1 Introduction

Cassava (Manihot esculenta Crantz) is a vital staple crop, ranking as the sixth most important food crop globally and serving as a primary food source for approximately 800 million people worldwide (Amelework and Bairu, 2022). In Africa, cassava holds significant importance, being the second most crucial food crop after maize, with the continent being the largest producer of cassava (Amelework and Bairu, 2022). Its resilience to harsh climatic conditions and ability to thrive in nutrient-poor soils make it an indispensable crop for food security, particularly in sub-Saharan Africa (Obata et al., 2020). The crop's adaptability and high productivity under challenging environmental conditions underscore its critical role in sustaining the livelihoods of millions of smallholder farmers.

Despite its importance, cassava cultivation faces several challenges that hinder its potential yield and quality. One of the primary issues is the limited understanding of the metabolic processes that contribute to its high productivity, including photosynthesis efficiency and source/sink limitations (Obata et al., 2020). Additionally, cassava breeding programs encounter difficulties due to the crop's genetic complexity and the presence of various genetic groups with heterogeneous linkage disequilibrium (Ogbonna et al., 2020). These challenges are compounded by the need for improved resistance to pests and diseases, as well as enhanced nutritional content and processing qualities. Addressing these issues requires innovative breeding techniques and a comprehensive understanding of the genetic and metabolic traits that influence cassava's performance.

This study highlights advancements in cassava genetic analysis and breeding efforts, examines the genetic diversity and population structure of cassava germplasm to inform breeding strategies, identifies key metabolic traits and genetic markers that can be targeted for enhancing cassava yield and quality, and discusses the potential of doubled haploids and genetic engineering in overcoming the current challenges in cassava breeding and cultivation. By synthesizing the latest research findings, this study seeks to provide a comprehensive understanding of how modern breeding techniques can be leveraged to enhance cassava's productivity, resilience, and nutritional value, ultimately contributing to global food security.

2 Traditional Breeding Techniques in Cassava

2.1 Conventional breeding approaches

Conventional breeding approaches in cassava primarily involve sequential self-pollination to produce inbred or homozygous lines. This method is essential for exposing useful recessive traits and enhancing the breeding value of progenitors. However, the process is notably time-consuming, often taking 10-15 years to achieve the desired homozygosity through successive self-pollination cycles (Lentini et al., 2020).

Another traditional method includes cross-pollination, which leverages the genetic diversity of cassava to introduce new traits. However, the heterozygous nature of cassava and its long breeding cycle pose significant challenges to rapid genetic improvement (Baguma et al., 2019b). Cross-pollination is often followed by selection and backcrossing to stabilize the desired traits, which further extends the breeding timeline.

2.2 Limitations of traditional breeding

The primary limitation of traditional breeding techniques in cassava is the extensive time required to produce true-breeding lines. The sequential self-pollination method, while effective, is slow and labor-intensive, taking over a decade to achieve homozygosity (Lentini et al., 2020). This slow pace is a significant bottleneck, especially in the context of rapidly changing environmental conditions and the urgent need for climate-resilient cassava varieties.

Moreover, the heterozygous nature of cassava complicates the breeding process. The genetic variability inherent in cross-pollinated progeny makes it challenging to stabilize desired traits quickly (Baguma et al., 2019b). This variability necessitates multiple generations of backcrossing and selection, further delaying the development of new varieties.

Additionally, traditional breeding methods are limited in their ability to introduce and stabilize complex traits, such as disease resistance and drought tolerance, which are polygenic and require precise genetic manipulation. The reliance on natural genetic variation and the slow pace of conventional breeding make it difficult to keep up with the evolving challenges posed by pests, diseases, and climate change (Baguma et al., 2019b; Lentini et al., 2020).

In summary, while conventional breeding approaches have been foundational in cassava improvement, their limitations in terms of time efficiency and genetic precision underscore the need for innovative breeding techniques, such as doubled haploids and genetic engineering, to accelerate the development of improved cassava varieties.

