OPTIMISING SOMATIC EMBRYOGENESIS OF C. ARABICA HYBRIDS IN KENYA USING TEMPORARY IMMERSION SYSTEM
CHAPTER ONE
- Introduction
The agricultural sector is a major contributor to Kenya’s economic growth accounting for 25.4% (indirectly) and 27% (directly) of the country’s Gross Domestic Product (ICO, 2019). The sector concurrently provides employment to 80% of the rural workforce and is responsible for 65% of the total exports earnings (ICO, 2019). The coffee sub-sector is among the contributors to the growth of Kenya’s GDP through foreign exchange, income generation, employment opportunities and food security. The sub-sector accounts for 5.5% of the country’s total exports, with a share of 0.22% of the GDP equivalent to Kshs. 23 billion per annum (ICO, 2019). The production capacity has consequently decreased from 120,000 metric tons in the 1980s to 40 000 metric tons in 2018 (Monroy et al.; 2013, ICO 2019). The decline in coffee production is the result of decreased competitive global prices, poor management of the sub-sector, inadequate value addition policies and poor production practices including inadequate control of coffee diseases in the country (Monroy et al., 2013). Despite the decrease in production, coffee remains a major cash crop in the country, providing employment to about 800 000 farmers, small-holder farmers (ICO, 2019).
In a bid to revamp the coffee subs-sector, the National Government put in place a coffee revitalisation program under the National Task Force on the Coffee sub-sector. The program seeks to revive the sub-sector through the support of Vision 2030 platform under the agricultural pillar. Under the program, the emphasis has been put on the increase in acreage of coffee alongside improved management strategies for small-scale farmers. Primarily, the approach targets the increase in production through the adoption of elite coffee hybrid varieties that are disease resistant and high yielding crop varieties Ruiru 11 (Agwanda et al., 1997). The sector is yet to meet the threshold of the objectives with production recorded between 40,000 and 50,000 metric tons since 2013 (ICO, 2019). Part of the slow growth in coffee hectarage, productivity and overall production is due to inadequate planting materials. The underlying factor is that production of planting materials of the superior varieties is inadequate to meet the growing demand for the elite coffee varieties.
For a long time, access to planting materials has largely depended on conventional propagation techniques which rely on hand pollination and vegetative propagation (VP) to produce seed and seedlings respectively. With production primarily undertaken at the Coffee Research Institute (Ruiru, Kenya), the Institute is unable to meet the current demand due to a number of reasons. The seed and seedling production methods used are inept as they are characterised by limitations such as dependency on weather factors for growth, risks of spreading pests and diseases through VP, labour intensiveness and high cost of production. Conventional methods are complex, thus, prove insufficient on return on investment. Adoption of improved technology through tissue culture promises potential in increased production capacity through direct somatic embryogenesis. The method provides the best avenue for the propagation of Ruiru 11, which has otherwise proved difficult to propagate via traditional methods due to the aforementioned difficulties, including the risk of loss of genetic fidelity. The ability to meet the demand for seedlings of the hybrid is still insufficient. The Institute is only able to propagate a maximum of 0.1 million seedlings per annum against a demand of one million. The proposed adoption of automated technology, Bioreactor Systems, the Temporary Immersion Systems, with high throughput presents a viable option to improve the production capacity of the elite variety.
- Background of Study
Bioreactor, an automated system, is a powerful technology that improves production capacities in coffee tissue culture laboratories. A bioreactor provides a biological culture environment that sustains the growth and development of tissues and cells. The system has been shown to significantly improve the production of coffee plants in tissue culture (Watt 2012; Krishnan, 2012; Georgiev et al., 2014; Etienne et al., 2018). The improved production is associated with increased nutrient intake via liquid media and reduced soma-clonal variation rates (Etienne et al., 2018).
Various bioreactor technology exists, based on the objective of use, desired products and mode of operation. In coffee tissue culture, Temporary Immersion Systems (TIS) with specificity to RITA® have gained prominence in relation to somatic embryogenesis (Ahloowalia et al., 2004; Watt, 2012; Etienne et al., 2018). In C. arabica, for example, Sondhal (1992) reported production of 45,000 embryos using 5-L stirred bioreactors, Etienne and Berthouly (2002), on the other hand, produced 15,000-50,000 in 1-L RITA® and de Feria et al. (2003) produced 70,000 in a 2-L bioreactor, while Ducos et al. (2007) report on increased embryo production ranging 200,000-400,000 in Robusta in mechanically agitated bioreactors.
