Male sterility (MS) is the inability of the plant to produce functional pollen although female fertility remains unaffected. Utilizing MS line in hybrid seed production reduces time, cost, and energy related to emasculation procedures. MS system is a multiline maintenance strategy and is broadly divided into Genic male sterility (GMS) and Cytoplasmic male sterility (CMS). GMS mechanisms contribute to tapetal cell malformation that hinders pollen development while CMS can be explained using models like the cytotoxicity model, lack of energy model, asynchronous programmed cell death model, and retrograde regulation model. Availability and maintenance of CMS sources are the major limiting factors in conventional MS system3.

Genetic engineering is the use of molecular biology to modify DNA sequence(s) in genomes, using a variety of approaches. In plant species, male sterility has been introduced by genetic engineering methods like Barnase-Barstar system, however, its use is limited due to biosafety issues. To tackle these pitfalls, genome editing (GE) comes into the picture. GE methods, in particular gene knockout by CRISPR/Cas-related tools, have resulted in flexible and successful strategies to alter the function of key genes, regulating many biological processes including MS. The gene responsible for fertility is identified using mutagenesis approach, TILLING, transcriptomics, proteomics, and molecular processes regulating anther and pollen development. The identified genes will be knocked out to induce male sterility. However, generating CRISPR-edited DNA-free plants is difficult and complex using the conventional method. To overcome this, DNA vectors through Agrobacterium infiltration, Ribonucleoprotein using biolistic method, nanotubes, virus transfection, and PEG-calcium methods can be used. In wheat, rice, maize, tomato, soybean, melon, watermelon, etc., CRISPR/Cas system was successfully used to induce male sterility3.

In one such study, CRISPR/Cas technology was used in maize to target the MS8 gene to produce male sterile lines. Vector was constructed and transformed by Agrobacterium-mediated method. Mutation in MS8 gene and male sterile phenotype was inherited in Mendelian fashion. Transgene-free ms8 lines were obtained by screening the F2 generation of male sterile plants2. In tobacco, CMS associated gene (mtatp9) was cleaved using mitoCRISPR/Cas9 with a mitochondrial localization signal. Transcriptomic analyses of the male sterile lines showed inhibition of pathways related to aerobic respiration that includes glycolysis, tricarboxylic acid cycle, and oxidative phosphorylation pathway in stamens1. Genome editing experiments conducted so far have resulted in potential male sterility which can be further tested and used in hybrid breeding in the years to come.

References:

1. CHANG, Y., LIU, B., JIANG, Y., CAO, D., LIU, Y. AND LI, Y., 2023, Induce male sterility by CRISPR/Cas9-mediated mitochondrial genome editing in tobacco. Funct Integr Genomic, 23(3): 205.

2. CHEN, R., XU, Q., LIU, Y., ZHANG, J., REN, D., WANG, G. AND LIU, Y., 2018, Generation of transgene-free maize male sterile lines using the CRISPR/Cas9 system. Front. Plant Sci., 9: 1180.

3. FARINATI, S., DRAGA, S., BETTO, A., PALUMBO, F., VANNOZZI, A., LUCCHIN, M. AND BARCACCIA, G., 2023, Current insights and advances into plant male sterility: new precision breeding technology based on genome editing applications. Front. Plant Sci., 14: 1223861.