Effects of Foliar Application of Melatonin on Head Yield and Quality of Broccoli cv. Palam Samridhi

Broccoli [Brassica oleracea (L.) var. italica Plenk] is a relatively new vegetable for many Indians. It belongs to family Brassicaceae. Broccoli is an Italian word from the Latin brachium, meaning an arm or branch. The term sprouting as used in sprouting broccoli refers to the branching habit of this type, the young edible inflorescences often being referred to as sprouts. Broccoli has gained attention because of the struggles on patenting genotypes with high concentrations of glucosinolates showing positive effects in cancer treatment (Wolf et al., 2014). The sprouting broccolis are thought to have originated from the eastern Mediterranean then introduced into different parts of the world (Owis, 2015). The United States is the world’s largest producer of broccoli. The flowers of broccoli are borne on a faceted floral shoot so that the inflorescence terminates the axis of the plant. The inflorescence, which has been described as a corymb, a corymbose panicle or a modified racemose panicle, consists of functional floral buds, perfect flowers, stem and bracts. At the time of harvesting, the inflorescence is a growing, faceted axis bearing a large number of immature, stalked flowers, floral buds and varied bracts which are smaller and simpler in form than the vegetative leaves. The bracts are absent from the terminal portion of the inflorescence (Buck, 1956; Gray, 1982).

Melatonin (C13H16N2O2) is a low molecular weight organic compound ubiquitously available in almost all living organisms from bacteria to mammals and considered as the nature’s most versatile biological signal (Pandi-Perumal et al., 2006). Chemically, melatonin is N-acetyl-5-methoxytryptamine, an indolic compound derived from serotonin (5-hydroxytryptamine). Both biogenic amines are synthesized from the amino acid tryptophan through a well characterized  biosynthetic pathway (Arnao and Hernández-Ruiz, 2014; Lee et al., 2015). Phytohormone IAA (indolyl-3-acetic acid) bears some resemblance to melatonin since both are indole compounds and have a common biosynthetic pathway through the tryptamine  in  the  tryptophan  dependent  IAA  biosynthetic  pathway.Melatonin has been reported to involve in regulating plant growth and development and especially play critical role (s) in plant defense systems (Weeda et al., 2014). The present investigation was carried out toinvestigatethe effectof foliar application of melatonin on improving head yield andquality attributes of broccoli.

The investigation was carried out at the Division of Basic Sciences and Humanities, Faculty of Horticulture, SKUAST-Kashmir during the year 2017 and 18. One month old uniform and healthy seedling of broccoli cv. Palam Samridhi was collected from the nursery, Division of Vegetable Science (SKUAST-K) and transplanted at a spacing of 45 × 45 cm in a well prepared field. Different concentrations of melatonin (Mel) viz 0, 20, 40, 60 and 80 ppm were sprayed through hand spray on the foliage of plants at 15 days after transplanting (DAT). One plot constituted one sample unit and each treatment consisted of four plots with 6 plants per plot.

Head diameter (at spherical) was measured by measuring tape then average of five plants was computed to get the mean head polar diameter. Head diameter (at spherical) was measured by measuring tape then average of five plants was computed to get the head equatorial diameter. After harvesting, five broccoli head were weighed at pan balance and average individual head weight was computed in terms of grams. Total yield of broccoli head sprouts from each plot was taken in kilogram and then, it was also converted to yield in quintal per hectare.

For total soluble sugar 100 mg of dried sample was hydrolysed with 5.0 ml of 2.5N HCl by keeping it in a boiling water bath for 3 hours. After cooling at room temperature the content was neutralized with solid sodium bicarbonate until effervescence ceases and volume was to 100 ml. Centrifuged the content at 500 g for 10 minutes and supernatant was collected. The amount of total soluble sugars was estimated by phenol sulphuric acid method (Dubois et al., 1956) colorimetrically. Extraction of protein was done by grinding 0.2 g of sample with 0.1 M phosphate buffer (ph 7.5) in a mortar and pestle. Centrifuge it   and use the supernatant for protein estimation (Bradford 1976). Bovine serum albumin (BSA) was used as protein standard. Total phenol was determined by spectrophotometeric measurement of blue colured complex by the reaction of phenols with phosphomolybdic acid in Folion Ciocalteur in alkaline medium (Bray and Thorpe, 1954). The total anti-oxidant capacity was measured by the method described by Prieto et al., (1999). The assay is based on the reduction of Mo (VI) to Mo (V) by the extract and subsequent formation of green phosphate/Mo (V) complex at acid pH. Malondialdehyde (MDA) content generated as product of lipid peroxidation in the leaves of broccoli was estimated in different treatments according to Cakmak and Horst (1991). Leaf sample of 0.12 g were ground in 1.2 ml 0.1% (w/v) trichloroacetic acid (TCA), then centrifuged at 12000 g for 10 min. Then 0.3 ml 0.5% (w/v) thiobarbituric acid (TBA) was added to 0.3 ml of the supernatant. The resulting mixture was boiled at 100°C for 20 min. The reaction was stopped by placing the reaction tubes in an ice bucket. The absorbance values were measured at 532, 600 and 450 nm. The interference of soluble sugars in the samples at A₅₃₂ and A₄₅₀ was corrected by subtraction. The MDA content (μmol g^-1 FW) was calculated according to the formula:
 

