Đánh giá giống và xử lý hóa chất nâng cao năng suất và hàm lượng curcumin trong nghệ curcuma spp tt tiếng anh

MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY SUMMARY OF DOCTORAL THESIS MAJOR: CROP SCIENCE Code: 62 01 10 BUI THI CAM HUONG EVALUATION ON VARIETIES AND CHEMICALS TREATMENT TO IMPROVE THE CONTENT AND YIELD OF CURCUMIN IN CURCUMA SPP CAN THƠ - 2019 WORK DONE IN CAN THO UNIVERSITY Scientific supervisor 1: Assoc Prof Dr Le Vinh Thuc Scientific supervisor 2: Dr Luu Thai Danh The thesis will be defended in Can Tho university At , date month year Reviewer 1: Reviewer 2: This thesis will be submitted: LIST OF PUBLISHED PAPERS A survey of genetic diversity of Curcuma species from Southern - Vietnam using RAPD and ISSR markers Journal of Science, Can Tho University 2016 3:11-19 Effect of concentration and timing foliar application of phenylalanine on the growth, yield and curcumin content in turmeric (Curcuma xanthorrhiza Roxb.) Vietnam J Agri Sci 2017 15(6): 817-825 Effect of foliar fertilizer on growth curcumin content and yield of turmeric Curcuma xanthorrhiza Roxb Journal of Science, Can Tho University Special issue on Biotechnology 2019 55(1): 168-173 CHAPTER INTRODUCTION 1.1 THE NECESSITY Turmeric (Curcuma), a member of the ginger family (Zingiberaceae), is one of the important medicinal plants According to Sahoo et al (2017), the current published varieties are mainly based on morphological and biochemical characteristics Therefore, most of the varieties are the same without clear differences in morphology of leaves, flowers and rhizome, it caused confusion for researchers and users In different environmental conditions, turmeric quality is also different The combination of modern molecular techniques and biochemical targets has helped the selection and development of varieties based on morphological characteristics more effectively Currently, the world has applied molecular markers to evaluate genetic diversity in turmeric, in which, RAPD marker indicator (Syamkumar, 2008; Jan et al., 2011; Khan et al., 2013; Phurailatpam et al., 2013; Mohanty et al., 2014) and ISSR (Syamkumar, 2008; Taheri et al., 2012) are widely used due to simple and easy implementation Akamine et al (2007), Niranjan and Prakash (2008), Yue et al (2010), Hu et al (2015), Panahi et al (2015) suggested that curcumin, the main ingredient in turmeric, has many biological activities such as anti-oxidant, anti-mutant, anti-cancer, antiinflammatory, antibacterial, antifungal, and parasitic resistance and has Detoxification ability However, curcumin content in turmeric is very low at about 1-6% (Aggarwal et al., 2006 and Niranjan et al., 2013) According to Ishimine et al (2003 and 2004); Hossain et al (2005a and 2005b); Hossain and Ishimine (2005); Hossain and Ishimine (2007) have many determinants of curcumin growth, content, and yield on turmeric, in which seed plays the most important role (Singh et al., 2013) In addition to protein, phosphorus and potassium, the addition of trace elements such as iron, zinc and boron is also essential, contributing to the increase of curcumin content and yield in turmeric (Velmurugan et al., 2007; Singh, 2014; Datta et al., 2017) In addition, when adding phenylalanine, a precursor in the curcumin biosynthesis pathway, curcumin content can be increased in turmeric (Kita et al., 2008) due to the conversion of phenylalanine to cinnamic acid (mainly occurring in leaves) and through this pathway, it can lead to the synthesis of curcumin in turmeric (Neema, 2005) Besides phenylalanine, salicylic acid also plays an important role in the accumulation of secondary metabolites and activation of phenylalanine ammonia lyase, the first enzyme of the curcumin biosynthetic chain (Janas et al., 2002; Solecka and Kacperska, 2003; Zhao et al., 2005; Kita et al., 2008) In Vietnam, Škornič et al (2015) said that there are about 27 species of turmeric scattered from the North to the South, this is a valuable source of genetic material in the selection of new varieties However, nowadays, research on turmeric only focuses on morphological and biological activity, not many researches are conducted on seed and nutrition Therefore, the discovery of turmeric varieties with high content and curcumin yield on the basis of morphological, molecular and biochemical characteristics; at the same time, finding appropriate chemicals that affect growth, content and curcumin yield in turmeric is essential 1.2 OBJECTIVES OF THE STUDY Selecting turmeric samples with high curcumin content and yield based on morphological traits, molecular markers and biochemical Finding suitable chemical treatment for the growth, increasing curcumin content and yield in turmeric 1.3 OBJECT AND SCOPE OF THE STUDY Research subjects: 34 turmeric samples of local and imported turmeric were collected in Vietnam (32 samples), Indonesia (1 sample), Australia (1 sample) Scope of research: Research on the selection and impact of chemicals on increasing yield and content of curcumin in turmeric 1.4 CONTENTS OF THE STUDY * Content 1: Evaluating turmeric samples Collect 34 samples of local and imported turmeric Evaluate the diversity of 34 turmeric samples based on morphological traits, molecular markers and biochemical * Content 2: Study the effect of chemicals treatment on growth parameters, content and yield of curcumin in turmeric Effect of impact stages and concentration of phenylalanine (Phe); salicylic acid (SA); iron sulfate (FeSO4); zinc sulfate (ZnSO4); borax; then, the effect of chemical treatment on growth, content and yield of curcumin in turmeric 1.5 NEW CONTRIBUTIONS OF THE STUDY The thesis has selected C.34 sample with highest curcumin content and yield on dry rhizome (12,2%; 11,6 g) C.34 sample has leaf midrib; orange/orange-yellow rhizome colour, aroma of turmeric, little bitter taste of rhizome; colour of the coma bract is green/light pink/white, is C xanthorrhiza species Determination of genetic relationship of C.34 sample in comparison with the remaining samples on the basis of RAPD and ISSR molecular markers Spraying on the leaves of C xanthorrhiza with one of the substances such as Phe, SA 100 ppm, FeSO4, ZnSO4, borax 0.5% at 120 days after planting increased the content and yield of curcumin on dry rhizome weight Spraying with 100 ppm Phe or 0.5% FeSO4 on the leaves of C xanthorrhiza increased weight of fresh rhizome (1.63; 1.72 times); weight of dry rhizome (1.80; 1.78 times); curcumin content (1.39 times) and curcumin yield on dry rhizome (by 2.50; 2.48 times) in comparison with the control 