Selection of Promising Candidate of Malabar Tamarind [Garcinia gummi-gutta (L.) Roxb.]- a Multipurpose Fruit Tree

ABSTRACT Garcinia gummi-gutta is valued for its dried fruit rind used as a souring agent in traditional cuisine and for the extraction of natural hydroxy citric acid (HCA) in the formulation of anti-obesity nutraceuticals. The species is widely distributed in Kerala State but is not yet an economically profitable crop in the region. Promising germplasm screening based on the fruit mass traits of 180 trees was performed leading to the selection of 35 candidates plus trees (CPTs) characterized by the fewest fruits to weigh 1 kg (i.e., the largest fruits) with two fruit bearing seasons. Fruit mass data of 35 CPTs showed significant (P < .001) variations and highest fresh fruit mass (1091.27 kg), and dry rind mass (48.18 kg) recorded in CPT 6 and is 40-fold higher mass than mean fruit mass (25.4 kg) of 180 trees. Multivariate analysis and web diagram-based ranking supported selection of CPT 6 as a promising candidate tree. In the tree improvement programs through nutraceutical enrichment, the superior germplasm selected (CPT 6) in the present study can use effectively.


Introduction
Malabar Tamarind [Garcinia gummi-gutta (L.) Roxb.; Family Clusiaceae], is a large tree characterized by a dark green canopy with horizontal or drooping branches (Maheshwari, 1964). Dried fruit rind of G. gummi-gutta is a valuable resource and a Non-Timber Forest Product (NTFP), popularly known as Kudampuli or pot tamarind (Kumara and Santhosh, 2014). This large polygamodioecious tree is endemic to Southern Western Ghats of Peninsular India (Parthasarathy et al., 2013) and Sri Lanka (Orwa et al., 2009). Malabar tamarind is a well-accepted souring agent in traditional cuisines of South India, and thus in demand in the region (Sumayya et al., 2016). During the monsoon (mid of May to August), villagers collect ripened fruits to extract rind, and in the traditional way of processing, rinds are smoke-dried after removing pulpy seeds. The processing imparts brownish-black color and augments the flavor of the rind. Thereby the resource serves as the source of income to households of midlands and coastal areas in the Kerala State, South India.
An anti-obesity property of Malabar tamarind has been reported in the past due to richness of organic acid, namely hydroxy citric acid (HCA) in the fruit rind and fruit pulp (Lewis and Neelakantan, 1965). The HCA is a potent inhibitor of ATP citrate lyase activity; which thereby reduces the availability of Acetyl CoA units for fatty acid synthesis (Sethi, 2011), thus it effectively controls obesity. Because of this property, interest in the fruit rind has increased, especially in the last couple of decades, this was evident in the emergence of several nutraceutical formulations based on natural HCA extracted from the fruit rinds of G. gummi-gutta. In addition to HCA, the fruit rind contains other phytoconstituents viz., xanthones, garcinol, ascorbic acid, and flavonoids, which also increase its demand for organized cultivation of this tree crop. Previous records suggest a high degree of diversity exists in G. gummi-gutta, even from the same geographic area (Anilkumar et al. 2002;Joseph et al., 2007). Maintenance of diversity and improvement of the crops are primarily governed by the identification of individuals from hermaphrodite trees (Maheshwari, 1964), which takes 10-15 years to reach maturity (Joseph et al., 2014). Garcinia gummi-gutta is a cross-pollinated tree, and natural regeneration occurs only through seeds. These limitations make it challenging to apply conventional crop improvement practices to G. gummi-gutta. However, it is possible to apply horticultural techniques to bypass the long gestation period and to multiply identified superior trees by practicing softwood grafting methods (Nazeema, 1992;Nazeema et al., 1993). However, this approach is often delayed due to the unavailability of good-quality rootstocks, which limits the mass production of superior plants. In the tree selection process, multiple parameters are often employed to capture the important economic traits of the tree. Such an approach necessitates the selection of superior germplasm from the natural stands to ensure rapid gains on yield and quality of the crop.
The present study sought to identify the best combinations of phenotypes among Malabar tamarind accessions; based on fruit mass gain traits and total acidity. Selected superior germplasm, therefore, can be conserved and appraised for the cloning and tree improvement programs aimed at nutraceutical enrichment.

