Cassava Biotechnology and Soil Modification

#### **Chapter 4**

## Plant Regeneration from Cassava Protoplasts

*Wen Feng, Hai-Tian Fu, Yan-Chun Luo and Jian-Qi Huang*

#### **Abstract**

Cassava is an important crop for food, feed, and industrial raw materials. Given that traditional conventional breeding is restricted by various factors, biotechnology breeding has become an important breeding method. Tissue culture regeneration is the basis of biotechnology breeding. This chapter reviews the establishment and development of cassava tissue culture and regeneration systems and the technical processes of tissue culture and regeneration starting from the induction of explants of tissue-cultured cassava plantlets to embryogenic calli, isolation to protoplasts, culture to embryogenic calli followed by differentiation into embryos, and then sprouting, stemming, and rooting into complete plants. This chapter focuses on the technical processes from protoplast to complete plant and summarizes the important influencing factors of protoplast regeneration, which is the key and difficult point in the entire regeneration process of cassava protoplasts. This chapter aims to provide technical guidance for cassava protoplast regeneration, offer useful inspiration and reference for cassava tissue culture, and lay a foundation for the genetic improvement of cassava.

**Keywords:** cassava, biotechnology, tissue culture, friable embryogenic callus, protoplast, somatic embryogenesis

#### **1. Introduction**

Cassava (*Manihot esculenta* Crantz), a perennial shrub of the Euphorbiaceae family, is widely cultivated in tropical and subtropical regions. It is a root crop that is a staple food for approximately 800 million people worldwide [1, 2]. It is also an important raw material for the production of starch, processed food, and biofuels [3, 4]. Its tubers, tender branches, and leaves are commonly used as animal feed [5]. Cassava also has some advantages, such as tolerance to adverse environmental conditions, adaptation to poor soils, flexible harvest times, and the capability for growth under marginal conditions [6].

Viral diseases, insect pests, toxic cyanogenic glucosides, postharvest physiological deterioration, and low root protein content roots are problems in cassava cultivation [7, 8]. Traditional conventional cassava breeding is restricted by several problems, such as high genotype heterozygosity, long life cycle, low natural fertility, poor seed set, and low seed germination rates [9–11]. Biotechnology breeding is a supplement to traditional breeding methods. With the development of molecular breeding and

genetic engineering, biotechnology breeding requires an effective regeneration system [12, 13].

This chapter provides a review of the establishment and development of cassava tissue culture and regeneration systems and the tissue culture and regeneration technology of cassava starting from the induction of the explants of tissue-cultured cassava plantlets into embryogenic calli and then into protoplasts, followed by culturing into complete plants in the cyclic process of plant regeneration from cassava protoplasts. It also provides information on experiences and skills in protoplast regeneration to lay a foundation for the genetic improvement of cassava.

#### **2. Establishment and development of cassava tissue culture and regeneration systems**

Cassava tissue culture and regeneration technologies have been continuously developed since Kartha et al. cultured the apical meristem of cassava and obtained complete plants of five varieties for the first time in 1974 [14].

#### **2.1 Organogenesis**

Cassava axillary buds were cultured on medium with a high concentration of 6-benzylaminopurine (6-BA, 10 mg L−1) to form a round compact bulb-like structure and then transferred onto medium with a low concentration of 6-BA (1 mg L−1) for multiple shoot production; this approach was an efficient mass propagation system for cassava [15, 16].

On the basis of the establishment of the plant regeneration pathway of cassava somatic embryogenesis, cotyledons formed from primary embryos, secondary embryos, and circulating embryos could regenerate plants through organogenesis in medium containing 1.0 mg L−1 6-BA and 0.5 mg L−1 IBA [17]. The primary embryo has low ability for cotyledon organogenesis, whereas the circulating embryo has the highest ability for cotyledon organogenesis [18]. Cassava explants for organogenesis could be derived from the axillary buds, stem tips, young leaf lobes, and cotyledons of primary, secondary, and circulating embryos.

#### **2.2 Somatic embryogenesis**

Somatic embryogenesis has been widely developed since Stamp and Hemhaw first reported in 1982 that embryoids could be successfully induced from the cotyledons and hypocotyls of cassava zygotic embryos [19]. The four-step method of the somatic embryogenesis and plant regeneration of cassava has been established as follows: (1) induction of somatic embryos on medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) and other auxins; (2) maturation or germination on medium containing a low concentration of 6-BA; (3) growth on medium containing a high concentration of 6-BA and development into stems; and (4) rooting in low naphthaleneacetic acid (NAA) concentration or hormone-free medium [20–22]. In steps 1 and 2 of circulation, secondary and cyclic somatic embryos could be generated, forming a cyclic somatic embryogenesis system, and cyclic embryo explants could be induced into embryos more significantly [20, 22].

Since then, most studies on somatic embryogenesis performed optimization in accordance with different factors, such as genotype, explant type, and hormone type. *Plant Regeneration from Cassava Protoplasts DOI: http://dx.doi.org/10.5772/intechopen.110081*

Explants have also been developed from the hypocotyls and cotyledons of the initial zygotic embryo and the apical buds, young leaf lobes, axillary buds, flower tissues, and cotyledons of primary, secondary, and cyclic somatic embryos. The development of somatic embryogenesis and plant regeneration laid a foundation for the induction of friable embryogenic callus (FEC) [23] and genetic transformation in cassava [24].

#### **2.3 Friable embryogenic callus (FEC) induction**

FEC is an important research material in genetic and cell engineering. It has been developed for plants, and zygotic embryos are usually the preferred explant materials for FEC induction [25]. The zygotic embryos of cassava have extremely heterogeneous and unclear genotypes and are therefore unsuitable as explants.

Taylor et al. described the initiation of FEC for the first time. They utilized young leaves to induce embryogenic calli [23]. After embryogenic calli were generated, high-quality embryonic tissues were continuously subcultured on Gresshoff and Doy (GD) medium containing picloram to produce a small cell cluster that was composed of dozens of cells with diameters of 1 mm; these tissues were considered as fragile embryogenic calli from which highly totipotent embryogenic suspension cultures were established [23].

Since then, many reports on FEC induction have been published [26–29]. However, considering that cassava is a gene-dependent crop, FEC cannot be induced successfully for every variety [10, 30].

Successful FEC induction has laid a foundation for genetic transformation [28, 31, 32], CRISPR/Cas9 genome editing technology [33, 34], protoplast culture and regeneration [35, 36], and somatic hybridization [37].

