(Tiểu luận) morphological and biochemical diversity vietnameses basil (ocimum sp ) germplasm under conventional production

VIETNAM GENERAL CONFEDERATION OF LABOUR TON DUC THANG UNIVERSITY FACULTY OF APPLIED SCIENCES Biotechnological Research Project MORPHOLOGICAL AND BIOCHEMICAL DIVERSITY VIETNAMESE'S BASIL (OCIMUM SP.) GERMPLASM UNDER CONVENTIONAL PRODUCTION Advisor: Ph D PHAM THI THU HA Student: VAN PHAM NHU NGOC ID Number: 618H0045 Class: 18H60302 Course: 2018-2022 HO CHI MINH CITY, 2021 ii CONTENT LIST OF CONTENTS LIST OF TABLE………………………………………………………………….ii ABBREVIATIONS……………………………………………………………….iv CHAPTER INTRODUCTION……………………………………………….1 1.1 Introduction ………………………………………………………………… 1.2 Main objective …………………………………………………………………2 1.3 Signification……………………………………………………………………2 CHAPTER OVERVIEW OF RESEARCH………………………………… 2.1 Subspecies…………………………………………………………………… 2.2 Distribution……………………………………………………………………3 2.3 Chemical components in basil…………………………………………… … 2.4 Morphological diversity of basil plants………………………………… … 2.5 Biochemical diversity……………………………………………………… CHAPTER MATERIALS AND METHODS……………………………… 3.1 Materials…………………………………………………………………… 3.1.1 Plant materials……………………………………………………………8 3.1.2 Equipment…………………………………………………………… 3.2 Methods………………………………………………………………… … 3.2.1 Morphological diversity……………………………………………… …9 3.2.1.1 Morphological variation for colour scale development………………9 3.2.1.2 Morphological variation for yield traits………………………………9 3.2.2 Biochemical diversity…………………………………………………… 3.2.2.1 Plant extraction……………………………………………………… iii 3.2.2.2 Gas Chromatography Coupled To Continuous Mass Spectrometry… 3.2.3 Cluster Analysis………………………………………………………… 10 CHAPTER EXPECTED RESULTS……………………………………… 11 4.1 Output …………………………………………………………………… 11 4.1.1 Grouping basil by color scale………………………………………… 11 4.1.2 Grouping according to agronomic characteristics…………………… 11 4.1.3 Evaluate the biochemical diversity of basil varieties through GC/MS results……………………………………………………………… .11 4.1.4 Making a grouping diagram based on two 3.2.1.1 and 3.2.1.2 experiments, evaluating the morphological diversity of basil varieties in Vietnam………… 11 4.2 Work plan ………………………………………………………………… 11 CHAPTER 5: CONCLUSIONS AND RECOMMENDATIONS ………… 12 REFERENCES ……………………………………………………………… 13 iv ABBREVIATIONS GC/MS Gas Chromatography Coupled To Continuous Mass Spectrometry EO Essential Oil DNA Deoxyribonucleic acid CHAPTER INTRODUCTION 1.1 Introduction Common basil (Ocimum basilicum L.) is one of the most essential aromatic perennial herbs belonging to the Lamiaceae (Labiatae) family (Pistelli et al 2020; Ha et al., 2021) It comprises around 150 species of herbs and shrubs from the tropical and subtropical regions of Asia, Africa, and Central and South America (Barley et al., 1924) However, most of their taxa are essentially based on leaf morphology and color, which frequently depend on environmental conditions ( Gautam et al., 2009) For instance, leaves of the best-known species, O basilicum, and of its close relatives vary from small and ligniform to large and round and colors vary from yellow-green to grey-green, to red, or to almost black Different species and forms of Ocimum species vary in growth habit, color, and aromatic composition, making the true botanical identity of basil difficult (Chavan and Nikam, 1982) Several types of basil essential oils produced from different cultivars or chemotypes of O basilicum are of considerable importance in the international trade market (Avetisyan et al., 2017) Essential oils extracted from Ocimum have been reported to possess interesting therapeutic and biological properties by various researchers from the world over There are significant differences in the chemical composition and amounts and kinds of aromatic components in the essential oils of basil depending on the species/variety and environmental conditions of the cultivation locations (Mehta and Mehta,1943) The differences in the oil composition could be due to several factors such as climatic conditions, geographical locations, stage of development, processing of plant material, extraction method, drying, and storage conditions The Reunion type from Comoro Islands, Thailand, Madagascar, and Vietnam is characterized by its high concentrations of methyl chavicol (Dambolena et al., 2010) The still existing uncertainty in the classification within the genus depends on the fact that species identification relies on morphological