In vitro fertilization (IVF): production of distant hybrids with suitable genome combination

Abstract

Hybridization generates biodiversity and wide hybridization plays a pivotal role in enhancing and broadening the useful attributes of crops. Hybridization can be achieved through sexual or somatic hybridization between distant species; however, sexual hybridization between intergeneric or inter-subfamily combinations often results in unsuccessful fertilization/embryogenesis due to pre- and/or post-fertilization barriers. In addition, as cytoplasmic organelles are maternally transmitted to the zygote via sexual reproduction, cytoplasmic genome hybridization is rare. Although somatic hybridization offers distinct advantages over sexual hybridization, the isolation and culture of protoplasts are not feasible in most plant species, including vital crops. Recently, allopolyploid wheat–rice or wheat–maize hybrid zygotes were produced by in vitro fertilization (IVF) using isolated wheat, rice, and maize gametes, and these hybrid zygotes developed into wheat cytoplasmic hybrids (cybrids) with wheat–rice or wheat–maize hybrid mitochondria. These indicate that the IVF system with isolated gametes provides a solution to limitations of fertilization barriers and one-way cytoplasmic inheritance in sexual and somatic hybridizations, and that the present rice–wheat cybrid, ^OryzaWheat, and maize–wheat, ^ZeaWheat, will provide a new horizon for the utilization of inter-subfamily genetic resources.

Introduction

Hybridization is a key strategy in plant breeding, as this process can increase genetic variation through sexual or somatic crosses between distant species. Sexual hybridization is generally conducted through cross-pollination, and the resulting hybrid zygote, which possesses totipotency for embryogenesis, can transmit heterogeneous genetic material to progeny. However, sexual hybridization between intergeneric or inter-subfamily combinations often results in unsuccessful fertilization/embryogenesis due to pre- and/or post-fertilization barriers (Haig and Westoby 1991, Morgan et al. 2010). In addition, as cytoplasmic organelles are maternally transmitted to the zygote during sexual reproduction, cytoplasmic genome hybridization is rare.

Meanwhile, somatic hybridization offers distinct advantages compared to sexual hybridization. This process involves fusing somatic protoplasts from different species, resulting in hybrid somatic cells with nuclear and cytoplasmic genomes from the respective species. However, isolation and culture of protoplasts is not feasible in most plant species. In addition, in the majority of monocotyledonous plants, including vital crops such as rice, maize, and wheat, regeneration from protoplasts remains either unattainable or inefficient (Davey et al. 2005, Eeckhaut et al. 2013).

Recently, using in vitro fertilization (IVF) with isolated wheat and rice gametes, we produced allopolyploid wheat–rice hybrid zygotes that developed into wheat cytoplasmic hybrids (cybrids) with wheat–rice hybrid mitochondria (Fig. 1; Maryenti et al. 2021, 2024). In addition, cybrids with wheat–maize hybrid mitochondria were also recently created in our laboratories (Onda et al. 2025). These indicate that the IVF system with isolated gamete provides a solution to limitations of fertilization barriers and one-way cytoplasmic inheritance in sexual and somatic hybridizations, and serves as a bridge between sexual crossing and somatic hybridization. Thus, after an explanation of the IVF system for plant gametes, this review addresses the current status of hybrid production via IVF. Thereafter, possible new plant cytoplasmic breeding via the IVF system and future perspectives for cybrid research are discussed.

Fig. 1.

Cytoplasmic hybrids, ^OryzaWheat and ^ZeaWheat, across three major crops belonging to different subfamilies.

In vitro fertilization with plant gametes

Fertilization is the key event in the life cycle of higher organisms. In angiosperms, the female gametophyte, also called the embryo sac or megagametophyte, develops in the ovule, and fertilization and subsequent events, such as embryogenesis and endosperm development, occur within the embryo sac, which is deeply embedded in the ovular tissue (Raghavan 2003, Russell 1992). Meanwhile, difficulties in directly researching the biology of the embedded female gamete, zygote, and early embryo have impeded investigations into the molecular mechanisms of fertilization and embryogenesis. Therefore, such studies have been conducted predominantly by analyzing Arabidopsis mutants or transformants, coupled with live imaging (reviewed in Berger 2011, Hamamura et al. 2012, Maruyama et al. 2015). Alternatively, direct analyses using isolated gametes or zygotes are possible because procedures for isolating viable gametes have been established, and IVF systems using isolated gametes can be used to observe and analyze fertilization and post-fertilization processes directly (reviewed in Wang et al. 2006).

