NTT and Tokyo Metropolitan University Develop the World's First Multi-Variety Hybridization Technology Enabling Simultaneous Hybridization of Four Rice Varieties

— Enabling the combination of the four varieties-derived desirable traits to develop a novel variety with superior yield and enhanced functional properties —

Key Points:

Figure 1. Overview of the study

Background

Under its environmental and energy vision, "NTT Green Innovation toward 2040"², established in September 2021 to achieve both zero environmental impact and economic growth, NTT has been advancing research and development of plant breeding technologies that support sustainable, low-environmental-impact agriculture.

As part of this initiative, a joint research team from NTT and Tokyo Metropolitan University has been developing plant breeding technologies aimed at producing crop varieties with higher yields, enhanced functional properties, greater carbon dioxide (CO₂) absorption capacity, and reduced requirements for chemical fertilizers and pesticides.

In plant breeding, two conventional techniques have been used: hybridization, which combines two different varieties through artificial pollination (Figure 2, Conventional Method 1), and polyploidization³, which increases the number of genome⁴ sets mainly through chemical treatment (Figure 2, Conventional Method 2).

The first-generation hybrid (F1) produced through hybridization can exhibit superior growth, yield, and tolerance to environmental stress compared with either parent. This phenomenon, known as heterosis, has been widely utilized in crop breeding. Numerous commercial crop varieties have been developed using heterosis, and many vegetables currently distributed in Japan are F1 hybrids⁵.

Polyploid plants often exhibit enlarged organs, improved tolerance to environmental stress, and increased levels of beneficial compounds, making polyploidization another long-established breeding strategy. For example, cultivated strawberries have four times as many genome sets as their wild ancestors, producing larger fruit with superior flavor and aroma.

Figure 2. Overview of conventional breeding methods: hybridization and polyploidization

Despite their widespread use, each of these conventional breeding approaches has inherent limitations. Conventional hybridization is generally limited to two parental varieties at a single crossing. As a result, the resulting progenies retain the same number of genome sets as their parents and cannot benefit from the advantages associated with polyploidization. Conversely, polyploidization through chemical treatment duplicates the genome of a single variety, preventing the combination of genetic material from multiple parental lines and therefore limiting the benefits of hybridization.

A breeding approach capable of simultaneously combining genomes from multiple parental varieties while inducing polyploidization could integrate the advantages of both hybridization and polyploidization. Such a technology has the potential to establish a new breeding strategy for developing crop varieties with characteristics that are difficult or impossible to achieve using conventional methods.

Technical Highlights

Researchers at NTT and Tokyo Metropolitan University have developed the world's first method for producing autopolyploid rice through the simultaneous hybridization of four rice varieties. The approach utilizes in vitro fertilization (IVF)⁶ system, a micromanipulation technique in which isolated plant egg cells⁷ and sperm cells⁸ are artificially fused.

A system for regenerating plantlets from IVF-derived fertilized eggs has been established for the world's three major cereal crops, including rice, wheat, and maize. A key advantage of the IVF method is its flexibility in the number and combination of egg and sperm cells for fertilization to control hybrid and ploidy levels.

In this study, using rice as a model crop, the research team applied the IVF system to generate fertilized eggs by fusing egg and sperm cells derived from four different rice varieties (Figure 3). The resulting fertilized eggs were then cultured to regenerate rice plants carrying genomes from all four parental varieties while possessing twice the normal number of genome sets. This achievement demonstrates a new breeding approach that combines the advantages of multi-parent hybridization and polyploidization in a single process.

Figure 3. Overview of the multi-variety hybridization technology for simultaneous hybridization of four rice varieties

Experimental Results

1. Generation of Hybrid and Ployploid Rice through Simultaneous Hybridization of Gametes form Four Varieties Using the IVF system

In this study, the research team applied the IVF system to generate rice carrying genomes derived from four parental varieties (Figure 4). The parental varieties consisted of the diploid japonica⁹ cultivars Nipponbare and Taichung 65, the aus¹⁰ cultivar Kasalath, and the indica¹¹ cultivar IR26.

Egg and sperm cells were isolated from each parental variety and fused by electrical stimulation to generate fertilized eggs consisting of egg and sperm cells derived from all four varieties. The resulting fertilized eggs were cultured in multiple media to induce callus formation, followed by regeneration into plantlets.

The analysis using flow cytometry¹² confirmed that the regenerated plants were tetraploid rice with twice the normal number of genome sets. In addition, genome-wide analysis using numerous variety-specific single nucleotide polymorphism (SNP) markers¹³ demonstrated that the regenerated plants retained genomes from all four parental varieties in approximately equal proportions.

These results demonstrate that the IVF system can be used to generate a tetraploid hybrid rice carrying genomes derived from four parental varieties. The four-parent derived hybrid rice was designated as "Quad".

Figure 4. Generation of a tetraploid four-parent rice hybrid (Quad) using the IVF method

2. Evaluation of Phenotipic Traits in the Four-Parent Hybrid Rice

Quad exhibited characteristics that differed substantially from those of its parental varieties (Figure 5). The Quad plants showed vigorous growth, and at the grain-filling stage¹⁴, both plant height and flag leaf¹⁵ length were comparable to or greater than those of the largest parental variety.

Quad also exceeded all parental varieties in seed length, width, and thickness. The weight of ten brown rice grains was approximately 1.7 times the average of the four parental varieties. Furthermore, Quad showed a significantly greater ten-grain weight than a conventional tetraploid hybrid derived from only two parental varieties, Nipponbare and Kasalath.

