Mutation Breeding: A Tool in Nutritional Improvement of Cassava

Abstract

Cassava is an important food security crop worldwide with a lot of unexploited potential. More than 60% of global production is used for human consumption, while lesser quantity is used in livestock and Pharmacia industries. Improvement through hybridization and selection have been exploited but is limited by inter-specific and intra generic crop boundary, irregular flowering and low spontaneous mutation rate which cannot be depended on considering the high demand on the crop. Induce mutations has continue to remain an alternative tool for cassava improvement. The cytology analysis carried on five cassava varieties using varying levels of colchicine showed that the mutagen has significant aberration effect at (p < 0.05), with a Mitotic Index (MI) of (132.14), an error in cell divisions as shown in the positive increase yield of both parents and progeny of the cassava varieties evaluated. An epidermal-polyploidy change induced includes laggard, bridges, fragments, stickiness, vagrant and crises-cross at various concentrations. A required aberration was observed in the result. This shows significant difference in the mitotic index in a decreasing order with an increase in level of mutagen (132.14, 65.21 and 42.60) respectively. This result showed the mutagenic potentialities of colchicine in cassava induction and improvement.

1. Introduction

Cassava is a prominent root crop, which plays important role in the food security of many countries in the tropics, especially in sub-Sahara Africa. Every part of the plant is important but of most economic importance is the root. It is efficient in carbohydrate production and provides cheap source of calories for millions of people in Sub-Sahara. The largest producer of cassava in Africa is Nigeria and the third largest producer in the world after Brazil and Thailand [1]. Cassava is rated as the major staple crop in Nigeria feeding about 70% of the populace (FAOSTAT 2021). It has a fresh root starch content of about 30–40%, and the crop gives the highest yield of starch per unit area of both cereals and root and tuber crops [2, 3], but highly deficient in essential micronutrients, and extremely low in protein content which range between 1 and 3%. Although cassava is relatively rich in vitamin C, its content of iron, phosphorus, calcium and other minerals are in trace amount [4]. These micronutrient levels are not sufficient to take care of the micronutrient requirement of the low-income group of people who depend on root and tuber crops as their staple food. The inadequate intake of essential micro nutrients such as vitamin A, zinc and iron has been identified as the major causes of hidden hunger in the world practically in Nigeria. This has made malnutrition an immediate global challenge that requires urgent attention, and the global scale challenge posed by hidden hunger (micronutrient deficiency) informed its inclusion as one of the Sustainable Development Goals of the United Nations – SDG Goal 2: Zero Hunger, which has as one of its aims: to insure that enough nutritious foods are available to people by 2030 in a sustainable manner. Stephen in his health care nutrition analysis stated that malnutrition cannot be sustainably achieved through supplements and drugs, rather through a combination of options led by bio fortification of staple foods to produce whole and organic food; Food base approach is a more sustainable approach to attaining micronutrient adequacy compared to other methods [5, 6]. Bio fortification is a current complementary strategy to obtain and maintain adequate supply of essential micronutrients particularly among cassava products consumer.

2. The crop Cassava

1. Morphological Description of Cassava: (Manihot esculenta, with common names; cassava, manioc and tapioca Brazilian [7] is a procumbent and semi-herbaceous sub shrub plant in the family Euphorbiaceae (spurge family). It is native to South America but now grown in tropical and sub-tropical areas worldwide for the edible starchy roots. It can grow up to 7 m in height with a diameter of up to 20 cm, [8, 9], though most current varieties are hardly more than 3 m long. It is generally divided into two distant parts: the vegetative and the underground part. The vegetative part is made up of woody stem(s), which can be straight or branched. The outer bark is smooth, light brown to yellowish gray. The inner bark is cream-green with thin exudates, watery latex or sap but often bluish-gray when young. The branching pattern is typically dichotomous or trichotomous with branch-lets light green to tinged reddish having at the branching point a terminal inflorescence. Leaves are spirally arranged on petiole of up to 30 cm long, often reddish-purple. The root system is made up of the adventitious and the storage roots. The adventitious root is normally used for anchorage and absorption, while the storage root is the edible and the most important part of the plant. The tuberous edible root grows in clusters of 4–8 at the stem base. Roots are from 1 to 4 inches in diameter and 8–15 inches long, although roots up to 3 feet long roots up to 3 feet long have been found [10]. Some are usually cylindrical and tapered, some are irregular, while others are oval. They are covered with a thin reddish brown fibrous bark, which protects it from rodents and pest.

