Eluciadation of the Plant Morphological and Biochemical Characterization of Bhela (Semecarpus Anacardium L.): An Underutilized Plant of Tropics

Semecarpus anacardium L. is a potential underutilized edible, highly nutritious fruit crop with ample medicinal properties grown in some localized pockets of India. Being a hardy crop, it can be easily used for climate resilient horticulture adaptation. But due to inadequate knowledge it is remains in underused position. Therefore the investigation was carried out to study the morphological and biochemical characteristics of the plant which will help in further improvement of the crop. The plant followed quadratic growth curve in different vegetative characters and leaf chlorophyll in both the years. Positive correlation was observed in different vegetative characters with different weather parameters during rst year whereas in second year negatively correlation was recorded with sunshine hours only. The vegetative growth almost ceased during winter season, slow to moderate growth during summer and rapid growth was noticed from rainy to autumn season during experimentation. Leaf chlorophyll content followed an increasing trend during April to November and whereas a decreasing trend from December-March. It bears only terminally in older shoot from May to June with very lower fruit set and retention yield. The ripened fruits of Bhela showed high (23.94 ºbrix) TSS, total sugar, protein (21.08%), total carbohydrate, crude fat (34.91%) and food energy value (445.43 kcal/g). The observed performance of the crop with regards to plant morphology, growth rate and fruit quality was indicative for commercial exploitation in future. was found statistically signicant in 1st year, 2nd year and pooled data. Results showed that it was increased throughout the experimental period and reached at peak after rainy season in autumn (1.49, 1.52 and 1.50 mg/g). In case of monthly variation (Table it was noticed that leaf chlorophyll was decreased from December to March (1.31–1.29, 1.38–1.36, 1.35–1.32 mg/g) in 1st year, 2nd year and of pooled data. After that it was increased from April to November (1.31–1.50, 1.38–1.52 and 1.34–1.51 mg/g) in 1st year, 2nd year and pooled data respectively. and negatively with sunshine hours (r=-0.14). Plant spread (E-W) had high and positive correlation with trunk girth (r canopy volume (r and leaf chlorophyll (r = 0.96). Plant spread (E-W) was positively correlated with different weather parameters like average temperature (r = 0.30), relative humidity (r = 0.58), total rainfall (r = 0.41) and negatively with sunshine hours (r=-0.18). Trunk girth was positively and highly correlated with canopy volume (r = 0.75) and leaf chlorophyll (r = 0.77). Trunk girth was positively correlated with different weather parameters like average temperature (r = 0.21), relative humidity (r = 0.56), total rainfall (r = 0.43) and negatively with sunshine hours (r=-0.24). Canopy volume was highly and positively correlated with leaf chlorophyll (r = 0.96) and positively correlated with different weather parameters like average temperature (r = 0.31), relative humidity (r = 0.59), total rainfall (r = 0.40) and negatively with sunshine hours (r=-0.19). Leaf chlorophyll had positive correlation with different weather parameters like average temperature (r = 0.35), relative humidity (r = 0.75), total rainfall (r = 0.56) and negatively with sunshine hours (r=-0.37).