3 Doubled Haploids in Cassava Breeding

3.1 Concept and methods of producing doubled haploids

Doubled haploids (DH) are plants that are completely homozygous, achieved by doubling the chromosome number of haploid cells. This technique is highly valuable in plant breeding as it accelerates the development of pure lines, which are essential for hybrid production and genetic studies. The primary methods for producing doubled haploids include anther culture, microspore culture, and in vitro fertilization techniques. These methods have been successfully applied in various crops such as barley, pepper, rapeseed, rice, sugar beet, and wheat (Niazian and Shariatpanahi, 2020; Srividya et al., 2023).

In cassava, the potential for producing doubled haploids through gynogenesis has been explored. Gynogenesis involves the in vitro culture of unfertilized ovules or embryos. In a study, female flowers of cassava were bagged to prevent pollination, and early embryo rescue and ovule culture were performed (Figure 1). Although the study did not result in doubled haploids, it provided significant insights into cassava flowering biology and laid the groundwork for future protocol development (Baguma et al., 2019a).

Figure 1 Comparison of developmental stages in non-pollinated and self-pollinated cassava flowers (Adopted from Baguma et al., 2019a) Image caption: (A) ovule at 7 DAA in a non-pollinated flower showing a degenerating egg apparatus in embryo sac; (B) ovule at 7 DAP in a self- pollinated flower showing cell proliferation in embryo sac; (C) ovule at 21 DAA in a non-pollinated flower showing a disorganizing embryo sac; (D) ovule at 21 DAP in a self-pollinated flower showing an organizing embryo sac; E) ovule at 28 DAA in a non-pollinated flower showing degenerated embryo sac (white arrow); F) ovule at 28 DAP in a self-pollinated flower showing embryo and surrounding tissues developing (white arrow). ES=embryosac; NU=nucellus; OI=outer integument; II=inner integument (Adopted from Baguma et al., 2019a)

Baguma et al. (2019a) investigates flower and fruit production across different branching levels, revealing inconsistent trends in non-pollinated flowers, with the lowest and highest fruit set at the second and seventh levels, respectively. Open-pollinated flowers generally show a declining trend in fruit set with increasing branching, except at the seventh level, and exhibit higher overall fruit and seed set compared to non-pollinated ones. Significant differences are observed in fruit length, width, and survival rates between the two pollination methods. At 42 days after anthesis, 47 unique embryos were rescued, leading to seven unique plantlets, with NASE 19 having the highest success rate. Early ovule culture faced challenges, with limited callus formation and degeneration of embryo sacs in non-pollinated flowers.

3.2 Applications in cassava improvement

The application of doubled haploids in cassava breeding holds great promise for accelerating genetic gains and developing superior varieties. Doubled haploids can facilitate the rapid production of homozygous lines, which are crucial for hybrid breeding and trait introgression. This technique can significantly shorten the breeding cycle, allowing for faster development of new cassava varieties with desirable traits such as disease resistance, improved yield, and stress tolerance (Li et al., 2020; Srividya et al., 2023).

In other crops, doubled haploids have been used to enhance genetic diversity and improve adaptation to changing environmental conditions. For instance, in maize, doubled haploid technology has been integrated with genomic selection to optimize hybrid breeding, resulting in increased efficiency and genetic gain (Li et al., 2020). Similar approaches could be applied to cassava to enhance its breeding programs and develop varieties that are better suited to diverse growing conditions.

3.3 Challenges and opportunities

Despite the potential benefits, there are several challenges associated with the production of doubled haploids in cassava. One of the main challenges is the recalcitrance of cassava to in vitro culture regeneration, which has been a significant bottleneck in developing efficient doubled haploid induction protocols (Baguma et al., 2019a; Mabuza et al., 2023). Additionally, the low efficiency of haploid induction and the occurrence of albinism are common issues that need to be addressed (Patial et al., 2022).

