Cocoa Genetic Resources and Their Utilization in Palm-Based Cropping Systems of India

Cocoa ( Theobroma cacao L.) became an integral part of palm-based cropping systems of India. It is being grown profitably as a mixed crop under arecanut ( Areca catechu L.), coconut ( Cocos nucifera L.), and oil palm ( Elaeis guineensis Jacq.) gardens of the southern states Karnataka, Kerala, Tamil Nadu, and Andhra Pradesh. It is well adapted to the humid tropics with high rainfall and short dry spells as well as irrigated gardens of tropical belts, utilizing the shade provided by the palms. Research efforts of more than four decades at the ICAR-CPCRI (Indian Council of Agricultural Research-Central Plantation Crops Research Institute) and KAU (Kerala Agriculture University) have allowed efficient utilization of conserved cocoa genetic resources by farmers to provide additional income through multispecies cropping systems in the spices and plantation sector. National Horticulture Mission of Govt. of India identified cocoa as a potential crop for area expansion and development to meet both internal and export demands. Improved varieties were developed with high pod yield, bean quality, suitable to different agro-climatic zones and to tackle major biotic and abiotic stress. This chapter out-lines the contributions of cocoa breeding efforts at the research institutes and State Agricultural Universities; developmental programs of Ministry of Agriculture and procurement and processing facilities to the growth of the cocoa sector in India.


Introduction
Cocoa was introduced into India way back in 1798 at Courtallam in Tirunelveli District of the Old Madras State (Tamil Nadu) by East India Company [1]. Cocoa was distributed then in the agro-climatic region covering Western Ghats Hills and Plains of Malabar (Kerala) and Mysore (Karnataka) states, having more rainy days and short dry periods [2]. Commercial cultivation of cocoa as a plantation crop under palms started in early 1970s, and the current area under cocoa is 82,940 ha with a production of 18,920 tonnes of cocoa beans [3]. From the traditional hilly regions, cocoa production has shifted and expanded to coconut gardens of nontraditional areas of Tamil Nadu and Andhra Pradesh states utilizing the 50% shade available in the gardens and irrigation. Safe conservation of genetic resources of this introduced crop and its utilization in breeding program is a top priority considering its perennial nature, adaptability, and compatibility with palms. Cocoa tree has a

Introduction of germplasm
The basic step in any breeding program is the introduction of germplasm from both the primary and secondary centers of origin and distribution. Cocoa in its native zones of South and Central America and other major producing countries of Africa is suffering with many debilitating diseases caused by both viral and fungal pathogens and serious pests. To safe guard the germplasm exchange program, Intermediate Cocoa Quarantine Centre (ICQC, R) was established in Europe. The University of Reading took over the responsibility for cocoa quarantine in 1987 [8]. The center routinely collects important clones from international and national gene banks, wild collections and conducts virus indexing. It conducts strict quarantine for other major diseases and pests using the guidelines for safe movement of germplasm formulated by FAO/Bioversity International [9,10]. Clones that have been cleared at the quarantine are supplied as bud sticks, with proper import permits and sanitary certificates to cocoa researchers over 20 countries. It also maintains the International Cocoa Germplasm Database to know about the gene banks, clones, traits, and even SSR/SNP profiles [11]. CPCRI also utilizes facilities offered by ICQC, R and collected exotic clones with desirable traits for specific research pur-