The IVF system used for angiosperms involves a combination of three basic microtechniques: (i) the isolation and selection of male and female gametes, (ii) the fusion of pairs of gametes, and (iii) single-cell cultures (Fig. 2; Kranz 1999). Procedures have been established in a wide range of plant species for the isolation of viable gametes, including both monocotyledonous and dicotyledonous plants (reviewed in Kranz 1999 and in Okamoto 2011). As for gamete fusion, the fusion procedure can be performed electrically (Kranz and Lörz 1993, Uchiumi et al. 2007) or chemically via calcium (Faure et al. 1994, Khalequzzaman and Haq 2005, Kranz and Lörz 1994), polyethylene glycol (PEG) (Sun et al. 1995, Tian and Russell 1997), or bovine serum albumin (BSA) (Peng et al. 2005), as plasma membranes of isolated gametes are partly or largely exposed, and plasma membranes between the isolated gametes can attach. Investigations with calcium-based fusion of maize gametes have shown that calcium influx is triggered by gamete fusion and that cell wall formation, an event known as egg activation, is induced by the calcium influx (Antoine et al. 2001). Moreover, fusion behavior and gamete interaction have been traced using a PEG-mediated gamete fusion procedure (Sun et al. 2000). However, as presented in Table 1, zygotes produced by calcium-, PEG-, or BSA-fusion appeared to become arrested in development (Kranz and Lörz 1994, Peng et al. 2005, Sun et al. 2000, Tian and Russell 1997). Importantly, among these four different procedures, only zygotes produced by electro-fusion are known to divide and develop into embryo-like structures and plantlets.

Fig. 2.

Schematic illustration of procedures for an in vitro fertilization system with wheat gametes. (A) Gametes, an egg cell and a sperm cell, are isolated from an unpollinated flower and transferred into a droplet of mannitol solution overlaid with mineral oil on a coverslip. The gametes are positioned in the center of a droplet, fixed at one of the electrodes under an alternating current field, and fused by multiple direct current pulses. The zygote produced in vitro was cultured on the membrane of a Millicell insert containing N6Z medium and feeder cells. (B) A zygote divides into a compact embryonic callus through early embryonic development in the Millicell insert. (C) A compact embryonic callus develops on the callus induction medium. (D, E) Thereafter, the callus is transferred onto regeneration and rooting medium to obtain regenerated shoots and plantlets, respectively.

Table 1.Procedures of in vitro fusion of isolated gametes and the developmental potential of the produced zygotes

Fusion procedure	Species	Developmental progression of fused gametes (zygotes)					References
		Gamete fusion	Karyogamy	Two-celled embryo	Cell mass	Plantlet
Calcium-fusion	Maize	○	○	—	—	—	Kranz and Lörz 1994
	Maize	○	—	—	—	—	Faure et al. 1994
	Rice	○	—	—	—	—	Khalequzzaman and Haq 2005
PEG-fusion	Maize	○	—	—	—	—	Sun et al. 1995
BSA-fusion	Maize	○	—	—	—	—	Peng et al. 2005
Electro-fusion	Maize	○	○	○	○	○	Kranz and Lörz 1993
	Rice	○	○	○	○	○	Uchiumi et al. 2007
	Wheat	○	○	○	○	○	Maryenti et al. 2019

Circles indicates the developmental stages into which zygotes produced by respective fusion procedures develop. Dashes means the developmental stages into which zygotes produced by respective fusion procedures cannot develop.