However, recent advancements in biotechnological tools and genome editing offer new opportunities to overcome these challenges. The use of CRISPR/Cas9 for targeted gene editing and the development of haploid inducer lines could significantly improve the efficiency of doubled haploid production in cassava (Hooghvorst and Nogués, 2020a; 2020b). Moreover, optimizing culture conditions and exploring the use of chemical agents and stress treatments could enhance the induction and regeneration phases of doubled haploid production (Niazian and Shariatpanahi, 2020).

In conclusion, while there are challenges in producing doubled haploids in cassava, the potential benefits for cassava breeding are substantial. Continued research and the integration of innovative technologies will be crucial in developing efficient protocols and realizing the full potential of doubled haploids in cassava improvement.

4 Genetic Engineering in Cassava

4.1 Overview of genetic engineering techniques

Genetic engineering in cassava involves the manipulation of its genetic material to introduce desirable traits such as increased yield, disease resistance, and improved nutritional content. Techniques such as CRISPR/Cas9 genome editing have been pivotal in this field. The Cassava Source-Sink project exemplifies the integration of genetic engineering with conventional breeding strategies to enhance cassava's storage root and starch yield. This project employs a multi-national pipeline for genetic engineering, covering gene discovery, cloning, transformation, and field trials (Figure 2) (Sonnewald et al., 2020). Additionally, the development of a haploid-inducer mediated genome editing system in maize demonstrates the potential for similar approaches in cassava, where genome-edited haploids can be generated to accelerate breeding cycles (Wang et al., 2019).

Figure 2 Development of cassava fibrous and storage roots (Adapted from Sonnewald et al., 2020) Image caption: Cassava is typically propagated using stem cuttings. Nodal-derived fibrous roots emerge within 2~4 days after planting and exhibit primary vascular anatomy with a central vascular cylinder containing star-shaped primary xylem alternating with primary phloem. Around 25–30 days post-planting, secondary root growth begins. These fibrous roots develop a vascular cambium, leading to the formation of new xylem and phloem cells, which eventually break the central vascular cylinder, indicating secondary growth. By 30–40 days, slightly enlarged storage roots with a well-organized vascular cambium between the phloem and xylem can be observed, along with the periderm. Longitudinal vascular rays derived from the cambium bridge the phloem and xylem cells, facilitating the exchange of water, nutrients, and carbohydrates. Storage roots continue to enlarge, forming xylem parenchyma cells for starch and other molecule storage (Adapted from Sonnewald et al., 2020)

Sonnewald et al. (2020) examines cassava's source-sink metabolism, highlighting the process from C3 photosynthesis to the development of storage roots. Photoassimilates from leaves are apoplasmically loaded into the phloem and transported to storage roots, where they follow a symplasmic unloading route facilitated by vascular rays. The expression of genes responsible for starch biosynthesis in cassava is similar to those in potato tubers. Challenges in transgenic approaches stem from the complexity of source-sink relationships, single-gene reliance, and environmental variability. However, advancements in mathematical modeling and multi-gene targeting offer new strategies to improve cassava yield. Integrating developmental and metabolic processes is crucial for enhancing source-sink interactions and overall root productivity.

4.2 Target traits for genetic improvement

The primary target traits for genetic improvement in cassava include increased storage root yield, enhanced starch content, and resistance to pests and diseases. The Cassava Source-Sink project focuses on improving the source-sink relations in cassava to boost its yield potential (Sonnewald et al., 2020). Moreover, genetic engineering efforts aim to address the challenges posed by the crop's long breeding cycle and heterozygous nature. For instance, interspecific pollination with castor bean has been explored to induce and regenerate cassava doubled haploids, which could significantly speed up the breeding process (Baguma et al., 2019a). These efforts are crucial for adapting cassava to changing environmental conditions and ensuring food security in regions dependent on this staple crop.

4.3 Regulatory and ethical considerations

The application of genetic engineering in cassava, like in other crops, is subject to regulatory and ethical considerations. Regulatory frameworks ensure that genetically modified organisms (GMOs) are safe for human consumption and the environment. Ethical considerations include the potential impact on biodiversity, the rights of farmers, and the accessibility of genetically engineered crops to smallholder farmers. The Cassava Source-Sink project, for example, operates within a framework that includes confined field trials to assess the safety and efficacy of genetically engineered cassava varieties (Sonnewald et al., 2020). Additionally, the development of genome editing technologies such as CRISPR/Cas9 must consider off-target effects and the long-term implications of genetic modifications (Wang et al., 2019). These considerations are essential to balance the benefits of genetic engineering with the need for responsible and sustainable agricultural practices.

