that decreases. The most abundant fatty acids in somatic embryos of *Picea abies* are linolic, oleic, palmitic and 5,9-octadecanioc acids [53]. There is a quantitative difference in the composition of the fatty acids in *in vitro* and *in vivo*

When a somatic cell enters into embryogenic state it starts to modify the gene expression level. There are number of genes involved in somatic embryogenesis, showed increase expression. These can be categorized in various groups [55]. These are hormone responsive genes, house-keeping genes, genes expressed during maturation, genes coding for extracellular proteins, homeotic genes, HSPs,(Heat-Shock Proteins) germins, zygotic mutants and genes for signal transductions, except these there are the genes of transcription factors. The various transcription factors regulate several genes that showed expression in the somatic embryo induction [15]. In *Arabidopsis thaliana* the basic pattern of embryo formation is the polarity of apical-basal axis and other perpendicular to the axis. It consists of shoot meristem, cotyledons, hypocotyl, root, and root meristem along the apical–basal axis and a concentric arrangement of epidermis, subepidermal ground tissue, and central vascular cylinder along the radial axis For the apical-basal axis formation asymmetric *PIN7* and *WOX2* expression is important to establish apical cell identity at the apical and basal cell of the zygote while another *gnom* mutant argues against the possibility that the different sizes of the daughter cells per se are important for apical–basal development [56]. There are some genes that interact with one another and control the pattern formation. The mutant analysis showed that the resulting mutant disturbed the pattern formation related to the primary shoot and root meristems [57]. Some embryogenesis-related genes similar expression pattern in both in vitro and in vivo embryogenesis and these were *LEA* (Late Embryogenesis Abundance) genes, *SERK*, (*SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE*), *AGL15 (AGAMOUS-LIKE15*), *BBM* (BABY BOOM gene*), LEC1*(LEAFY

COTYLEDON*)*, *FUS3* (FUSCA3, B3-domin transcription factor) and *AB13*

*(ABSCICSIC ACID INSENSETIVE3)* [58]. There are about 250 EMB genes required for the normal development of the embryo and for complete seed development additional 219 genes product required [11, 59]. A dataset of about 510 *EMBRYO*-*DEFECTIVE* (*EMB*) genes were identified in Arabidopsis [60]. The TARGET OF RAPAMYCIN (TOR) kinase has been recognize as a key developmental regulator in both plants and animals which integrates environmental and nutrient signals to direct growth and development [61]. The rapamycin kinase receptor gene in

(AtRaptor1) is responsible for the early development of embryo [62]. The genes also essential for the post-embryonic plant growth because the AtRaptor1A, AtRaptor1B double mutants are defective in meristem driven growth during post

In near future, the techniques of somatic embryos induction, and embryo rescue in *Brassica* can be further improved so that there should be about 99% chances of hybrid embryos survival. This will not only increase their survival rate but number of plantlets regenerated also improved, especially in those intergeneric crosses where the hybrid embryos survival rate is very low. This will give an opportunity to increase the productivity of the *Brassica* crop through various crop improvement

**62**

programs.

*Arabidopsis.*

embryonic stage [63].

In this chapter, the somatic embryos and zygotic embryos with embryo rescue techniques are discussed. The hybrid embryo rescue technique in *Brassica* and other plants provide a useful tool to developed intergeneric and interspecific hybrids. The modification of the culture conditions, use of plant growth regulators and other complex medium components, the immature hybrid embryo can be successfully rescued at an early stage. The hybrid embryo in *Brassica* directly gives rise to new plants or some time it is desired to generate secondary embryos. The cytology of the somatic and zygotic embryos and the genes involved are also briefly discussed.

### **Acknowledgements**

The author acknowledges Prof. M. Z. Abdin lab, Jamia Hamdard, New Delhi for carried out research work on somatic embryogenesis in *Brassica juncea* L.

### **Acronyms and abbreviations**

### **Author details**

Mohammad Akmal

Advanced Plant Physiology Laboratory, Department of Botany, M. L. K. PG. College, Balrampur, India

\*Address all correspondence to<EMAIL_ADDRESS>
© 2021 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.

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[2] Pen S, Nath UK, Song S, et al. Developmental stage and shape of embryo determine the efficacy of embryo rescue in introgressing orange/yellow color and anthocyanin genes of *Brassica* species. Plants (Basel). 2018;7(4):99. DOI: 10.3390/

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