A complete IVF system that involves maize gametes and electrical fusion was first developed by Kranz and Lörz (1993), and rice and wheat IVF systems were also established by Uchiumi et al. (2007) and Maryenti et al. (2019), respectively, to take advantage of the abundant resources stemming from rice and wheat research. These electrofusion-based IVF systems have been successfully utilized to observe and investigate post-fertilization events, such as karyogamy (Faure et al. 1993, Ohnishi et al. 2014), egg activation and zygotic development (Kranz et al. 1995, Nakajima et al. 2010, Sato et al. 2010, Sukawa and Okamoto 2018), paternal chromatin decondensation in zygote nuclei (Scholten et al. 2002), microtubular architecture in egg cells and zygotes (Hoshino et al. 2004), ROS and mitochondrial dynamics (Aini et al. 2022, Rattanawong et al. 2021), fertilization-induced/suppressed gene expression (Okamoto et al. 2005, Toda et al. 2025), the contribution of parental genomes to zygotic development (Deushi et al. 2021, Toda et al. 2016, 2018), epigenetic resetting in early embryos (Jahnke and Scholten 2009), and the synergistical function of parental genomes (Akter et al. 2025, Masuo et al. 2025, Rahman et al. 2019).

In addition to investigations of post-fertilization events, IVF systems can be applied to generate new genotypes and/or hybrids, as gametes isolated from different species, such as maize and wheat, can be fused to produce distantly related hybrid zygotes beyond the reproductive barrier. Additionally, the combination and number of gametes for electro-fusion can be set according to the species and ploidy of interest. The following three sections describe the recent progress in cytoplasmic hybrids between “rice and wheat” and “maize and wheat” across subfamilies.

Overcoming the hybridization defect between wheat and rice, and production of the rice–wheat cytoplasmic hybrid, ^OryzaWheat

Based on the availability of rice and wheat IVF systems, rice–wheat hybrid zygotes were produced by fusing a rice egg cell (Re) and a wheat sperm cell (Ws). Although the ReWs zygotes underwent several rounds of cell division, their developmental progressions ceased at the embryo-like stage (Maryenti et al. 2021). Fluorescence in situ hybridization (FISH) analyses using probes for the rice centromere sequence and/or the wheat genome were employed to determine the chromosome composition of cells in ReWs hybrid embryo-like structures. Notably, rice centromeres were not observed or detectable at the periphery or outside the nuclei in these cells. In contrast, the wheat-specific probe signals were stable and uniformly detected in all hybrid nuclei. This suggests that rice chromosomes are eliminated during the early developmental stage of the ReWs hybrid zygotes, and that the selective rice chromosome elimination in ReWs embryos resulted in cells possessing the cytonuclear environment of the wheat nucleus and rice cytoplasm (Fig. 3A). This allogenic cytonuclear environment was presumed to be the reason that ReWs embryos arrested at globular-like embryo stage because dysfunctional interactions between a cytoplasmic genome from one species and a nuclear genome from another often result in organelle dysfunction and, consequently, hybrid breakdown (Burton et al. 2013, Johnson 2010, Nakamura 2023). Therefore, an additional wheat egg cell was further fused with a ReWs zygote to supply wheat cytoplasmic genome into the hybrid zygote and to establish a nuclear–cytoplasmic (NC) relationship between wheat nucleus and wheat cytoplasm, possibly rescuing the developmental defect of ReWs embryos triggered by dysfunction of the NC relationship between wheat nucleus and rice cytoplasm (Fig. 3B).

Fig. 3.

Schematic representation of the effects of gamete combination on the developmental profiles of wheat–rice hybrid zygotes (A–C) and mature plants regenerated from the hybrid zygotes and harvested seeds (D). (A) Hybrid zygotes derived from one rice egg (Re) and one wheat sperm (Ws) cease their development at the globular embryo-like structure stage after a selective elimination of rice chromosomes. (B) Hybrid incompatibility of the ReWs zygote presented in panel (A) can be overcome by additional fusion of one wheat egg cell (We) to the wheat–rice hybrid zygote. (C) Fusion of wheat and rice gamete sets, We, Ws cell, Re cell, and rice sperm cell (Rs), results in fusion products between wheat and rice zygote. The wheat–rice double zygote (DZ) also develops and regenerates into a mature plant via possible elimination of rice chromosomes. (D) Representative image of the regenerated plants and harvested seeds. Scale bar in D, 5 mm.