5 Case Study: Successful Applications of Innovative Breeding Techniques in Cassava

5.1 Overview of the case study

Cassava (Manihot esculenta Crantz) is a staple crop in tropical and subtropical regions, crucial for food security and economic stability. Traditional breeding methods in cassava are time-consuming and often inefficient due to the crop's genetic complexity and long breeding cycles. Innovative breeding techniques, such as doubled haploids (DH) and genetic engineering, have shown promise in accelerating the development of improved cassava varieties. This case study explores the successful application of these techniques in cassava breeding programs.

5.2 Implementation of doubled haploids

Doubled haploid technology has been recognized for its potential to rapidly produce homozygous lines, which are essential for breeding programs. In cassava, the induction of doubled haploids through gynogenesis has been a focal point of research. Studies have demonstrated the feasibility of generating doubled haploids by culturing unpollinated ovules, leading to the formation of embryos from egg cells without fertilization (Baguma et al., 2019a; Lentini et al., 2020). Although the efficiency of these methods varies, the development of protocols for gynogenesis in cassava marks a significant step forward. For instance, a study involving the bagging of female flowers and subsequent embryo rescue resulted in the regeneration of plantlets, although achieving true doubled haploids remains a challenge (Baguma et al., 2019a).

5.3 Application of genetic engineering

Genetic engineering has also played a pivotal role in cassava improvement. Techniques such as CRISPR/Cas9 have been employed to introduce desirable traits and enhance genetic diversity. The integration of haploid inducer-mediated genome-editing systems has shown potential in producing haploid plant material, which can then be doubled to create homozygous lines (Liu et al., 2019; Hooghvorst and Nogués, 2020a). This approach not only accelerates the breeding process but also allows for precise genetic modifications, thereby improving traits such as disease resistance, yield, and stress tolerance.

5.4 Impact and implications

The application of doubled haploids and genetic engineering in cassava breeding has profound implications. These techniques significantly reduce the time required to develop new varieties, thereby accelerating genetic gains and enhancing the crop's adaptability to changing environmental conditions. The ability to produce homozygous lines rapidly through doubled haploids facilitates the incorporation of beneficial traits and the development of superior cassava varieties (Lentini et al., 2020; Srividya et al., 2023). Moreover, genetic engineering offers a precise and efficient means to introduce and stack multiple traits, further enhancing the crop's resilience and productivity (Liu et al., 2019; Hooghvorst and Nogués, 2020a). The continued refinement and integration of these innovative breeding techniques hold promise for the future of cassava breeding, ensuring food security and economic stability in regions dependent on this vital crop.

6 Comparative Analysis: Doubled Haploids vs. Genetic Engineering

6.1 Efficiency and effectiveness

Doubled haploid (DH) technology and genetic engineering are both pivotal in advancing cassava breeding, but they differ significantly in their efficiency and effectiveness. DH technology accelerates the production of homozygous lines, which traditionally takes 10-15 years through successive self-pollination, by generating pure inbred lines in a single generation (Lentini et al., 2020; Srividya et al., 2023). This method has been successfully applied in various crops, although its application in cassava is still in the experimental stages (Baguma et al., 2019a). The efficiency of DH production in cassava is currently limited by the challenges in haploid induction and chromosome doubling, with recent studies focusing on optimizing these processes (Baguma et al., 2019a; Hooghvorst and Nogués, 2020a).

On the other hand, genetic engineering, particularly through CRISPR/Cas9, offers precise and targeted modifications of the cassava genome. This method can introduce or knock out specific genes to enhance desirable traits such as disease resistance, drought tolerance, and nutritional content. The haploid inducer-mediated CRISPR/Cas9 system represents a breakthrough in combining genome editing with haploid induction, potentially increasing the efficiency of producing genetically modified cassava plants (Hooghvorst and Nogués, 2020a).