#### **2.4 Protoplast culture and regeneration**

#### *2.4.1 Culture and regeneration of mesophyll protoplasts*

Protoplasts were separated from the leaves of tissue-cultured seedlings and cultured in a double-layered solid and liquid medium inserted with short glass rods evenly and vertically. No glass rod was inserted in the control culture, and the remaining cultures were all the same. The protoplasts divided continuously to form visible calli only in the medium inserted with glass rods [38].

After a long time, plant regeneration from the leaf mesophyll-derived protoplasts of cassava was reported in 2022 [39]. Prior to this report, only one successful report of plant regeneration from protoplasts isolated from cassava leaves had been published [40], and other scholars could not repeat this process [37, 38].

#### *2.4.2 Culture and regeneration of protoplasts from embryos and embryogenic calli*

The protoplasts separated from secondary embryos were tested in more than 50 media to form visible calli, a small number of which formed adventitious roots but never formed adventitious buds or embryos [41].

At present, most methods for protoplast regeneration involve inducing FEC and establishing an embryogenic suspension culture for the separation of protoplasts. In the 1990s, a breakthrough was made in the research on cassava protoplast regeneration. On the basis of establishing FEC induction technology and its suspension culture system, research on the isolation, purification, and culture of cassava protoplasts from FEC was carried out, and plants were regenerated. However, regeneration efficiency was low mainly due to the low efficiency of protoplast-derived calli for differentiating into somatic embryos and the low germination efficiency of mature embryos, which is the bottleneck in cassava protoplast regeneration [35].

Subsequently, cassava protoplast regeneration was not reported for a long time. In 2012, Wen et al. improved the yield and activity of isolated protoplasts on the basis of their predecessors. Callus-derived protoplasts were first subjected to suspension culture in suspension culture medium (SH) liquid medium, and then cultured in somatic embryo emerging medium (MSN) solid medium [36]. The bottleneck mentioned by Sofiari et al. [35] was broken, and the regeneration efficiency of protoplasts was greatly improved. On the basis of the established protoplast regeneration technology system, tetraploid cassava plants were regenerated *via* protoplast electrofusion [37].

#### **3. Plant regeneration from cassava protoplasts**

In this section, cassava tissue culture and regeneration technology is mainly reviewed starting with the induction of explants of tissue-cultured cassava plantlets into embryogenic callus; followed by the isolation of protoplasts; the culture and differentiation into embryos of embryogenic calli; and sprouting, stemming, and rooting into a complete plant. This section focuses on the technical process from the isolation of protoplasts to the generation of a complete plant (**Figure 1**).

**Figure 1.** *Cyclic process of plant regeneration from cassava protoplasts.*

#### **3.1 Explants**

Young leaf lobes of tissue-cultured cassava seedlings with an area of approximately 1 cm2 and approximately 1 cm of stem cuttings with buds of tissue-cultured seedlings were used as explants.

#### **3.2 Embryogenic callus induction**

Embryonic calli were induced from young leaf lobe explants of tissue-cultured plantlets on somatic embryo induction medium (CIM) containing 12 mg L−1 picloram and were produced after 2–3 weeks. Embryogenic calli were picked out with tweezers and cultured continuously on CIM for 6–8 weeks for propagation. The medium was refreshed every 2 weeks. All cultures were kept in a growth chamber in the dark at 25°C. If the explants were stem cuttings with buds, they were cultured on axillary bud enlargement medium (CAM) for 2–4 days for bud enlargement before being cultured on CIM. The later cultures were the same as those used to culture young leaf lobe explants.

#### **3.3 FEC induction**

Embryogenic calli were cultured on GD for 2–4 weeks. The fine particles generated on the surfaces of embryogenic calli were separated and then propagated continuously on GD. FEC formed after 2–4 weeks of continuous circulation culture on GD. The medium was refreshed every 2 weeks. All cultures were kept in a growth chamber in the dark at 25°C.

#### **3.4 Suspension culture**

Cell suspension cultures were initiated by transferring approximately 1 g of FEC into a 100-mL flask with 30 mL of SH. The flask was agitated on a rotary shaker at 110–130 r min−1. The liquid medium was refreshed every 2–3 days. All cultures were kept in a growth chamber at 25°C with a 12 h photoperiod and irradiance of 45 μmol−2 s−1. Protoplast isolation was performed through 5 days of suspension culture in SH.

#### **3.5 Protoplast isolation and purification**

#### *3.5.1 Enzymolysis*

Large particles were removed from 5-day-old cell suspension cultures in SH with tweezers, and the liquid medium was aspirated out with a straw. Approximately 1 g of tissue was placed in a Petri dish (9 cm diameter) with 12 mL of cell digestion solution. The cell digestion solution contained a mixture of enzymes (10 g L−1 cellulase R-10, 400 mg L−1 macerozyme R-10, and 100 mg L−1 pectolyase from Yakult, Japan) and 1 mg L−1 NAA, 1 mg L−1 2,4-D, 740 mg L−1 KNO3, 368 mg L−1 CaCl2, 34 mg L−1 KH2PO4, 492 mg L−1 MgSO4·7H2O, 19.2 mg L−1 Na-EDTA, 14 mg L−1 FeSO4·7H2O, 91 g L−1 D-mannitol, and 0.5 g L −1 MES. The suspension tissues were incubated in the enzyme solution for 18 h on a shaker at 40 r min−1 and 25°C in the dark.

#### *3.5.2 Purification*

Protoplasts were purified through the gradient centrifugation method. The digested tissues were filtered through a 45 μm stainless steel mesh to remove undigested cell clumps and debris. The filtrate was transferred into 10-mL centrifuge tubes and centrifuged for 6 min at 960 r min−1. The supernatant was removed with a Pasteur pipet. The pellets were gently resuspended in 1.0–1.5 mL of 13% mannitol solution containing CPW nutrients (27.2 mg L−1 KH2PO4, 250 mg L−1 MgSO4, 100 mg L−1 KNO3, 150 mg L−1 CaCl2, 0.2 mg L−1 KI, 0.003 mg L−1 CuSO4). Then, the protoplast-containing 13% mannitol solution was slowly pipetted onto the top of 3–4 mL of 26% sucrose solution containing CPW nutrients while avoiding mixing and centrifuged for 6 min at 960 r min−1. A band of viable protoplasts formed at the interface between the two layers. The protoplasts were carefully removed from the interface with a Pasteur pipet and resuspended in protoplast culture medium (TM2G). The protoplasts in TM2G were centrifuged for 6 min at 960 r min−1. The supernatant was removed with a Pasteur pipet, and the protoplasts were resuspended in TM2G with 0.36 mol L−1 glucose at a density of 5 × 105 protoplasts mL−1.