Breeding through selection
The presence of genetic variability offers wide scope in selection of potential types. Yield is the main selection criterion. An easy approach in yield improvement is to select superior plants and subsequently developing them into clones. The major selection criteria followed in cocoa are, trees yielding 100 pods/tree/year, pods weighing 350-400 g or more with 35-40 beans having a fermented single dry bean weight of 1 g. Dry bean production is in general considered as a combination of three yield components: bean weight per pod, number of pods per tree, and number of trees per hectare. It is expected to be 1 kg and above per tree in a year, and productivity is usually assessed over 6 years in varietal trials after stabilization. At CPCRI, seven high yielding clones VTLC-1, VTLC-5, VTLC-7, VTLC-8, VTLC-9, VTLC-11, and VTLC-30 were selected and utilized as parents in the breeding programs as well as in establishment of seed gardens [16]. KAU identified seven clones CCRP 1 to 7 and released these selections for cultivation. Though individual tree selections are being made from seedling progenies, they have to be further evaluated in clonal trials for confirmation. Heritability for yield is low or average when based on single tree harvests but higher when based on yields from plots containing several trees, and so clonal varieties are gaining importance [17]. To assess the phenotypic value of genotype even in hybrid selection programs, clonal trials are advised [18]. From the clonal trials, three varieties VTLCC-1, VTLCS-1, and VTLCS-2 have been released for commercial cultivation in different agro-climatic zones in the countryn ( Table 1). Genetic analysis on 17 plant, pod, and bean characters in 44 exotic cocoa clones resulted in the selection of superior genotypes for higher performance with traits of high heritability and genetic advance. Based on the pod yield, VTLC-25, VTLC-15, VTLC-18, VTLC-36, VTLC-13, VTLC-37, and VTLC-17 have been identified as heavy bearers with an optimal canopy. These clones recorded single dry bean weight of more than 1 g, 10-15% shell, high nib recovery 85-90%, and more than 50% butter fat content making them suitable for industries as well [19,20].
In the palm-based inter cropping systems, the pod yield in general is expressed with respect to the canopy area which is mainly maintained as cone/ umbrella shaped single tier architecture. In the evaluation trials of Trinidad cocoa and Wayanad collections, 5 clones each are selected for high pod and dry bean yield ranging from 2.2 to 3.3 kg/tree/year [21,22] with an optimal canopy of 15-20 m 2 . Trait specific improvements are being taken up in the current breeding programs. A bean index of 100 beans/100 g, that is, dry bean weight of 1 g is considered as a standard for grade I beans [23], and so selections are aimed at a larger bean size of 1 g and above, which will have high butter content suitable for chocolate industry. The bean size is influenced by genotype, environment, and also the processing methods. Box, basket, and tray methods are being examined by research institutes as well as by farmers. Variation in bean indices has been observed across collections and the single dry bean weight ranged from 0.7 to 2.5 g. To improve the qualitative parameters, Criollo cocoa is used in hybridization programs [24], and white bean genotypes are being evaluated for quality parameters. Cocoa is considered as functional food, and so dark chocolates are consumed for their health benefits. Catechin, epicatechin, and procyanidines are the main polyphenols present in cocoa contributing to bitterness, astringency, and the organoleptic quality of cocoa. Cocoa beans of different clones evaluated for polyphenols and antioxidant activity exhibited distinct differences. Polyphenols ranged from 82 to 136 mg/g, procyanidin 49 to 64 mg/g, fat content of 24-55%, and antioxidant activity of 77-98% among cocoa clones studied. In general, cocoa beans with high polyphenol and procyanidin contents exhibited high antioxidant activities which are utilized for qualitative improvement [25]. Cocoa butter with free fatty acid (FFA) content of 1% or less together with acceptable flavor is the best indication of good quality beans. The type of fatty acids in 18 hybrids and 10 elite clones has been assessed, and from the profile, it is now known that 11 fatty acids are involved in the quality of cocoa beans. The fatty acids palmitic, stearic, oleic, linoleic, and arachidic acids present in all the genotypes invariably. The percentage of stearic acid is the highest in a range of 30.5% in VTLCP-7 to 44.2% in VTLCP-1. The other fatty acids differed among the genotypes in the percentage of expressio.

Breeding through hybridization
Hybrid vigor between parents showing good combining ability is readily exploited in cocoa improvement programs along with inter-population heterosis. Most commonly adopted method is developing hybrids between two distant genotypes.

Method of hand pollination and fruit set
For production of true hybrids with specific objectives and to confirm the compatibility reaction, hand pollination is routinely practiced. In artificial pollination, a flower bud, which will open the following day, recognized by its whitish color and swollen appearance, is selected. The bud is covered with a hood of plastic tube of 5 Â 1.5-2.0 cm size, which is sealed to the bark using materials like plasticine/glaze putty. The tube is covered with muslin cloth at the top, kept in place with a rubber band. Opened flowers are collected from the desired male parent, and stamens are carefully taken out by pushing the corresponding petal. One entire anther with a part of the filament is deposited on the stigma. The pollinated flowers are labeled using tin foil pieces fixed in the cushion using ball pins. The hoods are removed 24 hours after pollination, and in 3-5 days, fertilization is confirmed by the visual swelling of the ovary [26].