The allopolyploid rice egg (Re)–wheat sperm (Ws)–wheat egg (We) hybrid zygotes, produced through the fusion of an extra We cell with a ReWs, were successfully developed and regenerated into the possible hybrid plantlets (Fig. 3B). In addition to the gamete combination of ReWsWe hybrid zygotes, alternative allopolyploid hybrid zygotes, termed double zygotes (DZs), consisting of a Re, a rice sperm (Rs), a We, and a Ws, also bypassed the hybridization defect in ReWs hybrid zygotes (Fig. 3C). Possible hybrid plants derived from ReWsWe zygotes and DZ zygotes were morphologically equivalent to wheat plants and fertile, and viable seeds were successfully harvested (Fig. 3D). In addition, flow cytometric analyses and chromosome number counting for these plants resulted in equivalent values with wheat, suggesting that the regenerated ReWsWe and DZ plants are equivalent to wheat plants (Maryenti et al. 2021).

Wheat-rice hybrid mitochondria genome in ^OryzaWheat and stable transmission of the hybrid genome across generations

To examine the genomic status of the possible wheat–rice hybrids, short-read sequencing of genome DNA isolated from seven possible wheat–rice hybrid plant lines was conducted, and the genomic origin of the sequence reads was estimated (Maryenti et al. 2024). The results indicated that possible hybrids possess partial regions derived from rice mitochondrial genome, as well as whole wheat nuclear and cytoplasmic genomes (Fig. 4A). Interestingly, the retention rate of rice mitochondrial DNA ranged from 11 to 47% depending on the hybrid line. Long-read sequencing of the genome DNA and subsequent in silico analyses suggested that each partial DNA region with >2000 bp of rice mitochondrial genome is inserted at the putative hotspot region of wheat mitochondrial DNA (Fig. 4B). The hybrid mitochondrial DNA were visualized by FISH analyses with specific fluorescent probes for rice and mitochondrial DNA to root cells of the wheat–rice hybrids (Fig. 4C). These genomic and cytogenetic results indicate that the rice–wheat hybrids are cytoplasmic hybrid wheat possessing rice mitochondrial genome DNA regions. Therefore, we termed the cytoplasmic hybrid as ^OryzaWheat.

Fig. 4.

Genome composition of the rice–wheat hybrid. (A) Mapping pattern of the short-read sequences derived from the genome DNA of a possible rice–wheat hybrid onto rice (upper panel) and wheat (bottom panel) mitochondrial genomes. Eight DNA regions in the rice mitochondrial genome are numbered as regions I–VIII. (B) Putative insertion sites in the rice mitochondrial DNA regions in the wheat mitochondrial genome. Five putative hotspot sites where the rice mitochondrial genome tends to be inserted into the wheat mitochondrial genome are indicated by the black triangles, and the putatively inserted DNA regions from the rice mitochondrial genome are shown in parentheses after each possible hotspot. (C) Fluorescence in situ hybridization (FISH) analyses for rice and wheat mitochondrial genomes in cells of hybrid plants. FISH analyses using specific fluorescent probes for rice and wheat mitochondrial genomic DNA were conducted on root cells of rice–wheat hybrid plants (^OryzaWheat), rice plants, and wheat plants. Scale bars, 10 μm.

The transmission status of the rice mitochondrial genome DNA was examined in F₂ and F₃ generations using genomic PCR with primer sets specific to each mitochondrial region. The results suggested that the rice mitochondrial genome in ^Oryzawheat was maintained and transmitted to their progenies (F₂ and F₃), although the retention profiles of the F₂ and F₃ ^Oryzawheat generations displayed a possible loss of the insertional regions.

Production of maize–wheat cytoplasmic hybrid, ^ZeaWheat

Wheat belongs to the subfamily Pooideae (C3 plant) and grows in temperate and subarctic zones (Erenstein et al. 2022a, Huang et al. 2022). In contrast, maize, a C4 Poaceae plant belonging to the subfamily Panicoideae, exhibits high photosynthetic efficiency and the ability for growing in tropical zones (Erenstein et al. 2022b). Despite the valuable genetic resources in maize offers for enhancing the physiological and agricultural traits of wheat, transferring genetic material between wheat and maize remains challenging due to the hybridization barrier posed by chromosome elimination. Therefore, wheat and maize IVF systems were used to produce maize–wheat hybrids according to the procedures for ^OryzaWheat production.