Introduction
Neglected and wild edible fruits were the important sources of food for mankind before dawn of civilization. The tribal groups inhabit in the forests depend on these fruits. They passed on valuable information on utility of fruits from generation to generation. Under estimation of their potential use, non-availability of their complete botanical information, inadequate research on their commercial exploitation, lack of knowledge on their food and nutrition potentiality and fast disappearance of ecosystem and habitat destruction make them remains as an underutilized (Dandin and Krishna, 2016). These crops are locally marketed after harvesting. They are not only medicinally and nutritionally rich but also thrive well under adverse climatic conditions. It can be the considered as a potential source of several desirable traits particularly for resistance to biotic and abiotic stresses. To recover the present crisis of nutritious food supply due to increasing population rate, exploitation of neglected horticultural crops can become a solution for malnutrition and hidden hunger problem (Dandin and Krishna, 2016). Bhela (Semecarpus anacardium L), commonly known as 'Marking nut' or 'Oriental cashew', an important underutilized plant species (family Anacardiaceae) with a boon of medicinal and nutritional properties grown in sub-Himalayan region, tropical and central parts of India and western peninsula of East Archipelago, Northern Australia.
Medical industries extensively utilized its medical properties like antiatherogenic, antiin ammatory, antioxidant, antimicrobial, hypoglycemic, anticarcinogenic etc (Semalty et al., 2010). The crop improvement programme mainly depends on the superior mother plants for hybridization and the knowledge of inheritance pattern. Characterization of germplasm is essential to maximize its utility on yield and quality traits, as well as descriptions of available variation and estimates of trait heritability and encourage the development of e cient ex-situ conservation (Rubenstein et al., 2006, Anumalla et al., 2015, Hammer et al., 2003and De Vicente et al., 2006. Phenotypic characters (Mostly quantitative) are mainly in uenced by environments and plant developmental stages and genotypic character is based on the quality of the plant materials which is required for testing the varieties. In this study, an effort has been made to generate and identify Bhela plants with special emphasis on morphological and biochemical traits which is necessary for effective crop improvement programmes. Comprehensive information regarding variability as required by the breeder for evaluation and characterization of various traits, to enhance its productivity and commercial value is lacking. The best efforts were put in describing the underutilized plant taking into consideration all the important morphological and biochemical traits.

Experimental details
The experiment was done with 4 (four) seasonal treatments viz. -Winter, Summer, Rainy and Autumn and 5 ( ve) replications in 5 years old plant.

Observation recorded
In the present study a total of 59 morphological (32 qualitative and 27 quantitative) characters, which correspond to the different plant characteristics and plant parts, including leaves, owers and in orescences, fruits, nut, and overall architecture have been established. The qualitative characters are follows as-type of planting material, tree habit, tree nature, foliage density, leaf shape, leaf apex shape, leaf base shape, dots on leaf, leaf pubescence, leaf brittleness, young leaf colour, mature leaf colour (above and beneath), young shoot colour, in orescence position, bearing habit, type of in orescence, in orescence shape, petal colour, ower fragrance, pedicel surface, pedicel thickness, pulp texture, pulp taste, mature apple skin colour, apple shape, apple surface, attachment of nut to apple, nut shape, nut base shape, harvesting time, productivity status and biotic stress susceptibility. Quantitative characters which are observed are-tree height, trunk girth, tree spread, canopy volume, leaf length, leaf breadth, leaf petiole length, curvature of the secondary veins to the midrib, internodal length, start of owering, end of owering, owering duration, number of owers/plant, days taken from owering to fruit maturity, fruit set, fruit retention, number of fruits harvested per plant, fruit yield, fruit weight, fruit length, fruit breadth, apple weight, apple length, apple breadth, nut weight, nut length and nut breadth. Different characters which were observed were presented pictorially in Fig. 7 The total soluble solids (°Brix) content of fruit was determined with the help of a hand refractometer calibrated at degree Brix (0 to 32oBrix) and the values were corrected at 20oC with the help of a temperature correction chart (Mazumdar and Majumder, 2003).

Titratable acidity
Five grams of fresh fruit pulp was dissolved in fty ml of distilled water by macerating in mortar with pestle. That was then ltered and ten ml of the ltrate was titrated against (N/10) NaOH solution using 1% phenolphthalein as an indicator and was expressed in percentage in terms of anhydrous citric acid (Rangana, 1977).
Titratable acidity = (Titre value X Normality of alkali X Volume made up X Equivalent weight of citric acid X 100) / Weight of sample X Aliquot X 1000.

TSS-Acidity ratio
TSS-Acidity ratio was calculated by dividing the mean value of TSS by the mean value of titratable acidity.

Total sugar and reducing sugar
The total sugar and reducing sugar content were estimated with the help of freshly made mixture containing equal volumes of Fehling's solution A & B by copper reducing method (A.O.A. C, 1984) using methylene blue as an indicator and was expressed in percentage (Mazumdar and Majumder, 2003).