The yield of obtained protoplasts (cells g−1) was calculated by using the following formula: N × 5 × 104 × V/m; where N = number of protoplasts counted in a hemocytometer chamber; V = volume of diluted protoplasts; and m = fresh weight of plant material for protoplast isolation.

#### *3.5.3 Viability test*

The viability of the obtained protoplasts was checked with fluorescein diacetate (FDA). A total of 12 μL of 5 mg mL−1 FDA solution was added to 0.5 mL of the protoplast suspension. After 5 min, the protoplasts were examined with an Olympus IX71 inverted fluorescence microscope (green fluorescence, Olympus, Japan). The viability of obtained protoplasts (%) was calculated as follows: number of protoplasts with green fluorescence/Total protoplasts in the field × 100.

#### **3.6 Protoplast culture**

Initially, protoplasts were cultured through the thin liquid layer culture method in 1.5 mL of TM2G with 0.30–0.36 mol L−1 glucose in a 6-cm plastic Petri dish in the dark at 28°C. The medium was refreshed every 10 days: twice with TM2G with 0.30–0.33 mol L−1 glucose, then twice with a medium with reduced levels of glucose (0.27–0.30 mol L−1). It was refreshed again two times with reduced glucose levels (0.25 mol L−1 glucose). The osmotic pressure of the culture was reduced by gradually reducing the glucose concentration of the TM2G medium to promote cell division.

#### **3.7 Suspension culture**

Protoplasts were cultured in TM2G with gradual dilution for approximately 6–10 weeks. Then, protoplast-derived compact calli were transferred into SH for suspension culture, and the other calli were cultured further. The liquid medium was refreshed every 7–15 days, and the calli were propagated in SH for 2–3 weeks.

#### **3.8 Somatic embryogenesis**

For embryo differentiation, the calli propagated in SH were transferred to MSN under light. The differentiated embryos were cultured on embryo maturation medium (CMM) for 1–3 weeks to develop large green cotyledon embryos. Then, the mature large green cotyledon embryos were transferred to shoot elongation medium (CEM). Shoot elongation began after 4–8 weeks. When the length of the elongated shoot reached 2–3 cm, the shoot was cut off for rooting on Murashige and Skoog (MS) medium. Rooting could occur in 7 days, and the protoplasts usually took 5–7 months to develop into complete plants.

#### **3.9 Influencing factors of plant regeneration from cassava protoplast**

#### *3.9.1 State of FEC*

The isolation of high-quality protoplasts is a prerequisite for protoplast culture, and the state of FEC directly affects the quality of isolated protoplasts, including yield and activity. FEC is characterized by a loose structure, the presence of spherical particles on its surface, and a milky white or yellow color. It can be used to establish suspension systems. When suspended in SH, numerous fine particles are dispersed.

In general, subculturing FEC on GD for 15–20 days results in FEC in the best state, i.e., friable and loose, and increases the yield to the maximum. After suspension culture, protoplasts are separated from FEC. This approach is conducive to plant regeneration. The protoplasts isolated from the embryogenic callus suspension of cassava subcultured for 5–15 days have high activity and few impurities.

#### *3.9.2 Protoplast extraction and purification*

The extraction and purification of cassava protoplasts separated in cell digestion solution are a key step. The cell digestion solution may not flow automatically when it is filtered through a stainless steel screen due to the effect of its surface tension, and filtration generally takes a long time. The longer the protoplasts stay in the enzyme solution, the lower their activity and the higher their impurity content. Therefore, the enzyme solution should be filtered through a stainless steel screen immediately. An external force can be exerted on the stainless steel screen to enable the enzyme solution to flow down and filter quickly. Purification through gradient centrifugation provides protoplasts with high yield and activity.

#### *3.9.3 Protoplast culture*

Cassava protoplasts were cultured in TM2G at a density of 5 × 105 pieces mL−1. The initial concentration of glucose in TM2G can be within the range of 0.30– 0.36 mol L−1. Subsequently, the medium must be constantly refreshed and its glucose concentration must be reduced gradually to promote cell cluster division and growth. After protoplasts were cultured in TM2G for 6–8 weeks, 1–2 mm compact calli visible to the naked eye were selected, and other calli were used for further culture.

#### *3.9.4 Embryo differentiation and germination*

Sofiari et al. [35] believed that the differentiation of cassava protoplast-derived calli into embryos and the germination of embryos constitute the bottleneck of plant regeneration from protoplasts. Therefore, somatic embryogenesis is a key step in plant regeneration from cassava protoplast. The medium is an important factor in this process.

Given that the compact callus of protoplast origin is in the same state as the FEC used for cassava genetic transformation, compact callus of protoplast origin and FEC are considered as cell clusters, and the MSN used as the medium for genetic transformation is also used as the medium for embryo differentiation.

Before the differentiation of embryos on MSN, compact calli are first suspended in SH for 2–4 weeks. The compact calli become loose after being cultured in SH. This effect is advantageous for further somatic embryogenesis or proliferation on MSN or GD.

#### **3.10 Composition and function of cassava culture medium**

Nine kinds of cassava media are discussed in this chapter. **Table 1** shows the functions of nine kinds of media, and **Tables 1–3** present the composition of the nine kinds of media.

Cassava culture media containing MS, CAM, CIM, MSN, CMM, and CEM have basically the same compositions and differ only by hormone type or dosage. They are all composed of MS salt and vitamins, plus 20 g L−1 sucrose, 8 g L−1 agar, and 2 μM CuSO4 (or not). They play different roles in the tissue culture and regeneration of cassava due to the different kinds or dosage of hormones that they contain (**Table 1**).

Although CIM and MSN media could be used to induce cassava embryos, they induce different explants. The explants often induced on CIM can be young leaf lobes, apical buds, and axillary buds used for the induction of primary, secondary, and circulating somatic embryos, which are in the beginning stages of somatic


### **Table 1.**

*Medium components and function.*

*Plant Regeneration from Cassava Protoplasts DOI: http://dx.doi.org/10.5772/intechopen.110081*


#### **Table 2.**

*MS salts and vitamins.*

embryogenesis and can grow into different types of embryoids, such as globular, torpedo, and cotyledon embryos. The explants often induced on MSN are FEC or calli derived from protoplast division. When cultured on MSN under light, they can grow into different types of embryoids, such as globular, torpedo, and green cotyledon embryos. CIM is used as a somatic embryo induction medium under dark conditions, whereas MSN is used as a somatic embryo induction medium under light conditions.