When a hybrid zygote was produced by fusing a maize egg cell (Me) with a wheat sperm cell (Ws) and cultured, the MeWs zygotes arrested at the early cell mass stage (Fig. 5A; Onda et al. 2025). The developmental arrest of the MeWs embryo will also be attributable to the elimination of the maize genome during MeWs zygote development, as the coexistence of the maize nuclear genome with that of wheat results in the elimination of the maize genome during the development of wheat–maize hybrid zygotes (Laurie and Bennett 1988, Mochida et al. 2004). Developmental arrest of MeWs embryos by possible dysfunction of the NC-relationship between wheat and maize is consistent with that between wheat and rice in rice egg-wheat sperm (ReWs) hybrid embryos.

Fig. 5.

Developmental profiles of wheat–maize hybrid zygotes (A, B) and harvested seeds (C). (A) MeWs zygotes developed into globular-like embryos and early cell masses. Thereafter, the early cell masses became highly vacuolated and arrested. (B) MeWeWs zygotes developed into globular-like embryos, early cell masses, cell masses, compact calli, and calli. After regenerated shoots and the roots were formed from the calli, the regenerated plantlets grew into mature plants with fertile flowers. (C) Harvested seeds from six independent plants derived from hybrid zygotes. DAF: day after fusion. Scale bars in C, 1 cm.

The developmental arrest was overcome by the fusion of an extra wheat egg cell (We) to MeWs zygotes probably via coexistence of wheat cytoplasm (Fig. 5B). In addition to the gamete combination of MeWeWs hybrid zygotes, alternative allopolyploid hybrid zygotes, termed DZs, consisting of a Me, a maize sperm (Ms), a We, and a Ws cell, were also bypassed the hybridization defect in MeWs hybrid zygotes (Onda et al. 2025).

All MeWeWs and DZ regenerated plants were morphologically similar to wheat plants, and flow cytometric analyses of these plants indicated that their DNA content was equivalent to that of wheat. Of the 40 tested plants (21 MeWeWs and 19 DZ plants), 37 were fertile, and viable seeds were successfully harvested (Fig. 5C), whereas the remaining three plants were infertile with a dwarf phenotype.

Genome composition and photosynthetic type of ^ZeaWheat

Using short-read sequencing of genome DNA isolated from eight possible maize–wheat hybrid plant lines, we found that the possible hybrid plants possess the whole wheat nuclear and cytoplasmic genomes and rice mitochondrial genome (Fig. 6A); this led us to name the cytoplasmic hybrid ^ZeaWheat. Interestingly, the coverage rate of the maize mitochondrial genome in MeWeWs and DZ plants ranged from 30% to 93% (average: 75%), and the coverage rate in the ^ZeaWheat hybrid was much higher than that in ^OryzaWheat (11% to 47%, average: 27%). Moreover, the maize mitochondrial DNA regions were found to be stably transmitted across generations (Fig. 6B). The FISH analyses of ^ZeaWheat root cells with specific probes for maize and wheat mitochondrial DNAs indicated that maize and wheat mitochondria coexist, and that fusion between the maize and wheat mitochondria and subsequent recombination between the maize and wheat mitochondrial DNA was found to occur in the cybrid cells (Onda et al. 2025).

Fig. 6.

Mitochondrial genome composition of wheat–maize hybrid plants (A), transmission of maize mtDNA (B), and photosynthetic rate and stable carbon isotope ratios in wheat–maize cybrid plants (C, D). (A) Mapping profiles of sequence reads from the genomic DNA of a hybrid plant onto the maize mitochondrial genome. The positions of the PCR primer sets used in panel B were indicated by red-colored numbers. (B) Genomic PCR using gDNA from hybrid plants at F₁ and F₂ generations. (C) Response of photosynthetic rate to intercellular CO₂ concentration (Ci) at 23°C and 1800 μmol m^–2 s^–1 light intensity for a wild-type maize (C₄), wild-type wheat (C₃), and two F₃ cybrids. The low CO₂ portion of the A versus Ci response is shown in the bottom panel. (D) Stable carbon-isotope ratios of wheat–maize F₁ cybrid plants. Dunnett’s test, *p < 0.05, **p < 0.01, ***p < 0.001 vs. Fielder. wheat: n = 6, maize: n = 5, MeWeWs: n = 42, DZ: n = 20.