1.6 APPLICATIONS AND SUGGESTIONS FOR FURTHER STUDY For science, the research on breeding and chemical treatment experiments to increase the growth, content and productivity of curcumin in turmeric shows that: (1) Molecular markers and biochemical analysis help select better turmeric sample; (2) Beside the breed, the pathway of biosynthesis of curcumin is affected by the addition of nutrients (FeSO4) and amino acid (phenylalanine); (3) The results of the thesis can be supplemented to the curriculum and reference materials for further research on medicinal plants For life, the results of the research can be effectively applied in production as a basis for orientation, stable planning and sustainable development of key domestic production areas Chapter RESEARCH MATERIALS AND METHODS 2.1 RESEARCH INSTRUMENTS 2.1.1 Research location and time 2.1.1.1 Research time The dissertation has been carried out from 2014 to 2018 2.1.1.2 Research location Laboratory of Crop Science Department, Genetics and plant breeding Department - College of Agriculture and Applied Biology; Can Tho University and experimental soil at Phong Dien District and Binh Thuy District, Can Tho City 2.1.2 Research materials and instruments 34 samples of local and imported turmeric, 32 Vietnamese samples; Indonesian sample; and Australian sample 2.2 RESEARCH METHODS 2.2.1 Content 1: Evaluating turmeric varieties 2.2.1.1 Collecting turmeric samples 34 samples of local and imported turmeric were planted in Binh Thuy District, Can Tho City The experiment has been carried out from 2014 to 2015, samples turmeric was grown in a Randomized complete block design (RCBD), with three replications and each lot was separated by m2 on sand foundation mixed with soil which has good water drainage and nutrients are easily washed off * Cultivation techniques: (applied for all samples) It is to select secondary rhizomes turmeric with uniform weight from 30-40 g (Hossain et al., 2005) to plant Seed-rhizomes were treated by chlorine 0.5% in 30 minutes, setting aside to dry and incubating for week before cultivating Rhizomes were incubated in shade on high foundation which has good water drainage and covered by a layer of straw ash It was sufficiently watered to germinate After 7-10 days, rhizomes were planted at the depth about 7-8 cm with spacing of 25x25 cm (Mohamed et al., 2014) Water was applied adequately every day to maintain optimum soil moisture level for proper seedling emergence and plant growth When the samples were young with small leaf canopy, the light helped grass developed well It should be weed to prevent from the competition of light and nutrient towards young plants, weeding two weeks/time Fertilizers: Mai Van Quyen et al., (2007) with an improvement that the application of fertilizer is divided into three times: application of NPK for the first time (16-16-8-13S): 100-150 kg ha-1; application of NPK for the second time (16-16-8-13S): 150-200 kg ha-1; application of NPK for the third time (16-16-8-13S): 100-150 kg.ha-1 The application was conducted at 60 days after planting (DAP - at the stage of 2-3 leaves - the phase representing growth), 120 DAP (characterized by initiation of finger and maximization of shoot growth) 180 DAP (the period dominated by rhizome growth) (Ravindran et al., 2007) 2.3.1.2 Morphological characteristics Morphological characters such as leaf form, flowering, color of flower, form of rhizome and color of rhizome…and other growth characteristics were recorded at full maturity at 200-210 days after planting (Syamkuma, 2008), it was described in details by Pham Hoang Ho (2003), Vo Van Chi (2003) Quality characteristics were recorded at harvesting (240 days after planting) 2.3.1.3 Molecular characteristics * Collecting leaf samples: Choose young turmeric leaves and not get pests Each sample takes about 2-3 leaves Put turmeric leaves in polyethylene wrap, sealed and labeled Then, store leaf samples in a refrigerator of 4oC for use in DNA analysis * Isolation of DNA Isolation and purification of DNA Leaf tissue of 34 samples were isolated and purified by using the modified CTAB method (Doyle and Doyle, 1990) Checking DNA by agarose gel electrophoresis PCR reaction PCR reaction for each DNA turmeric samples were reacted with markers of 20 primers RAPD (OPA02, OPA03, OPA04, OPA10, OPA13, OPB07, OPB10, OPD02, OPD03 and OPD07); and ISSR (ISSR1, ISSR2, ISSR5, ISSR6, ISSR 7, ISSR10, ISSR12, ISSR14, ISSR17 and ISSR18) (were produced by Phu Sa Biochemical Company - Vinh Long, Vietnam) Electrophoresis of PCR components An electrophoresis were carried out for PCR components with 1% agarose gel in TAE 1X by an electrophoresis apparatus with 42V in 30 minutes and 60V in 65 minutes Ethidium bromide (1 mg/L) was added and poured to gel in 20 minutes, washed with water and used an UV transilluminator to capture gel The appearance of bands were the products of PCR amplification which was shown on agarose gel to distinguish the diversification of tumeric species 2.3.1.4 Biochemical characters The analysis of biochemical characterization was to analyze quality characteristics in morphological characters 2.3.2 Content 2: Chemicals treatment on growth parameters, content and yield of curcumin in rhizomes C.34 turmeric sample (selected in content 1) of Curcuma xanthorrhiza having high curcumin content and yield were selected to be the research material for experiments in this content Chemicals: Phenylalanine (C9H11NO2) - Sigma (Germany), salicylic acid (China), Iron (II) sulfate heptahydrate (FeSO4.7H2O) China, Zinc sulfate heptahydrate (ZnSO4.7H2O) – China and Borax (Disodium tetraborate decahydrate - Na2B4O7.10H2O - China) 2.3.2.1 Experiment 1: Effect of foliar application stages and concentrations of phenylalanine on growth, curcumin content and yield in rhizomes turmeric * Experimental design: The experiment was laid out in randomized complete block design that factor A was stages of spraying (90, 120, 150 and 180 days after planting) and factor B was concentrations of phenylalanine (0, 50, 100 and 200 ppm), including 16 treatments with replications 2.3.2.2 Experiment 2: Effect of foliar application stages and concentrations of salicylic acid on growth, curcumin content and yield in rhizomes turmeric * Experimental design: The experiment was laid out in randomized complete block design that factor A was stages of spraying (90, 120, 150 and 180 days after planting) and factor B was concentrations of salicylic acid (0, 50, 100 and 200 ppm), including 16 treatments with replications 2.3.2.3 Experiment 3: Effect