The regeneration processes of globular, torpedo, and cotyledon embryos induced on CIM and MSN are the same. They all undergo and complete maturation, bud elongation, and rooting on CMM, CEM, and MS, respectively. These processes should be conducted under light conditions.


*Cassava – Recent Updates on Food, Feed, and Industry*


#### **Table 3.**

*GD, SH, TM2G components.*

GD can be used for the induction, maintenance, and proliferation of cassava FEC. SH can also participate in the maintenance of the embryogenic proliferation of FEC. GD is a solid medium, whereas SH is a liquid medium. FEC can be converted between GD and SH cultures, and its properties do not change. FEC cultured on SH has better cell consistency and faster proliferation than that cultured on GD. TM2G is used as a medium for culturing cassava callus protoplasts. Its osmotic pressure is reduced during culture by decreasing its glucose concentration gradually to promote cell division.

### **4. Conclusion**

Cassava is a food crop, and the biotechnology research on cassava lags behind that on major food crops, such as rice and wheat. The establishment and development

*Plant Regeneration from Cassava Protoplasts DOI: http://dx.doi.org/10.5772/intechopen.110081*

of cassava tissue culture and regeneration technology have promoted the application of biotechnology techniques, such as genetic transformation, genome editing, and somatic hybridization, to cassava. However, deficiencies remain. Cassava tissue culture and regeneration technology still need development and optimization to establish an efficient regeneration system without genotype dependence.

In cassava, protoplast regeneration technology could be applied to somatic hybridization and protoplast transformation. Somatic hybridization technology could break through the barriers of sexual hybridization and represents a direction for cassava breeding with protoplast regeneration technology as the basis. The disadvantages of cassava protoplast regeneration technology are high technical requirements and time consumption. We hope the chapter will be beneficial for the genetic improvement of cassava.

#### **Acknowledgements**

This work was supported by the Guangxi Scientific and Technological Development Subject, China (AB21196071), and the Basic Scientific Research Fund of Guangxi Academy of Agricultural Sciences, China (2021YT149).

#### **Abbreviations**


### **Author details**

Wen Feng\*, Hai-Tian Fu, Yan-Chun Luo and Jian-Qi Huang Guangxi Subtropical Crops Research Institute, Nanning, P.R. China

\*Address all correspondence to: wenfengw83@163.com

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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[36] Wen F, Xiao SX, Nie YM, Ma QX, Zhang P, Guo WW. Protoplasts culture isolated from friable embryogenic callus of cassava and plant regeneration. Scientia Agricultura Sinica. 2012;**45**:4050-4056. (In Chinese)

[37] Wen F, Su W, Zheng H, Yu B, Ma Z, Zhang P, et al. Plant regeneration via protoplast electrofusion in cassava. Journal of Integrative Agriculture. 2020;**19**(3):632-642

[38] Anthony P, Davey MR, Power JB, Lowe KC. An improved protocol for the culture of cassava leaf protoplasts. Plant Cell, Tissue and Organ Culture. 1995;**42**:229-302

[39] Mukami A, Juma BS, Mweu C, Ngugi M, Oduor R, Mbinda WM. Plant regeneration from leaf mesophyll derived protoplasts of cassava (*Manihot esculenta* Crantz). PLoS One. 2022;**17**(12):e0278717. DOI: 10.1371/ journal. pone.0278717

[40] Shahin EA, Shepard JF. Cassava mesophyll protoplasts: Isolation, proliferation and shoot formation. Plant Science Letters. 1980;**17**:459-465

[41] Sofiari E. Regeneration and Transformation of Cassava. Netherlands: Wageningen Agricultural University of Wageningen; 1996

#### **Chapter 5**

## Mutation Breeding: A Tool in Nutritional Improvement of Cassava

*Amanze Ngozi Joan and Abah Simon Peter*

#### **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.

**Keywords:** cassava breeding, induced mutation, karyotype, progenies, micronutrients, colchicine

#### **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**


*Mutation Breeding: A Tool in Nutritional Improvement of Cassava DOI:http://dx.doi.org/10.5772/intechopen.110362*

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].

iii.*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

i.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. Cassavabased 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.

ii.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 [33–36].

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

**Figure 1.** *Cassava root generated from the treated materials.*

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**).

(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**).

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:

$$\text{Mitotic Index} \left( M \right) = \frac{\text{Number of dividing cell}}{\text{Total number of cells counted}} \tag{1}$$

$$\% \text{Abberrant cells} = \frac{\text{Total absent cell}}{\text{Dividing cells}} \times 100 \tag{2}$$

<sup>−</sup> <sup>=</sup> <sup>×</sup> Mitotic index of control Mitotic index of treated Mitotic inhibition <sup>100</sup> Mitotic index of control (3)

**Figure 3.** *(a) Cassava plant with capsule or fruit (b) Seeds harvested from induced parents.*


**Table 1.** *Cytological effects of colchicine on cells of* Manihot esculenta*.*

*Mutation Breeding: A Tool in Nutritional Improvement of Cassava DOI:http://dx.doi.org/10.5772/intechopen.110362*

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**.

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 physicochemical 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,

**Figure 4.** *Cassava root generated from F1 seeds.*

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**).

#### **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.

*Mutation Breeding: A Tool in Nutritional Improvement of Cassava DOI:http://dx.doi.org/10.5772/intechopen.110362*

#### **Author details**

Amanze Ngozi Joan1 \* and Abah Simon Peter2

1 Department of Crop Production, National Root Crops Research Institute, Umudike, Abia State, Nigeria

2 Department of Biotechnology, National Root Crops Research Institute, Umudike, Abia State, Nigeria

\*Address all correspondence to: amanzengozi@gmail.com

© 2024 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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#### **Chapter 6**

## Improving Cassava Cultivation as an Industrial Raw Material on Acid Soil in Indonesia

*Bariot Hafif, Yulia Pujiharti, Alvi Yani, Noveria Sjafrina, Robet Asnawi, Nendyo Adhi Wibowo, Andri Frediansyah, Neneng Laela Nurida and Ai Dariah*

#### **Abstract**

Cassava is grown nowadays for use in food, feed, and industrial purposes. It is believed that the agro-industrial sector, which uses cassava as a raw material, has more advanced farming technology for improving cassava production. Lampung province in Sumatra Island, Indonesia, is one of the cassava production centers for industrial raw materials, with a planted area of 256,632 ha in 2018. The planting areas are acid soils of Ultisols, Inceptisols, and Oxisols with pH levels ranging from 4.5 to 5.0. Acidic soils have a complicated set of plant growth-limiting constraints. Essential nutrients for plant growth, such as N, P, and K, as well as other cations, are often low due to leaching, nutrients fixed by Fe/Al oxides of clay minerals, and low soil cation exchange capacity. In these acid soils, cassava production ranges from 8 to 15 t ha<sup>1</sup> for traditional farming, 20–24 t ha<sup>1</sup> for semi-developed farming to 25–35 t ha<sup>1</sup> for advanced farming. Meanwhile, with numerous technological advancements, cassava productivity can reach 40–50 t ha<sup>1</sup> . Aside from improving varieties, technological updates being pursued include increasing the accuracy of mineral fertilizer dosage, improving planting system technology, bio-fertilizer technology, and *in situ* organic C enrichment of acid soils.

