Effects of Grafting on Volatile Compounds in Bitter Gourd Fruit

Ziji LIU, Yu NIU, Renbo YU, Yan YANG

Abstract [Objectives] This study was conducted to explore the effects of grafting on volatile compounds in bitter gourd fruit.

[Methods] The volatile compounds and relative contents of grafted and non-grafted fruit were analyzed by headspace solid phase micro-extraction with gas chromatography-mass spectrometry.

[Results] There were 59 volatile compounds in Haiyan No.2S， including six unique compounds. There were 58 volatile compounds in Haiyan No.2J， including five unique compounds.

[Conclusions] This study provides a scientific basis for further analysis of bitter gourd flavor regulation mediated by grafting.

Key words Bitter gourd; Grafting; Volatile compounds; Headspace solid phase micro-extraction （HS-SPME）; Gas chromatography-mass spectrometry （GC-MS）

Received： April 8， 2022  Accepted： June 10， 2022

Supported by Hainan Science and Technology Project （No.ZDYF2020229）; Scientific Research Project of Key Laboratory for Quality Regulation of Tropical Horticultural Plants of Hainan Province （No.HNZDSYS（YY）-03）.

Ziji LIU （1982-）， male， P. R. China， researcher， devoted to research about genetic breeding of vegetable.

*Corresponding author. E-mail： catasvegetable@163.com.

The bitter gourd （Momordica charantia L.） is an annual trailing herb belonging to the Cucurbitaceae family[1-2]. As one of the main vegetable crops grown in the winter in Hainan Province and transported to the north， bitter gourd is not only rich in nutritional value， but also have hypoglycemic action[3-4]， anti-tumor[5] and anti-inflammatory effects due to its pharmacologically active ingredients[6-7].

Headspace solid-phase microextraction （HS-SPME） has been successfully applied to the analysis of volatile flavor compounds in plants such as melon[8]， plum blossom[9]， onion[10]， olive[11] and selaginella[12]. Continuous cropping obstacles and soil-borne diseases are the main factors restricting the high-yield and high-efficiency production of bitter gourd. Grafting is an effective method to improve the yield and stress resistance of bitter gourd[13]. There are few reports on the effect of grafting on the flavor substances of bitter gourd at home and abroad. In this study， the differences in the types and relative contents of volatile flavor compounds in the fruit of non-grafted and grafted plants of Haiyan No.2 bitter gourd were determined by solid-phase microextraction-gas chromatography-mass spectrometry， aiming to provide a scientific basis for the study of flavor regulation of bitter gourd.

Material and Methods

Materials

The bitter gourd variety tested was Haiyan No.2， and the rootstock material was Haizhen No.1 white-seed pumpkin. Both were provided by Vegetable Research Institute of Hainan Academy of Agricultural Sciences. The non-grafted plants were marked as Haiyan No.2S， and the grafted plants were marked as Haiyan No.2J. The materials were planted in the test base of the fifth team of the Chinese Academy of Tropical Agricultural Sciences at the end of October 2019， and the same cultivation and management measures were adopted. Each material was harvested in mid-March 2020， and five pieces of fruit with uniform maturity and size were homogenized for the determination and analysis of volatile substances.

Methods

Instruments

HP6890/5975C gas chromatograph-mass spectrometer （GC-MS） was purchased from Agilent， USA. The manual solid-phase microextraction device was purchased from Supelco， USA， and the extraction fiber head was 2 cm-50/30 μm DVB/CAR/PDMS StableFlex.

Determination of volatile flavor substances

First， a 10 g of mixed sample was added into a 10 ml sampling bottle of solid phase microextraction instrument， which was inserted into a manual injector equipped with a 2 cm-50/30 μm DVB/CAR/PDMS StableFlex fiber head， and extracted with heating at 60 ℃ on a heated plate for 50 min. Then， the extraction head was removed and immediately inserted into the injection port of the gas chromatograph （temperature 250 ℃）， followed by thermal desorption for 5 min.

The chromatographic column was FLM FB-5MS （30 m×0.25 mm×0.25 μm） elastic quartz capillary column， the temperature of which was held for 2 min at 37 ℃， which was then increased at 3 ℃/min to 160 ℃， which was then increased at 6 ℃/min to 202 ℃. The separation and determination conditions were as follows： run time 40 min， temperature of the vaporization chamber 250 ℃， high-purity He （99.99%） as the carrier gas， pre-column pressure 6.89 psi， flow rate of carrier gas 1.0 ml/min， splitless injection， solvent delay time 1 min， EI ion source， ion source temperature 230 ℃， quadrupole temperature 150 ℃， electron energy 70 eV， emission current 34.6 μA， multiplier voltage 1 565 V， interface temperature 280 ℃， and mass range 29-500 amu.

