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prueba binomial, que determinó un mínimo del 30% de polinizaciones exitosas. El nivel de probabilidad de 0.05% se consideró como el umbral de compatibilidad de cada árbol. Para las progenies híbridas, al menos el 70% de sus individuos deben presentar esta característica, para lo cual se registraron datos a los tres, siete y quince días respectivamente, con el fin de tener mayor precisión en el proceso de desarrollo y rendimiento inicial del fruto. Los resultados mostraron que existía una amplia variabilidad en cuanto al nivel de autocompatibilidad de los individuos dentro de todas las familias estudiadas. Solo 3 de las 71 familias estudiadas tenían un número suficiente de individuos autocompatibles para ser clasificados de la misma manera. Sin embargo, su desempeño promedio no fue necesariamente superior al de otras familias que eran autocompatibles. Por otro lado, también se podría confirmar que la compatibilidad de un árbol, clon o cruce puede cambiar su porcentaje de fertilización según el tiempo y las condiciones ambientales.

Palabras clave: Compatibilidad sexual, cacao, progenie híbrida, productividad, fertilización, producir.

Introduction

Ecuador is the world’s leading producer of cocoa (Theobroma cacao L.) of high quality and commercial interest (Quiroz and Amores, 2002). As stated by Sánchez-Mora et al., (2015), in America 15% of world production is concentrated in the center and south of the continent, where Brazil and Ecuador are the largest producers. Cacao is a recognized product in international markets, and Ecuador is a supplier of 60% of the world’s cacao production (Anecacao 2017). In the country, hybridization is carried out in national cacao varieties in order to increase

production with “high” certification system, and thus this undoubtedly improves the economy of the small producing family’s dependent on this crop (Vera J. & Goya A. 2015). As stated by Loor-Solorzano et al., (2019), low yield is one of the main limitations that prevents improving cocoa production. The world average is around 0.5 t / ha, this is mainly attributed to the shortage of productive genetic materials. For Mena- Montoya et al., (2020), the great genetic variability of cocoa depends on the gene flow due to pollen dispersal, which is often substantial among its populations. The T. cacao presents incompatibility problems, mainly depending on the activity of pollinating insects, which ensure sustainable production. This crop reaches its productive maturity after three years of its establishment in the field, after which time its phenology is influenced by the humidity of the soil, distribution of rainfall,

shade and crop management, which affect the rhythm of the flowering and harvesting (Montero-Cedeño et al., 2019).

National type cocoa is attributed to the use of genotypes of unknown genetic origin, unselected, mixed and of low productivity (Sánchez-Mora et al., 2014). For León et al, (2016) to compensate for the reduction in the production of “high” cocoa, research has been intensified to improve and increase its production. In the country, hybridization is carried out in national cacao varieties in order to increase production with “high” certification system, and thus this undoubtedly improves the economy of the small producing families dependent on this crop (Anecacao, 2017). Obtaining varieties with greater economic value depends on genetic improvement and selection processes conducted in research centers such as the Pichilingue Tropical Experimental Station (EETP) of the National Institute for Agricultural Research (INIAP). In this experimental station, the country has a cocoa germplasm bank made up of several living collections, in addition to observation plots and new hybrid populations created during the last decades (Sotomayor et al., 2017; Tarqui et al., 2017).

Incompatibility is a genetic phenomenon, governed by a biochemical process in the recognition, acceptance or rejection of pollen, which occurs in the pollen tube of the recipient flower and in some cases in the stigma (Aránzazu et al., 2008). As indicated by Macoto et al., (2013), in 1960, after the discovery of heterosis in cocoa, the genetic improvement programs in cocoa focused on several countries for the production of interclonal hybrids. Most of the T. cacao accessions are self-incompatible due to a gameto-sporophytic self- incompatibility system. However, some domesticated varieties long ago, such as the creole varieties of Central America, the national of Ecuador and some of their hybrid forms are self-compatible. (Loor et al, 2012).