Non reducing sugar
Non-reducing sugar content of fruits were determined by subtracting the value of reducing sugar content from that of the total sugar and multiplying the value with 0.95 and was expressed in percentage (Mazumdar and Majumder, 2003).
Non-reducing sugar = (total sugar -reducing sugar) X 0.95 2.2.2.6 Sugar-Acidity ratio It was calculated by dividing the mean value of total sugar by the mean value of titratable acidity.

Ascorbic acid
Ascorbic acid content was estimated by using 2,6-dichlorophenol indophenols dye which was reduced by ascorbic acid to a colourless form (Rangana, 1977). Five grams of fruit pulp was grounded in a mortar with 25 ml of four percent oxalic acid, lter through Whatman no. 1 lter paper and the ltrate was collected in a fty ml volumetric ask and the volume was made up to fty ml with four percent oxalic acid. Five ml of the ltrate was taken with ml of four percent oxalic acid and was titrated against standard 2,6-dichlorophenol indophenols dye to a pink end point. Ascorbic acid was estimated following the method of A.O. A.C. (1984).
Ascorbic acid (mg / 100g pulp) = (Titre value X Dye factor X Volume made up / Aliquot of the of the extract Weight or volume of sample) X 100 2.2.2.8 Protein Protein content of the fruits was estimated based on the principle that the blue colour developed by the reduction of the phosphomolybdic-phosphotunstic components in the Folin-Ciocalteau reagent, purchased from SRL (Mumbai, India), by the amino acids tyrosine and tryptophan present in the protein. The colour developed by the biuret reaction of the protein with the alkaline cupric tartarate were measured colorimetrically at 660 nm (Lowry, 1951) and expressed in percentage.

Crude fat
Crude fat content of fruit was estimated based on the principle that ether is continuously volatilized, condensed and then allowed to pass through the sample to extract ether soluble materials. It was estimated by Soxhlet apparatus (Pelican Socsplus-SCS 04 R) using thimble size 25 mm X 80 mm. and expressed in percentage (AOAC, 1965).

Crude bre
Crude bre of fruits was estimated by the principle that a moisture-free and ether extracted sample is digested rst with a weak acid solution, then with a weak base solution. The organic residue was collected in a lter crucible. The loss of weight on ignition is crude bre and expressed in percentage (Sadasivam and Manikam, 1992).

Total ash content
Ash content of fruits was estimated by ignite the sample at 600 o C to burn off all organic material using mu e furnace following the method of AOAC (1965). It was expressed in percentage.

Total carbohydrate
Total carbohydrate of fruit was estimated based on the principle that carbohydrate is rst hydrolysed into sugars using dilute hydrochloric acid. In hot acidic medium glucose is dehydrated to hydroxymethyl furfural. This compound forms a green coloured product with anthrone, purchased from Merck (Mumbai, India), which was measured colorimetrically at 630 nm (Sadasivam and Manikam, 1992) using UV/Vis spectrometer and expressed in percentage.

Total phenol
Total phenolics of fruits was estimated based on the principle that phenols react with an oxidizing agent phosphomolybdate in Folin-Ciocalteu reagent under alkaline conditions and result is the formation of a blue coloured complex, the molybdenum blue which is measured at 650nm colorimetrically by UV/Vis spectrometer (Perkin Elmer, Lambda 25) and expressed as mg per 100g fruit pulp (Thimmaiah, 2004).

Statistical analysis
Analysis of variance (one way classi ed data) for each parameter was performed using op stat software (online version) and SPSS software. The statistical analysis was done by following completely randomized design (CRD) as per Gomez and Gomez (1984). The signi cance of different sources of variation was tested by error mean square by Fischer-Snedecor's 'F' test at probability level of 0.05 percent.
If the treatment regarding a parameter was found signi cant, Critical Difference (CD) value was calculated to compare the different treatments.
where, e.d.f = Error degrees of freedom.

Trend Analysis
Trend analysis was done using SPSS software.

