**Abstract**

Soybean, which has many foods, feed, and industrial raw material products, has relatively limited genetic diversity due to the domestication practices which mainly focused on higher yield for many centuries. Besides, cleistogamy in soybean plant reduces genetic variations even further. Improving genetic variation in soybean is crucial for breeding applications to improve traits such as higher yield, early maturity, herbicide, and pest resistance, lodging and shattering resistance, seed quality and composition, abiotic stress tolerance and more. In the 21st century, there are numerous alternatives from conventional breeding to biotechnological approaches. Among these, mutation breeding is still a major method to produce new alleles and desired traits within the crop genomes. Physical and chemical mutagen protocols are still improving and mutation breeding proves its value to be fast, flexible, and viable in crop sciences. In the verge of revolutionary genome editing era, induced mutagenesis passed important cross-roads successfully with the help of emerging supportive NGS based-methods and non-destructive screening approaches that reduce the timeconsuming labor-intensive selection practices of mutation breeding. Induced mutagenesis will retain its place in crop science in the next decades, especially for plants such as soybean for which cross breeding is limited or not applicable.

**Keywords:** soybean, mutation breeding, mutagens, induced mutagenesis, next generation sequencing

## **1. Introduction**

Soybean (*Glycine max* (L.) Merrill.) has a central position in agriculture along with barley, cassava, groundnut, maize, millet, potato, oil palm, rapeseed, rice, rye, sorghum, sugar beet, sugarcane, sunflower, and wheat which were considered as the most cultivated plants worldwide. Its central role is not only constituted due to the dense protein and high-quality oil contents but also industrial raw material supply. Tofu, soy milk, soy sauce, and miso are the main nutritious human soy products. Also, extracted soy oil, with over 75% oleic acid and under 10% polyunsaturated fatty acids, is one of the most preferred oils sold commercially in the United States today [1]. Long shelf-life required fry, spray, and ingredient oils should preferably contain higher oleic acid due to the better persistence to oxidation. Soy meal is also a major

source of protein used in pig and poultry industries. The companion animal industry prefers soy meal as a protein source in animal diet, especially for dogs. High-quality amino acid composition and highly digestible protein content leads to the use of soy meal in aquaculture diets [2]. On the other hand, soy oil has various industrial uses as pharmaceuticals, plastics, papers, inks, paints, varnishes, and cosmetics.

In the verge of global warming effects, renewable energy sources as an alternative to fossil fuel are getting importance. Soybean is also an important biodiesel crop in many countries along with maize, especially in South America countries [3]. Besides the alternative bioenergy crop role, it has also environmental effects as being capable of utilizing atmospheric nitrogen through biological nitrogen fixation and is therefore less dependent on synthetic nitrogen fertilizers. While drought is one of the most plant growth and development limiting factors in present days, nitrogen deficiency is equally crippling for plants, as well, due to its structural, genetic, and metabolic functions in crop yield. Highly stable and non-reactive N2 is the most abundant constituent of the Earth's atmosphere, still no eukaryotic organism can use it directly. Some members of *Leguminosae* (*Fabaceae*) family including soybean have adopted the ability to establish symbiotic interactions with diazotrophic bacteria known as rhizobia in evolutionary adaptations. By this means, a process called 'biological nitrogen fixation is a low-cost N source that sufficiently increases soybean yield with low environmental impact and avoids the use of synthetic N fertilizers [4].

Soybean (*G. max* (L.) Merr) as a member of the family *Fabaceae*/Leguminosae, subfamily *Papilionoideae,* and the tribe *Phaseoleae* contains two subgenera as *Glycine* which has 26 perennial species and *Soja* (Moench) F.J. Herm. having four annual species [5]. Domestication of cultivated soybean can be traced back to China in 5000 years ago, however, the geographical origin of *Glycine* genus can be traced back to putative ancestor (2n = 2x = 20) which was presumably migrated and formed unknown or extinct wild perennials (2n = 4x = 40) in China. Wild annuals (2n = 4x = 40; *Glycine soja*) and domesticated soybean (2n = 4x = 40; *G. max*) subsequently evolved [6]. The genetic diversity of *G. max* is assumed to regress due to man-made genetic bottlenecks through selection for high yielding lines in modern plant breeding applications. Indeed, yield is the backbone of the profitability and the feasibility. Varieties with other superior traits are not significant in industrial scale unless they have a high yield. As well as yield, maturity, herbicide, and pest resistance, lodging resistance, shattering resistance, seed quality and composition, abiotic stress tolerance are other breeding selection targets [7]. While the wild relative *G. soja* grows in various environmental conditions and have not been exposed to the selective bottlenecks, it retained significant genetic diversity over time.

On the other hand, soybean flowers represent cleistogamous characteristics. Cleistogamy, which is described as the production of both open (chasmogamous, CH) and closed (cleistogamous, CL) floral forms by one species, is very common among angiosperms. Soybean is pseudocleistogamous cleistogamy in which no morphological differences between CL and CH flowers occur other than a lack of expansion of petals and anthesis in CL flowers. It may also be induced by environmental stress factors, occasionally. Cleistogamy is observed both in cultivated soybean [*G. max* (L.) Merr.] and its wild relative [*G. soja* Sieb. & Zucc.]. Soybean usually produces both CH and CL flowers on the same plant. In these plants, fertilization occurs within closed petals of CL flowers [8–10]. The rates of natural cross-pollination have been observed between ranges of 0.03–1.14% in natural conditions for self-pollinating soybean plant [11]. Thus, cleistogamy may have influenced the genomic homogeneity and reduced genomic variation further in soybean along with domestication practices.

In this context, improving genomic variations is crucial in soybean breeding. This chapter will summarize present conventional and biotechnological methods in soybean breeding and emphasize on mutation breeding practices with the concluding discussion on future prospective.
