Keywords

Clones; Somatic embryogenesis; Cocoa; Mass propagation; Farmers

Introduction

The cacao tree is cultivated in the humid tropics and is a major source of income for small farmers as well as the main cash crop of several West African countries [1]. Cocoa is an essential ingredient for chocolate. Antioxidant and antiproliferative properties of the leaf, bark, husk, unfermented and fermented shell, pith, root, and Cherelle methanolic extracts of Theobroma cacao have been investigated [2]. Cocoa beans also contain many vitamins and minerals as well as healthy doses of potassium, copper, and iron which support cardiovascular health and transports of oxygen through the body respectively [3].

Several factors affect cocoa production such as pest and disease infestation, competition with other commodities and increasing agricultural input prices. This results in low yields per hectare (300 kg/ha/year to 450 kg/ha/year) with a negative impact on farmers’ revenue [4].

Indeed, the Cocoa mirids (Heteroptera: Miridae), also known as capsids, are the most economically important pests of cocoa in West and Central African countries. Yield loss may be as high as 75% in cocoa farms attacked by capsids which are left unattended for a period of over three years [5].

In West African producing countries, the two most disabling diseases of cocoa farming are black pods and swollen shoot. They are rife in and cause great anxiety to countries that cocoa represents an important part of their export products, i.e. 40% for Côte d’Ivoire.

Black pod disease is an important fungal infection in cocoa (Theobroma cacao L.) which causes high production losses [6]. In Cameroon, these losses reached 80% of cocoa production depending on ecological zones [7]. Several publications have reported on some researches done to understand and reduce black pod impact on farmers’ revenue [8-12].

Cocoa Swollen Shoot Disease (CSSD) is caused by the Cocoa Swollen Shoot Virus (CSSV), belonging to the genus Badnavirus [13]. CSSD only occurs in West Africa and has been reported in Cote d’Ivoire, Ghana, Nigeria, Sierra Leone, and Togo. CSSV is genetically diverse; different isolates/strains can cause different symptoms, including transient red veins and mottling in young leaves, different shades of chlorosis in mature leaves and pods, root atrophy and stunting, as well as root and stem swellings [14]. The only treatment for CSSVD-infected cocoa is to cut out visibly infected trees (eradication). Thereby, symptomatic trees are removed together with all adjacent, apparently healthy trees. When large outbreaks occur, extensive areas are cleared and replanted [15].

In addition to all previously described damages on the cocoa sector, cocoa cropping is also facing other different challenges such as climate change, deforestation, aging of farmers and plantations [16].

The propagation of cocoa trees with high yield and disease resistance is envisioned for the renewal of the old cocoa orchard. It is mainly through seeds, in vitro cultures such as somatic embryogenesis techniques or vegetative propagation and by micropropagation [17-19].

However, it showed that 70% of cultivated trees are from unselected material propagated by seeds [20]. Because of the high heterozygous nature of seedlings in a given field, only 2%-3% of the trees in a population of high-yielding families account for 60% of the yield [21]. The planting material, which has often undergone little improvement, is generally derived from seeds. Also, in West African producing countries, the dissemination of these plantlets obtained by seeds is limited compared to the needs [22].

A clonal propagation method such as somatic embryogenesis is one way of increasing yields, homogenizing cocoa production, increase smallholder farmers’ incomes, by propagating elite planting material. Since the production of the first cocoa somatic embryo [23], it has been possible to produce somatic embryos and plantlets from many genotypes [24-28].

The process of somatic embryogenesis involves four main steps:

• Primary Somatic Embryogenesis (PSE) where flower buds are cultured to obtain embryogenesis calli, which further develop into a primary somatic embryo

• Secondary Somatic Embryogenesis (SSE) in which cotyledons from the primary somatic embryo are re-cultured to obtain secondary somatic embryo

• Conversion, where mature embryos are converted in plantlets

• Acclimatization, where plantlets are acclimated into greenhouse or field conditions

Based on these techniques, plantlets were produced, and field trials were established to evaluate different criteria for this new planting materials acceptance in Côte d ’ Ivoire. Indeed, the introduction of plant material produced by new techniques raises several questions among researchers, propagators and especially from farmers.