Progeny trials
Different cross combinations have been tried with specific objectives for development of hybrids. At CPCRI, five progeny trials have been evaluated with 76 cross combinations during 1983-1994 at Vittal, Kidu, and Kasaragod centers with objectives of more number of pods, high dry bean yield, big bean size, and drought tolerance. Of these, 20 hybrids were identified as best hybrids and further evaluated as clones. Among them, 17 progenies exhibited high vigor and cropping efficiency even at early years of development [27,28]. From the progeny trials, four hybrids VTLCH-1, VTLCH-2, VTLCH-3, and VTLCH-4 have been released as improved varieties for cultivation in the country; in 2006, the Golden Jubilee year of CPCRI, RS, Vittal and VTLCH-5 is released as Nethra Centura for the centenary year of CPCRI, Kasaragod (Tables 2 and 3).
Hybridization program at KAU was initiated during 1983 and continued up to 1993. During this period, a total of 159 parents were included to produce 239 crosses, 187 pods, and 21,819 hybrid seedlings. Nursery evaluation of these hybrids is done with HD 2 (H-height and D-diameter) value. A total of 3126 superior seedlings were field planted in series I, II, III, and IV and progeny trials I, II, III, and IV, and 163 superior hybrids were selected, utilized in various breeding program for release of hybrids, and used as better combiners in clonal gardens. Three hybrids CCRP 8, CCRP 9, and CCRP 10 with high yield and good level of tolerance to Vascular Streak Dieback (VSD) have been released as varieties for cultivation in the country ( Table 4) [24].

Seed gardens/clonal orchards
Based on the compatibility reactions, self-incompatible but cross-compatible high yielding parents are selected and multiplied as clones through soft wood grafting and established as clonal orchards or seed gardens. Bi-clonal and poly-clonal gardens were

Inbreeding
Inbreeding forms a part of the breeding activities, not only to breed parents with some degree of homozygosity for the production of hybrids but also to breed materials homozygous for desirable traits like disease resistance. Existence of selfincompatibility makes inbreeding efforts in cocoa difficult. Since few selfcompatible trees are also identified in the populations, selfing is possible but it should be continued up to six to seven generations to attain homozygosity and thereafter to be utilized for crossing to exploit the hybrid vigor. KAU has taken up this task of selfing self-compatible plants and with over 20 years of continuous effort, maintains two genotypes of S4 generation, 5 of S3, 9 of S2, and 51 genotypes of S1 (Table 5) [24,26,31,32].

Comparative yield trial (CYT)
The clones and progenies developed through selection and hybridization programs are multiplied as clones and evaluated under comparative yield trials in different situations. Under high density planting in arecanut garden, five hybrids VTLCP-6, VTLCP-20, VTLCP-15, VTLCP-1, and VTLCP-19 have been identified as best performers even in their initial years of growth [28]. Comparative study of parents and progenies as clones resulted in identification of VTLCP-6, VTLCP-2, and VTLCP-20 and parents VTLC-1 and VTLC-56 as potential high yielders [33]. In another trial, clones suitable for both arecanut and coconut canopies have been identified [34] and released as varieties. Under coconut in double hedge system of planting, hybrids VTLCP-22, VTLCP-18, and VTLCP-1 showed the best performance with an optimal canopy and high yield [35]. Evaluation of clonal and seedling progenies of selected genotypes has resulted in identification of four hybrids and two clones for multiplication both as clones and seedlings for utilization in the area expansion program [36].

Multi location trial (MLT) and demonstration plots
To assess the survival and stability of hybrids and clones in different agroclimatic conditions, multi-location trials are important. Elite clones of cocoa are under evaluation in both traditional and nontraditional states, namely Karnataka, Kerala, Tamil Nadu, Andhra Pradesh, Maharashtra, West Bengal, and Assam, for studying genotype Â location Â environment interactions. Further, 115 front line demonstration plots were established under participatory research cum

Resistance breeding
Chemical control can be effective against fungal diseases but will pollute the environment and make the cultivation expensive. Integrated disease and pest management by the use of resistant materials and cultural and biological methods is probably the best way to contain pathogens and pests in the long run for sustainable crop protection. Breeding for resistance, therefore, became the primary objective for cocoa breeders worldwide. Sources of resistance have been identified for major diseases, and since cocoa genome is sequenced, it is expected to provide models for plant pathogen interactions and also facilitate identification of resistance genes. Many cocoa pests such as mealy bugs, aphids, caterpillars, and borers are also reported in India [37] are currently being managed with chemical control measures. Screening of available germplasm for prevailing diseases, existing and emerging pests will therefore be very important in light of seasonal weather variations.

