**6. miRNA-based strategies for improving plant crops**

miRNA functional analysis confirmed their essential role in different biological and metabolic pathways in economically important plants [37]. Several studies indicated that miRNAs are act as a riboregulatory which control gene expression during plant growth and development, and response to biotic and abiotic stress. Consequently, miRNA-based genetic engineering technology is one of the most important tools which can play an essential role to enhance agricultural production in order to generate superior crop cultivars [38]. miRNA-based regulation of gene expression manipulated by several transgenic methods like overexpression of miRNA resistant gene, the production of artificial targets [37] is used. When miRNA of interest possesses a negative control on stress factors that miRNA will be an outstanding way for crop improvement, in which transgenic plants overexpressing the semi RNAs are susceptible to stresses [39].

Additional approach in which artificial miRNAs (amiRNAs) designed and used to suppress protein-coding mRNA expression. This is an advance gene silencing technique at post-transcriptional level which has been used efficiently in different plant species [40]. The artificial miRNAs technology was used to suppress the expression of the cucumber mosaic virus suppressor 2b [18]. After efficiently inhibiting 2b expression it also enhances resistance to transgenic tobacco plants against this virus [41]. This method was found an essential way to generate advance transgenic plants with high yield and improving crop tolerance to biotic and abiotic stresses [42].

### **6.1 Improve crop tolerance to abiotic and biotic stresses using microRNA-based approaches**

Plant through molecular pathway replies to abiotic stresses involve interaction and interlinkage of different biosynthetic pathways involving gene expression regulation by miRNA and miRNA regulation [43]. Therefore, novel plant varieties produced with better environmental stress tolerance is essential for increasing crop productivity and quality. Previous studies proved that in tomato, the overexpression of miR169 increased water stress tolerance by inhibiting stomatal opening, which decreasing transpiration rate and water loss [44]. Salt stress tolerance in rice improved by decreasing expression of osa-MIR396c [45]. Similarly, transgenic rice lines produce by increasing the expression of miR398-resistant miRNA, due to this transgenic rice with more Cu- or Zn superoxide dismutases enzyme exhibited

more tolerance to high water and salinity stress [46]. Induced expression of miR319 improve cold stress tolerance and also modify leaf morphology in rice [47].

Modernisation and advancement in miRNA research have also contributed in biotic stress tolerance in several plant species. Some previous research investigated that the Osa-miR7696 miRNA overexpression produce blast resistance transgenic rice [48]. In several plant species it was studied that the overexpression of miR393 effectively hinders the microbial growth and providing a disease resistant tool [3]. Few investigations showed that the miR160a overexpression positively regulate callose deposition induced by MAMP, while miR773 and miR398b negatively control MAMP-induced callose deposition and provide specific protection to bacterial infection [49]. Numerous studies conducted on model plants like Arabidopsis to investigate the miRNA role in plants [30], but till now only few researches were done on the significant role of miRNAs. Therefore, it is concluded after detailed analysis of old literature that miRNAs and its regulation play a crucial role in stress tolerance in plants.

#### **6.2 microRNA-based approaches to improve plant growth and development**

miRNAs play a crucial role in plant growth and development pathways such as leaf morphogenesis, apical dominance and plant biomass. Several miRNAs based new method used for production of transgenic plants for improving growth and development like plant morphology, fruit quality improvement, grain yield and more shelf life [50]. Overexpression of miR319 caused continuous growth of leaf margins and larger leaflets in tomato [51]. Previous studies in *Arabidopsis* indicated that the miR156 overexpression results in the increase in number of leaves, shape and size which can be 10 times higher than normal wild-type [52]. Recent investigation on switchgrass showed that the overexpression of miR156 repressed apical dominance which results into the increase in biomass and number of tiller by 58–100% in genetic modified plants [53]. Correspondingly, in tomato the more expression of miR156, increased the number of branching and leaves, and further enhance the plant biomass but supress the apical dominance [54].

Overexpression of OsmiR397 microRNA enhance rice productivity by increasing number of panicle branching and grain size [55]. Overexpression of miR319 in rice also increases the number of small and longitudinal veins [47]. In rice overstimulation of miR390 miRNA increasing the lateral root formation [56] by decreasing the gene expression of several lateral root growth repressors such as ARF2, ARF3, and ARF4. In *Medicago truncatula*, it was studeid that the overexpression of miR160, regulate the expression of gene which were significant for gravitropic movement and root development and induce several defects in root growth, root apical meristem organization and root nodule formation [57]. It has also been well-documented that miRNAs also play an essential role in controlling transition from vegetative to floral meristem in few crop plants such as *Arabidopsis*, maize and rice. Glossy 15 (APETALA-like gene) in maize mainly control the transition from vegetative to reproductive phase along with the leaf morphogenesis. An additional study on maize demonstrated that the miR172 overexpression leads to the inhibition of glossy15 gene expression which delayed phase transition from vegetative to reproductive [58]. Furthermore, [59] demonstrates that overexpression of miR172 and miR156 promotes the adult reproductive phase however low miR172 and high miR156 expression promote juvenility. Till now various role of miRNAs are studied in economically important crop plants either by increasing or decreasing miRNA expression, the manipulation of miRNA expression can be used for confirmation of miRNA functions and provide an effective way for improving plant growth, development, fruit, and seed development as well as plant biomass and productivity [50].