characters whose expression is known to be affected by developmental and environmental factors ( Carovic-Stanko et al., 2011) To assist in classification a system of standardized descriptors based on volatile oil has been proposed by Lawrence (1993) and Grayer et al (1996) that classified the different basil chemotypes based on the prevalent aromatic compound or the components major than 20%, respectively Thus, a chemotaxonomic approach based on the essential oil composition of O basilicum generates several discussions among experts ( Javanmardi et al., 2002) However, research on basil plants has not received much attention in Vietnam, although they contain many good biochemical properties Since cinnamon in Vietnam has not been studied much about the diversity of phenotypes and biochemical characteristics Therefore, morphological, and chemical traits as characterization features for Genbank accessions can be used in breeding programs in Vietnam 1.2 Main objective The purpose of this study is to classify basil varieties by morphology in Vietnam And use gas chromatography-mass spectrometry (GC-MS) to detect compounds in basil, thereby showing the biochemical diversity of basil varieties found in Vietnam 1.3 Signification − Expanding knowledge on the morphological and biochemical diversity of basil plants − Investigation of compounds present in different varieties of basil − The diversity of plant genetic resources provides the opportunity for plant breeders to develop new and improved cultivars with desirable characteristics, including traits preferred by farmers (potential high yield, large seeds, etc.) and traits preferred by the breeder (pest resistance and photosensitivity, etc.) − This relationship will be considered in current breeding programs to select cultivars with high health-promoting properties Document continues below Discover more from: nghệ sinh Công học CNSH01 Đại học Tôn Đức… 249 documents Go to course Atsh - Đề cương 19 Cơng nghệ sinh học 100% (5) THÍ NGHIỆM SINH 31 25 HỌC TẾ BÀO Công nghệ sinh học 100% (5) Dược động - dược động học Công nghệ sinh học 100% (4) Seminar-SHTB26 Nhóm5 đại học tơn… Cơng nghệ sinh học 100% (4) Sinh đai cương tới 27 C13 Công nghệ sinh học 100% (3) Hoa - thực vật dược 39 Công nghệ CHAPTER OVERVIEW OF RESEARCH sinh học 100% (3) 2.1 Distribution The genus Ocimum belongs to the mint family (Lamiaceae), the name may be derived from ozo, ozein means fragrant, related to the subtle aroma of the plants of this genus; or because of the word okimon, okys means fast, fast because these trees grow very fast (Van Chi Vo, 2004) There are about 150 species, distributed mainly in tropical Africa and South America Holy basil is an ancient Asian tropical plant, scattered in China, Laos, and Thailand for medicinal and spice purposes In Vietnam, the new basil is grown only in family gardens or traditional medical treatment facilities The plant prefers hot and humid tropical climates; the annual average temperature is about 25 - 30oC; rainfall 1800 - 2600 mm/year In the high mountains with a subtropical and slightly cold climate, it is not grown (Van Chi Vo, 2004) In Vietnam, the genus Ocimum has species: • Ocimum americanum L - Wild basil, American basil in Hanoi and Cam Ranh area, Khanh Hoa province • Ocimum basilicum L from Ha Giang, Hanoi, Thua Thien Hue, Ninh Thuan, Ho Chi Minh City, and Long An, and they are also grown in many places • Ocimum gratissimum L - White basil, large leaves In our country, there are encounters from Ha Giang, Son La, Lang Son, Phu Tho, Vinh Phuc, and Hanoi to Ho Chi Minh City • Ocimum sanctum L (other name O tenuiflorum L ) - Perfume purple, E purple, E red, E forest In our country, there are meetings from Hoa Binh, Bac Giang, Hanoi, Ninh Binh to Khanh Hoa, Ninh Thuan, Ho Chi Minh City to An Giang, and they are also grown in many places 2.2 Chemical components in basil Based solely on chemical composition, many characterizations have been proposed (Bernhardt et al., 2014; da Costa et al., 2015; Grayer et al., 1996; Marotti et al., 1996; Telci et al., 2006) These characterizations are based on the prevalence of one or more chemical compounds in the oil above a fixed threshold The majority of researchers take into ac- count compounds present in concentrations higher than 10% (Carovic- Stanko et al., 2010; Liber et al., 2011) or 20% of the essential oil (Grayer et al., 1996; Labra et al., 2004) Different chemotypes have