of impact stages and level of FeSO4 on growth, curcumin content and yield in rhizomes turmeric * Experimental design: The experiment was laid out in randomized complete block design that factor A was impact stages (60, 90 and 120 days after planting) and factor B was levels of FeSO4 (2 concentrations of 0.5 and 1%; applications of 10, 20, 30 kg.ha-1 and control treatment), including 18 treatments with replications 2.3.2.4 Experiment 4: Effect of impact stages and level of ZnSO4 on growth, curcumin content and yield in rhizomes turmeric * Experimental design: The experiment was laid out in randomized complete block design that factor A is impact stages (60, 90 and 120 days after planting) and factor B is levels of ZnSO4 (2 concentrations of 0.5 and 1%; applications of 10, 20, 30 kg.ha-1 and control treatment), including 18 treatments with replications 2.3.2.5 Experiment 5: Effect of impact stages and level of borax on growth, curcumin content and yield in rhizomes turmeric * Experimental design: The experiment was laid out in randomized complete block design that factor A is impact stages (60, 90 and 120 days after planting) and factor B is levels of Borax (2 spraying concentrations of 0.5 and 1%; applications of 10, 20, 30 kg.ha-1 and control treatment), including 18 treatments with replications 2.3.2.6 Experiment 6: Effect of treated chemicals on growth, curcumin content and yield in rhizomes turmeric * Experimental design: The experiment was laid out in a randomized complete block design with factor, different foliar fertilizers treatments (results of the above experiments) and the control treatment of no spraying with replications The treatments were Phe 100 ppm; SA 100 ppm; FeSO4 0.5%; ZnSO4 0.5%; Borax 0.5% respectively and the control 2.3.3 Data analyzing methods Morphological characters were recorded and encoded under binary system: having characters = and having no characters = After recording all the above characters, collected data were stored in Excel software Cluster analysis (cluster) and evaluation of 34 turmeric samples based on Euclidean distances were conducted by using NTSYS pc 2.1 with UPGMA (Unweighted Pair Group Method with Arithmetic Mean) method Similarly, molecular characters in agarose gel electrophoresis were shown by the appearance or disappearance of a DNA band recorded and respectively Collected data were stored in Excel software Cluster analysis (cluster) and evaluation of genetic relationship among samples in similarity coefficient by using NTSYS pc 2.1 with UPGMA method The data were input and drawn in a graph by Microsoft excel, it was analyzed by SPSS 24.0 Chapter RESULTS AND DISCUSSION 3.1 EVALUATING VARIETIES TURMERIC 3.1.1 Morphological traits of 34 samples turmeric 3.1.1.1 Characteristics of pseudostem, leaf and flower Results showed that in 10 characteristics of pseudostem, leaf and flower of 34 samples of local and imported turmeric, there was no significant difference in color of leaf and spike position There was significant difference in characteristics: leaf blade shape, the appearance of petioles, leaf habit, color of midrib, color of pseudostem and color of the bract Most of turmeric samples had green pseudostem (79.0%), leaf petiole (85.0%), lanceolate leaf blades (73.0%), straight leaf (63.0%), green midrib (71.0%) and 100% leaf having special aroma and flowering Inferior bracts were green, and the superior ones were light green/ light pink/white which occupy high percentage (70%) 3.1.1.2 Morphological traits of rhizome The results showed that in morphological characteristics of 34 samples of local and imported turmeric, there was no significant difference in shape and taste of rhizome Characteristics of shape of mother rhizome, rhizome color, color and taste of the rhizome were the significant differences Most varieties have an oblong mother’s shape (73.0%), pale yellow rhizome (53.0%) and special smell of turmeric Colors of the rhizome were diversified and most of them were yellow (35.0%) (Fig 3.1) The results were appropriate to other studies According to Syamkumar and Sasikumar (2006), most of 15 surveyed turmeric samples had camphor aroma, bitterness or quite bitter (100%) According to Syahid and Heryanto (2017), in 12 varieties of white C zedoaria, most of them had white rhizome inner core (91.7%), except Curz10 which had yellow rhizome 3.1.1.5 The relationship between quality characteristics and morphological characters * The relationship between content of curcumin and morphological characters Samples having low content of curcumin (0.68±0.52%) had a light purple coloured leaf midrib; white or pale yellow rhizome or dark purple inner and pale purple outter, camphor aroma, bitter taste; dark purple bract or green inferior and reddish purple superior The characters of red brown midrib, dark purple rhizome, pale purple outter, reddish purple superior bract; was anthocyanine; the synthesis of curcumin (yellow) was not given priority so the content of curcumin of these samples were low According to Pham Hoang Ho (2003), Syamkumar (2008) and Lim (2016), these samples belonged to C aeruginosa and C zedoaria Samples having average content of curcumin (6.09±1.38%) had green midrib; yellow flesh, turmeric aroma, quite bitter and less metholated; color of superior bract was light green/ light pink/white With the above morphological characters, according to Syamkumar (2008) and Lim (2016), these samples belonged to C aromatica and C mangga Samples having high content of curcumin (10.5±1.11%) had green midrib; yellow or light orange yellow or dark yellow flesh, turmeric aroma, quite bitter and less metholated or no taste; color of superior bract was light green/ light pink/white According to Pham Hoang Ho (2003), Syamkumar (2008) and Lim (2016), these samples belonged to C longa and C xanthorrhiza * The relationship between yield of curcumin and morphological characters Samples having low content of curcumin (0.60±0.22) had features: reddish brown midrib; white or pale yellow flesh or dark purple inner and light purple outter, camphor aroma, bitter taste; dark purple bract or green inferior and reddish purple superior According to Pham Hoang Ho (2003), Syamkumar (2008) and Lim (2016), these samples belonged to C aeruginosa and C zedoaria Samples having average content of curcumin (5.14±1.33) had green midrib; yellow or light orange yellow, turmeric aroma, quite bitter and less metholated; color of superior bract was light green/ light pink/white According to Syamkumar (2008) and Lim (2016), these samples belonged to C aromatica