**3. Future perspectives for seed vigor improvement through** *-Omics* **results**

**2.3. Mapping the genes controlling seed aging/vigor**

270 Advances in Seed Biology

putative genes controlling various seed vigor traits in different crops.

BC3 F4

The use of molecular markers in modern plant breeding to increase selection efficiency through mapping genes to specific traits of interest was made possible by *-omics* precision tools. For many simply inherited traits of economic importance, fine-mapping and tagging with closely linked or gene-specific markers is straightforward simple. However, seedling vigor in crop plants is a complex quantitative trait under the control of large genotype and environment (GxE) effects. Hence, the advent of genomics tools for mapping and analyzing quantitative trait loci (QTL) is a major breakthrough for breeding seed vigor traits and gene identification for further experimentation. From many seed deterioration experiments, QTLs of seed longevity traits like LD50 in Arabidopsis seeds [21], germination of aged wheat seeds [48, 49] and half-life (P50) of aging barley seeds [50] have been mapped and linked to various genes. For the germination vigor of seeds, several seed priming experiments have found QTLs for germination of maize seeds [51] and QTLs for 30 vigor traits of rice seeds [52] to mention a few. These studies also provided useful information on chromosome regions and

QTL work on seed vigor began with the pioneering work of Clerkx et al*.* [21] on the model plant Arabidopsis, where QTL mapping was used to identify the loci controlling various aspects of seed longevity during storage and germination. Genotyping a recombinant inbred line population with 65 PCR-based markers and seed LD50 of phenotypic marker erecta, they identified three QTLs affecting seed longevity after controlled deterioration on chromosomes 1, 3, and 4 for Arabidopsis. Nagel et al*.* [50] also reported large QTL effects associated with seed half-life (P50) on chromosomes 5 and 7 in a doubled haploid mapping population of barley. Han et al*.* [51] found 65 QTLs in two maize populations mapped using single-nucleotide polymorphism (SNP) markers to four seed vigor traits under four germination treatment conditions. Integrating the QTLs into 18 meta-QTLs (mQTLs), 23 candidate genes associating with seed vigor phenotype coincides with 13 mQTLs controlling protein metabolism and the glycolytic pathway. They reported four seed vigor hotspots on chromosome regions for mQTL2, mQTL3-2, mQTL3-4, and mQTL5-2 with large QTL effects under various germination environments. There are a number of recent QTL studies on seed vigor of rice [52, 53]. Singh et al*.* [52] reported seed germination capacity of primed rice seeds derived from 253

 lines of crosses between Swarna and Moroberekan, phenotyped for early vigor and genotyped with 194 SNP markers. They identified six seed vigor genomic regions on chromosomes 3, 4, 5, and 6 [52]. Two of the QTL regions namely chr3 (*id3001701*-*id300833*) and chr5 (*wd5002636-id5001470*) were identified and tagged QTL hotspots because they were expressed consistently in field and glasshouse conditions. In the chr3 hotspot, most of QTLs identified for early vigor-related traits were *qEV3.1, qEUE3.1, qSHL3.1, qSL3.1, qSFW3.1, qTFW3.1, qRDW3.1* associated with early vigor, early uniform emergence, shoot length, stem length, shoot fresh weight, total fresh weight and root dry weight respectively. The QTL hotspot on chr5 includes almost similar seed vigor traits as the first hotspot except total fresh weight and root dry

weight but includes seed dry weight (*qSDW5.1*) and total dry weight (*qTDW5.1*).

From these QTL regions identified in the brief review above, putative candidate genes associated with many seed vigor traits in the hotspot QTL regions have been published for crops like This review has highlighted key advances provided by various *-omics* platforms for the dissection of the complex trait called seed vigor. With the current advances in *-omics* applications to seed vigor biomarkers, understanding of the regulatory mechanisms, gene mapping to traits, and genomic database resources for seed vigor, a unique platform for genetic manipulation of seed traits is emerging. We are moving towards a revolution of crop production that explores the complex traits of seed vigor for enhanced productivity in the face of environmental challenges, increasing human population and rising intensity of costs and land resources for food production.