also been classified based on their geographical origin, each with a specific chemical composition The European chemotype (Italy, France, Bulgaria) is characterized by linalool and methyl chavicol (estragole), as its main compounds The Tropical chemotype is rich in trans-methyl cinnamate and originates from India, Guatemala, and Pakistan The Reunion chemotype from Thailand, Madagascar, and Vietnam have high concentrations of methyl chavicol, while the chemotype from Russia and North Africa is eugenol-rich (Vernin and Metzger, 1984) Several essential oils and aroma compounds found in different basil lines, methyl chavicol, methyl cinnamate, citral, eugenol, linalool, and camphor are traded in the international essential oil markets (Caln-Snchez et al., 2012) The content and chemical composition of the Basil essential oil have been the subject of many studies The yield from different plant parts varies between 0.2-1.9% with the main components being linalool, methyl chavicol, eugenol, and methyl cinnamate, as well as 1,8-cineole, methyl eugenol, geraniol, geranial, neral, and α-bergamotene (Marotti et al.,1996; Labra et al., 2004; Sakkas and Papadopoulou, 2017) The essential oil of O basilicum obtained by distillation is used in several food products as a flavoring agent and is also used in perfumery thanks to its aromatic characteristics It contains cineol, pinene, methyl chavicol, d-camphor, and ocimene (Eltohami, 1997) The major aroma constituents of basil are 3,7-dimethyl-1,6-octadien-3-ol (linalool; 3.94 mg/g), 1-allyl4methoxy benzene (estragole; 2.03 mg/g), methyl cinnamate (1.28 mg/g), 4-allyl-2methoxyphenol (eugenol; 0.896 mg/g), and 1,8-cineole (0.288 mg/g) (Lee et al., 2005) The chemical composition of O basilicum essential oil (EO) differs according to the season These essential oils have oxygenated monoterpenes (60.768.9%), followed by sesquiterpene hydrocarbons (16.024.3%) and oxygenated sesquiterpenes (12.014.4%) Around 29 compounds representing 98.099.7% of the oil composition have been reported by Hussain et al.1 Linalool is the main constituent of essential oils (56.760.6%), followed by epiα-cadinol (8.61.4%), α-bergamotene (7.49.2%), γ-cadinene (3.35.4%), germacrene D (1.13.3%), and camphor (1.13.1%) In addition, components like methylchavikol, methylcinnamat, linolen, eugenol, camphor, cis-geraniol, 1,8-cineole, α-bergamotene, βcaryophyllene, germacrene D, γ-cadinene, epi-α-cadinol, and viridiflorol have been reported as important components (Tomaino et al., 2010) 2.3 Morphological diversity of basil plants The large morphological variations in the wide range of annual and perennial herbs and shrubs in the genus Ocimum (Carović-Stanko et al., 2010; Simon et al., 1990) are mainly due to geographic differences, polyploidy, interspecific hybridization, and generic description changes ( Nurzyńska-Wierdak, 2007; Paton et al., 1999) Basils vary in many characteristics including vigor, shape, plant height, branching, pubescence, leaf size, leaf shape, leaf texture, leaf color, leaf dimension, plant color, flower color, flowering time, flavor, and aroma (Marotti et al., 1996; Morales et al., 1993; Simon et al., 1990, 1999; Singh et al., 2002) recognizes more than 150 species in the genus However, most of their taxa are essentially based on leaf morphology and color, which frequently depend on environmental conditions For instance, leaves of the best-known species, Ocimum basilicum, and of its close relatives vary from small and uniform to large and round and colors vary from yellow-green to grey-green, to red, or to almost black More recently, (Paton et al., 1999) proposed that only 65 species of Ocimum should be retained and that other attributions should be considered synonyms or false attribution 2.4 Biochemical diversity Genotype characterization based on the chemical constitution of the essential oil has been used in several cultures such as Zingiber officinale (Liber et al., 2011) and Hyptis suaveolens (Azevedo et al., 2002), including plants of the genus Ocimum, with emphasis on O basilicum (Telci et al., 2006) Several studies assessing the chemical composition of 18 basil essential oils observed that the samples were distributed into seven distinct types, each one presenting as the major volatile compound among the following: linalool, methyl cinnamate, methyl cinnamate/linalool, methyl eugenol, citral, methyl chavicol (estragole), and methyl