The questions were turned around two major aspects 1) Does cocoa plantlets obtained from somatic embryogenesis technique affect the productivity of cocoa trees compared to plants provided from seeds which are usually distributed in each country 2) What are the features of planting material provided by a new propagation technology like somatic embryogenesis method.

Indeed, the level of management of cocoa farms improved the yield more than the genetic origin of the planting material [10]. These authors reported that out of 690 farmers interviewed, 87% were satisfied with the traditional varieties (TV), whereas only 39% of the producers were satisfied with the hybrid varieties (HV). An analysis of preferences towards the criteria of selection within each varietal type revealed that farmers considered yield as the criterion to be improved in TV, whereas the resistance to Phytophthora pod rot remains the most challenging within the HV.

We believe that the plants produced through somatic embryogenesis would positively respond to these concerns, but we need to evaluate before mass propagation. For that, a “juvenile” vigor and productivity parameters of trees were collected over five years to answer farmer’s needs in terms of plant quality acceptance.

To the best of our knowledge, this is the first study to evaluate agronomic performances combined to black pods disease evaluation of cocoa trees produced by somatic embryogenesis technique in Côte d’Ivoire and in West Africa countries. Different points of plantlet acceptance were screened to increase the probability distributing of SE trees to farmers for the first time in Cote d’Ivoire.

Therefore, the objectives of the present study were to:

• Show that the somatic embryogenesis technique has no negative effect on planting material genetic and agronomic traits, under local conditions

• Highlight the possibility to use somatic embryogenesis technique on cocoa mass propagation for old orchard renewal

Materials and Methods

Cocoa varieties evaluated

Five (5) cocoa clones provided from International Cocoa Germplasm were used for this study. These varieties were multiplied using somatic embryogenesis method, planted in fields and compared to a hybrid seed called “ Hybrid Mercedes”, usually produced and distributed to farmers in Côte d’Ivoire. The hybrid plantlets were produced by manual pollination processes by Dr. Tahi (CNRA, unpublished data). The embryogenic potential of the clonal material was also determined according to the technique described by Lopez- Baez et al. [18] and Florin et al. [27]. The agronomic and technical characteristics of the varieties used in this study were evaluated since 2000 in fields and data provided by the CNRA team are consigned in Table 1.

Table 1: Agronomic performances and technical characteristics of selected clones propagated by somatic embryogenesis (SE).

Plots design and location

Two plots with the same experimental design were implemented at two different locations: one in Nestlé Experimental Station at Zambakro and another one in CNRA Experimental Station at Divo. These two Ivorian locations are about 200 km apart. Divo town is a cocoa production area with rainfall of more than 1500 mm average per year in the last 5 years, whereas Zambakro is a region at the edge of the wet and warm zones where rainfall rarely exceeds 1100 mm average per year.

For this study, the plot designed is as follows: each clone is represented in the plot by 40 trees spread over two contiguous lines of four trees repeated 5 times. Five families of hybrids (Mercedes) considered as one variety were used as controls.

Measurement of different parameters

Three types of parameters were collected to estimate the field behavior of plantlets. The criterion was a) the vigor of plantlets, b) plants ability to produce cocoa pods: plots establishment and productivity, and c) pods resistance to black pods disease.

The vigor of plantlet

The vigor was determined by the growth of the tree. It is a measure of the difference in the heights of the crown over a period of six (6) months and by the magnification of the diameter of the trunk, over the same period.

Productivity

Planting material productivity was a main criterion of trees for farmers because it ’ s related directly to the farm profitability. It ’ s was estimated using the number of pods harvested during two years for each clone according to the following formula:

Pods morphological parameters were evaluated by average pod volume (Volcab), average pod cortex (Ecortex), and average bean per pod (NFN). The volume of a pod was determined by the following formula:

Behavior of trees produced by somatic embryogenesis against black pod disease

Evaluation of the impact of black pods disease on cocoa trees was measured by the rate of rotten pods, which is determined by the ratio of the number of pods rotten over the period to the total number of pods (healthy, gnawed and rotten) produced by the tree over the same period, according the formula described by Nyadanu et al. [29]. Indeed, the rate of rotten pods indicates the level of sensibility of planting material against the most widespread cocoa disease in West Africa producing countries.