Success in utilisation of Temporary Immersion Systems is determined by a number of factors including species of plant, culture parameters and genotype of the plant. Scientists at the Coffee Research Institute (CRI), Kenya, adopted the technology with urgency with the need to meet the increasing demand for coffee planting materials. However, realisation of the potential of TIS proved difficult given that the available protocols of solid-based media direct somatic embryogenesis were not fully adaptable to TIS techniques when integrating the elite coffee hybrids. To fully realise the potential of the TIS, optimisation of TIS protocols in somatic embryogenesis specific to F1 Hybrid, Ruiru 11 is required to improve efficiency in the adoption of the technology in Kenya. The specific factors on culture parameters, namely plant growth regulator types and concentration levels and cell densities, are necessary for optimum embryo production. The current research was designed with the above in mind.
- Problem Statement
Since 1950, coffee has maintained a central role in the economy of Kenya, despite the volatile international prices and declining production. It is among the leading foreign exchange earner for the Kenyan economy and is ranked third after tea and horticulture in the agricultural sector. The Kenya Vision 2030 recognises the importance of the coffee sector as part of the broader national initiative of employment creation, food and nutrition security, poverty reduction, industrial transformation and foreign exchange earnings. Key to the growth of the sector is increased sustainable production of coffee, which can be achieved by adopting high performing disease-resistant hybrid varieties. In line with this, several interventions by county government units, in addition to promotion and marketing efforts of the Coffee Directorate, is the dissemination of elite planting material which has led to an increase in the production area at 30% in the year 2015/2016. The increased impetus to enhance coffee production in the country catalysed the increased demand for elite hybrid planting material which has subsequently outstripped the supply. This is largely due to the use of inefficient propagation methods such as manual pollination and vegetative propagation. Overcoming the perennial shortage of planting materials of the improved varieties require the adoption of propagation technologies with high planting material throughput and are both technically and economically efficient. Tissue culture technique offers these traits and is currently being deployed for mass propagation of Ruiru 11 seedlings at CRI. However, the Institute still relies on direct somatic embryogenesis in solid media whose efficiency is limited to 0.1 million seedlings annually against the annual demand of one million seedlings. The modern automated Temporary Immersion Systems is a powerful alternative to the direct embryogenesis system and combines high prolificacy with safety against somaclonal variation and hence, is a superior tool in optimising the techniques of tissue culture for the mass production of Arabica coffee hybrid planting materials. It possesses inherent cost-effectiveness and is amenable to varying seedling production levels with minimum adjustments in labour, laboratory space and associated logistical factors. The advantages associated with TIS have, however, not been realised at CRI due to a number of protocol-based limitations which needs to be refined in order to achieve the desired level of production efficiencies. The current project proposal is informed by this need and is aimed at improving the efficiency of TIS using the RITA bioreactors.
- Justification
Traditional tissue culture is characterised by several constraints that limit the successful mass propagation of hybrid coffee varieties. Modern tissue culture methods offer improved alternatives and efficient techniques in mass propagation of planting material. Automation of micropropagation is a recent technique, that is more precise and efficient. However, the gradual adoption of this technology is due to limited or lack of precise protocols. Hence, the specificity of the Temporary Immersion Systems necessitates optimisation of the control parameters and protocol to successfully mass micro-propagate planting material. The study aimed at determining optimal plant growth regulator levels for induction of calli from leaf cultures. Combinations of plant growth hormones were used based on differential levels that include benzyle-amino purine (BAP), IBA, Kinetin and 2,4-Dichlorophenoxyacetic acid. Standard referencing involved the use of the plant growth regulators singly. Also, evaluate optimal cell density in the multiplication of somatic embryos in liquid bioreactors to improve on efficiency in rates of multiplication.
- OBJECTIVES
- Main Objective
The main objective of the project was to determine optimised somatic embryogenesis in Temporary Immersion based Systems for mass production of C. arabica hybrids in Kenya.
- Specific Objectives
Two specific objectives were pursued by the study as follows.
- To investigate the effects of different auxin and cytokinin combinations on calli induction in arabica hybrid Ruiru II.
- To determine optimal cell densities necessary for mass production of somatic embryos of Ruiru 11 coffee hybrids in temporary immersion system using the RITA bioreactors.
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