MDA (µ mol/g FW) = 6.45 × (A₅₃₂ - A₆₀₀) - 0.56 × A₄₅₀

 
Where,
A532, A600 and A450 represent the absorbance of the mixture at 532, 600 and 450 nm, respectively.

The leaf membrane stability index (MSI) was determined according to the method of Premachandra et al., (1990) as modified by Sairam (1994). Leaf discs (100 mg) were thoroughly washed in running tap water followed by washing with double distilled water. Thereafter, the discs were heated in 10 ml of double distilled water at 40oC for 30 minutes. Then electrical conductivity (C₁) was recorded by EC (Electrical Conductivity) meter. Subsequently, the same samples were placed in a boiling water bath (100^oC) for 10 minutes and their electrical conductivity was also recorded (C₂). The MSI was calculated as:
 

Membrane stability index (MSI) = [1- (C₁/C₂)] × 100

 
The data recorded on various variables were statistically analysed by using technique of analysis of variance and significance was determined as given by Panse and Sukhatme (1967).

Physical characters of broccoli head
 
Perusal of the data (Table 1) indicated that broccoli plant sprayed with 0 ppm melatonin (control) showed the minimum polar as well as equatorial diameter of 7.1 cm and 6.5 cm, respectively. However, there was significant improvement in polar as well as equatorial diameter of broccoli when sprayed with different doses of melatonin and Mel 60 ppm resulted in maximum diameter of 11.6 cm and 10.4 cm, respectively. The polar and equatorial diameter recorded with Mel 80 ppm (10.7 cm and 9.5 cm) was found at par with Mel 60ppm. Mel 40ppm resulted in the second highest head diameter (10.1 cm and 8.1 cm) followed by 9.7 cm and 6.9 cm polar and equatorial diameter in Mel 20 ppm, respectively. Results further indicated that maximum individual head weight and yield (240 g and 1.9 kg/plot) was recorded with Mel 60ppm which was at par with Mel 80 ppm (233.8 g and 1.8 kh/plot). The next highest head weight and yield (221.5 g and 1.7 kg/plot) was recorded with Mel 40 ppm followed by Mel 20 ppm (208.1 g and 1.6 kg/plot) against the minimum head weight and yield (199.9 g and 1.5 kh/plot) in Mel 0 ppm (control), respectively.



Enhanced head size, weight and yield in Mel treated broccoli may be attributed to improved photosynthesis, plant growth and vigour (Mansha Gul et al., 2018, 2021) that might have resulted in a better development of source leaves which synthesized and supplied more photosynthates to the sink for the formation of broccoli heads. Similar trend has also been noticed by Sermenli et al (2011) for main head size in broccoli.
 
Food reserves and pigmentation
 
Findings of the study (Table 2) showed that application of Mel 80 ppm resulted in maximum levels of sugar and protein contents (129.0 and 88.0 µg/ml), respectively of broccoli sprouts. Mel 60ppm was established as the second best treatment in terms of sugar and protein content (125.4 and 81.1 µg/ml) followed by 40 ppm (118.2 and 79.5 µg/ml) and 20 ppm (111.3 and 73.8 µg/ml) of Mel compared to minimum sugar and protein contents (105.0 and 68.0) in control. Perusal of the data again pointed out that amount of both chlorophyll and carotenoid present in head of control plants (0.31 and 0.012mg/gFW) was better when sprayed with different concentrations of melatonin wherein Mel 60ppm was established as the most superior treatment and produced the highest amount of chlorophyll and carotenoid content (0.79 and 0.028 mg/gFW). Mel 80 ppm was proved as the next superior treatment in producing these compounds (0.64 and 0.021 mg/gFW). Mel 40 ppm and Mel 20 ppm followed the above treatments in order and resulted in chlorophyll and carotenoid content of 0.55 and 0.017 and 0.42 and 0.014 mg/g.FW, respectively.