Black pod rot
India is free of most of the debilitating viral and fungal diseases known in cocoa. Since the current cocoa growing area comes under high rainfall zone and the main harvest season coincides with monsoon, incidence of black pod rot caused by Phytophthora palmivora is comparatively higher. Though the main harvest is safe guarded with systematic annual pruning, in the post monsoon period, the second season crop is still affected by pod rot. On field screening, clones have been categorized into having <10, 10-25, and >25% damage levels. In-vitro screening using isolates of P. palmivora, P. capsici, and P. citrophthora indicated that collections of Nigerian origin exhibit certain degree of tolerance [38]. Few Wayanad collections have also expressed field tolerance to pod rot when tested over three seasons. Further, 21 exotic clones collected exclusively for Phytophthora pod rot resistance was identified for utilization in cocoa hybridization program. The variety VTLCH-2, a combination of ICS 6 Â SCA 6 is found to be tolerant to black pod rot in India as well. KAU has taken up screening and hybridization program for combining desirable traits of CCRP released varieties and black pod resistance in cocoa.

Vascular streak die back
In India, vascular streak die back (VSD) caused by Oncobasidium theobromae was first reported from Kottayam, Kerala, and it began to spread to adjoining cocoa growing areas of the state. As this disease cannot be controlled effectively by the use of fungicides, KAU breeding program concentrated mainly on production of VSD resistant varieties. Hybrid seedlings were screened in the nursery by subjecting them to high inoculum load by keeping them in the midst of infected seedlings. The tolerant and vigorous seedlings were selected and established in field for evaluation. CCRP varieties have been especially released for VSD resistance and also have been utilized for establishment of clonal gardens for seedling supply [31].

Tea mosquito bug
Tea mosquito bug (TMB) (Helopeltis sp.) incidence became severe in the recent years in summer and post monsoon seasons. Helopeltis antonii, H. theivora, and H. bradyi are reported on cocoa in South India. Insect population is influenced by many factors like temperature, humidity, water stress, condition of cocoa tree, etc. The development and use of mirid resistant cocoa varieties is one of the alternatives to chemical control and resistance studies in cocoa have mostly concentrated on assessment of field damage [39]. Damage on flushes, cherelles, and pods of individual trees and different grade levels of infection on cherelles and pods are assessed to work out the TMB tolerance among genotypes. Penetrometer readings for determining the hardness of sclerotic layer, thickness at primary and secondary furrows of pod husk have been recorded in 100 cocoa genotypes and interpreted with reference to insect resistance [40]. Mechanism of plant resistance to insects is a complex phenomenon. Plant attractiveness to some extent affects the level of infestation, antixenosis prevents feeding, while antibiosis disturbs the pest development, and finally cocoa tolerance is assessed from the ability of a tree to contain damage and recover from it. Red colored pods with the smooth surface have been identified as tolerant to TMB damage among Wayanad collections.

