Carotenoids in Cassava (*Manihot esculenta* Crantz)

*Lovina I. Udoh, Josephine U. Agogbua*, *Eberechi R. Keyagha and Itorobong I. Nkanga*

## **Abstract**

Cassava is produced globally and consumed as an important staple in Africa for its calories, but the crop is deficient in micronutrients such as vitamin A. Pro-vitamin A carotenoids including β-carotene are precursors of vitamin A in the human body. Carotenoids are generally associated with colors of fruits and vegetables. Although most cassava varieties have white tuberous roots and generally accepted, naturally; some cassava roots are colored yellow and contain negligible amounts of vitamin A. Several genes have been identified in the carotenoids biosynthesis pathway of plants, but studies show that Phytoene synthase 2 (*PSY2*), lycopene epsilon cyclase, and β-carotene hydroxylase genes have higher expression levels in yellow cassava roots. So far, the *PSY2* gene has been identified as the key gene associated with carotenoids in cassava. Some initiatives are implementing conventional breeding to increase pro-vitamin A carotenoids in cassava roots, and much success has been achieved in this regard. This chapter highlights various prediction tools employed for carotenoid content in fresh cassava roots, including molecular marker-assisted strategies developed to fast-track the conventional breeding for increased carotenoids in cassava.

**Keywords:** cassava, carotenoids, marker assisted selection, molecular markers, vitamin A, biofortification, phenotyping

## **1. Introduction**

Cassava (*Manihot esculenta* Crantz) is an important crop globally, and in Nigeria, it is consumed as a staple by more than 100 million people every day [1]. Global production of cassava has been given at approximately 278.7 million tons, it was estimated to be 281 million tons and 288.4 million tons in 2015 and 2016, respectively [2]. Global cassava market in 2019 increased by 0.4% to \$164.1B, and consumption was peak at \$172.1B [3]. Nigeria stands out as largest producer as its progressive cassava pattern increased from 42.5 million tons in 2010 to 61 million tons in 2020; total production area in 2012 was 3.85 million hectares [4]. In Nigeria, the cost of cassava production per hectare is estimated to be 82,055 naira, with a profit of about 123,745 naira. Although, in Africa, 50% of the cassava produced is largely consumed as food after processing; 38% in fresh and/or cooked form; and 12% is utilized for animal feed [5]. The crop is

cultivated mostly by small scale farmers because it outperforms other staple food crops under long-term drought and poor soil conditions [6].

Commonly available white cassava can provide most of the body's daily energy needs, but it does not provide adequate protein, essential micronutrients, and vitamin A. Vitamin A deficiency makes the body susceptible to infection, especially among women and children [7]. It causes illness and eye defects that can lead to partial or complete blindness [7]. Most cultivars of cassava are white or off-white, and the roots of tubers are generally low in carotenoids [8]. Cassava varieties with colored pulp that may be rich in carotenoids are very rarely available and are not well known to the general public. Yellow flesh color of some cassava varieties is associated with the presence of carotenoids [9, 10], and the nutritive importance of carotenoids is attributed to its conversion to vitamin A when consumed. The consumption of tuberous roots of β-carotene-rich cultivars may contribute significantly to addressing vitamin A deficiency in sub-Saharan Africa.

