Selection Gain of Maize Haploid Inducers for the Tropical Savanna Environments

Lacking elite haploid inducers performing high haploid induction rate (HIR) and agronomic performance is one of fundamental factors hindering the rapid adoption of doubled haploid technology in maize hybrid breeding, especially under tropical savanna climate. Breeding haploid inducers for specific agro-ecology, thus, is indispensable yet challenging. We used temperate inducer Stock6 as genetic source for haploid induction ability and eight tropical maize genotypes as principal donors for agronomic adaptation. Three cycles of modified ear-to-row with 5% intra-family selection were applied in a population set of 78 putative haploid inducer families emphasized on agronomic performance, R1-nj anthocyanin intensity, and inducer seed set. Genetic gains, variance components, and heritability on given traits were estimated. Hierarchical clustering based on five selection criteria was performed to investigate the phenotypic diversity of putative families. Cycle effect was predominant for all observed traits. Realized genetic gain was positive for HIR (0.40% per cycle) and inducer seed set (30.10% or 47.30 seeds per ear per cycle). In this study, we reported the first haploid inducers for regions under tropical savanna climate. Three inducer families, KHI-42, KHI-54, and KHI-64, were promising as they possessed HIR about 7.8% or 14 haploid seeds per tester ear and inducer seed rate about 95.0% or 208 inducer seeds per ear. The breeding method was effective for enhancing the seed set and the expression of R1-nj anthocyanin marker of inducers, yet it showed a low effectiveness to improve haploid induction rate. Introgression of temperate inducer Stock6 into tropical gene pool followed by phenotypic selections through modified ear-to-row selection on inducer seed set and R1-nj marker did not compromise the agronomic traits of tropical inducer families. Implications and further strategies for optimizing genetic gain on HIR are discussed.

Loss-of-function alleles of ZmPLD3 cause haploid induction in maize

Doubled haploid technology has been widely applied to multiple plant species and is recognized as one of the most important technologies for improving crop breeding efficiency. Although mutations in MATRILINEAL/Zea mays PHOSPHOLIPASE A1/NOT LIKE DAD (MTL/ZmPLA1/NLD) and Zea mays DOMAIN OF UNKNOWN FUNCTION 679 MEMBRANE PROTEIN (ZmDMP) have been shown to generate haploids in maize, knowledge of the genetic basis of haploid induction (HI) remains incomplete. Therefore, cloning of new genes underlying HI is important for further elucidating its genetic architecture. Here, we found that loss-of-function mutations of Zea mays PHOSPHOLIPASE D3 (ZmPLD3), one of the members from the phospholipase D subfamily, could trigger maternal HI in maize. ZmPLD3 was identified through a reverse genetic strategy based on analysis of pollen-specifically expressed phospholipases, followed by validation through the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR–Cas9) system. Mutations of ZmPLD3 resulted in a haploid induction rate (HIR) similar to that of mtl/zmpla1/nld and showed synergistic effects rather than functional redundancy on tripling the HIR (from 1.19% to 4.13%) in the presence of mtl/zmpla1/nld. RNA-seq profiling of mature pollen indicated that a large number of pollen-specific differentially expressed genes were enriched in processes related to gametogenesis development, such as pollen tube development and cell communication, during the double-fertilization process. In addition, ZmPLD3 is highly conserved among cereals, highlighting the potential application of these in vivo haploid-inducer lines for other important crop plant species. Collectively, our discovery identifies a novel gene underlying in vivo maternal HI and provides possibility of breeding haploid inducers with further improved HIR.