Low moisture stress
Cocoa plants are susceptible to environmental conditions especially temperature and drought and considerably influences the pod yield [41]. Cocoa is very sensitive to water scarcity and undergoes a period of low moisture stress for 5-6 months in its current growing condition in India. Detailed study on climate change and weather variability over 43 years (1970-2012) at Vittal, which is located between 12°15 0 N latitude and 75°25 0 E longitude, showed 38% yield variability in cocoa [42]. The trends of temperature increase are +0.4°C for mean maximum (P < 0.001) and +0.4°C for mean minimum during the last decade. Breeding for drought tolerance is unique to our country and is taken up with systematic screening of available germplasm as well as hybridization programs. Screening of accessions is conducted for physiological parameters like stomatal resistance, chlorophyll fluorescence, proline accumulation under stress and by studies on seed germination under low osmotic potential, etc. A total of 216 cocoa genotypes have so far been screened for physiological and biochemical parameters under different trials [43]. In all these studies, field measurements were taken during unstressed (October) and stressed (March) conditions. Few Nigerian collections have been identified as drought tolerant and used for hybridization with high yielding Malaysian collections under two progeny trials. Two hybrids VTLCH-3 and VTLCH-4 have been released as varieties suitable for cultivation under water limited conditions in the country. Studies on leaf morphology, stomatal behavior, water relation components, and biochemical factors indicated that thick leaf, higher wax content, efficient stomatal closure, and high tissue elasticity are responsible for better adaptation of cocoa plants to drought conditions. The application of chlorophyll fluorescence as a tool to screen cocoa for drought tolerance has been confirmed with a series of genotypes. Recently, photosynthesis, chlorophyll fluorescence, and water potential under stress and nonstress conditions were estimated in 11 genotypes from different geographical origins, Columbia, Brazil, Peru, Mexico and Ecuador [44]. Seasonal and varietal differences were found, and transpirational water loss was found to be reduced with increased stomatal closure, which is considered as a favorable drought trait in any crop. Among the 52 new introductions, five Amazon and Pound collections have been found to be adaptable to water limited conditions [45] with high yields, which will be further utilized in the breeding program. Genotypic differences for morphophysiological criteria, potential antioxidant enzymes, and biochemical components depicting drought tolerance in young seedlings were determined with cocoa clones and hybrids under controlled low moisture stress conditions [46][47][48]. From these trials, standards and thresholds for several physiological parameters related to cocoa were established.
Hadley [49] detailed the visual estimates of physiological traits in cocoa, and the morpho-physiological parameters include measurements of flowering, fruiting, cherelle wilting, leaf flushing, branching and pruning intensity, canopy shape, density, and light transmission on different point scales. In order to understand and elucidate the optimum canopy shape and structure of cocoa, different spacing and canopy sizes have been studied at CPCRI [50], which showed significant differences in crop yield. In an experiment with grafts, the photosynthetically active radiation (PAR) and light interception varied significantly over the years with two spacings (2.7 Â 2.7 m and 2.7 Â 5.4 m) and three canopy sizes (small, medium, and large), and similar results were noticed with transpiration rate and stomatal conductance [51]. It is important to note that the maximum leaf area should be maintained, self shading of leaves should be avoided, and pruning should be done to the extent of retaining 20-30 leaves/developing pod to ensure the yielding potential of the genotype. With an annual pruning of single tier canopy, fertilizer dose of 100:40:140 g NPK in two splits with 20 L of water as drip is being practiced in maintenance of field gene bank under arecanut and coconutbased cropping systems.

Biotechnology and bioinformatics
DNA fingerprinting with RAPD markers has been done earlier on 76 collections, and the clones VTLC-11, 67, and 83 and VTLC-93 were identified as highly divergent. DNA extraction protocol of cocoa with fully expanded but soft leaves is standardized with the modified SDS method. Recently, 16 SSR primers specific to cocoa were used to assess diversity in 44 exotic clones, and both morphological and molecular diversity were assessed in detail [52]. An attempt has been made to identify the markers for drought sensitivity by utilizing susceptible and tolerant parents and progenies of cocoa [53]. About 75% of the genomic data of cocoa is available in the public domain which has paved the way for analyzing genes related to specific needs. CPCRI hosts one of the Agri Bioinformatics center under the Department of Information Technology and through bioinformatics tools, proteins involved in drought tolerance, Phytophthora resistance, and carotenoid biosynthetic pathways have been analyzed, and databases, CocoaESTdb, CocoaSTRESSdb, and a Standalone EST microsatellite mining and analysis tool (SEMAT) have been developed [54][55][56][57][58][59][60].

Future prospects
Cocoa improvement has attained a positive phase with the sequencing of its genome. Identifying genes responsible for incompatibility and disease resistance is the main concern of geneticists and molecular biologists. Expression of genes for resistance and quality parameters and their validation with trait specific germplasm is very important for future cocoa improvement program. Possible use of inbred lines will be taken up. Development of early selection, detection, and diagnostic methods for resistance will enable rapid screening of plant material and permit preselection activities. Because of the health benefits of dark chocolates, biochemical constituents and antioxidant properties of cocoa are to be given greater attention in the breeding programs. Farmers participatory plant breeding, in-situ conservation of land races, exploitation of flavor components from genotypes belonging to specific geographic region, varieties for changing climatic conditions, and environment-friendly management strategies will be considered. Adaptability of cocoa genotypes in traditional and nontraditional zones should be verified, and location specific varieties should be developed [61]. At the national level, expansion of cocoa cultivation with the quality planting material of elite clones, collaborative approach between research institutes, universities, state horticulture departments, and developmental agencies are required. At the international level, participation of India in cocoa genetic resources networking and regional breeding groups of both developed and developing countries is important. Collaboration of India with Asia Pacific regional countries, Malaysia, Indonesia, Philippines, Vietnam, and Papua New Guinea is essential with their common coconut-based cropping systems with known pest and disease problems. This will enhance region specific sustainability of cocoa cultivation.

Author details
Subbian Elain Apshara ICAR-Central Plantation Crops Research Institute-CPCRI, Vittal, Karnataka, India *Address all correspondence to: elain_apshara@yahoo.co.in © 2019 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.