**Keywords:** acid soil, cassava varieties, fertilizer, the plantation system, biofertilizer, Indonesia

#### **1. Introduction**

Cassava (*Manihot esculenta* Crantz) is the third staple food after rice and corn in Indonesia, having carbohydrate content 40% higher than rice and 25% higher than corn. Cassava has a starch content of about 24%, which gives it potential as a raw material for bioethanol [1]. It can be grown on marginal land and has a high tolerance to acid soil [2]. These advantages make cassava farming possible with no or low input to the soil.

Cassava cultivation exists throughout Indonesia, such as in large islands like Sumatra, Java, Kalimantan, Sulawesi, and Papua, and small islands like Bali,

Sumbawa, and Maluku, etc. Cassava is suitable to be developed in the wet tropical climate that dominates the territory of Indonesia and also grows quite well in the dryer part of Indonesia, like on the island of Nusa Tenggara [3]. Under high temperatures, high light intensity, and heavy rainfall in Indonesia, cassava for industrial raw materials requires a long maintenance time until harvest, about 9 to 10 months [4].

In Indonesia, planting cassava as a food ingredient is rarely expanded by farmers in large areas. Farmers grow cassava as a secondary crop, usually on narrow land (several hundred square meters), and in some areas, the cassava yield sometimes goes into food stocks as the mixing of rice during famine periods like at the end of the dry season or the beginning of the growing season. In places where cassava is grown as an industrial raw material, it is more common to grow it over a large area (> 1 ha). Lampung Province, in the southern part of Sumatra Island, Indonesia, is one of the production center areas. In Lampung, most farmers sell cassava yields to factories that process them for tapioca flour, feed, and bioethanol. At present, there are around 130 units of cassava processing factories in this area, with a demand of about 5 million tons of cassava per year [5]. In 2018, Lampung's total cassava farming land was around 295,548 ha. Other areas of Indonesia that develop cassava farming extensively are East Java Province, with approximately 157,899 ha, and Central Java Province, with 155,660 ha [6].

The type of soil for cassava farming in the three provinces of cassava production centers is quite different. In the provinces of Central Java and East Java, farmers are growing cassava on Alfisols, and Inceptisols, with slightly acidic to neutral soil pH (pH 5.0–6.5) [4, 7, 8], Meanwhile, in Lampung province, the soil is dominated by Ultisols, Oxisols, and Inceptisols, with soil pH ranging from 4.5 to 5.0 [9, 10].

#### **2. Acid soil and constraints for plant growth in Lampung**

Mulyani et al. [11] reported that acidic soils develop very widely in Indonesia, especially in wet climates, such as on the island of Sumatra. One of the areas on the island of Sumatra with extensive acidic dryland is the province of Lampung. The acidic dryland in this area reaches approximately 2.87 million ha and is dominated by the orders of Oxisols, Ultisols, and Inceptisols. Oxisols consist of the great group of Hapludox and Kandiudox, Ultisols consist of the great-groups Hapludult and Kanhapludult, while Inceptisols consists of the great-groups Dystrandept, Dystropept, and Eutropept [12]. The classification of acid soils in the Lampung area is following the soil classification as the soil classification of the USDA Taxonomy [13]. The profile of the prime acid soil orders found in the Lampung and their general properties are presented in **Tables 1**–**3**.

The low availability of phosphorus in tropical acid soils is due to P chelation by clay minerals, namely by amorphous and crystalline hydrous oxides of Fe and Al clay minerals, which is very conducive to happening in low pH soil. As shown in **Figure 1**, the forms of P chelated by clay minerals in acid soils are H2PO4 and HPO4 <sup>2</sup> [20]. The availability of K is low in acid soils, especially in those that have undergone advanced weathering, such as Ultisols and Oxisols. Rainfall and high temperatures speed up the release of K from rocks and other parent materials into the soil solution. Then, heavy rain keeps washing K out of the soil as low exchangeable K range of 0.03–0.11 meq 100 g<sup>1</sup> in the acid soils of Lampung in **Table 4**. In the study of cassava *Improving Cassava Cultivation as an Industrial Raw Material on Acid Soil in Indonesia DOI: http://dx.doi.org/10.5772/intechopen.109709*


**Table 1.** *The Oxisol profile and properties in Lampung Province, Sumatra Island, Indonesia [9].*


*Improving Cassava Cultivation as an Industrial Raw Material on Acid Soil in Indonesia DOI: http://dx.doi.org/10.5772/intechopen.109709*

**Table 3.** *The Inceptisol profile and properties in Lampung Province, Sumatra Island, Indonesia (private document of Hafif).*

#### **Figure 1.**

*Phosphorus (P) fixation in acidic tropical soil by amorphous and crystalline hydrous oxides of aluminum (AI) clay minerals. Source: Basak and Rakshit [20].*

cultivation on acid soil, the field experiments were conducted on four great groups of two soil orders, Ultisol and Oxisol. The great group of Oxisol was Hapludox, and the three great groups of Ultisol were Plinthudult, Kandiudult, and Kanhapludult. The great group of Oxisol, Hapludox, is the Oxisol has soil moisture regime udic (soil in a humid climate), with no other identifying horizons. The great groups Plinthudult are sub-order Udult, with plinthite (mixture of clay with other minerals riching iron and humus-poor) found in 150 cm horizon from the soil surface. The Kandiudult is a suborder Udult of the Ultisols, having a kandic horizon, and the Kanhapudult is the other sub-order Udult having a kandic horizon [13]. The general physicochemical characteristics of the acid soils used for cassava study in the field are presented in **Table 4**.