2. Karyotype and Origin of M. esculenta: Cassava geographically is a native of America with centers of diversity in Brazil and Mexico [11]. The crop has more than 98 species which resulted from natural and artificial interspecific hybridization among M. esculenta and wide species [12]. Among these species, only Manihot esculenta Crantz is the most important and widely cultivated in the tropics and sub-tropical regions. This specie has a wide range of genetic diversity of more 7000 cultivars and 16,000 accessions due to the weak reproductive isolation barriers. This attribute makes transfer of desirable genes easy and manipulation of traits possible. Genetically cassava is a typical diploid having a chromosome number 2n = 36 with only few with more chromosome numbers [12].

3. Nutritional Composition of Cassava: Cassava roots are very rich in carbohydrate. They contain significant amount of minerals - calcium (50 mg/100 g), phosphorus (40 mg/100 g) and vitamin C (25 mg/100 g) [1]. It is very rich in starch content, yielding more than 30% of starch per unit area [1]. However, they are poor in protein and other nutrients except the leaves, which are rich in protein (lysine), but deficient in other amino acids such as thiamine and tryptophan [13]. The protein content is extremely low and ranges between 1 and 3% [13, 14]. It has a large quantity of hydrocyanic, which makes it toxic, unhealthy to man, and animal. The quantity of this toxin depends on the variety, age of harvest and method of planting. However, this substance is very volatile and is removed to a safety level just by peeling, washing, grating and heating [14].

3. Production and utilization of Cassava in food and industrial

Global production and importance in the world economy: cassava is a universal crop produced either in subsistence level or in large scales. Production worldwide is contented in five countries namely Nigeria, Brazil, Thailand, Indonesia and Congo Democratic Republic cultivated under an average land area of 16.7 million hectares at the growth rate of 2.2%per annual Cassava. It is the most important staple food crop among the four major tropical root and tuber crops (cassava, yam, potatoes and cocoyam), providing basic diet for over half a billion people in the developing world [15, 16]. Globally, cassava is the second carbohydrate and starch source for food and industrial uses [17, 18] and the seventh most important food crop worldwide [19]. It is efficient in carbohydrate production and provides cheap source of calories for millions of people in Sahara, Sub-Sahara Africa. According to [20], more than 60% of global production is used as food for man, with lesser quantity being used for animal feed and agro based industries. In sub-Saharan Africa and Latin America, cassava is mostly used for human consumption [21], whereas in Asia and parts of Latin America, it is mostly used commercially for the production of animal feed and starch-based products [22]. Nigeria is the largest producer of cassava in the world producing more than

1. The roots can be processed into various forms of starch for domestic consumption, local and foreign market. It can also be utilized fresh, as in the case of sweet cultivars (low cyanorganic glycosides) or in processed forms as flour, starch and animal feed in the case of bitter cultivars (high cyanogenic glycosides [23]. Cassava fresh leaves are rich in crude protein (21.39%) and are utilized for human food as vegetable or as a constituent in the form of source eaten alone or with main staple [24]. The stems are used for propagation, staking and as firewood. In animal nutrition, it is either used in feed formulation or eaten fresh. The cassava water known as Manipulearia in Brazil is used for animal fattening and enhancement of milk production in dairy farming (unpublished lecture). They are also used in the production of local gin and liquor. Different countries make different types of alcoholic beverages from cassava: Caum and tiquira Brazil, nihamanchi South America, impata Mozambique and others. Cassava- based dishes are widely consumed wherever the crop is cultivated, and the food forms are either regional, or national [25]. Cassava has been found as source of alternative energy that is strong, renewable and sustainable. In some economies such as China, it has gradually become a major source of ethanol production (Business Green news, 2008). In addition to this, China-based Hainan Yedao Group invested $51.5 m (£31.8 m) to produce 33 million US gallons (120,000 m³) a year of bio- ethanol from cassava plants in 2008 [26]. It is an attractive fuel crop because it can give high yields of starch and total dry matter in spite of drought conditions and poor soil.