Correlation coe cient (r)
Correlation coe cients for all possible combinations were worked out utilizing the 1st and 2nd year data over two years as per Al- Jibouri et al. (1958).
Where, r (x1, x2) = correlation between x1, x2 V (x1) = variance of x1  Data on leaf shape of the plants were recorded visually on mature leaf as obovate to oblong and presented in Table 2. Poornima et al. (2013) also found oblong elliptic leaf shape in Semecarpus species. The report also nds support from Semalty et al. (2010).
3.1.1.7 Leaf base shape The shape of leaf base was noted visually on mature leaf as obtuse (Table 2).

Dots on leaf
Data pertaining to the dots on leaf presented in Table 2 showed absence of do on mature leaf (Table 2).

Leaf pubescence
Data pertaining to leaf pubescence presented in Table 2 revealed that it was sparsely present on lower part of mature leaf. Ghosh et al. (2017), Poornima et al. (2013) and Semalty et al. (2010) observed similar result (more or less pubescent beneath) in bhela. Brittleness of matured leaf was recorded as brittle and presenting in Table 3.

Young leaf colour
In case of young leaf, the leaf colour on both upper and lower surface was observed visually (Table 3)  3.1.1.12 Mature leaf colour (above and beneath) Mature leaf colour was presented in Table 3 and revealed that it was deep green above and pale green beneath on mature leaf (Table   3). Mature leaf colour in bhela is also noticed by Ghosh et al. (2017).

Young shoot colour
From the data represented in Table 3 revealed that the colour of young shoot was light green.
3.1.1.14 In orescence position Data on position of in orescence in the plants showed occurance of in orescence on older shoot (Table 3).

Bearing habit
From the data presented in Table 3 it was found that bearing in bhela was in terminal position of the shoot. Result was in agreement with the observation of Ghosh et al. (2017). In orescence shape was recorded after one month of owering and recorded as narrowly pyramidal (Table 4).

Petal colour
Data pertaining to petal colour of the owers was recorded as dull greenish yellow with purple inner base and presented in Table 4.
Similar result was also noticed by Semalty et al. (2010).

Flower fragrance
From the data presenting in Table 4 it was found that ower fragrance is mild in bhela.

Pedicel surface
Data on Pedicel surface of mature fruit was observed as moderately rough (Table 4). The thickness of pedicel was found to be thick on mature fruit (Table 5).

Pulp texture
Data regarding pulp texture was recorded as soft on ripe fruit and presented in Table 5. Premalatha (2000) and Ghosh et al. (2017) also reported similar pulp texture in Semecarpus anacardium.

Pulp taste
Data presented in Table 5 in respect to pulp taste was observed as sweet on ripe fruit. Ghosh et al. (2017) noted sweet taste of pulp in Semecarpus species.
3.1.1.24 Mature apple skin colour Observation on mature apple skin colour was recorded at peak harvesting stage as orange and presented in Table 5. Similar result was also reported by Motirauji (2012) and Ghosh et al. (2017).

Apple shape
Data pertaining to apple shape presented in Table 5 recorded as 'U' shaped at peak harvesting stage. Motirauji (2012) also noticed similar apple shape in Semecarpus anacardium. Observation regarding to apple surface was recorded at peak harvesting stage and presented as rough and dull in Table 6. Ghosh et al. (2017) also recorded apple surface of mature bhela fruit.

Attachment of nut to apple
Data on attachment of nut to apple was recorded at peak harvesting stage and revealed that nuts are tightly attached (Table 6) with the apples.
3.1.1.28 Nut shape Data pertaining to nut shape was recorded as broadly kidney shaped on mature nut (Table 6). Results are in agreements with the nding of Premalatha (2000).
3.1.1.29 Nut base shape From the Table 6 it was found that shape of nut base was obliquely ovoid on mature nut.  Table 7 in respect to time of harvesting exhibited peak harvesting was done between 3rd week of November to 1st week of December. This observation regarding time of fruit harvesting was in accordance to Ghosh et al. (2017).

Productivity status
Productivity status of the bhela plants was observed as low at the time of harvest (Table 7). It might be due to lower age of the plants which are just start to produce fruits.