Methods of data analysis

The comparison of averages between the different forms of planting material: Hybrid Mercedes (M) and somatic embryogenesis plants (CI), we're done with SAS SOFTWARE. The coefficient of variation (CV) was calculated to estimate the measure of homogeneity or dispersion between the plants. Excel files were also used for standard deviation calculation.

Results

Plots establishment

The ability of plot establishment within in vitro plantlets was determined by the rate of mortality after one year of planting. Results obtained in a different location are presented in Table 2. The mortality rate was around 9% for both planting locations (Divo and Zambakro). This is below the 12.5% standard critical level established for a cocoa plot (CNRA, unpublished data). The mortality rate of planting material derived from somatic embryogenesis (9.8%) is not significantly different from that of hybrid plants (7.5%) as shown in Table 2.

Table 2: Mortality rate (%) of different variety on the two locations.

Vigor of plantlets

The height of the crown (HC) and the diameter of the trunk (Diam) were measured as described in Materiel and Method paragraph § 2.3.1.

Height of crown (HC)

The measurements of HC from different varieties at both sites are presented in Table 3. These results highlighted that the average HC of planting materials from Divo is higher than those planted at the Zambakro site to 46 ± cm. The rank of the planting material showed that clones CI 02 and CI 03 were stables on each location (Divo and Zambakro). Indeed, it appears that CI 02 was at the top of the classification and CI 03 was at the bottom of the ranking. The control hybrid seeds "HM" was also stable for this character by presenting on both sites, a height of crown intermediate between those of CI 02 and CI 03.

Table 3: Comparison of five (5) clones from somatic embryogenesis for crown height measured two (2) years after planting at CNRA-Divo Station and Nestlé Experimental farm Zambakro.

The level of HC obtained from each plot (Divo: CV=24.87, Zambakro: CV=29.53) showed small heterogeneity between trees from all varieties. That difference could be explained by other factors and not related to the genetic of varieties.

Trunk diameter

Data on each trunk from both plots at Divo and Zambakro sites were collected. Results obtained were analyzed to verify if the effect of the position of the trees is linked to the trunk diameter for each planting material (Table 4).

Table 4: Analysis of variance of the trunk diameter (vigor) measured on the plant material (5 clones and HMCNRA Merc) test at CNRA station Divo and at Nestlé Experimental Farm Zambakro.

An interaction effect of the planting material with a position on trunk diameter was significant at Divo (Pr>F=0.0089) and not significant on the same character observed on trees at the Zambakro plot (Pr>F=0.86). The variability observed between each planting material does not mean that these materials are different. That could be linked to their position in the plot.

The comparable analysis of data collected on trunk diameter between the two sites, during a period of six (6) months, showed that the diameter is higher in the Zambakro site than Divo ones by 0.2 cm, only. Also, a coefficient of variation (CV) of trees from Divo (CV=33.4) was higher than CV=24.3 obtained at the Zambakro site. This fact translated a high heterogeneity of the tree's diameter growth inside each plot. This dynamic of growth demonstrated the vigor of clones which is comparable to hybrid Mercedes as control one.

Planting material productivity

The yield of different clones was obtained by the enumeration of the number of pods from each variety during a period of two (2) years. The potential productivity of each planting material is presented in Figure 1 and compared to those obtained by cuttings and evaluated since 2000 at Divo station.

Figure 1: Trees derived from somatic embryogenesis after the two first years of production.

This rank of the estimated production of SE trees is generally similar to the corresponded clonal planting material obtained by cuttings and evaluated over many years. The small difference observed at the level of clones arriving at the head could be explained by the precocity stage of production of the latter. This result highlighted that the somatic embryogenesis method does not modify the productivity behavior of planting material.

Pods analysis

The evaluation of morphological parameters of pods derived from somatic embryogenesis trees was determined compared to the control HM. Three parameters were evaluated: the average volume of a pod (Volcab), the average thickness of the cortex of a pod (Ecortex) and the average number of beans per pod (Table 5).