The incompatibility problem is general in all the provinces of Ecuador, where cocoa growns (Moreno, 1970). As stated by Aránzazu et al., (2008) currently faces the productive inefficiency of hybrid plantations, of advanced age and inadequate management, as well as the existence of a large percentage of unproductive or very low production trees, caused by the phenomenon known as “sexual incompatibility”. For (Quinaluisa 2010), this is because tree physiological conditions can change the compatibility system at several levels. As Montero-Cedeño et al. (2019) indicates, in many cocoa-producing regions, trees show an incompatibility of more than 50%, so there is more flowering than fruit production. The incompatibility of cocoa is more severe than in other fruit trees, since there are few species of

pollinating insects and they only transport pollen from one tree to another at a certain time. The productive capacity of T. cacao will depend on the reproductive efficiency of the formation of fruits, flowers, pollen and ovules, which may be specific to the genotype (García- Cruzatty et al., 2020).

It is known that the adult cocoa plant under normal conditions can produce 6000 to 10000 flowers per year, of which only 1% become fruit. The setting or fertilization may be even lower due to the lack of specific pollinating insects such as the fly (Forcypomnia spp.) (Vera et al., 2016). Compatibility is translated as the ability of a cocoa plant to produce seeds, either by self-pollination (AP) or cross pollination (PC), which, in both cases can be achieved by natural (insect) or artificial methods (human). However, knowledge of compatibility systems and the incompatibility genotypes of different experimental or commercial cocoa clones constitute important information for future use in hybridization programs (Mendoza, 2015; INIAP, 2016).

Artificial pollination is the procedure used to obtain seed trees, with outstanding characteristics in terms of production, early quality, and resistance to diseases or pests. Studies carried out by INIAP have allowed the procedure to be developed, which consists of manually pollinating fresh cocoa flowers, of national origin, with which it is possible to provoke a growing expression of being able to increase the yield of decadent traditional cocoa plantations, profitably, but has not been tested on self-compatible cocoas such as the CCN-51 clone. (Vera, J. & Vera, J., 2018).

It is important to mention that hybridization or natural combination of genotypes other than cocoa has occurred in nature for hundreds of years. This fact,

together with the existing bioclimatic conditions, has resulted in the current Ecuadorian cocoa maintaining its unique characteristics.

Based on the background described, the objective of this study was to estimate the level of compatibility of a group of hybrid progenies and its relationship with their performance.

Materials and methods

Experiment location and environmental conditions The research was conducted at the INIAP Tropical Pichilingue Experimental Station (EETP), located at 5th km of the Quevedo-El Empalme road, at an altitude of 85 meters above sea level. The geographical coordinates are 79 ° 2’ West Longitude and 1 ° 6’ South Latitude. The average temperature is 27.3° C, annual precipitation 1640.90 mm and 772.60 light hours day (INAMHI), 2018). The soil at the experimental site has a loamy texture with a good level of fertility and moderate drainage. This study was carried out between January 2008 to February 2010 and the climatic conditions for the period in question are: mean annual temperature of 25.06 ° C, mean annual rainfall of 1856

mm and heliophany 669.95 light hours

Genotypes

In the present study, 71 hybrid families were evaluated from which 333 trees were selected, using selection criteria such as productivity and health during 5 years of evaluation (2002-2007) and thus determine the level of self-compatibility per individual within a group of hybrid progenies. of cocoa, estimate the level of self-compatibility of the hybrid progeny under study and its relationship with yield and select self-

compatible trees with high productivity (Table 1). For each trial the number of treatments/trials, number of repetitions/trials, number of trees/treatment and total trees/trial are shown in Table 2.

Employed tools

For the execution of the experiment it was necessary to tubes Eppendorf, which served both for the isolation of floral buttons and the protection of pollinated flowers. The plastic tubes were 5 ml long by 1 ml in diameter, closed at one end and weighing 0.65 g.

Pollination technique

To determine self-compatibility, the following procedure was used: one day before carrying out manual pollinations, the flower buds that are physiologically

mature and ready to open the next day in the morning are selected, which are generally characterized by swelling that indicates the beginning of the opening of the button for the formation of the flower. Plastic tubes were placed on the selected buttons, this is done to ensure that at the time of opening the flower no pollinator has access to them and thus be sure that self- pollinations are carried out. This work was carried out in the afternoon of the day before the anthesis of the flower (Alvarado et al., 2017).

