Comparative effects of plant growth promoters and earthworms (Millsonia anomala) on rooting of cocoa orthotropic cuttings

Propagation of cocoa trees with high yield and disease resistance is envisioned for the renewal of the cocoa orchard. The dissemination of these elite varieties by orthotropic cuttings is strongly recommended to achieve quantitative and qualitative objectives. Some experiments reported that some Theobroma cacao clones produced by orthotropic cuttings do not root easily and the rate is very low, around 30 to 50% results. In this work, the impact of earthworms and chemical hormonal growth promoters on cocoa cuttings acclimatization were studied to increase the survival rate of cocoa stems during acclimatization step. The presence of earthworms identified as Millsonia anomala in acclimatization substrates allowed to convert more than 65% of stems into plantlets and exceeds 83% when the section of stems was a softwood part of branch. In the same profile, growth promoters as chemical hormonal solutions, regularly used in the process, are converting around 49.5% of stems into plantlets. In this study, a variability on the presence of taproots number across various parts of the branch were shown. Indeed, 69.2% of stems from hardwood, 64.3% stems from semi-hardwood and 56.4% of stems from softwood have developed at least two main roots. Indeed, this fact corroborates the capacity of these trees to able supporting the probable weight of cocoa pods and resists on wind which could appear on areas with bad weather.


INTRODUCTION
Cacao (Theobroma cacao) belongs to the genus Theobroma classified under the subfamily Sterculioidea of the mallow family Malvaceae.Cacao is one of 22 species of Theobroma.T. cacao L., exclusively cultivated in the inter-tropical area of the world, is a major source of income for developing countries (Alemano et al., 2007).
Cocoa is the essential ingredient for chocolate and the role as the antioxidant and antiproliferative activities of T. cacao leaf, bark, husk, unfermented and fermented shell, pith, root, and cherelle methanolic extracts were well screened (Baharum et al., 2014;Da Silva et al., 2014).
The cocoa cropping has great economic impact in Côte d'Ivoire and Ghana which produced around 70% of cocoa beans around the world.These crops contribute in 10 and 8.1% of Côte d'Ivoire and Ghana gross deomestic products (GDPs), respectively.Despite its importance in the country's economy, cocoa cropping is facing different challenges in Côte d'Ivoire among which climate change, deforestation (Kouadio and Desdoigts, 2012), disease and pests pressure (Despréaux et al., 1987;Babin, 2009), aging of farmers and plantations, but also low access to inputs as fertilizers and pesticides and good quality planting materials.This results in low yields per hectare (300 to 450 kg/ha/year) with a negative impact on farmers' revenue (IITA, 2009).Propagation of high yielding and disease resistant planting materials to farmers for renewal of the old plantations is one of the elements contributing to a sustainable cocoa cropping.
Propagation of cocoa planting material could be done by different methods (Niemenak et al., 2008).It is mainly through seeds, in vitro cultures such as somatic embryogenesis technics or vegetative propagation and by micropagation (Figueira et al., 1995;Lopez-Baez et al., 1993;Maxwell and Blake, 1984).However, in West African cocoa countries producers, 70% of cocoa plantlets are currently produced by seeds for plants propagation.The dissemination of these plantlets obtained by seeds is limited compared to the needs.One of the solutions could be to use orthotropic cuttings technique to address these problems.Indeed, promotion of cuttings methods particularly by orthotropic shoot growth has been achieved by the bending of mature trees or young orthotropic stems (Glicenstein et al., 1990;Bertrand and Dupois, 1992).Plants production by cuttings has an advantage in terms of capacity of production, insurance of genetic material contrary to the hybrids seeds that present a very strong heterogeneity at the level of production (N'Goran et al., 1994).
Plantlets from orthotropic shoots were obtained using branches harvested from trees bended on fields after a minimum of two months.These branches are divided into three parts to obtain stems and those planted in the polybags which contained substrates and placed under plastic tunnel during forty days.Indeed using this system, nearly 40 to 250 plants can be produced from each somatic embryo plant as mother tree per year.The conversion of cuttings into plantlets during a large scale production is a one major challenge for the success of the process.Indeed, cultural strategies have been tested to promote the growth of orthotropic material in clonal gardens with mixed success (Miller and Gultinan, 2003).
Several methods were used to improve the success rate of cuttings conversion into plantlets by acting on acclimatization environments or by adding growth substances in the media or soil substrates and improving micro environmental conditions (Niemanak et al., 2008;Tapi et al. 861 Tee and Lamin, 2011).As observed in the rooting of cocoa cuttings, the application of auxin in the basal region of stem has been used worldwide to promote the adventitious rooting of woody species (Schwambach et al., 2008;Wendling et al., 2010;Hartmann et al., 2011;Hunt et al., 2011), to influence the micro-cuttings survival and then the rooting of species considered recalcitrant to rooting (Bennett et al., 2003;Schwambach et al., 2005).Beside, many environmental and genetically effects such as nursery practice and source of generative and vegetative materials in different plant species were reported by Dilaver et al. (2015) and Yazici and Babalik (2016).
At the other hand, earthworms generally are assumed to be beneficial soil animals which are mainly based on the belief that they promote plant growth (Lee, 1985;Edwards and Bohlen, 1996).However, knowledge on effects of earthworms on plant growth is very biased; most studies investigated crop plants, particularly cereals, and pastures; very little is known on plant species in more natural communities (Scheu, 2003).Within soil organisms, earthworms are in terms of biomass and activity among the most important detritivores in terrestrial ecosystems (Edwards, 2004).Earthworms are known to impact plant growth, generally positively, via five main mechanisms: (1) an increase mineralization of soil organic matter, (2) the production of plant growth substances via the stimulation of microbial activity; (3) the control of pests and parasites; (4) the stimulation of symbionts; and (5) modifications of soil porosity and aggregation, which induces changes in water and oxygen availability to plant roots (Brown et al., 2004;Scheu, 2003).
Although, earthworms impact on plant growth through production of plant growth substances, this has never been tested on growth of cocoa cuttings.Therefore, in the current study, earthworm effects on cocoa cuttings root and shoot development were evaluated compared to classic plant growth promoters as chemical hormones.These cuttings are obtained from three types of stem: (a) hardwood (basal section of branch), (b) semi-hardwood (intermediate section), and (c) softwood (apical part of the branch).
The following hypotheses were tested: (1) Earthworms induce greater impact on root and shoot development of cocoa cuttings compared to plant growth promoters and (2) Earthworms exert greater impact on biomass production than do plant growth promoters, whatever the position of the stems on the branch (softwood, semihardwood and hardwood).