and C mangga Samples having high content of curcumin (10.0±1.09) had green midrib; orange yellow or dark orange yellow flesh, turmeric aroma, quite bitter and less metholated; color of superior bract was light green/light pink/white According to Pham 12 Hoang Ho (2003), Syamkumar (2008) and Lim (2016), these samples belonged to C longa and C xanthorrhiza According to Mayazaki et al (2014), samples having high content of curcumin, yield of curcumin depended on weight of dry rhizome; samples having low content of curcumin, yield of curcumin depended on the content of curcumin; samples having average content of curcumin, yield of curcumin depended on both of them Moreover, samples having low mass and high content of curcumin had orange flesh, samples having great mass and low content of curcumin had light yellow flesh Therefore, genotyes not only affected the content of curcumin but also the yeild of rhizome and curcumin In comparison with molecular marker technique, Beyene et al (2005) suggest that analyzing diversity based on morphological characters was simple and easy to conduct but it was quite inaccurate, less effective and it only used for preliminary analysis In the experiment results, the combination of morphological characteristics with biochemical indicators showed that it helped identify or distinguish the varieties/species However, these characteristics are very susceptible to genotype and environment Therefore, in order to more effectively assess the variety of varieties/species belonging to the genus of the topic, two molecular markers RAPD and ISSR were used 3.1.2 MOLECULAR MARKERS 3.1.2.1 RAPD markers 10 RAPD primers were used to analyze 34 samples of local and imported turmeric, showed that all bands were polymorphic 167 bands were recorded with a mean of 16.7 ± 2.75 band/primer; in which, 155 bands were polymorphic (a ratio of 90.7±18.5%) The number of polymorphic bands ranged from 04 bands (OPA02 primer) to 19 band (OPD02 primer) with a mean of 15.5±4.28 polymorphic bands per primer Most primers produced many bands and had high ratio of polymorphic band except OPA02 primer (only bands and ratio of polymorphic band was 40.0%) The PIC index ranges from 0.31 (primer OPA02) to 0.93 (primer OPB10) and averages 0,59±0,16 The MI index ranges from 6.78 (primer OPB10) to 12.3 (primer OPD02) and averages 10.4 ± 2.08 The Rp index ranges from 1.18 (OPA02 primer) to 12.8 (OPD02 primer) and averages 8.8 ± 3.33 (Tab 3.2) 13 Tab 3.2: Index of polymorphic analysis on 10 RAPD primers of 34 samples of local and imported turmeric No 10 Primer NB PB P (%) PIC MI Rp OPA02 10 40.0 0.31 7.00 1.18 OPA03 16 16 100 0.49 11.4 9.29 OPA04 18 18 100 0.55 12.1 10.8 OPA10 17 16 94.1 0.5 10.9 9.90 OPA13 15 14 93.3 0.7 10.9 6.10 OPB07 18 18 100 0.53 11.9 10.9 OPB10 17 17 100 0.93 6.78 6.94 OPD02 19 19 100 0.58 12.3 12.8 OPD03 20 17 85.0 0.73 8.88 9.00 OPD07 17 16 94.1 0.60 11.7 11.0 167 155 Total 16.7±2.75 15.5±4.28 90.7±18.5 0.59±0.16 10.4±2.08 8.8±3.33 Aver Note: NB: Number of bands; PB: Polymorphic bands; P: Polymorphism; PIC: Polymorphism information content; Marker index; Rp: Resolving power 4.2.3 ISSR markers 10 ISSR primers were used to analyze 34 samples, showed that all bands were polymorphic Total of 166 bands were recorded with a mean of 16,6 ± 3,31 bands/primer In which, 162 bands were polymorphic (with a ratio of 97,1±3,87%) Number of polymorphic bands ranged from bands (ISSR1) to 29 bands (ISSR10) with a mean of 16,2±3,61 polymorphic bands per primer Most primers produced many bands with high level of polymorphism 90,0% (Tab 3.3) Thus, using 10 ISSR primers to analyze genetic diversity in 34 samples of local and imported turmeric showed the results of higher level of polymorphism than in the study of Singh et al (2012), Taheri et al (2012) and Saha et al (2016) but lower than the study of Nguyen Loc Hien et al (2013) Tab 3.3: Index of polymorphic analysis on 10 ISSR primers of 34 samples of local and imported turmeric No Primer NB PB P (%) PIC MI Rp 10 ISSR1 ISSR2 ISSR5 ISSR6 ISSR7 ISSR10 ISSR12 ISSR14 ISSR17 ISSR18 10 17 16 20 14 22 16 16 19 16 17 15 20 14 22 15 15 19 16 90.0 100 93.8 100 100 100 93.8 93.8 100 100 0.50 0.67 0.5 0.84 0.63 0.79 0.6 0.7 0.69 0.68 11.1 12.3 11.1 9.9 12.4 10.8 11.2 10.8 12.0 12.6 5.60 11.1 9.00 11.7 9.12 12.9 9.70 9.50 11.8 11.8 166 162 Total 16.6 ±3.31 16.2±3.61 97.1±3.87 0.67±0.10 11.4±0.87 10.2±2.11 Aver Note: NB: Number of bands; PB: Polymorphic bands; P: Polymorphism; PIC: Polymorphism information content; Marker index; Rp: Resolving power 14 3.1.2.3 The combination of RAPD and ISSR markers The results in Tab 3.4 showed that the combination of 10 RAPD primers and 10 ISSR primers in 34 samples of local and imported turmeric had a total of 333 bands with 272 polymorphic bands (93,2%) The subgroup of 34 samples of local and imported turmeric by morphological characters in comparison with RAPD showed similar results (23/34 were same), with ISSR (17/34 were same) and with the combination of RAPD + ISSR (18/34 were same) In which, C.27 belonged to separate group Tab 3.4: Comparison of RAPD, ISSR and cumulative band data analyses in 34 samples of local and imported turmeric Markers RAPD ISSR *Cumulative Total no of primer 10 10 20 Total no of bands amplified 167 166 333 No of bands/primer 16.7±2.75 16.6±3.31 15.9±2.97 Polymorphic bands 155 162 317 Polymorphic bands/primer 15.5±4.28 16.2±3.61 15.9±3.87 Polymorphism (%) 90.7 97.1 93.9 * Cumulative: combined data of RAPD and ISSR Skornickova and Sabu (2005) stated that there was a wrong determination of C aromatica vand C zedoaria to C xanthorrhiza Similarly, Sabu (2006) stated that C aromatica had grey yellow but in previous report, other author recorded that C aromatica had the color of yellow to dark yellow (Sasikumar, 2005) The difference of morphology caused by the interaction between environment and genetic factors which were mainly by quantitative characters and rarely by quality characteristics In addition, the choice of plant varieties, or wrong identification of seeds, planting and tending techniques also affects The results showed that the use of RAPD and ISSR molecular markers is very effective and reliable in assessing genetic diversity, relationships and classification of 34 local and imported turmeric varieties In summary, using morphological characteristics, growth and quality parameters, molecular markers have identified the genetic relationship of C.34 turmeric sample (belonging to Curcuma xanthorrhiza species) with other turmeric samples 3.2 CONTENT 2: EFFECT OF CHEMICAL