Table 5: Technical parameters the five (5) clones derived from somatic embryogenesis.

It was observed that the control "HM" had the highest volume of the pod (16062.7 cm3), the thickness of the cortex (1.93 cm) and the average number of beans per pod (39.2 beans). It is followed in decreasing order of performance, CI 03 (12314 cm³) and CI 02 (12262.5 cm³) clones for the volume of pod, clones CI 03 (1.8 cm) and CI 07 (1.7 cm) for thickness of the cortex of pod and clones CI 07 (36.4 beans) and CI 01 (33.3 beans) for the number of beans per pod. These results indicate that the volume of HM pod is higher than pods obtained by the SE method. But it is interesting to note that the standard deviation is higher in the control "HM" than in each of the five (5) clones. This highlights a greater variability between trees within the control HM than between SE trees within clones.

Statistical analysis revealed a highly significant effect of the plant material (Pr>F<0.0001) on each of the three parameters evaluated (Table 6), indicating a difference between the clones for each trait. Plant material had more effect on the pod volume (F=67.1) and thickness of the cortex (F=67.8) than on the average number of beans per pod (F=36).

Table 6: Pods agromorphologic characters derived from somatic embryogenesis: Pod volume (Volcab). cortex thickness (Ecortex) and number of normal beans evaluated.

Black pod incidence of the cocoa varieties

The black pod is the most widespread cocoa disease in West African countries and particularly in Côte d ’ Ivoire. The tolerance of the planting material towards this disease is a quality criterion for the choice of the material which is being distributed, thus, we assessed its impact on the varieties studied as described in section 2.4. Results obtained on the five cocoa clones compared to the control are presented in Figure 2

The most resistant clones are CI 03 (2.8%), CI 14 (4.1%), CI 01 (4.5%) and CI 07 (4.6%). The most susceptible clone to the black pod was CI 02 with 16.2% while the black pod incidence rate was 5.9% for the control. The ability to classify the level of infestation of planting material from the most resistant to the most sensitive confirms the horizontal character of the clones to black pod disease.

Figure 2: Black pod diseases impact score on cocoa varieties (%).e

Discussion

Cocoa plots established using trees derived from somatic embryogenesis showed a relatively low mortality rate, which is under the critical standard. Indeed, after planting, the plant's survival rate depends principally on the plots maintenance and the adequate period of planting such as the rainy period and it is not directly linked to the genetic factors of the varieties.

The study of the general behavior of tree clones produced by the technique of somatic embryogenesis showed a low variability in the height of the crown and in the diameter of the trunk. These criteria were influenced by the position of each tree in the plot, and independent of the plants genetic. This variability could be explained by the local or microenvironmental conditions due to soil fertility or/and a shade cover around each tree. As demonstrated in this study, according to each position of trees in the plot, the vigor and the height of the crown are not the same for trees within the same clonal material. Indeed, some authors showed that in vine plants culture, soil properties, water, and plant nutritional status could impact certain morphological character [30]. In the same sense, the heterogeneity of cocoa trees during experimentation is due to the fact that the cocoa tree is shadeloving and their growth and fruiting capacity are very diversely affected by the cover [31]. Indeed, cocoa shows considerable genotypic variability on morphological and physiological traits associated with yield included vigor [32].

Our study showed that the production potential trend of clonal planting material obtained by the somatic embryogenesis technique was similar to those obtained by plants derived from other plant propagation methods: seeds or cuttings.

Heterogeneity was also observed on pods traits such as pod volume, cortex thickness between clonal trees and hybrid ones. However, this variation was lower in trees from clonal material than those provided from hybrid trees. The low variability of these characters within the same clone reproduced by somatic embryogenesis shows that this technique does not change the qualitative characteristics associated with clonal planting material, unlike hybrid seeds.