Cocoa planting material
Two cocoa accessions T79-501 and PA7 from the International Cocoa Germplasm Database were used in this work as they respond highly to in vitro somatic embryogenesis methods (unpublished results).The agronomic characteristics of these two clones are shown in Table 1, according to information provided from Dr. Tahi (CNRA cocoa breeder).
Immature flowers buds of these cocoa trees are collected and processed to produce in vitro cocoa plantlets by somatic embryogenesis technique (Lopez-Baez et al., 1993;Florin et al., 2009).After five or six months of acclimatization growing period in greenhouses, these plantlets were planted on the cocoa gardens in an experimental farm to produce stems by orthotropic cuttings techniques.

Orthotropic cuttings production from mother plants
Previous plantlets produced by somatic embryogenesis technique were used as mother plants for generating orthotropic planting materials.These mother plants were arched and fixed on ground with a solid wire during four months after planting.Several orthotropic buds appeared after three weeks along the main trunk length.Three months after bending, these buds have grown and became orthotropic branches measuring around 30 to 70 cm.These branches were harvested on the base of horizontal main trunk of mother plants, and orthotropic cuttings were obtained by cutting the main trunk of these branches in three parts.For this study, each branch was divided into three portions of around 5 to 8 cm: hardwood (basal section on the branch); semi-hardwood (intermediate section); and softwood (apical part of the branch).

Growing conditions and general cultural management
Stems (2880) of each part of the branch were used as orthotropic plantlets from the two clones previously described were produced.These stems were sanitized in a solution of 2.5 g/20 L of Benomyl powder (Louis Drefus Commodities, Côte d'Ivoire), an antifungal product, during 1 to 3 min.As presented in Table 2, six different treatments were applied to acclimatize stems.
The stems of each clone were dipped in the rooting hormone solutions during few seconds and planted in polybags which contained steppe black soil.
The other stems which were not dipped in the hormonal solutions were placed directly in the polybags which contained steppe black soil supplemented with three earthworms as Millsonia anomala (1 living organism/0.002m 3 of soil) and in steppe black soil also supplemented with Agrinos C (15 g/0.001 m 3 of soil).
After transplanting, cuttings in polybags were placed under a white plastic film with 70% of shade.Closing them with plastic films has elevated tunnels' relative humidity (RH) above 80% and increased temperature around 40°C.After 30 days, plastic tunnels were opened gradually during the 10 days.

