Milestones achieved in response to drought stress through reverse genetic approaches

Drought stress is the most important abiotic stress that constrains crop production and reduces yield drastically. The germplasm of most of the cultivated crops possesses numerous unknown drought stress tolerant genes. Moreover, there are many reports suggesting that the wild species of most of the modern cultivars have abiotic stress tolerant genes. Due to climate change and population booms, food security has become a global issue. To develop drought tolerant crop varieties knowledge of various genes involved in drought stress is required. Different reverse genetic approaches such as virus-induced gene silencing (VIGS), clustered regularly interspace short palindromic repeat (CRISPR), targeting induced local lesions in genomes (TILLING) and expressed sequence tags (ESTs) have been used extensively to study the functionality of different genes involved in response to drought stress. In this review, we described the contributions of different techniques of functional genomics in the study of drought tolerant genes.


Introduction
Nowadays, global food security has becomes a major challenge due to the extreme changes to the climate and increases in the global population 1 . Therefore, plants are growing under various kinds of unfavourable environmental stresses such as drought, salinity, heat, cold and oxidative stresses which are retarding the growth and yield 2,3 . Of these, drought stress is the most predominant abiotic stress making this situation worse. Over the last decade, climate change has been increasing the frequency drought conditions and reduced the crop yield (Table 1) by affecting the basic plant growth processes such as seed germination, photosynthesis, source sink relationships, turgor pressure, cell division and elongation, enzyme activities, and secondary metabolites production [14][15][16][17][18][19][20][21][22][23][24] . In addition, drought can also increase the production and accumulation of reactive oxygen species (ROS) in plants which leads to oxidative stress too 25,26 . Several genes that express under drought conditions are involved in the regulation of all these processes and pathways. In recent years, many drought tolerant genes have been identified in major food crops and still there are numerous genes taking part in drought stress whose functions are unknown. With the help of available genomic and transcriptomic data reverse genetic approaches accelerated the investigations of gene function under different abiotic stresses 27 .
From the perspective of crop improvement, transgenic approaches have been successfully used in many crops. However, development of stable transgenic lines is relatively expensive, time consuming and a laborious task. Moreover, it is not successful in many cultivated crops and slows down the investigations into specific gene 28 . In contrast, there are several techniques available for the study of these genes which give prompt results and have other advantages over transgenic techniques for analysis of target gene(s) such as virus-induced gene silencing (VIGS), clustered regularly interspace short palindromic repeat (CRISPR)-Cas9 system, targeting induced local lesions in genomes (TILLING) and expressed sequence tags (ESTs) 29-32 .

VIGS
It is a simple, rapid, reliable and cost-effective post-transcriptional gene silencing (PTGS) technique for the study of endogenous genes. It is a powerful tool for the mining and study of genes involved in drought tolerance (Table 2). In VIGS a 200-400bp long fragment of the target gene is selected and cloned into a

Conclusion and future perspectives
Severe droughts are becoming more common every year and are reducing crop yield considerably. There is an urgent need for drought tolerant varieties. Breeding and transgenic approaches could solve this problem but the knowledge of molecular mechanisms and genes taking part in drought tolerance is essential. Several reverse genetic techniques have proved their potential in many crops and some are still evolving. During the last decade, the genomes of several crops were successfully sequenced, various new VIGS systems have been developed for different crops [126][127][128][129][130][131] and CRISPR has become the most powerful tool for genome editing [132][133][134] . Thus, these techniques can play a pivotal role in crop improvement and can contribute highly in the development of drought tolerant varieties.

Data availability
No data are associated with this article

Competing interests
No competing interests were disclosed.

Grant information
The author(s) declared that no grants were involved in supporting this work.

2.
3. The review entitled "Milestones achieved in response to drought stress through reverse genetic approaches" by Singh presents recent advances in understanding the response of drought stress in et al., crop plants using reverse genetic technologies. It is well known that drought stress is major concern in the era of climate change. Therefore, it is important to address the recent updates on drought stress response in crop plants to scientific communities. The review article is written and organized very well but minor points need to be taken care.

Open Peer Review
Authors have explained several techniques available to study the functionality of different genes involved in response to drought stress but should also include the advantages of these techniques in monocots/dicots.
In VIGS, authors should explain the functional analysis of transcription factors using VIGS DREB technology citing some latest references.
In ESTs analysis, authors should include the analysis of drought responsive ESTs generated through chemical priming studied in crop plants. Authors should arrange them alphabetically Keywords:

Is the review written in accessible language? Yes
At the end of first paragraph, authors suggested about available transcriptomic data. I Introduction: believe there they can add some references of available transcriptomes related to the topic. Overall, it has been written fine.
Authors provided all the required information related to this technology. However, there are some VIGS: bioinformatics tools, which helps in the selection of target fragment within the gene. They can add these too. Ok

Conclusion:
To summarize, the review by presents our current knowledge of many genes deciphered by Singh et al. reverse genetic technologies: and The tables represents ample amount of VIGS, EST, TILLING CRISPR. information in a well-organised way. Overall, the Review provides a useful compilation of subject matter related to addressed topic in a coherent way.

Is the review written in accessible language? Yes
Are the conclusions drawn appropriate in the context of the current research literature? Yes No competing interests were disclosed.

Competing Interests:
I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.