In the morning of the next day when the flower was open, the pollinations were carried out manually, these were carried out between 7:30 am to 11:30 am. On rainy days it was waited until it stopped and continued later. 20 self-pollinations were carried out per tree, reaching a total of 6660 flowers self-pollinated. The

stamens of the flowers from the same tree were taken, after releasing the shell or cogulla. Holding it with the forceps, the anthers were rubbed on the stigma or style of the flower, with a smooth movement from the bottom up, until they were sure that enough pollen had been deposited on the female organ. The flower was immediately covered with the tube.

Data to evaluate

Self Compatibility

The evaluation was carried out at 3, 7 and 15 days after the manual self-pollinations were done, since within this period self-compatibility problems would be observed.

Fertilized flowers

In order to determine the number of fertilized flowers, the verification was made 15 days after the self-pollinations were carried out when the “fruit” had already taken shape.

These evaluations must be carried out in the field after 10 or 15 days of pollination to corroborate the take-off, the fall of the self-pollinated flowers begins after three (Arciniegas, 2005).

Unfertilized flowers

The registration of this variable was done jointly with the previous one, counting the number of pollinated flowers that had come off, or that remained in the tree in the process of physiological wilting (Cherelles wilt).

Fertilization percentage

This variable was estimated by counting the number of flowers fertilized at 15 days after pollinations. This calculation was made with the following formula.

In general, the interpretation of the self-pollination results was performed by transforming the values obtained in percentages considering the minimum fertilization (30%) which coincides with the Binomial Test.

Statistical analyzes based on the genotype selection criteria

Statistical analyzes were based on the analysis per family (% compatibility by family and individuals) and by each characteristic; rank/tree based on four selection criteria for the total years of evaluation:

•Potential fresh weight (PF Pot = (PF of MS + PF of ME)) = the estimated production based on total produced fruits

•Percentage of healthy (harvested) ears (% DM)

•Witch’s broom (WB) = total witch’s brooms

•Production efficiency = (PF/diameter3)

Progeny hybrid self-compatibility and performance For hybrid progenies at least 70% of their individuals must be self-compatible, before considering a progeny compatible. Said calculation was made with

the following formula:

The records of this variable were presented in terms of percentage to know the self-compatibility between hybrid families. Their performance was related to it based on the analysis / family, which is one of the selection criteria of this research.

Selection of productive and self-compatible trees This evaluation method was performed based

on the total performance during the evaluation years (2002-2007) of each selected individual. Being the most important selection criterion in the study, this selection criterion was related to the percentage of self- compatibility/tree where self-compatible trees superior to 30% compatibility were selected and with good potential performance.

Statistical test to determine the percentage of self- compatibility of hybrid progenies

The binomial test was used, which allowed determining a minimum of six successful pollinations (30% fertilization), the necessary limit before considering a tree as self-compatible. The 5% probability level was used following the below formula

P=probability of success q= Probability of failure

k= Number of desired successes n= Number of tests carried out

Results

Self-compatibility by individuals among one group of hybrid progenies

Table 3 shows the varying rates of fertilization and the presence of different degrees of self-compatibility and self-incompatibility in four trials of hybrid families, which means that each pollinated tree responded differently to this characteristic.

Of 333 pollinated trees, 154 were self-compatible, i.e. 46.25%. The pollination range of self-compatible individuals was within 27 to 83%. The 4 trials of

hybrid families showed different percentages of self- compatible trees. Trials 1, 2 and 3 recorded percentages of 67.50, 70.59 and 54.61%, respectively. While the group 4 presented the lowest percentage of self- compatible trees with 25.81%. The 53.75% of the trees studied were self-incompatible, with fertilization rates below 30%. The highest number of self-incompatible trees was recorded in trial 4 with 74.19%. Although pollinations were repeated, fertilization was absent in a total of 40 trees. More than half of these trees belong to group 4, followed by group 3 with 35% of trees that exhibited zero fertilization.

Self-compatibility level of hybrid progenies and their relationship with performance

Table 4 presents the productivity of the hybrid families that were self-compatible, through the selected trees and their yield of cocoa in fresh weight

(g). It was also included comparative purposes, i.e. the average fresh weight yield of the individuals who initially formed the family, before the pre-selection by performance.