As ^ZeaWheat possessed mitochondrial genomes of both C₃ plants (wheat) and C₄ plants (maize), the A/Ci response was measured to estimate the photosynthetic type of the cybrid plants. Although the maize plant performed at a better photosynthetic rate than wheat at low CO₂ concentrations, the ^ZeaWheat hybrid showed photosynthetic rates similar to those of wheat at low CO₂ concentrations (Fig. 6C), suggesting that ^ZeaWheat performs C₃-like photosynthesis even if the cybrids possess maize mitochondria. The δ¹³C was also analyzed using EA/IRMS to estimate the carbon fixation pathway in the wheat–maize cybrid plants. Maize and wheat showed a high δ¹³C (–10.3 to –9.2‰) and a low δ¹³C (–36.8 to –32.9‰), respectively, and ^ZeaWheat lines showed a low δ¹³C ranging from –37.8 to –31.3‰, equivalent to that of wheat (Fig. 6D), suggesting that the cybrid plants fix CO₂ via a C₃-type pathway.

Discussion and Future Perspectives

Mitochondrial hybridization across subfamily and physiological/agronomical traits

Cytoplasmic organelles, mitochondria and plastids, are essential for energy production, nutrient sensing, metabolism, and stress responses (Liberatore et al. 2016). It has been demonstrated that the cytoplasm evolves adaptively to the environment, conferring survival and fitness advantages (Bock et al. 2014, Leinonen et al. 2011, Sambatti et al. 2008), and that wheat cytoplasm substitution lines, in which Aegilops cytoplasm replaces wheat cytoplasm, exhibit distinct phenotypes, such as cytoplasmic male sterility (CMS), delayed heading, and parthenogenesis (Tsunewaki 2009, Tsunewaki et al. 2002). However, studies on plasmotypic effects have largely been limited to closely related species capable of cross-pollination. Therefore, examining the impact of cytoplasmic hybridization between distantly related species from different subfamilies is of considerable interest.

Cytoplasmic hybridization increases the nucleus-cytoplasm (NC) relationship and combination, and the variations in the NC relationship/combination are relevant from both agricultural and evolutionary perspectives (Bock et al. 2014, Levings 1990, Nakamura 2023). Moreover, the impacts of plasmotypic variation are particularly pronounced in fluctuating and stressful environments, because plasmotypes serve as reservoirs of variation that are predominantly expressed under certain conditions (Flood et al. 2020). These suggest that distant cytoplasmic hybridization among major crops will be highly important for broadening the variety of NC combinations and the subsequent physiological and agricultural traits of crops. To investigate the effects of the hybrid mitochondrial genome hybridization on the physiological and agronomic characteristics of cybrid wheat, comprehensive phenotypic evaluations and stress tolerance trials of the F₂ generation and beyond are currently underway in our laboratories. The cybrids display a wide range of phenotypic variation, with several lines outperforming control wheat in biomass accumulation, heading date, and salt resistance, suggesting that mitochondrial genome diversity generates phenotypic diversity (Ikpa-Agodo et al. 2025, Nowroz et al. 2025, Sugiura et al. 2025). Molecular machineries for hybrid mitochondria–nuclear interactions related to these characteristic traits will be elucidated in the near future.

Wheat as an ideal plant material for increasing cytoplasmic variety via the IVF system

It has been demonstrated that hybridization between wheat and distantly related species, such as Hordeum bulbosum, rice, maize, pearl millet, sorghum, Coix lacryma-jobi, and Imperata cylindrica, eliminates the non-wheat genome (Barclay 1975, Inagaki and Mujeeb-Kazi 1995, Ishii et al. 2010, 2016, Komeda et al. 2007, Maryenti et al. 2024, Mochida and Tsujimoto 2001, Mochida et al. 2004). The stable wheat genome characteristics upon hybridization are extremely suitable for generating cybrids that incorporate the mitochondrial genome of other species while retaining a wheat nuclear background. In addition to rice and maize, egg cells can be isolated from other Poaceae species, such as pearl millet, sugarcane, barley, sorghum, and setaria (Fig. 7), and these egg cells can also be utilized to create new cytoplasmic hybrid wheat.

Fig. 7.