2. Cassava production and utilization in Nigeria: Cassava (Manihot esculents crantz) cassava is staple crop of choice across Nigerian households, playing very significant role in the diets and income of the producing households [27] with less production cost per unit output than any other staple food crop, it is capable of growing well and giving reasonable yield on marginal soils because of its drought-tolerance Cassava is grown in virtually all the states in Nigeria but more prominently grown in the following states Benue, Kogi, Enugu, Imo, Cross-River, Ondo, Ogun, Delta, Anambra, Edo, and Taraba, States [28, 29]. It is the congregate of production from these areas that placed Nigeria the largest producer of cassava worldwide producing an estimate of 52million per annual Cassava is the subject of many expansion programmes in the Sub-Saharan African region, as commercializing cassava and do-mystically producing staple crops – in order to limit imports. It– remains a key objective of many West African governments. In Nigeria, the regional production leader, the “Anchor Borrower’s Programme” (ABP), initiated by the country’s Central Bank (CBN), and provides preferential loans to smallholder farmers who provide their product to the processing sector. However, while cassava is one of the many commodities listed in the programme, the implementation of ABP has, in effect, made rice more lucrative to cultivate. More so, the annual increase in demand for cassava flour in Nigeria to feed her local and foreign companies for bread production, sugar based additive, glues, plywood, textiles, paper, monosodium glutamate, drugs, bio-degradable products and as bio- energy production has made cassava production an economic necessity [28].

4. Cassava: a potential raw material in animal feed industries

Cassava is an ideal alternative crop as a source of energy in livestock industry. It has been used in various forms to feed livestock worldwide. All parts of the crop plants-the leaves, the stem and roots can be fed to animals processed or wholly, singly or mixed with other stuffs depending on the type of animal. For ruminates the whole plants can be chopped sun dried and fed. For the monogastric they are processed into palates or feed meals. Cassava can replace about 30 to 50 percent of corn in animal feed ration. However, this crop capable of providing high potential source of energy in animal feed production is limited by a number of problems, which reduces its use and utilization. The most common are the poor quality feed outcome of cassava mills due to lack of essentials micronutrients in cassava raw materials which calls for the use of additives (vitamins-minerals and amino acids.) additional cost, high moisture content, the present of toxic substance hydrocyanic acid and order. Presently several researches on the use of cassava as energy source replacing maize has shown that the above mention problems can be corrected through development and improvement programs which encourage the development of bio fortified cassava varieties that will supply those essential nutrients in a whole meal [29].

4.1 Cassava improvement

Early cassava breeding programs have largely focused on increasing cassava productivity and resistance to pest and disease through inter-specific hybridization, which led to the release of many varieties [30]. Later in the 70s focused on enhanced food qualities and traits for utility, which led to the release of high dry starch and dry matter content [31]. However it was identified that conventional breeding present’s limitation in the breeding of specific traits needed to move cassava utilization forward [32]. This therefore led to the development of other breeding technologies which could manipulate on the genes and genomes to identify gene of great importance to farmers such as Obasanjo, gain changers, and others in Nigeria m. Some advanced breeding platforms have also developed breeding pipelines for enhanced cassava nutrition – IITA and NRCRI breeding for enhanced pro-vitamin A. Allard [33] also identified wild species with high protein content and with the advancement in plant breeding techniques, the problems of poor micronutrients and other issues such as adaptation to arid and semi-arid conditions are being addressed [33, 34, 35, 36].

Some conventional and unconventional breeding approaches have been implode in the improvement – Several conventional breeding approaches implode in the improvement of cassava are hybridization which used inter-specific crosses in cassava breeding to develop recombinants between cultivated and wild cultivars from which clones with better characters were obtained Storey and Nichols. Through this breeding approach many hybrid cassava cultivars that are resists to prevailing disease and pest, have be developed and released in many countries [36]. However, hybridization and selection have been exploited but cannot work beyond the biological boundary of inter-specific and intra generic crop breeding, its time consuming, their high degree of long vegetative propagation with its associated low and irregular flowering, high heterozygosity and difficulty of selection of recombinant and the problem of transferring undesirable traits and un locking desirable traits in sterile crops could not always be dependable due to gene actions and involved for the trait and diverse genetic structure of the parent in case of brake down. These necessitate the exploration of other breeding tools in order to improve difficult traits in crops with high degree of long vegetative propagation or sterility.