One of the most important micronutrients with deficiency of high public health concern is vitamin A, followed by iron, zinc, and iodine [11]. The generic descriptor for compounds with the qualitative biological activity of retinol is vitamin A. It exists in the form of preformed retinoids that are preserved in animal tissues as pro-vitamin A carotenoids usually gotten from green, yellow, and/or orange plant tissues. A total of two-thirds of dietary vitamin A worldwide and more than 80% in the developing world have been said to come from carotenoids in vegetables [5]. The all-trans-βcarotene is observed to be the most abundant carotenoid in cassava together with isomers such as 9-cis β-carotene, 13 cis-β-carotene, and β-cryptoxanthin [5, 12, 13]. Several carotenoid biosynthesis genes and enzymes such as lycopene epsilon cyclase (*LCYε*), β-carotene hydroxylase (CHYβ), phytoene synthase 1 and 2, lycopene β-cyclase, and phytoene desaturase (*PDS*) have been identified for different plants including cassava [14–16]. Studies by Olayide et al. [13] detected more carotenoids and isomers in the leaves than roots. Phytoene synthase 2 (*PSY2*), *LCYε*, and CHYβ genes were mostly associated with β-carotene content in white and yellow roots, but they generally had higher expression in yellow root cassava [13]. To enhance markerassisted selection in the conventional breeding to increase carotenoids in cassava roots, six single-nucleotide polymorphisms (SNP) markers were designed on candidate genes and validated on 650 elite cassava accessions of which PSY2\_572 explained most of the phenotypic variation (R2 = 0.75) in root pulp color [12].

Limited access to diets that are rich in vitamin A is known to be the root cause of vitamin A deficiency in Africa and other vitamin A deficiency inflicted regions. Efforts are continually being made to improve the nutritional value of cassava through biofortification, which has led to an improvement of its carotenoid content. These improvements have been successful through the adoption of advanced breeding techniques, which involves the screening of large numbers of genotypes for nutritional quality, agronomic traits, yield traits, etc., in order to select progenies with the best traits for further breeding.

## **2. Origin and domestication of cassava**

Cassava (*Manihot esculenta* Crantz) is a dicotyledonous plant belonging to the Euphorbiaceous family known only in the cultivated form and was first domesticated by the Amerindians of South and Central America [17]. There is archaeological evidence of two major centers of origin for cassava, one in Mexico and Central America and the other in North-eastern Brazil. In sixteenth century, Portuguese navigators

*Carotenoids in Cassava (*Manihot esculenta *Crantz) DOI: http://dx.doi.org/10.5772/intechopen.105210*

took cassava from Brazil to the western coast of Africa [18] and later to East Africa in eighteenth century through island of Reunion, Madagascar, also Zanzibar as described by Iglesias et al. [18]. It was introduced in India in the nineteenth century. Cassava plantations were set up by the Portuguese, who colonized South American regions by 1500 A.D. They carried cassava from these plantations to other continents [19]; hence, cassava was first introduced to Africa and Asia in the late sixteenth century by the Portuguese travelers. It was initially planted around the Congo River basin from where it moved to West and Central Africa [17, 20]. Nigeria was among the first African countries to receive the crop in the eighteenth century. The cassava crop was perhaps introduced in southern Nigeria by freed slaves who returned from South America through Sao Tome and Fernando Po islands [21].

#### **2.1 Taxonomy**

Cassava, as it is called in English, is referred to as "manioc" in French, "yuca" in Spanish, and "mandioca" in Portuguese. Cassava comprises about 7200 species. It belongs to the following [22];

Kingdom – Plantae Subkingdom – Tracheobionta Super division – Spermatophyta Division – Magnoliophyta Class – Magnoliopsida Subclass – Rosidae Order – Euphorbiales Family – Euphorbiaceae Subfamily – Manihotae Genus – Manihot Species – *Manihot esculenta* Crantz

This family is characterized by lactiferous vessels composed of secretory cells [17]. A total of 98 *Manihot* species have been recognized with one species (*Manihotoides pauciflora*) known in the closest related genus [17]. A lot of its characteristics have not been identified in any *Manihot* species, which are its mono-flower inflorescences and leaves borne at the apex of short, condensed stems arising from branch-lets. *M. pauciflora* is suggested to be a possible progenitor of all the Manihot groups. Unfortunately, this species is on the verge of extinction [23], and cassava is the only species that is widely cultivated for food production [23, 24]. The cultivated species may be derived from the wild progenitor *M. flabellifolia* [17].

