Male sterility in plants: an overview of advancements from natural CMS to genetically manipulated systems for hybrid seed production.

KEY MESSAGE: The male sterility system in plants has traditionally been utilized for hybrid seed production. In last three decades, genetic manipulation for male sterility has revolutionized this area of research related to hybrid seed production technology. Here, we have surveyed some of the natural cytoplasmic male sterility (CMS) systems that existed/ were developed in different crop plants for developing male sterility-fertility restoration systems used in hybrid seed production and highlighted some of the recent biotechnological advancements in the development of genetically engineered systems that occurred in this area. We have indicated the possible future directions toward the development of engineered male sterility systems. Cytoplasmic male sterility (CMS) is an important trait that is naturally prevalent in many plant species, which has been used in the development of hybrid varieties. This is associated with the use of appropriate genes for fertility restoration provided by the restorer line that restores fertility on the corresponding CMS line. The development of hybrids based on a CMS system has been demonstrated in several different crops. However, there are examples of species, which do not have usable cytoplasmic male sterility and fertility restoration systems (Cytoplasmic Genetic Male Sterility Systems-CGMS) for hybrid variety development. In such plants, it is necessary to develop usable male sterile lines through genetic engineering with the use of heterologous expression of suitable genes that control the development of male gametophyte and fertile male gamete formation. They can also be developed through gene editing using the recently developed CRISPR-Cas technology to knock out suitable genes that are responsible for the development of male gametes. The present review aims at providing an insight into the development of various technologies for successful production of hybrid varieties and is intended to provide only essential information on male sterility systems starting from naturally occurring ones to the genetically engineered systems obtained through different means.

Ectopic expression of DnaJ type-I protein homolog of Vigna aconitifolia (VaDJI) confers ABA insensitivity and multiple stress tolerance in transgenic tobacco plants.

Reduced crop productivity results from altered plant physiological processes caused by dysfunctional proteins due to environmental stressors. In this study, a novel DnaJ Type-I encoding gene, VaDJI having a zinc finger motif in its C-terminal domain was found to be induced early upon treatment with heat stress (within 5 min) in a heat tolerant genotype of Vigna aconitifolia RMO-40. VaDJI is induced by multiple stresses. In tobacco, ectopic expression of VaDJI reduced ABA sensitivity during seed germination and the early stages of seedling growth of transgenic tobacco plants. Concomitantly, it also improved the ability of transgenic tobacco plants to withstand drought stress by modulating the photosynthetic efficiency, with the transgenic plants having higher Fv/Fm ratios and reduced growth inhibition. Additionally, transgenic plants showed a reduced build-up of H2O2 and lower MDA levels and higher chlorophyll content during drought stress, which attenuated cell damage and reduced oxidative damage. An analysis using the qRT-PCR study demonstrated that VaDJI overexpression is associated with the expression of some ROS-detoxification-related genes and stress-marker genes that are often induced during drought stress responses. These findings suggest a hypothesis whereby VaDJI positively influences drought stress tolerance and ABA signalling in transgenic tobacco, and suggests that it is a potential gene for genetic improvement of drought and heat stress tolerance in crop plants.

Improved photosynthetic characteristics correlated with enhanced biomass in a heterotic F1 hybrid of maize (Zea mays L.).

Heterosis is a phenomenon wherein F1 hybrid often displays phenotypic superiority and surpasses its parents in terms of growth and agronomic traits. Investigations on the physiological and biochemical properties of the heterotic F1 hybrid are important to uncover the mechanisms underlying heterosis in plants. In the present study, the photosynthetic capacity of a heterotic F1 hybrid of Zea mays L. (DHM 117) that exhibited a higher growth rate and increased biomass was compared with its parental inbreds at vegetative and reproductive stages in the field during 2017 and 2018. The net photosynthetic rate (Pn), stomatal conductance (gs), transpiration rate (E) as well as foliar carbohydrates were higher in F1 hybrid than parental inbreds at vegetative and reproductive stages. An increase in total chlorophyll content along with better chlorophyll a fluorescence characteristics including effective quantum yield of photosystem II (ΔF/Fm'), maximum quantum yield of PSII (Fv/Fm), photochemical quenching (qp) and decreased non-photochemical quenching (NPQ) was observed in F1 hybrid than the parental inbreds. Further, the expression of potential genes related to C4 photosynthesis was considerably upregulated in F1 hybrid than the parental inbreds during vegetative and reproductive stages. Moreover, the F1 hybrid exhibited distinct heterosis in yield with 63% and 62% increase relative to parental inbreds during 2017 and 2018. We conclude that improved photosynthetic efficiency associated with increased foliar carbohydrates could have contributed to higher growth rate, biomass and yield in the F1 hybrid.

A proteomic study of cysteine protease induced cell death in anthers of male sterile tobacco transgenic plants.

