**7. Male sterility**

The use of genetically engineered male sterility has a variety of applications, ranging from hybrid seed production to transgenic bioconfinement in genetically modified crops. The influence of this technique has aided in dealing with global food security concerns. The production of transgenic male sterile plants through the expression of a ribonuclease gene under the direction of an anther- or pollen-specific promoter has shown to be an efficient method of producing pollen-free elite cultivars.

#### **7.1 Male sterility due to mutation on nuclear genes**

Mariani et al. [162] achieved the first success in developing genetically engineered male sterility in crop plants by transforming tobacco and rapeseed plants with a chimeric dominant gene *barnase* (bacterial RNase from *Bacillus amyloliquefaciense*) driven by a tapetum-specific promoter (TA29) from tobacco. The coding sequences of RNase T1 from *Aspergillus oryzae* and *barnase* from *B. amyloliquefaciens* used to manipulate the trait were fused with the tapetum-specific TA29 promoter which is responsible for the expression of the *barnase* gene specifically to anther tapetal cells, causing selective destruction of the tapetal cell layer that surrounds the pollen sac by hydrolysing the tapetal cells, causing abnormal pollen formation (**Figure 2**). Male sterile anther carries empty exine [162]. Mariani et al. [163] demonstrated fertility

*Genetically Modified Crops and Their Impact on New Era of Agriculture DOI: http://dx.doi.org/10.5772/intechopen.105937*

restoration in TA29-barnase male sterile plants by gene encoding the barnase-specific RNase inhibitor called barstar which was isolated from same bacteria *B amyloliquefaciense*. When genetically engineered, male sterile plant is crossed with plant carrying TA29- barstar gene the F1 progeny shows co-expression of both genes in the anther of male fertile plants. In this system fertility restoration is due to the formation of tapetal cell-specific barnase/barstar protein complexes which completely inactivate the barnase enzyme [163]. This dominant nuclear genetic male sterility system faces same drawback as GMS system. During hybrid seed production the plants in female rows segregate in the ratio of 1:1 for male sterility and male fertility [164]. To counter the drawback of nuclear genetic male sterility system problem the *barnase* gene was linked to a dominant herbicide resistant gene (bar) under control of the constitutive promoter CaMV 35S which conferred resistance to broad-spectrum herbicide Basta (active ingredient is phosphinothricin or PPT). When seedlings are sprayed with Basta only the male sterile plants survive and the male fertile plants are killed as they lack bar gene.The use of bar gene allows elimination of male fertile segregants from female rows in the hybrid seed production plot thus assuring 100 per cent pure hybrid seed production [165].

#### **7.2 Male sterility due to mutation on chloroplast genes**

The genetic transformation of the plastid genome has various advantages, including high level transgenic expression, expression of multigene operons, transgene maternal inheritance, and expression of bacterial genes without codon optimization [166]. Ruiz and Daniell [167] elucidated that, with chloroplast transformation, hyper-expression of β-ketothiolase encoded by the phaA gene of Acinetobacter sp. in the leaves, flower, and anther of transgenic lines gets in the way of pollen development and results in male sterility. This was restored by exposing transgenic male sterile plants to continuous illumination, which allows acetyl CoA carboxylase (ACCase) to access acetyl CoA, restoring normal fatty acid synthesis and minimising PHB production through β-ketothiola.

#### **7.3 Male sterility due to altering metabolic process**

Callose is a plant polysaccharide comprised of β-1-3 glucan that is deposited around microspore tetrads during meiosis. Tight developmental regulation and the timing of callase activity are required for optimal microspore development. The expression of modified PR-b-1-3 glucanase in transgenic tobacco plants led in the premature disintegration of the microsporocyte callose wall, resulting in mild to total male sterility [168]. Chang et al. [169] created a rice hybrid breeding method employing the rice nuclear gene *Oryza sativa* No Pollen 1 (*OsNP1*), which encodes a putative glucose–methanol–choline–oxidoreductase with involvement in tapetum degeneration and pollen exine production. The ethyl methane sulfonate-induced rice mutant, *osnp1-1*, was completely male sterile.