4.2 Mutation breeding

Mutation breeding despite the few bottle necks associated with it was the earliest tool used by plant breeders to increase plant size, develop non exiting traits and generate variations in crops [37]. Plants are made up of genes, which are the molecular unit of heredity of every living organism. The nucleus of an organism’s cell contains a number of having normally (2n sets) of chromosomes of which if they appear in pairs, the organism is said to be in a diploid, while and if more than a pair, the organism any organism that has higher sets of chromosomes is regarded as a polyphoid [11].

Polyploidy, an accidental change in the cytology of an organism, can be brought about by spontaneous or induced mutation. Spontaneous Mutation breeding is a breeding tool that can be used to create genetic variation for traits that generally have low variability in seed and vegetative propagated crops. It has a great potential in genetic improvement of cassava, cocoyam and cereal such as rice and ray and other root and tuber crops. But Spontaneous Mutation breeding, however begin low cannot meet the ecological, industrial and economical challenge of time led to the diversification of technologies to increase cassava productivity (Figure 1).

Figure 1.

Cassava root generated from the treated materials.

(iii) Induction Mutation: In recent times, induction mutation and analyses of mutants have received great attention as alternative means of crop improvement [12]. Breeding for micro-nutrients in cassava- In order to produce cassava varieties with high micronutrient levels, different breeding methods and tools have been used such as biotechnology (genetic engineering and molecular breeding techniques), and tissue culture approaches such as soma clonal variation and somatic hybridization. Induction mutation using colchicine is an excellent improvement tool successfully used in the manipulation of plant genome for the development of traits in sterile and irregular flowering plants of importance, including in roots and tuber crops [38] (Figure 2).

Figure 2.

(a) Typical Variety TMS0505 plant (b) Manipulated/induced variety TMS0505 plant.

5. Creation of genetic diversity using mutagne Colchicine

However, there is still immense scope to enhance the mineral nutrient, essential amino acid altered protein and fatty acid profiles, physicochemical properties of starch, phyto-nutrients, reduce anti-nutritional factors in cassava for human and animal consumption through bio fortification [39]. Induced mutation was conducted in National Root Crops Research Institute (NRCRI), Umudike Research farm, with Five cassava varieties namely: TMS98/0505, TMS94/4479, TMS98/1632, TMS92/0057, TMS98/0581 treated with manipulating hormone at three levels of colchicine: 0, 2 and 4 ppm. (Parts per million) (Figure 3).

Figure 3.

(a) Cassava plant with capsule or fruit (b) Seeds harvested from induced parents.

The stakes were soaked in the solution for 30 minutes, air dried for 24 hours in the screen house, pre-sprouted in nursery bags and transplanted to the field at 3-leaf stage. At 7 months after planting, 25 pieces of 25 cm stake cuttings each of the cassava varieties were cut from the mature plants raised from colchicine treated materials and planted in a well harrowed and ridged field in a 4×5 randomized block design, at a spacing of 1×1 m intra and inter-row replicated three times.

The cytology evaluation was screened and calculated for chromosomal aberration using the following formulae:

$$\text{Mitotic Index}\left(MI\right)=\frac{\text{Number of dividing cell}}{\text{Total number of cells counted}}$$E1

$$\%\text{Abberrant cells}=\frac{\text{Total aberrant cell}}{\text{Dividing cells}}\times 100$$E2

$$\text{Mitotic inhibition}=\frac{\text{Mitotic index of control}-\text{Mitotic index of treated}}{\text{Mitotic index of control}}\times 100$$E3

The proximate analyses of the roots were carried out using AOAC (1990) methods. Observations made showed that the treatment with colchicine mutagen had significant effect on the sizes of the stomata with and physico composition of the parent and their progenies. The microscopic analysis showed that the mutagen has significant aberration effect on the varieties across the concentrations (p < 0.05), with a Mitotic Index (MI) value of (132.14) and this led to error in cell divisions as shown in the positive increase yield of both parents and progeny of the cassava varieties evaluated, while there was no chromosomal aberration in the control. The type of change induced by the colchicine in this study was epidermal-polyploidy change which includes laggard, bridges, fragments, stickiness, vagrant and crises-cross at various concentrations. Mutation frequency calculated reported significant difference in the mitotic index in a decreasing order with the increase in level of mutagen (132.14, 65.21 and 42.60) respectively as shown in Table 1.