6.2 Advantages and disadvantages

6.2.1 Doubled haploids:

Advantages: 1) Rapid generation of homozygous lines, significantly reducing breeding cycles (Lentini et al., 2020; Srividya et al., 2023). 2) Facilitates the exposure of recessive traits and enhances the breeding value of progenitors (Lentini et al., 2020). 3) Potential to integrate with other biotechnological advancements for improved efficiency (Srividya et al., 2023).

Disadvantages: 1) Technical challenges in haploid induction and chromosome doubling, especially in cassava (Baguma et al., 2019a). 2) Limited success rates in regenerating viable doubled haploid plants from cassava (Baguma et al., 2019a). 3) Requires extensive optimization and adaptation for different species (Hooghvorst and Nogués, 2020a).

6.2.2 Genetic engineering:

Advantages: 1) Precision in gene editing allows for targeted trait improvement (Hooghvorst and Nogués, 2020a). 2) Can introduce new traits that are not present in the existing gene pool, such as enhanced nutritional content or resistance to specific diseases (Hooghvorst and Nogués, 2020a). 3) Potential to combine with DH technology for rapid and precise breeding outcomes (Hooghvorst and Nogués, 2020a).

Disadvantages: 1) Regulatory and public acceptance issues surrounding genetically modified organisms (GMOs) (Hooghvorst and Nogués, 2020a). 2) Potential off-target effects and unintended consequences of gene editing (Hooghvorst and Nogués, 2020a). 3) Requires sophisticated infrastructure and expertise (Hooghvorst and Nogués, 2020a).

6.3 Synergistic potential

The integration of DH technology with genetic engineering holds significant promise for cassava breeding. By combining the rapid generation of homozygous lines through DH with the precision of genetic engineering, breeders can achieve faster and more targeted improvements in cassava varieties. For instance, the haploid inducer-mediated CRISPR/Cas9 system can be used to introduce specific genetic modifications in haploid plants, which are then doubled to produce homozygous lines with the desired traits (Hooghvorst and Nogués, 2020a). This synergistic approach can enhance the efficiency of breeding programs, reduce the time required to develop new varieties, and address the challenges posed by climate change and evolving pest and disease patterns (Hooghvorst and Nogués, 2020a; Lentini et al., 2020; Srividya et al., 2023).

In conclusion, while both DH technology and genetic engineering have their unique advantages and challenges, their combined application offers a powerful strategy for the rapid and precise improvement of cassava. Continued research and optimization in both fields are essential to fully realize their potential and address the specific needs of cassava breeding programs.

7 Future Directions and Prospects

7.1 Emerging technologies in cassava breeding

The future of cassava breeding is poised to benefit significantly from emerging technologies, particularly doubled haploid (DH) technology and genetic engineering. DH technology, which has already revolutionized breeding programs in crops like maize and barley, offers a promising avenue for cassava improvement by enabling the rapid production of homozygous lines. This technique can significantly shorten the breeding cycle, which traditionally takes 10~15 years through successive self-pollination (Lentini et al., 2020; Srividya et al., 2023). Recent advancements in DH technology, such as the development of novel haploid induction methods and chromosome doubling techniques, are expected to enhance the efficiency and applicability of this approach in cassava (Chaikam et al., 2019; Hooghvorst and Nogués, 2020a).

Moreover, the integration of genome editing tools like CRISPR/Cas9 with DH technology holds great potential. This combination can facilitate precise genetic modifications and the rapid fixation of desirable traits in cassava, thereby accelerating the development of improved varieties (Bhowmik and Bilichak, 2021; Mabuza et al., 2023). The use of viral vectors for delivering CRISPR components directly into plant cells, bypassing the need for in vitro culture, is a particularly promising development for recalcitrant species like cassava (Mabuza et al., 2023).