Experimental design for statistical analyses
Stems (1440) were produced per cocoa variety and the experimental design was done to establish the same experimental unit regardless of the three different positions on the branch and the six treatments.For that purpose, a split split plot with 4 replicates and 4 blocks was preferred (720 sections of stems by block 240 softwoods, 240 semi-hardwoods, and 240 hardwoods).
The following dependent variables were measured: survival rate of stems, number of plants with taproots, number of taproots, dry shoot and root dry biomasses for each treatment and taking into account the position of each stem.
Survival rate of stems was calculated on data collected after 40 days of acclimatization step under tunnels.The number of plants with taproots, shoot dry weight and root dry weight were also measured at the end of the experiment.All data were analyzed and compared with type 1 analysis of variance (ANOVA) using GLM procedure (SAS software 9.3).To determine the direction of significant effects, multiple comparison tests were used based on Least Significant Difference (LSD).General outcome of these comparisons without displaying them in detail was presented in Table 3.

Plant growth parameters
In this experiment, as can be seen in Table 3, treatments, section of branch and the section of branch×clone interaction were significant sources of variance for several variables.Importantly, earthworms and growth promoters (treatments) had significant effects on all of the parameters.The clone did not have a significant impact on all dependent variables measured.As shown in Table 3, this effect ranging from significant (0.01<P<0.05) for the rate of plants with one main root, to highly significant (P<0.01) for the survival rate, shoot dry weight and root dry weight.Furthermore, the section of stem also had a highly significant effect (P<0.01) on several dependent variables measured (the survival rate and number of taproots).Finally, a significant interaction (0.01<P<0.05) was observed between the section of branch and the clone (section of woods×clone) for the rate of plants with 1 main root, two main roots and highly significant (P<0.01) for the shoot dry weight, root dry weight, rate of plants with 4 main roots.

Effects of different treatments on the conversion of stems into plantlets
Stems were immersed in concentrated solutions of growth hormones and also were buried in different substrates formulations as described in previously.
The survival rate of stems according to each treatment was determined (Figure 1).These data showed a very high variability of parameters for each treatment.
The success rate of stems converted into plantlets was 65.8% when the humic soil contained earthworms.When stems are immersed in auxin solutions or introduced on humic soil which contains Agrinos C solutions, the survival rate was 42.4 and 41.04% in only humic soil substrate as a control.

Relation between the types of roots and treatments or stems position
To evaluate the influence of each treatment on the position of each stem, the rate of its conversion into plantlet was determined and presented as shown in Figure 2. Results obtained showed that the success rate was 44.4% when stems are made by hardwood, 42.4% when it is from semi-hardwood and 58.4% when softwoods are used to produce plantlets.
The success rate on stem conversion was around 83% when softwood part of branch was acclimatized in humic soil substrate which contained earthworms.
To correlate plantlets survival rate and corresponded treatments, roots and shoots dry weights parameters were calculated.Results obtained are as shown in Figure 3. Excepted substrates of only humic soil and humic soil contained agrinos C with 7.9 and 6.8%, respectively, no difference could be observed for root dry weights between other treatments (around 11%).
The development of roots is a major asset for the future tree, not only for nutrition system, but mainly to protect the trees against the vagaries of the weather as the wind in unfavorable areas.This criterion was also determined by the enumeration of the number of taproot of each plant   according to the treatment.Figure 4 showed results obtained after 6 to 8 weeks outside acclimatization tunnels.
All plantlets obtained by orthotropic cuttings were developed one or more taproots regardless to all treatments.Notwithstanding the type of treatment, the proportion of cuttings which developed one or two taproots is ranging between 40 and 80%.No significant differences between treatments could be observed for the development of two taproots for all stems.However, the probability to develop one taproot was higher when Agrinos C (15 g/L) treatment was applied on cuttings.
These results did not explain the preferential occurrence of a precise number of roots in relation to the type of treatment.Detailed analyses of these results in function of the position of stems are shown in Table 4.One taproot was developed by 43.6% of stems obtained with softwood, 35.7% of semi-hardwood stem and then 29.7% of stems from hardwood.More than 50% of stems produced by all sections (hardwood, semi-hardwood and softwood) produced two or three taproots.