#### **3. Plantation systems, fertilizer, and cassava production**

Generally, there are two systems in cassava plantations in Lampung Province, namely monoculture and intercropping. The study by Manihuruk et al. [21] found the factors influencing the farmers in choosing a plantation system were land area, the distance of the farming area from the processing factory, and the source of income. Farmers having small areas (< 1 ha) prefer the intercropping system because it generates more revenue than the monoculture system. Farmers with farming areas near processing plants prefer the monoculture system because the cost of production transport to the factory is relatively low. Meanwhile, farmers with other sources of income choose an intercropping system because the earnings from the monoculture cassava are lower than those from intercropping, especially if they have small areas (≤ 0.5 ha).

The planting distance of cassava in a monoculture system widely used by farmers is 1 m x 1 m or 1 m x 0.8 m. While in the intercropping system, the distance of cassava between double rows was 160 cm, and in double rows, it was 80 cm. In an intercropping system, the first step is to grow seasonal crops such as soybeans, peanuts, or corn on land. Then the cassava planting is carried out after the seasonal crops are 15–30 days old.


*Improving Cassava Cultivation as an Industrial Raw Material on Acid Soil in Indonesia DOI: http://dx.doi.org/10.5772/intechopen.109709*

#### **Table 4.**

*The physicochemical characteristics of some great-group acid soils at a soil depth of 0–20 cm in Lampung.*

Based on cassava farming patterns such as planting system, inputs to the land, seedling kind, and land area, Hafif [9] reported that there were three types of cassava farming in Lampung; traditional, semi-developed, and advanced. Farmers in the traditional type typically work on 1 ha or less of cassava land, using a monoculture or intercropping system, cultivating the land with family labor and livestock, using random seeds (derived from previously planted cassava), and using very little mineral fertilizer. The input to the land is only manure at a modest rate of around 1–1.5 t ha<sup>1</sup> , and sometimes a little urea is added (100 kg ha<sup>1</sup> ). Another habit is often harvesting too-young cassava due to pressing economic needs. When converted to hectares areas, traditional farmer's land produced cassava of around 8–15 t ha<sup>1</sup> (**Table 5**). Most of them consider cassava products as additional/side income.

Farmers in the semi-developed farming category prefer a monoculture system and have used complete chemical and organic fertilizers. They mostly used compound fertilizers (NPK 15:15:15%) with doses varying between 50 and 200 kg ha<sup>1</sup> and supported by manure ranging from 1 to 4 t ha<sup>1</sup> in a cassava planting area of 0.5–2 ha.


#### **Table 5.**

*Comparison of traditional, semi-developed, and advanced cassava farming characteristics in Lampung Province, Indonesia.*

For soil cultivation, the farmers used livestock or tractor services (hand tractor) and still relied on random seeds. Cassava production from semi-developed farming for monoculture patterns ranges from 20 to 24 t ha<sup>1</sup> (**Table 5**).

In advanced cassava farming, the farmers have considered the efficiency and effectiveness of a farm. In cultivating the land, farmers have fully used the services of a tractor to cultivate the land in a shorter amount of time, about 1–2 hours per ha or 7 ha per day, whereas it would take five days per ha to do the same work with livestock. Most cassava seeds are derived from outside the land, based on recommendations from extension workers or factories. The area of cassava planting ranges from 2 to 5 ha. Advanced farmers preferred the monoculture system and used fully compound fertilizers (NPK 15:15:15%), sometimes adding urea and SP-36. The amount of compound fertilizer applied is high, between 300 and 500 kg ha<sup>1</sup> , and manure as much as 2.5–4 t ha<sup>1</sup> . Advanced farmers harvest cassava until it is 9–10 months old, with cassava production ranging from 25 to 35 t ha<sup>1</sup> (**Table 5**).

#### **4. Cassava varieties as industrial raw matter**

Cassava of UJ-5, UJ-3, Malang 4, Malang 6, Ardira 2, Ardira 4, and Litbang UK 2 were among the new high-yielding varieties introduced by the Indonesian Ministry of Agriculture in Lampung since 2000 [4]. One of the varieties that are becoming a favorite and being developed by many farmers in Lampung is UJ-5. The Indonesian Legumes and Tuber Crops Research Institute was the inventor of the superior cassava varieties, especially for industrial raw materials.

The UJ-5 variety could meet the requirements as a fuel-grade ethanol (FGE) raw material, due to having the following properties; 1) high starch content, 2) high yield potential, 3) resistance to biotic and abiotic stresses, and 4) flexibility in farming and harvesting time [22]. According to the Indonesian Legumes and Tuber Research Institute, the UJ-5 could produce tubers in the range of 25–38 t ha<sup>1</sup> , had starch content of 20–30% fresh weight (FW), and HCN content >100 ppm (slightly bitter taste) and harvest age of 9–10 months. UJ-5 was relatively resistant to cassava

*Improving Cassava Cultivation as an Industrial Raw Material on Acid Soil in Indonesia DOI: http://dx.doi.org/10.5772/intechopen.109709*


#### **Table 6.**

*Comparison of some new high-yielding varieties and the properties of cassava as industrial raw materials and bioethanol in Lampung, Indonesia.*

bacterial blight (CBB) and had high dry matter (% DM), starch content from dry matter (% DM), sugar content (% FW), and amylose (%) of which was 43.9, 27.5, 39.1, and 22.4, respectively, and the conversion of fresh tubers to bioethanol was 4.5 kg liter<sup>1</sup> (**Table 4**) [22, 23]. Other beneficial properties of the UJ-5 are; 1) leaves do not fall quickly, 2) it can grow on low and high pH soils, 3) it can grow in high populations, and 4) they can develop in an intercropping system [24]. **Table 6** shows in more detail the benefits of the UJ-5 compared to several other types of industrial raw materials and bioethanol.

#### **5. Cassava yield quality**

The low available nutrient content in acid soils, especially P and K, was the cause of low cassava quality due to low starch content and high cyanogenic glucosides [25]. Another cause of low starch content was harvesting cassava before maturity (at 6–7 months old), which was common among farmers in traditional and semiadvanced farming. On average, the starch content of the cassava grown on the acidic soil of Lampung is around 18–22% (manufacturer's information), although the potential starch content of the UJ-5 variety can reach 30%. However, cassava yield factories may accept these starch levels and limit cassava purchases to only those with a starch content is at least 18% [9].