Conc (ppm)	No. of dividing cells	Chromosome aberration per 1000 cells						Total aberrant cells	Mitotic index	% aberrant cells	Mitotic inhibition
		Laggard	Bridge	Fragment	Stickiness	Vagrant	C-mitosis
0 ppm	660	0	0	0	0	0	0	0	132.14 ± 8.83	0.00 ± 0.00	0.00 ± 0.00
2 ppm	325	5	11	6	17	3	3	45	65.21 ± 8.18*	0.19 ± 0.01	0.51 ± 1.83*
4 ppm	213	7	19	7	24	5	7	69	42.60 ± 9.54**	0.32 ± 0.03	0.68 ± 1.71*

Table 1.

Cytological effects of colchicine on cells of Manihot esculenta.

* and ** mean significant at 5% and 1% respectively.

This result was advantageous in the induction of required changes in the studied cassava varieties and showed the mutagenic potentialities of colchicine. There was no significant difference among the three levels of concentration in most the physico-chemical compositions evaluated, but the concentration level 4 ppm gave highest ash content followed by level 2 and 0 ppm (2.437, 2.50 and 2.63%) respectively. On the other hand crude fiber was significantly affected by concentrations of colchicine as seen in the result (2.25, 2.46 and 2.65) and increased with increased level of colchicine from 0 to 4 ppm level. The starch content of the progenies evaluated according to levels (0, 2 and 4 ppm) were significantly different at (p < 0.05) with level 4 ppm higher than other levels (32.00, 29.44 and 34.03%) respectively, with an average of 31.70%, a value comparable to those of [36], who reported an average starch content of 32.6. Concentration level increased the major minerals of interest: zinc, iron and Magnesium significantly. Both zinc and iron were significantly affected by concentration level 4 ppm, while. Magnesium content at concentration level 2 ppm (0.58 mg/100 g) significantly differed from the other two concentration levels at (p < 0.05) of 4 ppm (0.48 mg/100 g) than followed by concentration level 0 ppm or control (0.41 mg/100) This result simply indicates that these essential nutrients can be enhanced using induced mutation and that the concentration levels has not been reached. The other mineral composition of the cassava materials evaluated: Nitrogen, Calcium, Potassium, Sodium and Phosphorous were not significantly affected. Some vitamins and amino-acids were significantly different among the three levels of concentrations evaluated. Concentration level 4 ppm affected hydrogen cyanide higher than other levels, followed by concentration level 2 ppm. While concentration level 2 ppm affected Phenol more than other levels (0.26 mg/100), followed by concentration level 4 ppm (0.17 mg/100), than 0 ppm (0.13 mg/100). Although Vitamin C was higher than other vitamins across level (22.51, 22.52, 22.51 mg/100 g), it was not significant. Thiamin, nicotinic, riboflavin (25.51, 22.51, 22.52 mg/100 g) was not only significant, there by proving that they were genetic in nature and can be improved through conventional breeding methods (Figure 4).

Figure 4.

Cassava root generated from F1 seeds.

6. Conclusion

Since the progressive increase level of colchicine continued to increase the level of some micro nutrients but did not in others, it is concluded that the nutritional values of cassava can be improved through mutation breeding using colchicine and suggest that level of application has not been exploited. Secondly, the fact that the present increase were lower than the Recommended Dietary Allowance of these nutrients [4], where the mean value range for zinc (1.08–2.52 mg/100 g) is low, iron (9.95–13.87 mg/100 g) is high, potassium (716.91–757.68 mg/100 g) is low, sodium (42.46–80.85 mg/100 g) magnesium (89.68–128.35 mg/100 g) is average, calcium (22.77–30.73 mg/100) is low and phosphorus (42.60–45.89 mg/100 g) there is still need to continue to increase the concentration of colchicine used in cassava improvement while animal sources be used as complements. For animal feed production, with enhanced botanical seed production through induced mutation, continuous and selection of variables for values of enhanced feed quality such as low moisture, low peel fiber in addition with the fortified micronutrient quality the goal of substituting higher percentage maize for animal feed production will be actualized.