TREATMENTS ON GROWTH PARAMETERS, CONTENT CURCUMIN AND YIELD OF TURMERIC C XANTHORRHIZA 3.2.1 Effect of phenylalanine on C xanthorrhiza Curcuma xanthorrhiza quality parameters over time and concentrations of phenylalanine showed that the interactions were significant (respectively at 1, 1, and 1%) (Table 3.5) 15 Tab 3.5: Quality traits of Curcuma xanthorrhiza at stages and spraying concentrations of phenylalanine (Phong Dien, Can Tho, 20152016) Phenylalanine (ppm) (B) 50 100 Weight of 200 fresh Aver (A) rhizome Sig (A) /clump (g) Sig (B) Sig (AxB) CV (%) 50 100 Weight of 200 dry Aver (A) rhizome Sig (A) /clump (g) Sig (B) Sig (AxB) CV (%) 50 Curcumin 100 content/ 200 weight of Aver (A) dry Sig (A) rhizome Sig (B) (%) Sig (AxB) CV (%) 50 Curcumin 100 yield/ 200 weight of Aver (A) dry Sig (A) rhizome Sig (B) (g) Sig (AxB) CV (%) Note: Values within each column same letter not statistically level; ns: no significance Parameters Stage (days after planting) (A) Aver (B) 90 120 150 180 164b 157c 161c 155b 159 184a 162c 180b 186a 178 aB aA aB aB 193 280 200 200 218 a b ab a 189 196 188 185 190 182 199 182 182 ** ** ** 4.90 39.4b 37.7c 38.7c 37.1b 38.2 44.2a 38.9c 43.2b 44.7a 42.8 aB aA aB aB 46.2 67.1 48.1 48.0 52.4 a b ab a 45.4 47.0 45.0 44.4 45.5 43.8 47.7 43.8 43.6 ns ** ** 4.92 d d 10.44 10.37 10.36d 10.38d 10.38 10.64c 10.78c 10.74c 10.68c 10.72 aB aA aAB aAB 12.16 12.40 12.32 12.27 12.26 bA bA bAB bB 11.22 11.36 11.20 11.04 11.18 A 11.12 11.23 11.16 11.09 ** ** * 0.80 d c 1.71 1.63 1.67d 1.61c 1.66 1.96c 1.75c 1.94c 1.99b 1.91 aB aA aB aB 2.34 3.47 2.47 2.45 2.68 AB bA bB bB 2.12b 2.23 2.10 2.04 2.12 2.03 2.27 2.05 2.02 ns ** ** 4.53 (lowercase) and row (uppercase) followed by the significant; * and **: at 5% and 1% significance The influence of phenylalanine concentration changes over time and opposite Therefore, averaging between the concentrations calculated based on the mean of all time, or the average between the times calculated based on the average of all treatment concentrations will not be effective The comparison between the averages of treatment concentrations at the same time of application or between the means of application time for each concentration of phenylalanine treated According to each treatment time, phenylalanine concentration was significantly different in all quality parameters For each treatment concentration, spray Phe 100 ppm, the time of 16 application was significantly different at all quality parameters Spraying 200 ppm, the time of application only differed in curcumin content and yield Spraying Phe and 50 ppm over time has no effect on turmeric quality In general, the interaction effect between time and treatment concentration or opposite, the combination of spraying treatment Phe 100 ppm at 120 days after planting always has the highest fresh weight, dry weight, curcumin content and yield (280 g; 67.1 g; 12.4% and 3.47 g; respectively) (Table 3.5) Curcumin was one of phenolic compound produced by phenylpropanoid Synthesizing curcumin from phenylalanine was a material chemical in the synthesis of flavonoid Phenylalanine was transformed into cinnamic acid by PAL enzyme (Nelson and Cox, 2012) According to Neema (2005), in turmeric, PAL was an initial enzyme in the synthesis of curcumin Besides that, according to Hancock (2012), enzyme BH4 (tetrahydrobiopterin enzyme) played an important role in the production of energy in photosynthesis and had a relationship with phenylalanine Activity of enzyme BH4 increased when the concentration of phenylalanine in turmeric increased and at 150 ppm phenylalanine, activity of enzyme BH4 stopped increasing (Gersting et al., 2010) As a result, phenylalanine directly affected turmeric leaf length and width which indirectly affected to the yield components of turmeric 3.2.2 Effect of salicylic acid on C xanthorrhiza Results Table 3.6 shows that the criteria of the quality of Curcuma txanthorrhiza over time and the concentration of salicylic acid treatment interactions were significant at 5, 5, and 5% respectively The effect of salicylic acid treatment changes over time and opposite According to each treatment time, salicylic acid concentration was significantly different at all quality parameters, except for the time of 180 days after planting For each treatment concentration, the application time was significantly different at all quality parameters, except for spraying SA and 200 ppm concentrations In general, spraying SA 100 ppm at 120 days after planting had the highest fresh weight, dry weight, curcumin content and yield (314 g; 75.3g; 12.54% and 3.94 g; respectively) (Tab 3.6) Ghasemzadeh and Jaafar (2012) realized that the supply of SA stimulated the synthesis of phenolic acid (cinnamic acid, vanillic acid ferulic acid and gallic acid) in Halia Bentong and Halia Bara According to Manoj (2017), spraying with 100 ppm SA 100 improved the content of curcumin in turmeric Therefore, spraying with 100 ppm SA at 120 days after planting was an appropriate stage to help turmeric growth and development, increased yield and quality of rhizome 17 Tab 3.6: Quality traits of Curcuma xanthorrhiza at impact stages and spraying concentrations of salicylic acid (Phong Dien Can Tho 2015-2016) Salicylic Impact stage (days after planting) (A) Aver acid (B) 90 120 150 180 (ppm) (B) 148b 156b 161b 167 158 50 156abB 183bA 185bA 194A 179 100 203aB 314aA 241aAB 197B 239 ab b b 200 198 205 194 190 197 Weight of fresh rhizome Aver (A) 176 214 195 187 /clump (g) Sig (A) * Sig (B) ** Sig (AxB) * CV (%) 16.2 35.4b 37.4b 38.6b 40.1 37.9 50 37.4abB 43.8bA 44.3bA 46.6A 43.0 aB aA bAB B 100 48.8 75.3 57.9 47.4 57.4 ab b a 200 47.4 49.3 46.7 45.6 47.3 Weight of dry rhizome/clump Aver (A) 42.3 51.5 46.9 44.9 (g) Sig (A) * Sig (B) ** Sig (AxB) * CV (%) 15.8 10.35c 10.30c 10.26d 10.39d 10.33 50 10.79bcB 11.49bA 10.95cB 10.84cB 11.02 aC aA aB aB 100 11.41 12.54 12.16 12.14 12.06 Curcumin 200 11.20ab 11.32b 11.25b 11.25b 11.26 content/weight Aver (A) 10.94 11.41 11.16 11.16 of dry rhizome Sig (A) ** (%) Sig (B) ** Sig (AxB) ** CV (%) 5.15 b b 1.53 1.60 1.65c 1.74 1.63 50 1.68abB 2.10bA 2.02bcA 2.11A 1.98 aB aA aB B 100 2.32 3.94 2.93 2.40 2.90 Curcumin a b b 200 2.22 2.32 2.18 2.14 2.22 yield/weight Aver (A) 1.94 2.49 2.20 2.09 of dry rhizome Sig (A) ** (g) Sig (B) ** Sig (AxB) ** CV (%) 17.0 Note: Values within each column (lowercase) and row (uppercase) followed by the same letter not statistically significant; * and **: at 5% and 1% significance level Parameter 3.2.3 Effect of FeSO4 on C xanthorrhiza The weight of fresh and dry rhizome at