Biotic Stress Susceptibility
Only few visible sign of leaf disease (burning of leaf tip and edges with mostly upward curling) was recorded in Semecarpus anacardium during the experimental period and grouped as 1 (Table 7).  Table 8. Results showed that plant height increased in all the seasons and reached peak in autumn after rainy season during two consecutive years and also in pooled data (3.02, 3.14 and 3.08 m). All the data regarding plant height was statistically non-signi cant in four seasons in both the year and also in pooled data. In case of monthly variation ( 1st year and pooled data and from July in case of 2nd year the height was increased in a rapid rate and reached in to a peak in October (3.02 m, 3.14 m and 3.08 m). After that it was again remain stable in November in both the year.

Trunk girth
Data recorded on trunk girth of bhela under different seasons presented in Table 8     year, 2nd year and pooled data respectively.

Leaf breadth
Data pertaining to leaf breadth presented in Table 13  3.1.2.9 Leaf petiole length Data on leaf petiole length presented in Table 13 exhibited 5.87 and 5.85 cm leaf petiole length in 1st year and in mean data respectively. Pal (2011) reported 1.2-3.8 cm petiole length in Semecarpus species.  (Table 14). Pal (2011) also observed 12-25 pairs of main nerves making a wide angle with the coasts in Semecarpus ancardium leaf.

Internodal length
Data in respect to internodal length was presented in Table 14. Result showed that it was 3.91 cm in both the year and mean data also.  Ghosh et al. (2017) and Motirauji (2012) also recorded owering initiation time in bhela.

13 End of owering
The data regarding time of end of owering was recorded in the 2nd week of June (Table 15) in both the year of investigation. Motirauji (2012) also observed owering time in bhela.

14 Flowering duration
The data presented in Table 15 revealed that owering duration continued for 39 days in both 1st year and 2nd year. The present result is in con rmity with the ndings of Ghosh et al. (2017) in Semecarpus species.   (Rajan, 2012). Improper pollination due to adverse weather also affects the fruit set in bhela.

Fruit retention
Result opined to percentage of fruit retention (Table 17) was revealed that it was 32 %, 32.42 % and 32.21 % in 1st year, 2nd year and mean data respectively. Lower retention in fruit might be due to fact that higher temperature causing altered hormonal status in the fruit which in turns affects the abscission zone (AZ) which ultimately leads shedding of fruit (Roemer et al., 2011).

Number of fruits harvested per plant
The data pertaining to number of fruits harvested per plant was presented in Table 17 and it was noted higher in 2nd year (734.80) as compared to 1st year (431.92). Increasement in number of fruit was might be due to increasing age of the plant.

Fruit yield
Observation in respect of yield per plant (Table 17) was revealed that yield in Semecarpus anacardium was increased in 2nd year (10 kg/plant) as compared to 1st year (5.87 kg/plant). It was might be due to increasing age of the plant which resulted in to harvesting of more number of fruits and yield was gradually increased. Fruit yield in bhela plant was also reported by Motirauji (2012). The data presented in Table 18 clearly revealed that fruit length varied from 12.72 to 12.78 g during the experimental period. The mean fruit weight was found to be 12.76 g. Results is in conformity with the ndings of Deshmukh et al. (2010).

Fruit length
Observation in respect of fruit length (Table 18) showed slightly higher value in the 2nd year (4.94 cm) as compared to 1st year (4.88 cm). The results are in agreement with the observation of Motirauji (2012).

Fruit breadth
Fruit breadth presented in Table 18 revealed slightly higher value in the 2nd year (5.10 cm) as compared to 1st year (5.06 cm). Similar result was also reported by Motirauji (2012).

Apple length
The data presenting in Table 19 clearly opined that apple length varied from 2.69 to 2.72 cm during the experimental period. The mean fruit length was found to be 2.71 cm.

Apple breadth
The data on apple breadth (Table 19) revealed slightly higher value in the 2nd year (2.78 cm) as compared to mean data (2.77 cm).
The results are in agreement with the ndings of Motirauji (2012).

Nut weight
Nut weight was recorded (Table 19) slight higher value in the 2nd year (3.92 g) as compared to 1st year (3.89 g). Deshmukh et al. (2010) and Motirauji (2012) also found similar nut weight in Semecarpus anacardium.