#### **2.2 Botanical description**

Cassava is propagated mainly from stem cuttings, thereby maintaining true-totype cultivars. Nevertheless, propagation by seed can take place naturally or during plant breeding procedures. When stem cuttings are planted in the moist soil under favorable conditions, they produce sprouts and adventitious roots at the base of the cuttings within a week. If propagated by seeds, it first develops into a tap root system. Cassava leaves are simple; it consists of a lamina and a petiole. Each leaf is subtended by two stipules, about 1 cm long. The petiole is between 5 and 30 cm long and varies from green to purple. The smooth margin of the lamina is palmate or lobed. The

lobes differ in number, ranging from 3 to 9, and are most of the time odd numbers. The lobe's vain color can differ from green to purple. Most cassava varieties grown in Africa have elliptical or lanceolated lobes [17, 25]. The arrangement of cassava leaves on a stem (phyllotaxis) is a 2/5 spiral, meaning that the position of five leaves turns twice spirally around the stem, then the next leaf comes just above the beginning of the other. Their stems are cylindrical and have a diameter, which varies between 2 and 6 cm. Cassava stems usually grow up to 4 m, but some genotypes may grow to only to a height of1 m. The older parts of the stems display prominent knob-like scars, which are leaf scars and their nodes [20, 25]. Cassava is a monoecious plant with male and female flowers located on the same plant. The inflorescences are produced at the reproductive branches [22].

Cassava is propagated from stem cutting or seed. In cassava, the fleshy part is the central portion of the tuberous root. Tuberous roots vary in shape and color, depending on the soil conditions and variety [25]. Cassava grows between 30°N and 30°S in areas where annual rainfall is greater than 500 mm and where mean temperature is greater than 20 °C. However, some cassava varieties grow at 2000 m altitude or in subtropical areas with annual mean temperatures as low as 16 °C. Cassava prefers a sandy or sandy loam soil, but all types of soils, except water logged soils, can be used. Cassava tolerates the high levels of aluminum and manganese often found in tropical soils [26].

## **3. Carotenoids biosynthesis in plants**

Exploitation of the diverse tropical cassava collection for development of high pro-vitamin A cassava cultivars entails understanding and application of knowledge derived from molecular and biochemical studies of carotenoids and their biosynthesis in plants. Carotenoids are naturally occurring organic pigments that are produced by plants and some photosynthetic organisms [27, 28]. They are characterized by their extensive conjugated double bond along their carbon backbone giving them the capability to absorb lights in the range of blue to green range of the visible spectrum [28]. In plants, carotenoids are present mainly as indispensable integral components of the chloroplast, providing multiple services to the photosynthetic machinery participating in the light harvesting process and guarding the photosystems from possible damages by quenching reactive singlet oxygens and radicals created during photooxidation [29–31].

The carotenoid biosynthesis pathway is extensively studied in plants [29–33] and is responsible for the biogenesis of about 600 40-carbon isoprenoid compounds broadly classified as xanthophylls and carotenes. The first reaction dedicated to siphoning substrates to the carotenoid biosynthesis pathway in plants is catalyzed by the enzyme phytoene synthase (*PSY*). In this reaction, two geranylgeranyl pyrophosphate molecules are condensed to produce the first colorless linear carotenoid compound, phytoene. Phytoene is then modified through a series of desaturation and isomerization reactions catalyzed by enzymes including phytoene desaturase (*PDS*) and carotenoid isomerase (*CRTISO*) yielding the red colored carotenoid, lycopene. Lycopene is the forking point in the pathway that leads to two separate downstream branches called α and β branches. In the α branch, carotenoids such as α-carotene and lutein are synthesized, while in the β branch, carotenoids such as β-carotene, β-cryptoxanthin, and zeaxanthin are generated following cyclization of the terminals of the linear structured lycopene. Key enzymes involved in the branched part of the pathway include lycopene epsilon α-cyclase (*LYCε*) and lycopene epsilon β cyclase

*Carotenoids in Cassava (*Manihot esculenta *Crantz) DOI: http://dx.doi.org/10.5772/intechopen.105210*

(LYCβ) and β-carotene hydroxylase. The *LYCβ* can add β-ionone rings in both ends of lycopene to give β-carotene; while *LYCε* can add ε-ring in one end only to give α-carotene [14, 30]. Hydroxylation at the C-3 position of each ring of β- carotene and α-carotene produces xanthophylls, zeaxanthin and lutein, respectively (**Figure 1**).