Data processing

The peaks in the total ion chromatograms were retrieved by the mass spectrometry computer data system and checked against the standard mass spectra of Nist 14 and Wiley 275 to determine the components of volatile substances， and the relative content of each component was determined by the peak area normalization method.

Results and Analysis

It can be seen from Fig. 1 and Table 1 that Haiyan No.2S and Haiyan No.2J contain a total of 64 kinds of volatile substances， including 15 kinds of alcohols， 7 kinds of ketones， 5 kinds of esters， 18 kinds of aldehydes， 13 alkanes， and 6 other components.

Analysis of volatile components in Haiyan No.2S

It can be seen from Table 1 that Haiyan No.2S contained 59 kinds of volatile substances， accounting for 98.92% of the total content of chromatographic effluent components. Among them， there were 15 alcohols with a relative content of 56.92%， and the relative content of myrtenol is the highest. There were 6 kinds of ketones， with a relative content of 5.15%， and the relative content of 1-penten-3-one was the highest， followed by 3-pentanone. There were 16 kinds of aldehydes with a relative content of 26.28%， and （E）-2-hexenal had the highest relative content.   Four kinds of esters were determined with a relative content of 0.51%， and the relative content of （Z）-3-hexenyl acetate was the highest. There were 12 kinds of alkanes with a relative content of 6.58%， and the relative content of cimonene was the highest. Six other components had a relative content of 3.48%， and the relative content of 2-ethylfuran was the highest.

Analysis of volatile components in Haiyan No.2J

It can be seen from Table 1 that Haiyan No.2J contained 58 volatile substances， accounting for 96.63% of the total content of chromatographic effluent components. Among them， there were 12 alcohols with a relative content of 52.08%， and the relative content of myrtenol was the highest， followed by （Z）-3-hexenol. There were 6 kinds of ketones with a relative content of 4.66%， and the relative content of 1-penten-3-one was the highest， followed by 3-pentanone. There were 17 kinds of aldehydes with a relative content of 28.08%， and the relative content of （E）-2-hexenal was the highest， followed by hexanal. There were five kinds of esters with a relative content of 1.27%， and （Z）-3-hexenyl acetate had the highest relative content. There were 12 kinds of alkanes with a relative content of 7.61%， and the relative content of cimonene was the highest. Six other components had a relative content of 2.93%， and the relative content of 2-ethylfuran was the highest， followed by 2-amylfuran.

Analysis of unique volatile components in Haiyan No.2S and Haiyan No.2J

There were obvious differences in the types and relative contents of volatile substances in Haiyan No.2S and Haiyan No.2J （Fig. 2）.  Haiyan No.2S contained six unique volatile components， namely 1-propanol， 4-hepten-1-ol， heptanol， 6-methyl-5-hepten-2-one， octanal and tridecane. Haiyan No.2J contained five unique volatile components， namely geranylacetone， 3-methyl-2-butenal， citral， 2-hexen-4-olide and 2-propenyl-cyclopentane.

Conclusions and Discussion

Volatile aromatic substances include alcohols， aldehydes， ketones， esters and sulfur-containing compounds， and these volatile components interact to form the flavor of bitter gourd. In this study， the differences in the contents of volatile flavor substances between the fruit of non-grafted and grafted plants of Haiyan No.2 bitter gourd cultivar were analyzed. Haiyan No.2S and Haiyan No.2J identified a total of 64 volatile flavor compounds. Haiyan No.2S included six unique ingredients， and Haiyan No.2J included five unique ingredients. Comprehensive comparison showed that there were significant differences in the amounts of volatile substances and the relative contents of various volatile flavor substances between the fruit of non-grafted and grafted plants. It might be due to the improved root vigor of grafted plants， which enhanced the ability of roots to absorb water and nutrients， improved the transport and distribution of assimilates in grafted plants， and coordinated the transport and synthesis of plant hormones， which thereby adjusted plant vegetative growth and fruit development and other indicators to affect the fruit flavor quality[14]. The establishment of flavor quality is a complex process， and how grafting regulates the metabolism of volatile flavor substances in bitter gourd needs to be further analyzed.

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