Selection of CPTs
Garcinia gummi-gutta (L.) Roxb. is a semi-domesticated tree crop grown extensively in the homesteads of Kerala for its fruits. The tree is easy to locate because of its characteristic conical shaped, dark green, and thick canopy. An extensive exploration survey was conducted during June -August 2018 and 2019 throughout Kerala State as a mission to select elite trees in the region. During the survey, 180 fruit-bearing trees having 20-35 years old were visited. Annual fruit yield per tree, no. of fruits required to weigh 1 kg were determined as the primary assessment of yield parameters and harvesting months of each accession in a year were included. Average precipitation data for each geographic location was collected from the report repository of India Water Resources Information System (IWRIS, https://indiawris.gov.in/wris/#/DataDownload). Trees showing a higher fruit mass than the mean of 180 trees were considered for the selection of Candidate Plus Trees (CPTs). Accordingly, 35 trees were selected as CPTs and assigned an accession code (Gar 1 to 35) based on their GPS location (Table 1).

Determination of Fruit Mass Traits
The approximate age of the tree was recorded as per the response of villagers and the diameter (cm) at breast height (DBH) of the tree was measured. Growth characters such as the number of main branches, the total number of primary branches (PB), sub-branches (SB), the total number of fruits/ branch (TNF/b) were recorded (Table S1). Fruit mass characters such as total number of fruit/tree (TNF/t) per season (total number of fruits per branch × total number of branches), the number of fruits per kilogram weight (NFkg), fruit mass per tree per year (kg), total no. of fruits per tree, no. of fruits weighing for 1 kg and rind mass per tree per year (kg) of CPTs were estimated.

Determination of Metric Fruit Characters
Quantitative characters of fruits traits, such as single fruit weight, rind weight, rind thickness, seed weight, fruit length and fruit diameter, nipple length and width, no. of ridges, fresh rind weight, dry rind weight and rind moisture content, no. of seeds and aborted seeds, were determined on 30 randomly selected fruits from each CPTs. This data was collected using an electronic balance, digital Vernier calliper and scale (Table S2). The fruit length: diameter ratio was used to estimate fruit shape HT-Habitat type (Ll -low land and Ml -midland), DBH-Diameter at breast height, NF/kg-Number of fruits required to weigh 1 kg, FM/t/y-Fruit mass/tree/year, DRM/t/y-Dry rind mass/tree/year and MP-Mean precipitation index (FSI). The rind: seed ratio by weight (R:S) was also determined. Rind dry mass was assessed as dry: fresh rind weight. The percentage of fresh rind (FR %) and dry rind (DR %) was calculated as rind mass/total mass × 100. The real fresh rind value, RFRV as %FR x fresh rind mass/100 and real dry rind value, RDRV as % DR × dry rind mass/100 were also estimated (Soloviev et al., 2004;Van den Bilcke et al., 2014).

Determination of Acid Value
Total acid was extracted by heating 25 g dried fruit rind along with 50 ml distilled water taken in a beaker and placed in a pressure cooker (121°C, 15psi) for 20 mins, cooled and filtered. The pressure cooker-based extraction was repeated twice for getting maximum extract. Combined supernatants were decolorized by treating with 1 g activated charcoal. After filtration, it was concentrated to 25 ml in a using a rotary evaporator (Rotavap, Superfit™ PBU 6, Mumbai, India) under reduced vacuum. Pectinaceous materials were removed by treating with 50 ml ethanol and centrifuged at 5000 rpm for 5 mins (Hermle Z 36 HK, Wehingen, Germany). Under reduced pressure, the supernatant was concentrated and the pale-yellow colored residue was stored at 4°C for further use. Acid extract (10 ml) was measured and taken in an Erlenmeyer flask and one or two drops of phenolphthalein was added. It was titrated against 0.1 N sodium hydroxide from a burette. The end point was the appearance of a permanent pale pink color (Gauri, 1966;Horwitz and Latimer, 2000). The experiment was repeated for concordant burette readings. Acid value was calculated using the following formula and presented as percentage acidity.

Statistical Analysis
Data on various yield traits were statistically analyzed using SPSS Statistics version. 22 (SPSS Inc. Chicago, IL, USA) and MVSP 32 statistical software (Kovach Computing Services, Wales, UK). Oneway ANOVA was performed to determine the overall significance of variations among traits and Duncan's multiple range test (DMRT; P < .05) was followed to mean separation. In order to group the 35 CPTs on the basis of similarity/dissimilarity pattern, cluster analysis was conducted using Unweighted Pair Group Method with Arithmetic mean (UPGMA) and based on Euclidean distance, a dendrogram was plotted (MVSP software v3.22). All the measured fruit traits were further subjected to correlation analysis (MVSP software v3.22) based on Pearson's coefficient of correlation to evaluate the relatedness of various traits. In addition, a web diagram was created in XLSTAT, after screening the CPTs, following a ranking method (Paudyal and Haq, 2008) where fruit weight, rind thickness, total acidity, dry rind mass, and real dry rind value along with annual fruit and rind mass were considered for the effective selection of elite tree to rank.