The low quality of cassava causes the price of this commodity to fluctuate. From 2011 to 2016, the average cassava price decreased by around 2.38% per year [6]. The decline in prices caused some cassava farmers to switch farming to other commodities resulting in a reduction in the cassava planting area of 10.8% per year in Lampung [5]. However, with improvements in cultivation technology and growing superior varieties, cassava productivity was indicated to increase. In 2018, the cassava productivity of Lampung Province was about 26.04 t ha<sup>1</sup> , which was better than the average

national productivity of 24.39 t ha<sup>1</sup> , and the selling price of cassava at the farmer level has continued to improve [6].

#### **6. Technology improvement of cassava cultivation on acid soil**

#### **6.1 Mineral fertilizers**

The study by Wargiono [26] on the acid soil in Lampung found that cassava in an intercropping system with rainfed rice gave the best result with the application of 90 kg N, 50 kg P2O5, and 90 kg K2O per ha. The results of Ernawati's research [27] on Kanhapludult acid soil found the application of a mixture of urea, SP36, and KCl fertilizer in a ratio of 2:1:1 or the equivalent of a mix of 90 kg N: 36 kg P2O5: 60 kg K2O with an application dose starting at 40 g plant<sup>1</sup> , 80 g plant<sup>1</sup> , 120 g plant<sup>1</sup> , and 160 g plant<sup>1</sup> or the equivalent of 400 kg ha<sup>1</sup> , 800 kg ha<sup>1</sup> , 1200 kg ha<sup>1</sup> and 1600 kg ha<sup>1</sup> if the cassava population was 10,000 ha<sup>1</sup> , the yield of cassava was not significantly different, namely 54 kg plant<sup>1</sup> or 54 t ha<sup>1</sup> . That means the lowest dose of mixed fertilizers, 400 kg ha<sup>1</sup> , was sufficient for cassava planted on a ha of acid soil.

KCl application of as much as 300 kg ha<sup>1</sup> on acid soil in Lampung increased the weight of cassava tubers by an average of 1.98 kg plant<sup>1</sup> compared to an average of 1.45 kg plant<sup>1</sup> by application of 200 kg KCl ha<sup>1</sup> [28]. Meanwhile, the study of Hafif [9] found that the application of straw compost (2 t ha<sup>1</sup> ), each enriched with 50 kg KCl, 100 kg KCl and 200 kg KCl, to acid soil in Lampung, significantly increased the weight of tubers of cassava from 7.35 kg plant<sup>1</sup> (without enrichment) to 7.97, 8.26 and 8.42 kg plant<sup>1</sup> (**Table 4**), respectively, and the same treatments increased tuber starch content from 30.1% to 30.9%, 32.3%, and 33% (FW), and decreased total cyanogen content by 13.8%, 26.4%, and 28% from 246 ppm (**Table 7**).

To increase cassava production on acid soils, it is necessary to solve some problems such as Al toxicity [29], low P and K availability [2], and aggregate instability due to low soil organic matter content [30]. Although cassava is a tolerant plant for marginal lands, without fertilizer application, the yield of cassava was far from the target. Even soil fertility under cassava plants will rapidly decline due to the high nutrient uptake


*The column means followed by the unequal letter are significantly different at an LSD of 0.05. Source: Hafif [9].*

#### **Table 7.**

*The application effect of straw compost (2 t ha<sup>1</sup> ) enriched by 50, 100 dan 200 kg KCl on stem diameter, tuber weight, tuber number, starch, and the total cyanogen of cassava in acid soil in Lampung.*

of cassava [31]. Howeler [32] reported the plantation of cassava for eight years consecutively without fertilization, which caused cassava production to decrease from 22 tons ha<sup>1</sup> to 13 tons ha<sup>1</sup> . Therefore, to get a high yield of cassava on marginal land, one should apply sufficient NPK and organic matter [25, 30]. Among the macronutrients, the K mineral is the one that plays a principal role in increasing the quantity and quality of cassava in acid soils [4, 24].

#### **6.2 Improvement of the plantation system**

Intercropping and monoculture systems are two options for cropping systems developed and used by Lampung cassava farmers. Research by Asnawi and Arief [33] found that cassava productivity could increase if the monoculture cropping system was changed to a monoculture with a double-row system. The monoculture system with a double row was different from the monoculture system of farmers, especially in terms of spacing, population per hectare, and fertilization rate. In the monoculture system of farmers, the spacing varies, namely 60 x 70 cm, 70 x 80 cm, or 80 x 80 cm. The total population of cassava in this monoculture system ranges from 15,000 to 20,000 plants per ha<sup>1</sup> . Fertilization is usually only 75–100 kg of urea plus a little SP-36 (50 kg ha<sup>1</sup> ) and manure of about 1 t ha<sup>1</sup> .

The monoculture system with double rows changed the spacing to 160 x 80 x 80 cm so that the cassava plant population per hectare is only around 11,200 plants. This system recommended a fertilizer dose of 100 kg Urea +150 kg NPK + 100 kg KCl and manure to be 5 t ha<sup>1</sup> . Cassava yields can reach 50–60 t ha<sup>1</sup> with this system [33]. In addition, the system with double rows can join the intercropping system by planting annual crops in the 160 cm space between the double rows. This method is even considered more profitable. The first step in the intercropping system with double rows was to grow seasonal crops such as soybeans, corn, peanuts, and green beans on the space between double rows (160 cm), then plant cassava when the crop was two weeks to a month old. The performance of the intercropping system with double rows and corn as an intercropping crop is shown in **Figure 2**.

#### **Figure 2.**

*Design of the intercropping double-rows system (a) and performance of the intercropping double-rows system in the field (B). Source: Robet and Arief [33].*

#### **6.3 Bio-fertilizer of mycorrhizae (arbuscular mycorrhiza)**

According to Howeler [34], another ingredient that also had the potential to improve the growth, yield, and yield quality of cassava is *Arbuscular mycorrhizae* (AM). Cassava can grow well on acid soils with low P contents because it has a very efficient symbiosis with AM, which occurs naturally. Cassava is most dependent on AM. At low concentrations of P in acidic soils, the growth and branching of AM hyphae will increase. The AM performed a symbiosis with the cassava roots, as illustrated in **Figure 3** [9].

One way of enriching soil mycorrhiza is through the application of biofertilizers. When AM and plant roots form a symbiotic mutualism, the plant roots will supply exudate to AM, and vice versa, AM will help deliver nutrients and water to the roots. AM hyphae will extend the root system of plants up to 100 times and help plants absorb more nutrients and water, especially in soil with less available nutrients like P and microelements like Zn, Mo, and Cu. AM also increases plant tolerance to drought, high temperatures, infections from fungal diseases, and even high soil acidity. Good plant growth with the help of AM is easier to see in the crops planted in acid soils with a high level of weathering, low base cations and P, and high Al content [35].