An elevated level of fruit sugar and protein contents has been reported by Debnath et al., (2018) in tomato. Yang et al., (2019) stated that melatonin induced significant accumulations of fructose, glucose and sucrose in apple through the down regulation of FRK2 expression, a gene encoding fructokinase (FRK).

Increased amount of chlorophyll due to foliar application of Mel may be attributed to its role in delaying Chl breakdown and simultaneously accelerating its de novo synthesis (Szafranska et al., 2017). Tal et al., (2011) stated that melatonin can also preserve chlorophyll content in plants by the radical rummaging activity of indoleamine of melatonin. Melatonin-induced increase in carotenoid content has been attributed to the expression of genes related to carotene biosynthesis (Sun et al., 2020). Results of the present study that increasing chlorophyll contents also showed a similar trend with respect to carotenoid also conforms the findings of earlier research (Ranmalbhai, 2014).
 
Antioxidant and membrane characters
 
Analysis of different antioxidant and membrane characters (phenol, total antioxidant, MDA and MSI) of broccoli sprouts under the influence of foliar spray of Mel (Table 3) indicated that the Mel 60 ppm resulted in highest value of phenol content in broccoli sprouts (178.2 mg/100 g DW) compared to the lowest value (151.9 mg/100 g DW) in control. The highest value of phenol in Mel 60 ppm was followed by Mel 80 ppm (170.1 mg/100 g DW), Mel 40 ppm (160.9 mg/100 g DW) and Mel 20 ppm (160.9 mg/100g DW). Data regarding total antioxidant showed that Mel 80ppm resulted in highest value of antioxidant (365.4 mg GAE 100 g DW). However, the values of antioxidant recorded with Mel 40 ppm (358.2 mg GAE 100g DW) as well with Mel 60 ppm (362.3 mg GAE 100 g DW) were found at par with Mel 80 ppm. These values were significantly followed by Mel 20 ppm (336.8 mg GAE 100 g DW) while as untreated plants showed the minimum antioxidant value (319.7 mg GAE 100 g DW) than all other treatments. Perusal of the data further revealed that the amount of malondialdehyde (MDA) measured in the broccoli sprouts was found minimum (0.12 µ mol MDA/g FW) with Mel 60ppm which was also at par (0.13 µ mol MDA/g FW) with the plants treated with Mel 80 ppm. The minimum MDA value obtained was followed significantly in ascending order by Mel 40 ppm (0.16 µ mol MDA/g FW), Mel 20 ppm (0.18 µ mol MDA/g FW) and maximum (0.21 µ mol MDA/g FW) in control. Measurement of antioxidant potential in terms of membrane stability index (MSI) revealed that Mel 80 ppm exhibited highest MSI (46.22%) which was found at par with MSI value of 45.38% under Mel 60 ppm treated plants. However, these values were significantly followed by Mel 40 ppm (39.56%) and Mel 20 ppm (32.22%) while the least value of MSI (27.16%) was recorded with control.



The application of exogenous melatonin alleviated reactive oxygen species and cell damage induced by means of repairing mitochondria (Sharif et al., 2018). Melatonin is known to play an important role in the detoxification of reactive oxygen and free radicals and functions as an antioxidant in living organisms. As a broad-spectrum antioxidant, melatonin can directly eliminated ROS and the subsequent products, its derivatives, AFMK (N1-acetyl-N2-formyl-5-methoxyknuramine) and AMK (N1-acetyl-5-methoxykynuramine), can also scavenge ROS and further terminate the cascade reaction of lipid peroxidation. Thus, one molecule of melatonin may eventually scavenge ten molecules of radicals at least. Melatonin treatment can markedly decreased the content of ROS and thus alleviate oxidative damages induced by the excessive ROS accumulation (Huang et al., 2019).

Foliar application of Mel (@ 60 ppm) at 30 days after transplanting resulted in significant improvement in head diameter and head weight. This treatment also resulted in highest chlorophyll and carotenoid contents in broccoli sprouts. However, total sugar and protein contents of broccoli sprouts were significantly highest with Mel 80 ppm.A significantly highest level of phenols and MSI were evident with Mel 60 ppm while as the level of total antioxidants was at par with Mel 60 and 80 ppm. An inverse trend was recorded in case of MDA level of broccoli sprouts.