treatment times of FeSO4 was not significantly The weight of fresh and dry rhizome at levels of FeSO4 treatment was statistically different at 1% level The weight of fresh rhizome was highest in the treatments of 20 and 30 kg ha-1 (211 and 209 g, respectively); dry rhizome weight was high at 30 kg.ha-1 (51.4 g) The results also showed that there was no interaction between time and the level of FeSO4 treatment on the weight of fresh 18 and dry rhizome (Table 3.7) The interaction between time and FeSO4 treatment level on curcumin content and yield, the experimental combinations were statistically different at the 1% significance level The influence of FeSO4 treatment level changes over time and opposite The spray FeSO4 0.5% at 120 days after planting had the highest curcumin content and yield (14.51% and 5.53 g, respectively) (Tab 3.7) Tab 3.7: Quality traits of Curcuma xanthorrhiza at impact stages and levels of FeSO4 (Phong Dien Can Tho 2015-2016) Parameter Weight of fresh rhizome/clump (g) Weight of dry rhizome/clump (g) Curcumin content/weight of dry rhizome (%) Curcumin yield/weight of dry rhizome (g) Treatment FeSO4 (B) Impact stage (DAP) (A) 60 90 120 159 160 159 179 177 177 172 168 167 185 186 186 210 212 212 207 208 211 185 185 185 ns ** ns 8.79 38.3 36.7 34.3 46.9 46.9 44.5 41.6 35.9 42.6 45.2 47.5 47.1 46.5 51.5 48.1 52.9 53.8 47.4 45.2 45.4 44.0 ns ** ns 10.6 cd b 10.31 10.26 10.30e 10.89bC 13.12aB 14.51aA 12.71a 12.90a 13.12b 10.19d 10.34b 10.62de cdB bB 10.27 10.51 11.42cA 10.63bc 10.62b 10.89cd 10.83 11.29 11.81 ** ** ** 7.07 3.88c 3.74d 3.77c 5.11aB 5.62aA 5.53aA a c 5.21 4.64 5.59a 4.74ab 4.70bc 5.22b abB abA 4.78 5.39 5.49aA 4.50bC 5.70aB 5.02bA 4.71 4.97 5.11 ** ** ** 5.81 Control Foliar spray 0.5% Foliar spray 1.0% Soil application 10 kg.ha-1 Soil application 20 kg.ha-1 Soil application 30 kg.ha-1 Aver (A) Sig (A) Sig (B) Sig (AxB) CV (%) Control Foliar spray 0.5% Foliar spray 1.0% Soil application 10 kg.ha-1 Soil application 20 kg.ha-1 Soil application 30 kg.ha-1 Aver (A) Sig (A) Sig (B) Sig (AxB) CV (%) Control Foliar spray 0.5% Foliar spray 1.0% Soil application 10 kg.ha-1 Soil application 20 kg.ha-1 Soil application 30 kg.ha-1 Aver (A) Sig (A) Sig (B) Sig (AxB) CV (%) Control Foliar spray 0.5% Foliar spray 1.0% Soil application 10 kg.ha-1 Soil application 20 kg.ha-1 Soil application 30 kg.ha-1 Aver (A) Sig (A) Sig (B) Sig (AxB) CV (%) Aver (B) 159d 178bc 169cd 186b 211a 209a 36.5c 46.1b 40.0c 46.6ab 48.7ab 51.4a 10.29 12.84 12.91 10.38 10.73 10.71 3.80 5.42 5.15 4.89 5.22 5.08 Note: DAP: days after planting; Values within each column (lowercase) and row (uppercase) followed by the same letter not statistically significant; **: at 1% significance level; ns: no significance Time of FeSO4 impact did not affect the growth index of turmeric However, levels of FeSO4 affected the growth, weight of fresh rhizome, content and yield of curcumin in turmeric Spraying or fertilizing FeSO4 also had higher weight of fresh rhizome and 19 curcumin yield Spraying FeSO4 at 120 days after planting had high curcumin yield and content According to Marschner (2012), Fe was a microelement affected the process of photosynthesis such as the synthesis of chlorophyll electron transport in photosynthesis enzyme catalysis in photosynthesis and carotenoid synthesis Lack of Fe reduced the activities of photosynthesis caused the repression of growth and development If there was no Fe, the growth of crop such as number of leaves, root, shoot, young plant, weight of fresh and dry rhizome reduced As per the results of analyzing the relationship between curcumin content and the growth characteristics showed that samples having orange yellow flesh had high content of curcumin Therefore, the study results showed that Fe participated in enzyme catalysis in the activity of carotenoid synthesis increased curcumin content in turmeric 3.2.4 Effect of ZnSO4 on C xanthorrhiza Table 3.8 shows that the weight of fresh and dry rhizome at treatment times of ZnSO4 was not significantly different The weight of fresh and dry rhizome at levels of ZnSO4 treatment was statistically different at 1% The weight of fresh and dry rhizome was high in all ZnSO4 treatments The results also showed that there was no interaction between time and ZnSO4 treatment level weight of fresh and dry rhizome The interaction between impact time and levels of ZnSO4 had significant difference towards curcumin content at 5% This proves that the influence of ZnSO4 treatment level changes over time and opposite The spray ZnSO4 0.5% at 120 days after planting had the highest curcumin/dry weight (13.1%) (Table 3.8) According to Naeem et al (2017) content of 10-300 ppm Zn in soil Spraying ZnSO4 on the leaves was an effective supply of Zn and improved the content of Zn in grain (Stomph et al., 2011) According to Rethinam et al (1994) and Dixit and Srivastava (2000), lack of Fe and Zn affected the development of turmeric The development of rhizome and curcumin accumulation depended on the transformation of metabolism from leaves A part of metabolism from leaves transferred to rhizome and affected the size yield as well as curcumin synthesis and accumulation Datta et al (2017) studied on ZnSO4 and microelement on turmeric variety Suranjana in Terai in West Bengal, India Zn2+ was supplied content of chlorophyll increased Leaf quality was improved and increased CO2 transfer of leaves, increased photosynthesis intensity In the experiment of the effect of zinc on turmeric growth, growth index (leaf area, weight of fresh rhizome, weight of dry rhizome) increased in the treatments of supplementing zinc Besides that, quality index (content of essential oil and total curcumin in rhizome) increased in comparison with the 20 control When leaf area increased, pseudostem required more nutrients from leaves than root Photosynthesis products increased, the process of synchronizing and accumulating essential oil content and total curcumin in rhizome was greater than in treatments of no zinc supplementation (Srivastava et al., 2006) Tab 3.8: Quality traits of Curcuma xanthorrhiza at impact stages and levels of ZnSO4 (Phong Dien, Can Tho, 2015-2016) Impact stage (DAP) (A) Aver (B) 60 90 120 Control 158 163 165 162b Foliar spray 0.5% 170 175 173 173ab Foliar spray 1.0% 175 173 174 174ab Soil application 10 kg.ha-1 171 173 172 172ab Weight of Soil application 20 kg.ha-1 184 183 186 184a fresh Soil application 30 kg.ha-1 184 182 183 183a rhizome/clump Aver (A) 174 175 176 (g) Sig (A) ns Sig (B) ** Sig (AxB) ns CV (%) 7.06 Control 38.7 39.9 40.6 39.8b