According to the findings of Hafif [9], the use of AM bio-fertilizer combined with zeolite as a carrier was able to enhance cassava yield from 7.1 kg plant<sup>1</sup> to 8.8 kg plant<sup>1</sup> , and the amount of starch produced increased from 29.5% to 32.1% (FW) or 75.1% to 76.7% (DW) (**Table 8**).

#### **6.4 In situ enrichment of soil organic C with root exudates of Brachiaria**

The research on degraded soils in Madagascar showed that Brachiaria grass, as the source of nutritious feed for livestock in the tropic, planted as an intercrop between cassava, had a good effect on cassava production, namely being able to increase cassava yield from 4 to 13 t ha<sup>1</sup> to 11–30 t ha<sup>1</sup> or an average increase of 240% [36]. The beneficial effects of Brachiaria root exudates include their ability to improve soil aggregates, nutrient cycles, and organic carbon levels [36–38].

A study conducted by Hafif [9] demonstrated that the roots of signal grass (*Brachiaria decumbens*) released low molecular weight organic acids into the

**Figure 3.**

*Symbiosis mutualism between a*rbuscular mycorrhiza *(AM) and cassava roots. Source: Hafif [9].*

*Improving Cassava Cultivation as an Industrial Raw Material on Acid Soil in Indonesia DOI: http://dx.doi.org/10.5772/intechopen.109709*


*The column means followed by the unequal letter are significantly different at an LSD of 0.05. Notes: FW = fresh weight, M0 = no mycorrhiza, M1 = with mycorrhiza. Source: Hafif [9].*

**Table 8.** *The effect of AM bio-fertilizer on quantity and quality of cassava yield on acid soils.*

rhizosphere. These acids included citric, malic, and oxalic. When compared to results obtained without the presence of root exudate from Brachiaria grass, the organic acids secreted by Brachiaria roots were able to chelate aluminum with a significantly higher organic aluminum content. Research on acid soil in Lampung found the root exudates of Brachiaria could reduce the amount of exchangeable aluminum by up to 33%. A decrease in exchangeable Al by root exudates will increase P mobilization in acid soil by inhibiting P fixation by the Al oxide-hydroxide adsorption complex [39]. Planting Brachiaria grass as an intercrop between cassava on acid soils in Lampung (**Figure 4**)

#### **Figure 4.**

*Brachiaria grass performance as an intercrop between cassava in field lab (A) and farmer's field (B) on acid soil in Lampung, Indonesia. Source: Hafif [9].*


#### **Table 9.**

*The effect of Brachiaria root exudates on the yield quantity and quality of acid soil Lampung Indonesia.*

increased yield and cassava starch. Brachiaria grass increased cassava tuber weight from 7.1 kg plant<sup>1</sup> to 8.2 kg plant<sup>1</sup> and starch content from 29.2% to 31.0% (FW) and reduced total cyanogen from 214 ppm to 195 ppm (**Table 9**).

#### **7. Conclusion**

Cassava in a tropical climate like Indonesia is one of the principal food sources, especially for marginalized people in rural areas. However, in certain areas, such as Lampung Province, cassava, which initially received little attention from farmers, has instead developed into one of the leading commodities. This positive development started in 2005, along with the rapid development of cassava processing factories in this region [5].

Farmers in Lampung are not discouraged from growing cassava because of the acidic soil. Cassava planting on acid soil with little external input could produce around 8–15 t ha<sup>1</sup> . However, if the next planting still has low input, then cassava production will decrease because cassava absorbs soil nutrients very highly. Based on that experience and supported by intensive counseling from the factory officer and agricultural extension from the local and central governments, the way farmers cultivate cassava is improving. In semi-developed and advanced cassava farming, cassava can produce 20 to 35 t ha<sup>1</sup> .

In Lampung, the average productivity of cassava is still around 17.53 t ha<sup>1</sup> [5]. This productivity is far from optimal because the experimental results can reach 40– 50 t ha<sup>1</sup> . The productivity of cassava on acid soils can increase if farmers improve or adopt cultivation technologies such as planting superior varieties, increasing the doses of mineral fertilizers and organic fertilizers, and improving cropping systems. In the future, it is necessary to encourage the use of biological fertilizers of mycorrhiza, organic C enrichment, and increased mobilization of soil nutrients in situ by planting intercrops that produce root exudates like Brachiaria among cassava plants.

On the other hand, the slow absorption of cassava cultivation technology in Lampung was due to several factors. One of the most influential is the unstable and relatively low selling price of cassava at the farmer level. Low prices make it difficult for farmers to survive in cassava farming. As a result, from 2011 to 2016, the cassava planting areas in Lampung decreased by 10.8% per year because farmers switched their farming to other commodities. However, since 2018, the price of cassava has continued to improve, and this is the hope that farmers will get excited again about growing cassava in Lampung [6].

### **Author details**

Bariot Hafif<sup>1</sup> \*, Yulia Pujiharti<sup>2</sup> , Alvi Yani<sup>3</sup> , Noveria Sjafrina<sup>3</sup> , Robet Asnawi<sup>4</sup> , Nendyo Adhi Wibowo<sup>5</sup> , Andri Frediansyah<sup>5</sup> , Neneng Laela Nurida<sup>6</sup> and Ai Dariah<sup>1</sup>

1 Research Center for Horticulture and Plantation, National Research and Innovation Agency (BRIN), Cibinong Bogor, Indonesia

2 Research Center for Macroeconomics, and Finance, National Research and Innovation Agency (BRIN), Jakarta, Indonesia

3 Research Center for Agroindustry, National Research and Innovation Agency (BRIN), Indonesia

4 Research Center for Behavioral and Circular Economic, National Research and Innovation Agency (BRIN), Jakarta, Indonesia

5 Research Center for Food Technology and Processing (PRTPP), National Research and Innovation Agency (BRIN), DI. Yogyakarta, Indonesia

6 Research Center for Food Crops, National Research and Innovation Agency (BRIN), Cibinong Bogor, Indonesia

\*Address all correspondence to: bari002@brin.go.id

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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*Improving Cassava Cultivation as an Industrial Raw Material on Acid Soil in Indonesia DOI: http://dx.doi.org/10.5772/intechopen.109709*

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Section 3