Among all carotenoid compounds, only β-carotene has full vitamin A activity due to its doubly ended β-ionone rings, while carotenoids that have single β ring, such as α-carotene and β-cryptoxanthin, have half vitamin A activity of β-carotene [18, 30, 34–36]. Although the mechanism of regulation of the carotenoid biosynthesis is still not fully understood, a lot of progress has been made in this regard [30, 37].

#### **3.1 Genes associated with carotenoid in cassava**

Studies by Iglesias and Chavez et al. [10, 18] reported that relatively few major genes are involved in the determination of carotenoid accumulation in cassava roots. Thus, the trait can be improved to a significant level through the process of selection and recombination. In other crops, genes such as phytoene synthase (PSY), β-carotene hydroxylase, lycopene β, and ε cyclase have been reported to play a role in increasing levels of carotenoids [36, 38]. In cassava, Arango et al. [39] observed three *PSY* genes, one of which was discovered to be associated with stress in the Poaceae homologs. However, the two remaining *PSY* genes contributed differentially to carotenoid accumulation in leaves, roots, and flower parts of cassava. So far, the *PSY* gene has been identified as the key gene associated with carotenoids in cassava [12, 40, 41]. Olayide et al. [13] observed that carotenoid synthesis genes were expressed in both white and yellow cassava roots, but the following genes had higher expression in yellow roots, including phytoene synthase 2, lycopene epsilon cyclase, and β-carotene hydroxylase.

#### **3.2 Breeding for increased carotenoids in cassava roots**

Cassava is an highly important diet not only for humans but also in animal diet especially poultry, due to its availability and calories [24, 42, 43]; thus, the need arose

**Figure 1.**

*A simplified diagram of the carotenoid biosynthetic pathway in plants, showing major genes and enzymes involved Figure 1.*

to fortify the crop with micronutrient to improve its nutritional status. Some cassava varieties originally have yellow root color (**Figure 2**) meaning they have negligible amount of pro-vitamin A [18, 23]. Total carotenoid concentration in fresh yellow cassava ranges from 1 to 100 μg/g (fresh weight), primarily as all-trans-β-carotene, and is located in the parenchyma cells, the storage cells of the roots, and isomers such as 9 and 15 cis β-carotene and β-cryptoxanthin have also been detected [5, 18]. Carotenoid concentration is a stable trait and is influenced more by genotype than by its environment. Studies showed that retention of carotenoids differs not only per processing and storage method for a certain variety [10] but also within a variety, and this might be due to the variable distribution of dry weight matter within a root [44]. Retention varies between 10% for heavily processed and roasted cassava granules and 87% for boiling [10, 45].

Genetic improvement for this crop has employed crossing the wild yellow cultivars with elite breeding lines through recurrent selection and recombination [46]. This is accompanied by extensive field evaluation (phenotyping), including observations of disease and pest resistance, plant architecture, flowering ability, and performance in storage root [47]. Recently, rapid cycling recurrent selection was employed, which is able to cut down on the number of breeding cycles [9, 44]. The color of fruits and vegetables is associated with the presence of carotenoids, and the tuber-flesh color of some cassava accessions is yellow [23]. This indicates that naturally in the gene pool there are accessions with negligible amount of carotenoids [48], and this is currently being utilized in breeding. Breeding to biofortify cassava with pro-vitamin A will have a significant positive impact on nutrition and overall health, especially among poorer communities.