Selection of CPTs
The survey identified that 35 out of 180 trees having higher fruit mass than the mean fruit mass (25.4 kg) and fewer than 35.7 fruits per kg. Similar strategies have been used to assess the productivity of certain domesticated fruit crops like Theobroma cacao L. (Jagoret et al. 2017), Phoenix dactylifera L. and Ficus carica L. (Goldschmidt, 2013). The 35 best trees designated as CPTs, and their various attributes duly recorded (Table 1). A high tree to tree intraspecific variation even within the same geographical zone has been previously reported for G. gummi-gutta (Abraham et al., 2006;Parthasarathy and Nandakishore, 2014). The phenomenon is also known in other less known fruit trees like Prunus amygdalus L. (Khadivi et al., 2019), Adansonia digitata L. (Simbo et al., 2013), Nephelium lappaceum L. (Muhamed et al., 2019) and Scelorcarya birrea (Leakey et al., 2005a;Leakey et al., 2005b;Leakey, 2005c). Geographic variation in fruit mass and morphometric traits between sites are not significant. Our records on the natural distribution of G. gummi-gutta suggest that the highlands, coastal plains, and river banks are suitable habitats, as the most of the CPTs selected from low land and midland of Kerala (Table 1). However, the trees from river banks have been reported to be more productive than trees growing in other habitats (Orwa et al., 2009). The occurrence of this tree in canal bunds is very common and is considered as one of the preferred habitats. These trees are popularly known as 'thottu puli' in the regional language Malayalam (thottu means canal, puli means sour fruit). In the case of two other underutilized edible fruit trees viz., Calamus meghalayensis (Becc.) A.J. Hend. and Prunus undulata Buch.-Ham. from Eastern Himalayas, the excellent fruit mass of trees on river banks has been explained by water availability and nutrientrich soil (Rymbai et al., 2016). Apart from its distribution in marginal lands, G. gummi-gutta is maintained in home-steads, especially in low lands (altitude <10 m asl) of rural and semi-urban areas. Tree distribution in lowlands, especially in Alappuzha district was higher than in other regions surveyed. Trees ranging 20-35 years-old, recorded remarkable fruiting (Table 1). Conversely, beyond 50 years, it is noticed that the number of fruits, as well as fruit size, was lower, perhaps due to reduced age-related fertility. Unlike other accessions, Gar 6 has two harvesting seasons, that is, June-August (monsoon type) and October-December (winter type). Year round harvesting property was reported for tropical fruit trees like Morinda citrifolia (Lin et al., 2014), Persea americana (Hurtado-Fernández et al., 2018) and Ficus carica (Mühlbauer and Müller, 2020), even though no species of Garcinia exhibited year round fruit bearing nature. The normal ripening and harvesting period of the fruits are confined to southwest monsoon period (June to August) in Kerala. An annual rainfall of 2515.73 mm (23.34% excess than mean rainfall) in 2018 and 2309.8 mm (12.72% excess than mean rainfall) in 2019 were recorded in the state. It was the highest precipitation among the decade (https://mausam.imd. gov.in/). However, Gar 6 gained the lowest seasonal rainfall during 2018 and 2019 (Table 1). The dried fruit rinds are economically important as Garcinia andamanica (Bohra et al., 2021), the winter-type harvesting, moderately lesser precipitation, and bi-seasonal bearing natures of Gar 6 facilitate fast and efficient drying process of fruit rind.