More than 70% of the individuals selected for their performance in the families, EET-577 X EET-534 and EET-547 X EET-534, were self-compatible with 83.33;

71.42and 71.42%, respectively while such families had the characteristic of self-compatibility. The EET-577 X EET-534 Family did not have the highest percentage of self-compatible trees (71.42%) although it contains the largest number of trees selected (14). In contrast, the EET-552 X EET-547 Family with the lowest number

(6) of selected trees reached the highest percentage of self-compatible trees (83.33%). The cumulative fresh weight yield of six years for the EET-577 X EET-534 family was 1226 g, which was higher than the EET- 552 X EET-547 and EET-547 X EET-534 families with

11.27and 27.01%, respectively. As expected, the trees with the highest yield belonged to the families with the highest average cumulative yield, i.e. EET-577 X EET-534 with 1226 g and EET-552 X EET-547 with

1088 g. In general, the highest productivity of such individuals was consistent throughout all the years in which data were recorded. The distance among annual average fresh weight yield and less productive tree (318 g), and the most productive one (2042 g) was 1724 g, equivalent to 542%. The least productive tree belonged to the family EET-547 X EET-534 with 318 g. The variability of performance among individuals of each family was quite noticeable.

Selection of productive and self-compatible trees Table 5 demonstrates cumulative dry weight of six

years of evaluation (2000-2007) of self-compatible and productive trees was in the range of 1924-5562 g. Tree 3 of repetition I of the EET-462 X EET-534 junction, tree 9 of repetition II of the EET-48 X EET-85 junction, tree 1 of repetition V of the CCN-51 X EET-534 junction, the tree 8 of the repetition IV of the CCN-51 X EET-233 crossing and the tree 9 of the repetition I of the EET-62 X EET-103 crossing, exceeded the average weight of 800 g/year. The average annual yield of these four materials was 856 g. If 1111 trees were planted in one hectare (3 x 3m) at eight years, the equivalent of

20.9quintals/ha/year could be expected. This figure is quite acceptable considering that the experiment was conducted under rainfed conditions.

Some trees had no production during the first years of evaluation and were those that generally coincided with those with less production. It was also observed during 2006 when the yield of any tree was superior to that of 2007, with two exceptions. However, it is notorious that the variability observed for the yield between trees in 2006 was not consistent with the variability between trees for the dry weight accumulated during the six years of evaluation (2002-2007).

In general, the trees with more yield showed a greater number of healthy ears, less number of diseased ears and percentages of diseased ears less than 10%. It is evident that the percentage of diseased ears went from low to medium (from 0 - 21.4%), in the group of

Quinaluisa et al., 2021

trees identified as self-compatible.

Discussion

Self-compatibility levels

The self-compatible materials presented fertilization percentages that varied from 30 to 100%, which shows that there were different ranges of self- compatibility and each tree responded in a different way to this characteristic. As reported by Quiroz (1990), this could be due to the fact that environmental conditions influence the physiology of the tree and at certain times of the year, trees tend to vary their compatibility. Moreno (1970) showed that all clones do not give equal results, because the flowers of some curdle more than the others, with which different degrees of compatibility occur. Loor (1998) stated that this situation may be due to genetic, physiological or environmental factors that include considerably in the pollination process. For Godoy et al. (2009) sexual incompatibility is one of the factors that contribute to the low productivity of cocoa; other factor that can influence the genotype is the variability in pollen yield. In most of the self- incompatible trees, especially those in which no flower was fertilized, all flowers with manual pollination fell before the seventh day, showing a strong reaction of self-incompatibility. On the other hand, Pastorelly (1992) reported that in some plantations there are unproductive or very low production trees, which form cobs due to their genotypic constitution but do not mature as there can also be trees that do not form fruits. The main idea is to have a basic knowledge of this characteristic in order to establish the way of planting the different cocoa materials.

In other trees it was observed that many flowers fell after 10 days of self-pollinated. Among the main factors that reduce cocoa production is the problem of the auto-incompatibility of certain trees in the plantations, a condition that according to Loor (1998), may be due to the ovarian stimulation caused by the rubbing of the anthers on the stigma. Zambrano (2000) pointed out that this abnormality can manifest up to 20 days.

It was also observed that in some trees a good number of cobs from manual pollination were affected by the phenomenon of physiological wilting or “ Cherelles wilt “, mainly when they were between one and two months old, which suggests a response of the tree to an overload of ears, thus regulating the number of fruits can hold.