DISCUSSION
The stems from cocoa branches were better converted into plantlets when the substrate contained a humic soil with earthworms than other chemical growth hormones and also than results shown by Gehlot et al. (2014) on cuttings treated with auxins.However, all results obtained, in this study on the different rooting substrates, are higher than those obtained by Tee and Lamin (2011) on vermiculite (32.6%), perlite (23.2%) and then coconut coir fibre (21.5%).
Indeed, in the cases of auxin application, great variation was observed among the concentrations, formulations and forms of application of plant growth regulator (Wendling et al., 2000;Fogaça and Fett-Neto, 2005;Wendling and Xavier, 2005;Almeida et al., 2007;Schwambach et al., 2008) as well as other factors considered intrinsic based on the genetics (Stape et al., 2001;Bennett et al., 2003;Corrêa and Fett-Neto, 2004).
The presence of earthworms in certain rooting substrates could probably play a major role in the transformation of inorganic materials in organic matters during stems acclimatization.In their studies to investigate the effects of earthworms on N cycling processes and microbial activity, Bolhen and Edwards (1995) indicate that living organisms increased the amounts of extractable N by feeding on the microbial biomass, and increasing the turnover and mineralization of microbial   tissues.Indeed, Ribeiro et al. (2008) showed that in certain cocoa genotypes, increasing levels of applied N improved growth (stem girth, dry weight of shoot and roots and shoot/root ratio), and concentration and uptake of N.
The results obtained on the relation between the position of stem and stems conversion are reported earlier.The position of stem on the branch used for producing the plant has an influence on its survival rate and showed that softwood section was easily rooted than other section.
However, the production of softwood stock plants requires a higher level of cultural management and propagation technique than those for bent semihardwood stock plants (Miller, 2009).Indeed, it is difficult to separate the three part of branch during cocoa trees mass propagation.
To correlate plantlets survival rate and corresponded treatments, roots and shoots dry weights parameters were determined.This showed that the root development is practically the same when earthworms were used and also chemical growth promoters too.The formation of adventitious roots is a high energy requiring process, which involves cell division, in which predetermined cells switch from their morphogenetic path to act as mother cells for the root primordia; hence, need more reserve food material for root initiation (Aeschabacher et al., 1994).No difference could be observed between IBA and NAA hormones on cocoa orthotropic cuttings rooting systems contrary to the assertion of Ghelot et al. ( 2014) when these two promoters were applied to produce Azadirachta indica trees.However, the measurement of shoots dry weights rate showed that it is higher (62.3%) for substrates which contained earthworms than other ones.These results showed that earthworms could be involved in enriching substrates on nutrients and does not probably implicate in root systems development during the acclimatization of stems.The efficiency of earthworms tested in this study resides rather in the provision of useful nutrients for leaves development.This impact positively stems transformation into plantlets around 65.8% independent of the position of stems used.Indeed, the survival rate of stems is high when roots and shoots dry weights are high.
In this work, the presence of one or more taproots on plants obtained by orthotropic cuttings methods was observed.However, the probability of the number of taproots by type of treatment must be relativized in view of the high absolute value of the standard deviation.The presence of several main roots should be confirmed to the adult age of the tree because of these results were obtained after 6 or 8 weeks of plantlets growth.
Finally, the use of earthworms proved better compared to the tested auxins on specific concentrations.However, the direct effects of earthworms could be relativized.Indeed, Laossi et al. (2010) demonstrated that the influence of Lumbricus terrestris earthworm varied with the soil type and this earthworm species did not have any significant effects on Trifolium dubium if the soil is poor in nutrients.

Conclusion
These studies demonstrated the ability to double the survival rate of plantlets produced by orthotropic cuttings from 49 to 83%.This was achieved (1) by improving the quality of acclimatization substrate with the use of earthworms specifically M. anomala and (2) using preferentially a softwood part of branch to produce cocoa plantlets.
However, the advantage of the presence of earthworms on substrate media, could only be observed if the soil is rich in organic material to start with.This is not the case for poor soils present in certain part of Côte d'Ivoire's lands.The use of living organisms such as earthworms that have the ability to turn the inorganic matter into nutrients is useful to initiate rooting system for cuttings during the first 8 weeks of growth.
The understanding of earthworm mechanisms could be deepened by the fine analytical study of the soils (pH, conductivity, porosity and microflora) used as substrates to root cocoa stems.
Cocoa plants propagation in a large scale by orthotropic cuttings methods with the use of humic soil as substrate with content earthworms will be a successful pathway of Tapi et al. 867 increasing more the efficiency of this technique.

Figure 3 .
Figure 3. Determination of roots and shoots dry weights parameters according stems survival rate treatments.

Figure 4 .
Figure 4. Effect of treatments on the number of roots×survival rate after 6 to 8 weeks stems growth after an acclimatization period of 40 days under tunnels.

Table 1 .
Agronomic characteristics of cocoa clones used in this study.

Table 2 .
Different treatments done for stems acclimatization.

Table 3 .
Analysis of variance F-values for ten parameters measuring plant produced by orthotropic shoot under effects of earthworms and growth promoters.

Table 4 .
Relation between the number of taproots and the position of stem on the branch.