#### **7.4 Conditional male sterility**

Conditional male sterility is a situation in which plants are typically fertile, but when a specific circumstance is applied, male sterility occurs. Hawkes et al. [170] revealed the use of inactive D-glufosinate as a male sterility inducer in transgenic plants expressing a modified (F58 K, M213S) version of *Rhodosporidium toruloides* Damino acid oxidase (DAAO) that converts oxidised D-glufosinate to its 2-oxoderivative (2-oxo-4-methyl phosphiny to create transgenic plants, the modified DAAO encoding gene was coupled with the TAP1 promoter from *Antirrhinum majus* and transformed into tobacco plants. When D-glufosinate was sprayed on these transgenic plants, it caused full male sterility that lasted two or more weeks while having no effect on female fertility [170]. Guerineau et al. [171] expressed the temperature-sensitive diphtheria toxin A-chain polypeptide gene sequence under the tapetum-specific A9 promoter and generated transgenic Arabidopsis plants that were fully fertile at 26 C, but when the temperature was decreased to 18°C, male sterility was induced [171].

#### **7.5 Male sterility through post transcriptional gene silencing**

Jasmonic acid (JA), a plant hormone, is involved in several developmental signalling events in plants, including senescence, fruit ripening, anther dehiscence, and pollen maturation [172, 173]. Bae et al. [174] reported inducing male sterility by inhibiting OsAOS1 and OsAOS2 activity with the promoters of the anther-specific genes Osc4 and Osg6b, respectively. RNAi (pSK124) constructs were designed and converted into rice calli independently, concluding that the OsAOS2-RNAi vector driven by Osg6b promoter is potent enough for generating male sterility in rice.

#### **7.6 Male sterility through modification of flavonoids**

Any disruption in flavonoid production changes pigmentation and causes male sterility in plants. Fischer et al. [175] discovered the expression of a stilbene synthase (STS) gene from grape vine (*VstI*) driven by a 35S RNA promoter with duplicated enhancer region and a tapetum-specific promoter (Tap1) of *A. majus* produced male sterility strives for the substrates,4-coumaroyl CoA and malonyl CoA, which are required for sporopollenin and fatty acid biosynthesis, and hypothesised that there was a decrease in p-coumaroyl availability, resulting in impaired sporopollenin production and pollen wall formation, causing male sterility(**Figure 3**).

*Genetically Modified Crops and Their Impact on New Era of Agriculture DOI: http://dx.doi.org/10.5772/intechopen.105937*

#### **Figure 3.**

*Simplified version of male sterility through modification of flavonoids.*

#### **7.7 Male sterility through RNA editing**

Nucleotide alterations or insertion of a nucleotide, leading to a change in the sequence of amino acid in polypeptide denotes RNA editing. Male sterility in CMS plants is connected to mitochondrial DNA rearrangement, causing the formation of novel chimeric open reading frames (ORFs), resulting in mitochondrial malfunction, such as the chimeric gene pcf-S of petunia, ORFB and ORF224 of polima in rapeseed [176]. The overexpression of unedited mitochondrial orfB gene in a transgenic strain of indica rice led to a decrease in activity of ATPase in F1F0-ATP synthase resulting in dose-dependent male sterility [177].

#### **7.8 Heterologous male sterility**

The association of CMS and new chimeric ORFs in mitochondrial DNA sequences, as well as mitochondrial dysfunction, is documented. Nizampatnam et al. [178] engineered transgenic tobacco plants to produce orfH522, a pet1-CMS-associated mitochondrial gene from sunflower that is driven by the TA29 promoter. Approximately 35% of the modified tobacco plants were completely sterile. Subsequently, by decreasing orfH522 transcripts using the RNAi approach, male fertility was restored [179].

### **8. GE for yield contributing characters**

To address growing food demand as well as the challenges posed by climate change, major increases in yields of vital food crops employing transgenic technology are required. Using CRISPR-Cas9, researchers were able to increase grain number, dense erect panicles, and grain size in rice by disrupting the DEP1, Gn1a, IPA1, and GS3 genes, which are regulators of grain number, panicle architecture, grain size, and plant architecture [180]. CRISPR-Cas9 gene deletion targets the wheat genes TaGW2-B1, TaGW2-D1, and TaGW2-A1 that govern grain weight and protein content, leading to an increase in grain weight and protein content [181]. In maize, the gene ARGOS8 responded to water stress by increasing grain output [182].

#### **Figure 4.**

*Simplified version of comparison between normal mosquito population and genetic drive mosquitos' population. There is a huge discrepancy among the rapid adoption of GM crops for production, global markets, and consumer approval. However, the following is a list of transgenic crops that have been worldwide authorised and released for various characteristics (adopted from ISAAA database).*