Foliar spray 0.5% 41.6 42.9 42.3 42.3ab Foliar spray 1.0% 43.0 42.5 42.7 42.7ab Soil application 10 kg.ha-1 41.9 42.4 42.2 42.2ab Soil application 20 kg.ha-1 45.1 44.8 45.6 45.1a Weight of dry Soil application 30 kg.ha-1 45.0 44.7 44.8 44.9a rhizome/clump (g) Aver (A) 42.6 42.9 43.0 Sig (A) ns Sig (B) ** Sig (AxB) ns CV (%) 12.6 b b Control 10,23 10,18 10,17c 10,19 Foliar spray 0.5% 10,98bB 12,33aA 13,10aA 12,14 a a ab Foliar spray 1.0% 12,30 12,04 12,28 12,21 Soil application 10 kg.ha-1 10,31b 10,51b 10,67c 10,50 Curcumin Soil application 20 kg.ha-1 10,34bB 10,72bAB 11,26bcA 10,77 content/weight Soil application 30 kg.ha-1 10,75b 10,93b 10,79c 10,82 of dry rhizome Aver (A) 10,82 11,12 11,38 (%) Sig (A) ** Sig (B) ** Sig (AxB) * CV (%) 4.58 Control 3.78 3.71 3.82 3.77D Foliar spray 0.5% 5.27 5.28 5.96 5.50A Foliar spray 1.0% 5.18 4.94 5.71 5.28AB Soil application 10 kg.ha-1 3.91 4.38 5.31 4.53C Curcumin Soil application 20 kg.ha-1 4.22 4.63 4.96 4.61C yield/weight of Soil application 30 kg.ha-1 4.63 4.89 4.82 4.78BC dry rhizome Aver (A) 4.50B 4.64B 5.10A (g) Sig (A) * Sig (B) ** Sig (AxB) ns CV (%) 6.78 Note: DAP: days after planting; Values within each column (lowercase) and row (uppercase)followed by the same letter not statistically significant; * and **: at and 1% significance level; ns: no significance Parameter Treatment ZnSO4 (B) 21 3.2.5 Effect of Borax on C xanthorrhiza Results Table 3.9 showed that the weight of fresh and dry rhizome at treatment stages of borax was not significantly different The weight of fresh and dry rhizome at levels of borax treatment was statistically different at 1% Tab 3.9: Quality traits of Curcuma xanthorrhiza at impact stages and levels of borax (Phong Dien Can Tho 2015-2016) Impact stage (DAP) (A) Aver (B) 60 90 120 Control 155 157 153 155b Foliar spray 0.5% 167 174 174 172ab Foliar spray 1.0% 168 177 174 173ab Soil application 10 kg.ha-1 177 180 188 181a Weight of Soil application 20 kg.ha-1 195 182 185 187a fresh Soil application 30 kg.ha-1 198 182 187 189a rhizome/clump Aver (A) 177 175 177 (g) Sig (A) ns Sig (B) * Sig (AxB) ns CV (%) 12.6 Control 38.0 38.5 37.6 38.0b Foliar spray 0.5% 40.8 42.7 42.6 42.0ab Foliar spray 1.0% 41.2 43.3 42.6 42.4ab Soil application 10 kg.ha-1 43.3 44.1 46.0 44.5a Soil application 20 kg.ha-1 47.7 44.5 45.4 45.9a Weight of dry Soil application 30 kg.ha-1 48.6 44.4 45.7 46.3a rhizome/clump (g) Aver (A) 43.3 42.9 43.3 Sig (A) ns Sig (B) ** Sig (AxB) ns CV (%) 9.87 b b Control 10.26 10.18 10.28c 10.24 Foliar spray 0.5% 10.78bB 12.10aA 12.87aA 11.91 a a b Foliar spray 1.0% 11.97 11.80 11.80 11.85 Soil application 10 kg.ha-1 10.10b 10.29b 10.43c 10.27 Curcumin Soil application 20 kg.ha-1 10.17b 10.39b 11.04c 10.53 content/weight Soil application 30 kg.ha-1 10.70b 10.42b 10.63c 10.58 of dry rhizome Aver (A) 10.66 10.86 11.17 (%) Sig (A) ** Sig (B) ** Sig (AxB) ** CV (%) 3.64 Control 3.89 3.92 4.04 3.95B Foliar spray 0.5% 4.87 4.97 5.69 5.17A Foliar spray 1.0% 4.91 5.07 5.65 5.21A Soil application 10 kg.ha-1 3.74 4.22 4.90 4.28B Curcumin Soil application 20 kg.ha-1 4.86 4.93 5.40 5.07A yield/weight of Soil application 30 kg.ha-1 4.90 4.96 5.32 5.06A dry rhizome Aver (A) 4.53B 4.68 AB 5.16A (g) Sig (A) * Sig (B) ** Sig (AxB) ns CV (%) 6.60 Note: DAP: days after planting; Values within each column (lowercase) and row (uppercase) followed by the same letter not statistically significant; * and **: at and 1% significance level; ns: no significance Parameter Treatment borax (B) The weight of fresh and dry rhizome was high in borax treated treatments The results also showed that there was no interaction 22 between time and borax treatment on fresh and dry rhizome weight Results Table 3.9 showed that the interaction between time with borax treatment level on curcumin content/weight of dry rhizome, the different treatment combinations was significant at 1% This proves that the effect of borax treatment level changes over time and opposite Spraying with borax 0.5% at 120 days after planting had the highest curcumin/dry rhizome weight (12.87%) The time and concentration of borax treatment on curcumin yield/dry rhizome weight were statistically different at 1% level Time of 120 days after planting for the highest curcumin yield (5.16 g); borax spray of 0.5; 1%, apply borax 20; 30 kg ha-1 gave the highest curcumin yield (5.17; 5.21; 5.07 and 5.06 g, respectively) (Table 3.9) The results also showed that there was no interaction between time and borax treatment to curcumin yield/dry rhizome weight Dirceu (2006) showed that fertilizing boron in soil helped plant absorb about 60-65%, while spraying on the leaves was only 10-20% Fertilizing 10 kg ha-1 boron helped increase ginger yield (Singh et al., 2007 and 2009), fertilizing 25 kg ha-1 boron helped increase turmeric yield (Datta et al., 2017) Besides that boron participated in glucose transport, lack of boron changed CO2 transfer in photosynthesis into secondary metabolism, it was relevant to curcumin and essential oil accumulation in C domestica (Dixit et al., 2002) Thus, spraying with borax 0.5% at 120 days after planting increased content and yield of curcumin in turmeric 3.2.6 The effect of foliar fertilizers on C xanthorrhiza 3.2.6.1 Content of PAL enzyme in turmeric leaves Content of PAL enzyme at spraying foliar fertilizers had a trend to gradually increase from 120 to 150 DAP and decrease at 180 DAP At 120 days after planting, content of PAL enzyme among treatments no significant difference, but at 150 and 180 DAP, it had significant difference at 1% At 150 DAP, content of PAL enzyme was highest at the treatment of spraying with FeSO4 0.5% (3580 µM.mg1 minute-1); followed by spraying with SA 100 ppm, Phe 100 ppm borax 0.5%, ZnSO4 0.5% (3388, 3312, 3043 and 2855 µM.mg1 minute-1, respectively); the control was lowest (1814 µM.mg1 minute-1) (Fig 3.4) At 180 days after planting content of PAL enzyme was high at treatments of spraying with borax 0.5%; SA 100 ppm (1536; 1527 µM.mg-1 minute-1), followed by spraying with FeSO4 0.5%; Phe 100 ppm (1511; 1502 µM.mg-1 minute-1), spraying with ZnSO4 0.5% (1447 µM.mg-1.minute-1); the control was lowest (1234 µM.mg1 minute-1) (Fig 3.4) 23 µM Trans-cinnamic acid/mg Protein/phút 4000 120NST 150NST 180NST 3000 2000 1000 Phe ( SA FeS ZnS Bor Đ ối a (100 O4 ch ứ 100 0,5% O4 0,5% x 0,5% ng ppm ppm ) ) Fig 3.4: The content of PAL enzymes in leaves when spraying foliar fertilizers varied at stages growth of C xanthorrhiza (Binh Thuy Can Tho 2017-2018) 3.2.6.2 Curcumin content and yield of C xanthorrhiza Curcumin content among