Genetic Approaches to Enhance Multiple Stress Tolerance in Maize

The multiple-stress effects on plant physiology and gene expression are being intensively studied lately, primarily in model plants such as Arabidopsis, where the effects of six stressors have simultaneously been documented. In maize, double and triple stress responses are obtaining more attention, such as simultaneous drought and heat or heavy metal exposure, or drought in combination with insect and fungal infestation. To keep up with these challenges, maize natural variation and genetic engineering are exploited. On one hand, quantitative trait loci (QTL) associated with multiple-stress tolerance are being identified by molecular breeding and genome-wide association studies (GWAS), which then could be utilized for future breeding programs of more resilient maize varieties. On the other hand, transgenic approaches in maize have already resulted in the creation of many commercial double or triple stress resistant varieties, predominantly weed-tolerant/insect-resistant and, additionally, also drought-resistant varieties. It is expected that first generation gene-editing techniques, as well as recently developed base and prime editing applications, in combination with the routine haploid induction in maize, will pave the way to pyramiding more stress tolerant alleles in elite lines/varieties on time.

Callose depositions underlie the incompatible reaction in intergeneric crosses of rice

Distant hybridization of cereals is often impaired by fertilization barriers. Haploid induction through intergeneric crossing is well developed in wheat but has not been successful in rice due to incompatibility issues. The present study was thus undertaken to identify fertilization barriers that hinder the compatibility of the rice cultivar Punjab Rice 121 with maize and pearl millet lines as pollinators. A total of 37,357 spikelets were pollinated, yielding 494 caryopses upon supplementation with auxins. The resultant caryopses, arising from true intergeneric crosses, lacked embryos. Imaging of the pollinated pistils at different intervals indicated that intense callose depositions block the release of generative nuclei to the ovule in these wide crosses. Rice spikelets pollinated with rice pollen (cis-generic crosses) exhibited positive indicators of fertilization reaction at the micropyle. While the cis-generic crosses initiated true caryopsis formation after 24 h, no comparative reaction was observed in the intergeneric crosses. The current survey underlines that the rice female gametophyte presents a strong pre-fertilization barrier to foreign pollen. This barrier may be modulated in the future by altering genotype and auxin combinations.

Embryo-gynogenic induction of Indonesia shallot (Allium cepa L. Aggregatum group) cultivars using three different protocol unpollinated flower cultures

Embryo-gynogenic induction was an important step in haploid induction. The research was determined the best method of unpollinated flower culture of Indonesia shallot varieties. Three methods were used including method 1 with two-step cultures using BDS as basic medium supplemented with polyamines 2 mM putrescine on the first step and spermidine 0,1 mM at the second step, method 2 using BDS as basic medium supplemented with 2,4-D 2 mgL−1 and BA 2 mgL−1 on the first step and NAA 1 mgL−1 and 2iP 2 mgL−1 on the second step and method 3 using B5 medium supplemented with 2,4-D 2 mgL−1 and BA 2 mgL−1. Six shallot cultivars used as flower donor were Katumi, Bima Brebes, Tajuk, Trisula, Superphilip, and Bauji. Flower umbel was collected and pretreated using Yoshida liquid medium then placed on incubator at four °C for overnight. The results showed that all flowers cultured could anthesis properly in the first week of cultured. The embryos could emerge from ovaries from week 7th of cultured in all methods. However, the highest percentage of embryos gynogenic developed to seedlings was achieved using method 3 in Superphilip (6.98%) followed by method 2 in Tajuk (4.44%).

Advances in Gene Editing of Haploid Tissues in Crops

Emerging threats of climate change require the rapid development of improved varieties with a higher tolerance to abiotic and biotic factors. Despite the success of traditional agricultural practices, novel techniques for precise manipulation of the crop’s genome are needed. Doubled haploid (DH) methods have been used for decades in major crops to fix desired alleles in elite backgrounds in a short time. DH plants are also widely used for mapping of the quantitative trait loci (QTLs), marker-assisted selection (MAS), genomic selection (GS), and hybrid production. Recent discoveries of genes responsible for haploid induction (HI) allowed engineering this trait through gene editing (GE) in non-inducer varieties of different crops. Direct editing of gametes or haploid embryos increases GE efficiency by generating null homozygous plants following chromosome doubling. Increased understanding of the underlying genetic mechanisms responsible for spontaneous chromosome doubling in haploid plants may allow transferring this trait to different elite varieties. Overall, further improvement in the efficiency of the DH technology combined with the optimized GE could accelerate breeding efforts of the major crops.