Our results obtained on cocoa trees derived from SE are like those obtained by many authors in other locations. An author demonstrated after a five-year field test at Union Vale Estate, Sainte Lucia (2001 to 2006) that no significant differences in growth parameters among the different genotypes and propagation methods evaluated [33]. In a similar work carried out in Ecuador from SE trees generated in the laboratory at Nestlé’s Research and Development Centre in Tours (France), Fontanel et al. [25] showed a normal development and beans production similar to the control trees in the test. In the same sense, Maximova et al. [34] showed that no differences could be detected in the average trunk diameters and crown formation between SE trees and trees propagated by seeds during agronomic performance and conformity of SE plants in two locations in Ecuador.

The behavior of cocoa variety evaluated in this study against black pod disease could be classified into two different groups:

• The first one consisting of CI 02 and Hybrid Mercedes (HM) were susceptible. Indeed, the infection rate of the pods of these two materials was greater than 5%, in absolute value

• The second group of clones which contained the clones CI 01, CI 03, CI 07 and CI 14 expressed relative resistance to black pods disease through analysis of their pods which were inferior to 5%, in absolute value

The presence of cocoa trees produced by somatic embryogenesis techniques in both groups showed that this plant propagation technique does not influence the behavior of cocoa planting material against black pod disease. Indeed, during the pods anatomical study of 8 cocoa genotypes showed that the arrangement of cells in epicarp and mesocarp were implicated on the behavior of cocoa pods against black pod disease infestation [29]. These authors demonstrated that lower epicarp thickness and the higher number of vascular bundles were observed in susceptible genotypes suggesting their porosity to Phytophthora species. Many authors have also demonstrated that the infestation mechanism by Phytophthora species is related to foliar and pods components [35-37]. All these authors showed the correlation between cocoa pods as physical barrier protection and black pod disease.

This confirms our assumption that plant production techniques could not be related to pods infestation by black pods disease. And in this work, we showed that there is no correlation between cocoa somaclonal and black pods disease infestation (Figure 3).

Figure 3: Relation between cocoa varieties somatic embryogenic score and their corresponding Pods Infestation Rate (PIR).

Indeed, the behavior of cocoa planting material multiplied by the somatic embryogenesis technique is governed at the cellular level. Indeed, some authors have demonstrated that the somatic embryogenic potential is related to the presence of phenol content, acidic peroxidase and IAA-oxidase [38] while the behavior of plants against black pod disease is depended to pod cortex as physical barrier protection.

The originality of our work resides in the fact that it’s the first time that the study of cocoa plants derived from SE propagation methods evaluated in fields was correlated with cocoa disease infestation, particularly black pod, which remains the most affected disease in Côte d’Ivoire. Indeed, Pokou et al. [11] showed that the predominant species of the pathogen, Phytophthora palmivora, cause pod losses of 10%-15% but the more aggressive Phytophthora megakarya present in the eastern part of the country causes losses of 40%-60%.

In the end, somatic embryogenesis is a process by which somatic cells are transformed into somatic embryos, which resemble zygotic embryos morphologically since they are carriers of typical embryogenic organs; however, they develop in a different way [39].

Conclusion and Perspectives

As demonstrated in this study, the somatic embryogenesis technique has no significant impact on the vigor of trees and their growth. Production of cocoa pods by trees was not affected, when, somatic embryogenesis technique was used for their multiplication. The little variation observed within clonal planting material on some parameters, specifically, the height of the crown is due to environmental factors mainly to the position of trees. Indeed, the heterogeneity of soil fertility balance in the plot and the cover are largely contributed to that variability.

In conclusion, the somatic embryogenesis technique could be used to mass propagate cocoa trees without affecting the major criteria of cocoa farmers’ preferences, because:

• SE trees had an agro-morphological architecture as seed trees usually propagated in Côte d’Ivoire

• SE trees were vigorous and more homogeneous, develop cocoa pods as seed trees and not increase their sensibility to black pods disease, as well

Acknowledgment

This work is supported by Nestlé R and D Abidjan and le Centre National de la Recherche Agronomique (CNRA, Côte d’Ivoire). The authors are grateful to all field workers involved in this work and from both teams: Nestlé R and D and CNRA. We also thank Dr. Joycelyn K. Quansah from the University of Legon-Ghana for re-reading this work.

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