Self-compatible hybrid progenies and their relationship with performance

With regard to the results obtained from the self-

compatible hybrid families (Table 3), it suggests that this genetic characteristic is present in a dominant state in each of the individuals belonging to these families. Based on this, it is worth to mention that it would be the cause of large losses for farmers if the selected and recommended hybrid progeny were self-incompatible. For this reason, it is recommended to select self-compatible trees in cocoa breeding programs. In relation to the cumulative and average six-year fresh weight yield of the selected individuals, including all progeny trees, it can be noted that the self- compatible crossover EET-577 X EET-534 maintained its superiority of production during the evaluation years. Solís et al. (2015) observed a significant variation between hybrid plants in terms of the variable dry cocoa yield, which allowed the selection of high production trees. This corroborates with what was found by Saucedo (2003), who indicated that based on many researchers at different sites there is a relationship between yields and their degrees of compatibility, achieving self-compatible trees and low-productivity self-incompatible productivity.

Within the self-compatible families EET-552 X EET-547, EET-577 X EET-534 and EET-547 X EET- 534, there were individuals who excelled in their accumulated performance during the evaluation years. Thus, within each of the self-compatible families there was a tree that stood out for producing 2.2% of the production, corresponding to 6 to 10% of the total accumulated weight of each of the families. Probably this superiority of the genotype is because of the genetic value of the material under study.

Selection of productive and self-compatible trees It was found that tree 3 of repetition I of the EET-

462 X EET-534 junction, tree 9 of repetition II of the EET-48 X EET-95 junction, tree 1 of repetition V of the CCN-51 X junction EET-534, tree 8 of repetition IV of crossing CCN-51 X EET-233 and tree 9 of repetition I of crossing EET-62 X EET-103, were self-compatible and the most productive with 927, 890, 853, 804 and 801 g of dry cocoa/tree/year, respectively. Possibly this is due to the genetic recombination of favorable genes of their parents. In Trinidad, Pound (1934) established basic criteria for the selection of cocoa trees with high yields. He proposed that data should be collected for 2-3 years about the total production of ears, fresh and/ or dry weight of almonds that are important factors in estimating the value of selection of a tree. Within the selection indices, the number of ears and the fresh and/or dry weight of almonds are considered the most important for the performance measurement. Eskes (1999) stated that yield efficiency is a relationship that is calculated between tree production and vigor. Campi

(2013) expressed that the number of healthy ears is decisive for yield key, which has direct proportionality with the variable total fresh weight. According to Barrón et al. (2014), the genetic improvement of cocoa is a strategy to obtain more productive trees.

Table 5 also shows the absence of diseased ears in the six years of evaluation for the hybrids CCAT- 21-19 X EET-574, EET-574 X EET-547, EET-48 X EET-95, EET-574 X EET-577 and CCN-51 X EET- 534, possibly due to genetic resistance of the selection and genetic improvement processes. In the same way, it was observed with the data of Solís et al, (2015) who conducted a four-year research study, where the agronomic management of 11 hybrid families of cocoa resistant to moniliasis was studied in plants obtained by controlled pollination of low clones in humid tropic conditions where a general average of sick ears of 12.83 were presented. Espín (2017) evaluated 31 Interclonal hybrids of cocoa where he observed that the highest number of sick cobs was for T1. For DYRCYT-H-272 with 6.33 ears, the minimum values were reflected in the T30 and for DYRCYT-H - 301 they were 0.33 sick ears. Pastorelly (1992) showed that these low levels of infection of diseased ears could be because of the action of environment or some genetic factor of resistance or tolerance to this disease. In another study, the genetic improvement of cocoa tree was seen as an alternative to address these pathogens by conferring increasing levels of resistance (Tirado-Gallego et al., 2016).

Conclusions

Wide variability was observed regarding the level of self-compatibility of individuals within all the families under study. The compatibility of a tree,

clone or cross can vary the percentage of fertilization according to time and environmental conditions, even a self-compatible one at certain times of year can become self-incompatible in other situations. Only 3 of the 71 studied families presented a sufficient number of self-compatible individuals to be classified as such EET-552 X EET-547, EET-577 X EET-534 and EET- 547 X EET-534. However, their average performance was not necessarily higher to other families that were self-incompatible. Self-compatibility is a genetic feature that greatly influences the good agronomic and productive behavior of cocoa cultivars.

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