chavicol/citral The chemical characterization of 38 basil genotypes resulted in seven groups: linalool (19-73%); linalool/eugenol (28-66% linalool and 5-29% eugenol); methyl chavicol (20-72% methyl chavicol); methyl chavicol/linalool (8-29% methyl chavicol and 8-53% linalool); methyl eugenol/linalool (two accessions with 37% and 91% methyl eugenol and 60% and 15% linalool); methyl cinnamate/linalool (9.7% methyl cinnamate and 31% linalool); and bergamotene (one accession with bergamotene as the major constituent) (Zheljazkov et al., 2008) A study by Marotti et al (1996) showed that the EO content in herbs of 10 Italian basil cultivars ranged from 0.3 to 0.8 % In a large study on 270 basil accessions in Germany, oil content varied from trace to 2.65 % A basil from Fiji has EO content only 0.2 % (dominant compounds: linalool 22.3 %, methyleugenol 24.7 % and methylcinnamate 23.6 %) Basil originated from Cuba has a much higher EO content of 1.9-2.5 % (dominant compounds: methylchavicol 66.8 %, 1,8-cineole 5.4 % and linalool 5.0 %), while basil from Burkina Faso has oil content of 0.7-1.8 % (dominant compounds: 1,8-cineole 60.2 %, α-terpineol 6.5 % and β-pinene 5.7 %) (Keita et al., 2000) 7 CHAPTER MATERIALS AND METHODS 3.1 Materials 3.1.1 Plant materials Basil seeds are collected from the provinces: Ben Tre, Long An, Vinh Long, Ho Chi Minh, Hanoi and planted on soil under normal conditions The experiment to investigate the phenotypic and biochemical diversity is carried out by bringing > 20 varieties of basil planted in earthen pots under the same environmental conditions After 60 days, samples were taken from plant parts to conduct experiments for biochemical analysis and physiological characteristics 3.2 Methods 3.2.1.1 Morphological variation for colour scale development The experiment is conducted in a covered garden house The seeds are planted in separate pots about 25cm in diameter, spaced 30cm apart Soil for the experiment used prepackaged organic clean soil Sow 2-3 seeds in each pot until the seeds germinate and then remove them, leaving only one plant Let plants grow and develop for 20 days under normal conditions After 20 days, observe the plant samples and evaluate the indicators: leaf color (1), colour of flowers (2), tree color(3) Then proceed to the group based on the assessed morphological characteristics ( Dou et al., 2018) 3.2.1.2 Morphological variation for yield traits After evaluating and grouping according to the above characteristics, continue to plant under normal conditions for another 40-45 days for full growth Add a little organic fertilizer to the plants to have enough nutrients Then, observe and evaluate agronomic parameters: plant height (A), leaf width (B), number of leaves (C), number of flowers (D) After that, continue to classify according to the parameters just evaluated on 20 planting samples ( Ehleringer and Sandquist, 2010) 3.2.2 Biochemical diversity Plant extraction After 60-65 days of planting, leaf samples were collected for biochemical analysis The fresh leaf samples will be dry at a temperature of about 50 oC for days Then, the dry sample is pulverized (100g) and soaked in ethanol at room temperature (29-30 °C) Filter the residue and remove the solvent using a rotary evaporator, collecting a liquid sample dissolved in absolute ethanol (96%) 3.2.2.2 Gas chromatography-mass spectrometry (GC-MS) Using gas chromatography coupled to continuous mass spectrometry with Thermo TSQ 8000 Evo triple quadrupole system GC-MS/MS, along with capillary column HP-5MS (30 m × 0.25 mm, 0.25 μm film thickness), as described by (Filip, 2017) with some modifications Helium is used as the carrier gas and a split ratio of 1: 49 is employed The carrier gas flow rate is 1.0 ml/min The oven initial temperature 60 oC for min, with an increase of 5°C/min to 210°C, ending with isothermal at 210°C electron energy: 70eV The total GC running time is 34 The relative % amount of each component is calculated by comparing its average peak area to the total peak area The compounds were identified by comparing their mass spectra to those from NIST/NBS libraries, using different search engines 3.3 Statistical analysis The morphological, and chemical traits for each measured parameter among germplasm are tested using variance analysis with one effect using the program SAS, procedure ANOVA (SAS, 1990) Averages of parameters were compared using Duncan’s multiple range test (at P