The work of Xu et al*.* [31] on *PIMT* encoded genes (*PIMT1* and *PIMT2*), which display distinct expression patterns but similar biochemical properties of repairing IsoAsp accumulation in seed proteins is blazing a trail which researchers have validated for a number of crops [14, 16, 22]. For example, Rajjou et al*.* [20] confirmed that transgenic *Arabidopsis* seeds over-expressing *NnMT2a* and *NnMT3* displayed a remarkably improved resistance to accelerated aging treatment, indicating their significant roles in seed germination vigor. Wei et al*.* [16] worked on one of the two *PIMT* genes from rice (*Oryza sativa* L.) and found that over-expression of *OsPIMT1* in transgenic rice seeds reduced the accumulation of isoAsp-containing protein in embryos, and increased embryo viability. Petla et al*.* [14] also reported that transgenic rice constitutively over-expressing *OsPIMT1* and *OsPIMT2* exhibited improved seed vigor and longevity. These data indicated that engineering *OsPIMT*-related seed longevity improvement is a feasible option for producing enhanced vigor GMO seeds through target-gene methods. A way forward from understanding the clear role of *PIMT* in seed vigor improvement is the application of these findings for genetic engineering of *PIMT* towards improving seed vigor. Wu et al*.* [34] summarized current knowledge on *PIMT* gene modifications, specific genetic engineering methodologies and their outcomes for seed vigor improvement in three different crops (**Table 2**). Altering *PIMT* accumulation in seeds shows various effects of physiological significance in the various studies opening opportunities for genetically manipulating seed vigor. While *PIMT* offer opportunity for producing high vigor seeds through the repair mechanism, other candidate genes utilizing alternative strategies to producing seed vigor phenotypes are also waiting to be explored. Examples are the LEA and HSP proteins that use the protective gene mechanism, the detoxification mechanism that uses the ROS scavenging gene action [43] and the AQP water uptake mechanism to enhance seed vigor [46]. This leaves a wide research gap that are indeed opportunities to explore towards mapping and genetic engineering of these classes of proteins already identified as implicated in enhancing inherent seed vigor. The obvious research questions raised from this review are whether other single-gene manipulations methods can also produce such effects as *PIMT.* Other research concerns might be the investigation of the effects of enhanced expression of seed vigor genes/proteins on other seed traits like nutrient value, potential health risk as food and feed and ethical issues of GMO seeds for innate vigor.

Since 2013, newer *-omics* tools that allow genome-editing and gene targeting are poised to contain ethical concerns of GMOs because of its capacity for precise modulation of traits of interest with unprecedented control and efficiency. A set of techniques called clustered, regularly interspaced, short palindromic repeat (CRISPR) technology capable of making precise targeted changes in the genome of living cells appeared recently [57], and can be the next great opportunity for genetic manipulation of seed vigor. Coming out of this is the CRISPR-Cas9 which is the latest borderline technology based on a bacterial CRISPR-associated protein-9 nuclease (Cas9) from *Streptococcus pyogenes* [58]. This has already been successfully used to target important genes in many cell lines and organisms. The simplicity of this method lends it to wide applications in biology, currently, it is possible to introduce single point mutations (deletions or insertions) in a target DNA with a guide RNA (gRNA) [59]; and induce large genomic re-arrangements, such as inversions or translocations with a pair of gRNA-directed


**Table 2.** Seed vigor outcomes of different PIMT gene alteration methodologies from different reference sources in various crop species [34].

Cas9 nucleases [60]. Proteins can also be targeted for transcriptional regulation using dCas9 version of the CRISPR-Cas9 system [57].

For seed vigor improvement, CRISPR-Cas9 can be used to manipulate gene functions that directly regulate DNA repair pathways like nucleotide and base excision repair, the nonhomologous end joining and homologous recombination all of which play notable roles in seed vigor development [3]. Furthermore, the capacity of CRISPR-Cas9 to enable rapid genome-wide study of gene function by generating large gRNA libraries for genomic screening offer opportunity for large scale deployment of precision *-omics* technology for genetic engineering of seed vigor. Of particular relevance of the new technology to crop breeding is the possibility of removing targeted gene constructs by conventional breeding in subsequent generations of the modified plants, thus addressing concerns of GMO contaminations.