Manifestation of male sterility in plants is an important requirement for hybrid seed production. Tapetum cell layer of anther is a primary target for genetic manipulation for male sterility. In our previous report, the targeted expression of Arachis cysteine protease in tapetum led to premature degeneration of tapetal layer that resulted in complete male sterility in transgenic tobacco plants. To correlate cysteine protease mediated cell death of tapetum, transmission electron microscopy (TEM) and proteomic pattern of anthers of cysteine protease induced male sterile plant were compared with the untransformed control plant. TEM study revealed the abnormal growth of tapetal cells exhibiting excessive vacuolization that synchronized with irregular exine wall formation of the microspores. In anther proteome, a total 250 protein spots were detected that were reproducible and exhibited similar distribution pattern. Further, anther proteome of male sterile plant showed the significant upregulation (≥ 1.5) of 56 protein spots. Using Mass spectroscopy (MALDI TOF/TOF), we have identified 14 protein spots that were involved in several processes such as energy metabolism, protein synthesis, plastid protein, lipid metabolism, and cell wall assembly. Upregulation of patatin-like protein-2 homolog, carboxylesterase 17 and dicer like protein-4 in male sterile anthers that have been demonstrated to induce cell death, suggesting that cysteine protease mediated premature tapetal cell death might involve the lipid peroxidation pathway in coordination with gene silencing mechanism.

Genome-wide characterization of ALDH Superfamily in Brassica rapa and enhancement of stress tolerance in heterologous hosts by BrALDH7B2 expression.

Aldehyde dehydrogenase (ALDH) carries out oxidation of toxic aldehydes using NAD+/NADP+ as cofactors. In the present study, we performed a genome-wide identification and expression analysis of genes in the ALDH gene family in Brassica rapa. A total of 23 ALDH genes in the superfamily have been identified according to the classification of ALDH Gene Nomenclature Committee (AGNC). They were distributed unevenly across all 10 chromosomes. All the 23 Brassica rapa ALDH (BrALDH) genes exhibited varied expression patterns during treatments with abiotic stress inducers and hormonal treatments. The relative expression profiles of ALDH genes in B. rapa showed that they are predominantly expressed in leaves and stem suggesting their function in the vegetative tissues. BrALDH7B2 showed a strong response to abiotic stress and hormonal treatments as compared to other ALDH genes; therefore, it was overexpressed in heterologous hosts, E. coli and yeast to study its possible function under abiotic stress conditions. Over-expression of BrALDH7B2 in heterologous systems, E. coli and yeast cells conferred significant tolerance to abiotic stress treatments. Results from this work demonstrate that BrALDH genes are a promising and untapped genetic resource for crop improvement and could be deployed further in the development of drought and salinity tolerance in B. rapa and other economically important crops.

Targeted expression of a cysteine protease (AdCP) in tapetum induces male sterility in Indian mustard, Brassica juncea.

The development of male sterile plants is a prerequisite to developing hybrid varieties to harness the benefits of hybrid vigor in crops and enhancing crop productivity for sustainable agriculture. In plants, cysteine proteases have been known for their multifaceted roles during programmed cell death, and in ubiquitin- and proteasome-mediated proteolysis. Here, we showed that Arachis diogoi cysteine protease (AdCP) expressed under the TA-29 promoter induced complete male sterility in Indian mustard, Brassica juncea. The herbicide resistance gene bar was used for the selection of transgenic plants. Mustard transgenic plants exhibited male sterile phenotype and failed to produce functional pollen grains. Irregularly shaped aborted pollen grains with groove-like structures were observed in male sterile plants during scanning electron microscopy analysis. The T1 progeny plants obtained from the seed of primary transgenic male sterile plants crossed with the wild-type plants exhibited segregation of the progeny into male sterile and fertile plants with normal seed development. Further, male sterile plants exhibited higher transcript levels of AdCP in anther tissues, which is consistent with its expression under the tapetum-specific promoter. Our results clearly suggest that the targeted expression of AdCP provides a potential tool for developing male sterile lines in crop plants by the malfunction of tapetal cells leading to male sterility as shown earlier in tobacco transgenic plants (Shukla et al. 2014, Funct Integr Genomics 14:307-317).

Molecular Approaches for Manipulating Male Sterility and Strategies for Fertility Restoration in Plants.

Usable pollination control systems have proven to be effective system for the development of hybrid crop varieties, which are important for optimal performance over varied environments and years. They also act as a biocontainment to check horizontal transgene flow. In the last two decades, many genetic manipulations involving genes controlling the production of cytotoxic products, conditional male sterility, altering metabolic processes, post-transcriptional gene silencing, RNA editing and chloroplast engineering methods have been used to develop a proper pollination control system. In this review article, we outline the approaches used for generating male sterile plants using an effective pollination control system to highlight the recent progress that occurred in this area. Furthermore, we propose possible future directions for biotechnological improvements that will allow the farmers to buy hybrid seed once for many generations in a cost-effective manner.