7.2 Integrating traditional and modern breeding approaches

The integration of traditional breeding methods with modern biotechnological tools is essential for the sustainable improvement of cassava. Traditional methods, such as sequential self-pollination, have been the cornerstone of cassava breeding but are time-consuming and labor-intensive (Lentini et al., 2020). By incorporating DH technology, breeders can achieve homozygosity in a single generation, thus expediting the breeding process (Sen et al., 2020; Srividya et al., 2023).

Additionally, marker-assisted selection (MAS) and genomic selection (GS) can be combined with DH technology to enhance the precision and efficiency of breeding programs. These approaches allow for the early identification of desirable traits, reducing the time and resources required for field evaluations (Li et al., 2020; Lantos et al., 2022). The integration of these technologies can lead to the development of superior cassava varieties with improved yield, disease resistance, and adaptability to changing environmental conditions.

7.3 Global collaboration and research priorities

Global collaboration is crucial for advancing cassava breeding research and addressing the challenges posed by climate change, pests, and diseases. Collaborative efforts can facilitate the sharing of knowledge, resources, and technologies, thereby accelerating the development and dissemination of improved cassava varieties (Sen et al., 2020; Patial et al., 2022). International research networks and partnerships can also help in standardizing protocols and methodologies, ensuring the reproducibility and scalability of breeding innovations.

Research priorities should focus on optimizing DH induction protocols for cassava, exploring novel chromosome doubling agents, and integrating genome editing tools with traditional breeding methods. Additionally, efforts should be directed towards understanding the genetic mechanisms underlying important agronomic traits and developing robust phenotyping platforms for large-scale screening (Hooghvorst and Nogués, 2020a; Bhowmik and Bilichak, 2021). By aligning research priorities with global collaboration, the cassava breeding community can effectively address the challenges and harness the full potential of emerging technologies for crop improvement.

8 Concluding Remarks

Innovative breeding techniques such as doubled haploids (DH) and genetic engineering have shown significant potential in enhancing cassava breeding. The doubled haploid technique, which involves generating haploid plants followed by chromosome doubling, has been extensively studied and optimized in various crops. Despite the challenges, recent advancements in DH technology, including the use of new antimitotic compounds and CRISPR/Cas9 genome-editing systems, have improved the efficiency of haploid induction and chromosome doubling. In cassava, efforts to induce doubled haploids through gynogenesis have provided valuable insights, although successful regeneration of DH plants remains limited. The integration of DH technology with genomic selection and other biotechnological tools has the potential to accelerate breeding cycles and enhance genetic gains in cassava.

The application of doubled haploid technology in cassava breeding could revolutionize the development of new varieties by significantly reducing the time required to achieve homozygosity. Traditional methods of self-pollination to produce inbred lines are time-consuming and labor-intensive, often taking over a decade. The successful implementation of DH technology in cassava would enable the rapid production of homozygous lines, facilitating the incorporation of desirable traits and improving genetic diversity. Additionally, the use of genome editing tools such as CRISPR/Cas9 in conjunction with DH technology could further enhance the precision and efficiency of cassava breeding programs. However, the challenges associated with haploid induction and chromosome doubling in cassava need to be addressed through continued research and optimization of protocols.

The integration of doubled haploid technology and genetic engineering holds great promise for the future of cassava breeding. While significant progress has been made in understanding and optimizing DH techniques in other crops, the application in cassava is still in its nascent stages. Continued research and collaboration among scientists will be crucial in overcoming the current limitations and fully realizing the potential of these innovative breeding techniques. The advancements in DH technology and genome editing not only offer opportunities for rapid genetic improvement in cassava but also provide valuable insights into the fundamental processes of plant reproduction and breeding. As these technologies continue to evolve, they will undoubtedly play a pivotal role in meeting the growing demand for improved cassava varieties adapted to changing environmental conditions and agricultural challenges.

Acknowledgments

Authors sincerely thank all the experts and scholars who reviewed the manuscript of this study. Their valuable comments and suggestions have contributed to the improvement of this study.

Conflict of Interest Disclosure

Authors affirm that this research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest.

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