treatments of spraying foliar fertilizers had significant difference at 1% Curcumin content was highest at spraying with Phe 100 ppm and FeSO4 0.5% (both accounted for 14.7%); 1.39 times; followed by SA 100 ppm and borax 0.5% (14.24; 14.29%); 1.39 times; then spraying with ZnSO4 0.5% (13.8%); 1.31 times higher than the control (Tab 3.10) Tab 3.10: Curcumin content and yield/dry weight of C xanthorrhiza when when spraying foliar fertilizer varies according to the growth time (Bình Thủy Cần Thơ 2017-2018) Curcumin content /dry weight Curcumin yield/dry weight (%) (g clump-1) (•) (•) Phe (100 ppm) 14.7a 1.39 17.2a 2.50 SA (100 ppm) 14.2b 1.34 15.1b 2.20 FeSO4 0.5% 14.7a 1.39 17.0a 2.48 ZnSO4 0.5% 13.8c 1.30 10.2c 1.49 Borax 0.5% 14.3b 1.35 15.2b 2.22 Control 10.6d 6.86d Aver 13.7 13.6 Sig ** ** CV (%) 4.04 9.0 Note: (•): increase compared to control; Values within each column followed by the same letter not statistically significant; **: at 1% significance level; ns: no significance Treatment Similarly, curcumin yield among treatments of spraying foliar fertilizers had significant difference at 1% Curcumin yield was highest at spraying with Phe 100 ppm (17.2 g clump-1), was no significant difference in comparison with spraying with FeSO4 0.5% (17.0 g clump-1) Curcumin yield at spraying with SA 100 ppm had no significant difference in comparison with spraying with borax 0.5% 24 (56.0 g); spraying with borax 0.5% the lowest was the control (6.86 g clump-1) (Tab 3.10) The experiment results showed that spraying with 100 ppm Phe/SA 100 ppm as well as spraying with FeSO4/borax 0.5% was related to activity of PAL enzyme in turmeric leaves PAL enzyme was a beginning enzyme in the synthesis of curcumin (Neema 2005) In turmeric, curcumin content was synthesized at 100 days after planting; with the growth, content of curcumin decreased in leaves and increased in rhizome The synthesis of curcumin started from leaves and moved to rhizome (Ravindran et al., 2007) Curcumin, a phenolic compound was a production of phenyl propanoid The synthesis of curcumin started from Phe, precursor substance in synthesizing flavonoid PAL enzyme was the first catalysis enzyme in the synthesis of phenolic When increasing activity of PAL enzyme, it shall speed up next processes in phenylpropanoid path (inluding phenol and flavonoid) Wada et al (2010), in stress conditions Endogenous SA and activity of PAL enzyme increased Mitra (2015), Fe was a microelement which was necessary for the metabolism, systhesis and maintain of chlorophyll in plants Most of enzyme having Fe participated in oxidation reaction in respiration and photosynthesis (Naeem et al., 2017) Fe and Zn were in the list of unharmful substance towards human health (JETRO, 2011) Content of Fe, Zn and B in C zedoaria was 348 108 and 166 ppm respectively (Tanzima et al., 2011) Content of Fe and Zn in turmeric was 53.6 and 17.3 mg.kg-1 respectively (Andriamisetra 2014); in C longa was 10.3 and 9.1 mg.kg-1 respectively (Francisconi et al., 2013); 327 and 15.8 mg.kg-1 (Silva et al., 2016); in C pseudomontana J Graham was 314.89 and 121.1 ppm respectively (Hiremath and Kaliwal, 2014) Chapter CONCLUSIONS AND RECOMMENDATIONS 4.1 Conclusions Thirty fours samples of local and imported turmeric are characterized based on morphological traits The dendrogram was split into three groups at similarity coefficient of 0.68 Group I: C zedoaria; group II: C aeruginosa; and group III: C xanthorrhiza, C longa and C mangga The research has selected the C.34 sample with highest curcumin content and yield per dry rhizome (12.2%; 11.6 g, respectively) C.34 sample has leaf midrib; orange/orange-yellow rhizome colour, aroma of turmeric, little bitter taste of rhizome; colour 25 of the coma bract is green/light pink/white, classified as Curcuma xanthorrhiza species The positive correlation between fresh rhizome and dry rhizome/clump; between content and yield of curcumin/dry rhizome; were significant (0.96 and 0.90 correlation coefficients, respectively) Regression models used fresh rhizome to estimate dry rhizome and curcumin content was used to calculate curcumin yield/dry rhizome were appropriate Thus, linear regression model explained 87% difference in dry rhizome/clump; 86% difference in curcumin yield/dry rhizome among samples Based on morphological characteristics, it is possible to explain the fluctuation in curcumin content as well as curcumin yield per dry rhizome of 34 turmeric varieties Analysis of genetic relationships based on RAPD, ISSR and the combination of RAPD with ISSR have divided 34 turmeric varieties into main groups, with average similarity coefficient of 0.71 The dendrogram based on morphological characteristics compared to the RAPD molecular markers showed similar results (23/34 samples), compared to ISSR (17/34 samples) and compared with the combination of RAPD + ISSR (18/34 samples) Spraying each Phe, SA 100 ppm, FeSO4, ZnSO4, borax 0.5% on the leaves of Curcuma xanthorrhiza at 120 days after planting increased the content and yield of curcumin per dry rhizome Spraying Phe 100 ppm or FeSO4 0.5% on the leaves of Curcuma xanthorrhiza increased weight of fresh rhizome (1.63; 1.72 times, respectively); weight of dry rhizome (1.80; 1.78 times, respectively); curcumin content increased (1.39 times) and curcumin yield per dry rhizome increased (2.50; 2.48 times) in comparison with the control 4.2 Recommendations It is necessary to replicate experiments on C.34 samples of Curcuma xanthorrhiza at different locations combined with analyzing growth index curcumin content and yield in turmeric in 1-2 consecutive years in order to increase the persuasion as well as the stability of the experiment results It is recommended to use the genetic sequence analysis of ITS or some specific genes located in mitochondria such as Cytb and control region; in chloroplasts such as matK, rbcL, atpβ, ndnF for further research on biotechnology to distinguish species of the turmeric 26 ... having high content of curcumin, yield of curcumin depended on weight of dry rhizome; samples having low content of curcumin, yield of curcumin depended on the content of curcumin; samples having... of curcumin content and yield in turmeric (Velmurugan et al., 2007; Singh, 2014; Datta et al., 2017) In addition, when adding phenylalanine, a precursor in the curcumin biosynthesis pathway, curcumin. .. (2009), content of curcumin of 67 Thailand turmeric varieties ranged from 0.32±0.44 to 10.13±1.27% Content of curcumin was one of three important biochemical components in Curcuma spp (Sasikumar,