Analysis of Fruit Mass Traits and Metric Characters of CPTs
Recorded data on metric fruit traits were statistically significant (P < .001) except for rind: seed ratio (Table 2). Among the collections, Gar 6 was superior in fruit mass, dry rind mass and had significantly fewer of fruits per 1 kg (Table 1). However, biochemical and metric fruit traits also contribute significantly to the attributes of promising germplasm as evident from the report on Garcinia mangostana (Mulyono et al., 2021) and Garcinia atroviridis (Bayu et al., 2018). Of these, total acidity is of prime importance as it largely determines the acceptance of Malabar tamarind in both industrial processing for the extraction of natural HCA and in various traditional cuisines of South India. The highest percentage of total acidity was found in Gar 7 and Gar 22 (Table 2). The total acidity of Gar 6 (24.96%) exceeded the mean of the 35 CPTs (23.73%), but previous reports indicated that the total acidity of Gar 6 was two times higher (Jayaprakasha and Sakariah, 1998;Karo-karo et al., 2019). In addition to acid content, accession Gar 6 showed superiority on some metric fruit traits, such as single fruit weight and real fresh rind value. Fruit rind thickness is an important trait that contributes to dry rind mass/tree/year, and also recognized as desirable in local markets, achieving a higher price than normal quality dry rind. Rind thickness varied significantly (P < .001) among the collections and was highest in Gar 3 followed by the Gar 6 ( Table 2). Fruit weight and rind thickness recorded in Gar 6 seems to be higher than in earlier reports (Abraham et al., 2006;Bohra et al., 2021;Shameer et al., 2016). Some of the metric traits, such as rind mass, percentage dry rind, real dry rind values, were high in Gar11 (Table 2). However, irrespective of its rind mass, real fresh rind value was high in Gar 6, indicating that rind thickness positively contributes to the rind value. Total number of fruits per tree was also high in Gar 6 (Table S1), as were traits, such as fruit diameter, fresh weight of rind, lesser number of aborted seeds per fruit (Table S2). Similar variations in fruit quality traits have been observed in other fruit trees like Cordia myxa (Sivalingam et al., 2012) and Arbutus unedo (Colak, 2019).

Correlation Analysis
Significant correlation was observed between some morpho-metric traits of the 35 accessions. Such associations between quality traits has been found important when screening germplasm in crops like Garcinia kola (Dah-Nouvlessounon et al., 2016), Jatropha curcas (Singh et al., 2016) and Picea mariana (Lenz et al., 2017). In the present study, Pearson's coefficient of correlation revealed that tree age is related to fruit mass parameters but not to DBH, which was not significantly correlated to number of fruit morphological traits (Table 3). This finding is in agreement with a report on Tamarindus indica L. (Van den Bilcke et al., 2014). However, tree age was significantly correlated to total number of fruits per tree, which is a prime factor determining the productivity of trees, such as apple (Forshey and Elfving, 1989). Total acidity was found to be independent of all other parameters recorded. A significant positive correlation between fruit morphological traits and fruit mass was noticed (Table 3). So, these traits can be used as a predictor of fruit production per tree and selection for better quality traits (Ugese et al., 2010;Van den Bilcke et al., 2014).

Selection of Promising Candidate Tree
The selection of 'elite' G. gummi-gutta trees was conducted by ranking the 35 CPTs based on commercial perspectives, as has been done for Garcinia mangostana (Syahputra et al., 2021), Mauritia flexuosa (Dos Santos Dias et al., 2017), Vitex doniana (Hounkpèvi et al., 2016), and for traits like pulp mass (Buchmann et al., 2010) and commercialization value (Tchoundjeu et al., 2008). Seasonal fruit mass analysis in the present study revealed that the number of fruits per 1 kg, mean fruit mass and dry rind mass were four times greater in Gar 6 than in Gar 3 and, 40fold greater that the average of the 180 trees (Table 1). Values of number of fruits per 1 kg, dry rind mass (Table 1), single fruit weight, rind thickness, total acidity, rind: seed ratio and real fresh rind values were superior to Gar 6 ( Table 2). Analysis and ranking, therefore, made Gar 6 the highest ranked accession for a combination of desirable qualities as depicted in the web diagram ( Figure 1). Generally, it is very difficult to achieve high yields by selecting high yielding clones of any plant species because of GxE interactions (Leakey, 2012;Leakey and Akinnifesi, 2008). Therefore, it is important to select for quality traits as reported (Leakey et al., 2004) in Dacryodes edulis and Irvingia gabonensis.

Conclusion
CPTs of Malabar tamarind collected from diverse localities of Kerala State, India showed significant variations in fruit mass traits and other metric traits. Based on the superiority of seasonal fruit yield, total acidity, fruit metric traits, and dual seasonal bearing nature, Gar 6 was ranked as a promising candidate tree among the collected accessions. Morphological and pomological qualities of elite G. gummi-gutta can be used for industrial extraction of HCA. Thus, selected promising germplasm can be used for the multiplication both through conventional and biotechnological tools.