Egg cells isolated from unpollinated flowers of rice (Oryza sativa L.), maize (Zea mays L.), wheat (Triticum aestivum L.), barley (Hordeum vulgare), pearl millet (Cenchrus americanus), sugarcane (Saccharum officinarum), Brachypodium distachyon, and Setaria viridis. Scale bars, 50 μm.

Importantly, hybrid plants produced by in vitro fusion of gametes from plant species within the same Poaceae family are free from legislative concerns related to genetically modified organisms (Jones 2015). This creates the possibility of cultivating these hybrid plants in open fields immediately after their production.

Research toward the elucidation of the function of the hybrid mitochondria in wheat cybrids

In addition to creating mitochondrial genome diversity using the IVF system with gametes from different species, the genome-editing technique can be utilized as a powerful tool to enhance or suppress the function of allogenic mitochondrial genomes. Indeed, we have developed a PEG–Ca²⁺-mediated transfection system to deliver macromolecules, including DNA, RNA, and proteins, into rice and maize zygotes produced in vitro and isolated from pollinated ovaries, respectively (Koiso et al. 2017). Importantly, genome editing of rice and maize has also been achieved via the delivery of a plasmid DNA vector with a CRISPR/Cas9-guideRNA expression cassette or CRISPR/Cas9-guideRNA ribonucleoproteins into rice zygotes (Toda et al. 2019) and maize zygotes (Yamada et al. 2024). However, the CRISPR/Cas9 system remains only adaptable to the nuclear genome. Therefore, an alternative nuclease, transcription activator-like effector nuclease tagged with the mitochondrial targeting sequence (mitoTALEN), has been used to edit mitochondrial DNA in plants and animals (reviewed in Arimura and Nakazato 2024). In angiosperms, open reading frame 79 (orf79) on the rice mitochondrial DNA was first successfully edited by Kazama et al. (2019). Delivering a DNA vector containing a mitoTALEN expression cassette into hybrid zygotes is a possible approach to edit hybrid mitochondrial DNA in cybrids toward elucidation of the function of hybrid mitochondria in cybrid wheat.

Application of IVF technology for production of doubled haploid and for regeneration of recalcitrant genotypes/cultivars

Production of haploid and subsequent genome doubling, resulting in emergence of doubled haploid, is an efficient approach for instant fixation of genome composition. Therefore, doubled haploid technology enabling efficient development of pure lines has been considered as an important procedure for crop breeding (Qu et al. 2024, Seguí-Simarro et al. 2021). Recently, we reported an alternative utilization of the IVF technology (egg cell isolation and subsequent egg cell culture) for production of haploid and doubled haploid rice plants. Upon cold treatment, egg cells isolated from japonica (cv. Nipponbare) and indica (cv. Kasalath) subspecies of rice (Oryza sativa) autonomously divided, developed and regenerated into doubled haploid or haploid plant with or without genome duplication, respectively, during egg cell culture (Rattanawong et al. 2026). These suggest that environmental stimuli such as cold stress, cell isolation and/or culture convert cell fate of egg cell from quiescence to proliferative state, and that direct production of doubled haploid form egg cell will become a powerful technology for haploid breeding. Although report for induction of autonomous development (parthenogenetic potential) of isolated egg cells has been still limited to rice, egg cells isolated from other crops can be subjected to abiotic stress treatment and subsequent culture to examine their parthenogenetic potentials.

Although cell/tissue culture and its subsequent regeneration into plantlets are mostly required for producing transgenic and/or genome-edited crops, these procedures are major technical hurdle for recalcitrant genotypes/cultivars. IVF technology may be useful for obtaining regenerated plantlets form recalcitrant genotype/cultivars, as genome edited maize plants were successfully obtained by culture of recalcitrant B73 maize zygotes, into which Cas9-gRNA ribonucleoproteins were delivered (Yamada et al. 2024).

Author Contribution Statement

T.O. wrote the manuscript.

 Acknowledgments

This study was partly supported by JSPS KAKENHI (Grant-in-Aid for Scientific Research(B), Grant No. 25K01990), the NEDO moonshot program (Grant No. 22101490-0), the JST-Mirai Program (Grant No. 23-231038606), F-REI (Grant No. JPFR25030101), and the Plant Science Core Alliance (PSCA) of the Joint Usage/Research Centers conducted at Tottori University (Grant No. PT#2605).

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