Nut length
The data presented in Table 19 clearly revealed that nut length varied from 2.19 to 2.22 cm during the experimental period. The mean nut length was found to be 2.21 cm. Results is in conformity with the ndings of Motirauji (2012).

Nut breadth
Observation regarding nut breadth was recorded (Table 19) slightly higher value in the 2nd year (2.32 cm) as compared to 1st year (2.30 cm). Similar observation was also reported by Deshmukh et al. (2010). Vegetative growth pattern of bhela during the years 2015-16 and 2016-17 over different months were recorded to trace the path of change in vegetative growth. Vegetative growth was examined through different linear and non-linear models (curves). Among the curves, linear, logarithmic, inverse, quadratic, cubic, exponential, compound, power, growth and sigmoid were explored and the best model was judged based on maximum R 2 and signi cance of the time coe cients. Ultimately, one best model in each case of each minor fruit crop was retained and explained.

Plant height
The trends in plant height of bhela during 2015-16 (Fig. 1a) showed a 2nd degree equation. The quadratic model (curve) ts the best.
For 2016-17 (Fig. 1b) the trend also showed a 2nd degree equation where quadratic model (curve) t the best (Table 20). The trends in plant spread (N-S) of bhela during 2015-16 (Fig. 2a) showed a 2nd degree equation. The quadratic model (curve) ts the best. For 2016-17 (Fig. 2b) the trend also showed a 2nd degree equation where quadratic model (curve) t the best (Table 21). The trends in plant spread (E-W) of bhela during 2015-16 (Fig. 3a) showed a 2nd degree equation. The quadratic model (curve) ts the best. For 2016-17 (Fig. 3b) the trend also showed a 2nd degree equation where quadratic model (curve) t the best (Table 22). The trends in trunk girth of bhela during 2015-16 (Fig. 4a) showed a 1st degree equation. The quadratic model (curve) ts the best.
The trend also showed a 1st degree equation in 2016-17 where quadratic model (curve) t the best (Table 23 and Fig. 4b). The trends in canopy volume of bhela during 2015-16 (Fig. 5a) showed a 2nd degree equation. The quadratic model (curve) ts the best. The trend also showed a 2nd degree equation where quadratic model (curve) t the best (Table 24 and Fig. 5b) during the year 2016-17. The trends in leaf chlorophyll of bhela during 2015-16 (Fig. 6a) showed a 2nd degree equation. The quadratic model (curve) ts the best. The trend also showed a 2nd degree equation in 2016-17 where quadratic model (curve) t the best (Table 25 and Fig. 6b). The data presented in Table 26  highly correlated with canopy volume (r = 0.96) and leaf chlorophyll (r = 0.93). Trunk girth was positively correlated with different weather parameters like average temperature (r = 0.02), relative humidity (r = 0.34), total rainfall (r = 0.08) and sunshine hours (r = 0.09). Canopy volume was highly and positively correlated with leaf chlorophyll (r = 0.95) and positively correlated with different weather parameters like average temperature (r = 0.15), relative humidity (r = 0.38), total rainfall (r = 0.13) and sunshine hours (r = 0.19). Leaf chlorophyll had positive correlation with different weather parameters like average temperature (r = 0.20), relative humidity (r = 0.59), total rainfall (r = 0.35) and sunshine hours (r = 0.02).

Conclusions
From the results it is concluded that weather parameters in uenced heavily on both vegetative and reproductive characters of the underutilized fruit crop (bhela). It provides fundamental basis for the classi cation based on vegetative, in orescence and fruit physico-chemical characteristics. The performance of crop with regards to productivity and fruit quality was quite satisfactory and indicative for commercial exploitation in future. This information can be applied in crop improvement, proper conservation and better use of this underutilized plant species. It is an essential work before carrying out comprehensive molecular studies. Accordingly, the results also provide baseline information on S. anacardium which will help to select superior germplasms of the crop.

Declarations
Fund The authors did not receive support from any organization for the submitted work.
Con icts of interest The authors declare that they have no con ict of interest.
Availability of data and material All the data analyzed during the study are available from the corresponding author on reasonable basis.    General view of the bhela plant in the experimental plot