These findings suggest that combining genomes from four parental varieties through tetraploidization has the potential to produce rice with greater yield than can be achieved using conventional breeding methods.

In addition, Quad developed red awns¹⁶, a trait that was not observed in any of the parental varieties. This finding suggests that combining multiple genetically distinct genomes within a single plant can generate novel traits beyond those represented by the parental lines.

Notably, autotetraploid rice produced using Nipponbare alone as the parental variety generated very few seeds, whereas Quad produced abundant seeds comparable to those of each parental variety. This difference in fertility indicates that polyploidization combined with multiple parental genomes may overcome the low fertility¹⁷ level of autotetraploid from a single rice variety.

Taken together, these findings demonstrate that the newly developed multi-variety hybridization technology has the potential to serve as a novel breeding strategy that integrates the advantages of both hybridization and polyploidization.

Figure 5. Comparison of traits between the four parental rice varieties and Quad

3. Definition of the "Polyparental Hybrid" Concept

In this study, the researchers introduce the term "polyparental hybrid" to describe a polyploid organism carrying genomes derived from three or more genetically distinct parents within the same species. This concept extends the conventional breeding framework, which has traditionally been based on hybridization between only two parental lines.

Figure 6. Definition of a polyparental hybrid

Roles of Each Organization

NTT: Leveraging its expertise in plant in vitro fertilization (IVF), cultivation, and phenotypic evaluation, NTT was responsible for planning and designing the study, generating the polyparental hybrids, and evaluating their characteristics.
Tokyo Metropolitan University (TMU): Developed the core technologies for plant IVF system and provided technical expertise throughout the study. TMU also applied its expertise in plant genome analysis to demonstrate that the generated polyparental hybrids retained genomes from all four parental varieties in approximately equal proportions.

Future Outlook

The research team will continue to conduct detailed analyses of the yield, environmental stress tolerance, and quality-related traits of the newly developed polyparental hybrid rice. These studies aim to clarify how polyparental hybridization influences crop yield, eating quality, and functional properties, while also demonstrating the potential of the technology for practical agricultural applications.

Although this study focused on rice, the technology is expected to be applicable to other plant species for which IVF systems have been established. Future research will focus on establishing IVF system for additional plant species and developing technologies to optimize combinations of parental varieties, thereby expanding the applicability of the technology and maximizing its breeding potential.

By leveraging these technologies, the research team aims to develop environmentally sustainable crops and trees that combine high growth and yield, enhanced functional properties, and superior carbon dioxide (CO₂) absorption capacity while requiring minimal fertilizer and pesticide inputs.

In the long term, the technology is expected to contribute to global food security by increasing and stabilizing agricultural production, while also supporting climate change mitigation through enhanced carbon sequestration in agricultural land and forests.

[Research Contacts]

Space Environment and Energy Laboratories, NTT, Inc.
Kazuma Sakoda, Associate Distinguished Researcher

Department of Biological Sciences, Graduate School of Science, Tokyo Metropolitan University
Takashi Okamoto, Professor
TEL：+81-42-677-2566 E-mail：okamoto-takashi@tmu.ac.jp

¹Gamete: A haploid reproductive cell that gives rise to the next generation.

²https://group.ntt/en/newsrelease/2021/09/28/210928a.html

³Polyploidization: The process by which the number of genome sets in an organism increases.

⁴Genome: The complete set of genetic information of an organism. It comprises the organism's entire DNA sequence and serves as the blueprint that determines its characteristics and biological functions.

⁵F1 hybrid: The first filial generation (F1) produced by hybridizing two different parental varieties. F1 hybrids often exhibit superior yield, quality, or other desirable traits compared with either parent and are widely used in crop breeding.

⁶In vitro fertilization (IVF): A technique in which reproductive cells isolated from plant reproductive organs (flowers) are artificially fused under a microscope using electrical stimulation. The resulting fertilized egg is then cultured and regenerated into a plantlet.

⁷Egg cell: The female gamete in plants.

⁸Sperm cell: The male gamete in plants.

⁹Japonica: One of the major groups of cultivated rice, widely grown in East Asia, including Japan. Japonica rice typically has short, round grains and a soft, sticky texture after cooking.

¹⁰Aus: A rice group primarily cultivated in South Asia. Aus varieties generally possess traits that enable adaptation to environmental conditions such as high temperatures and drought.

¹¹Indica: One of the major groups of cultivated rice, widely grown in South and Southeast Asia. Indica rice typically has long, slender grains and a less sticky texture after cooking.

¹²Flow cytometry: A technique that uses laser light to measure and analyze individual cells or their components, including DNA content and other cellular characteristics.

¹³ SNP marker: A genetic marker based on a single nucleotide polymorphism (SNP), which represents a variation at a single position in the DNA sequence. SNP markers are widely used to distinguish varieties and genetic lineages and to determine the parental origin of inherited genomes.

¹⁴ Grain-filling stage: The developmental stage after pollination and fertilization during which seeds develop and accumulate starch and other nutrients before reaching maturity.

¹⁵Flag leaf: The uppermost leaf on the stem of a cereal plant, such as rice. It plays a critical role in supplying nutrients to developing grains.

¹⁶Awn: A slender, bristle-like extension that protrudes from the tip of the husk of cereal grains such as rice and wheat.

¹⁷Fertility: The ability of a plant to produce viable pollen and seeds, thereby enabling successful reproduction and the production of the next generation.

About NTT

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