1 INTRODUCTION Introduction Talinum paniculatum is a herbaceous plant known for its high medicinal value Studies on the chemical composition of Talinum paniculatum show that in leaves and roots contain bioactive substances such as alkaloid, flavonoid, saponin, tannin, phytosterol, phytol; the content of phytol is very high (69,32 %) Since ancient time, T paniculatum has been used in traditional medicine, especially in the treatment of type diabetes, dermatitis, gastrointestinal disorders, physiological weakness and reproductive disorders Galactogue in leaves has anti-inflammatory effects, during the maternity has increased the use in stimulation of lactation, and has the ability to treat these ulcers The roots of T paniculatum are used to promote fertility and treat gynecological diseases such as menstrual cycle abnormalities Steroid saponin in the roots of Talinum paniculatum has the effect of preventing and treating arteriosclerosis, and is also a raw material to synthesize sex hormones Flavonoids play important roles in human health such as having an antioxidant effect, having hepatoprotective activities, antiinflammatory, anticancer, antibacterial However, no studies have been conducted to acquire flavonoids in T paniculatum because of the flavonoid content of species in the Talinum genus, including T paniculatum very low The problem is how to improve the content of flavonoids of the Talinum genus in general and T paniculatum in particular to be able to be used in public health care So far, there have been a number of major approaches applied to medicinal plants to increase flavonoid content It is to use selective methods from populations or experimental hybrid or mutant mutations, from which select lines of plants with high flavonoid content However, there have been no studies on the application of this method to improve flavonoid content; but the application of plant biotechnology such as gene transfer and cultivation plant cell and tissue in vitro in medicinal plants has been paid attention to and highly effective in the reception of bioactive substances, including flavonoids 2 In plants, flavonoids are synthesised by the phenylpropanoid biosynthesis pathway, converts the amino acid L-phenylalanine (L-Phe) into 4-coumaroyl CoA (or one thiol ester with the presence of a 4hydroxycinnamate) There are many enzymes involved in the synthesis of flavonoids pathway such as phenylalanine ammonia-lyase, cinnamate 4-hydroxylase, 4-Coumarate CoA ligase, chalcone synthase, chalcone isomerase In particular, Chalcone isomerase (CHI) is the key enzyme in flavanone biosynthesis that catalyses the intramolecular cyclization of bicyclic chalcones (i.e., naringenin chalcone) into tricyclic (S)flavanones (i.e., naringenin) The synthesis of naringenin, the first flavanone, directs the flavonoid pathway to the synthesis of other flavanones, flavonols and anthocyanins Thus, it is of great interest to improve its flavonoid content by overexpressing the CHI gene that encodes the key enzyme of flavonoid synthesis, and the GmCHI gene encoding chalcone isomerase, a key enzyme in metabolizing synthetic flavonoid in soybeans, was chosen In addition, T paniculatum is a plant with tuberous roots, many secondary compounds concentrated in the roots, including flavonoids Therefore a method has been proposed for enhancing flavonoid content in Talinum paniculatum plants by applying tissue culture technique to produce the hairy roots to enhance biomass When plant tissue (leaves, lateral shoot bud, cotyledons ) is infected with Agrobacterium rhizogenes, T-DNA in Ri-plasmid structure carries rol genes and auxincoding genes of IAA type to be transferred into plant tissue Simultaneous expression of rol genes and auxin synthesis genes will result in a pattern of hairy roots in plant tissue that are infected with A rhizogenes For the above reasons, we conducted the research entitled “A study on the expression of GmCHI gene involved in flavonoid synthesis and hairy root induction of Talinum paniculatum plants” Aims of the study Creating GmCHI transgenic T paniculatum lines has higher flavonoid content than wild-type plants and determine suitable conditions in induction of hairy roots in vitro of T paniculatum 3 Objectives of the study (i) Study on identification of T paniculatum samples collected in some localities by comparative morphological method, combined with molecular classification methods based on some DNA barcodes (ITS region; partial sequences of matK, rpoB, rpoC1) (ii) Study on GmCHI gene transfer and creation of transgenic T paniculatum lines Analysis of the recombinant CHI protein expression in the transgenic T paniculatum lines in the T1 generation (iii) Study on establisment of hairy root lines in T paniculatum plants via Agrobacterium rhizogenes New contributions of the dissertation The dissertation is a systematic study, from the identification of T paniculatum samples collected in some localities to create the initial material source for in vitro culture to transfer the structure of carrying GmCHI gene into T paniculatum and analysis of soybean GmCHI gene expression in transgenic T paniculatum plants The main results are as follows: 1) Identifying samples of T paniculatum collected in localities in Vietnam belong to T paniculatum species, Talinum genus, Portulacaceae family 2) For the first time, the GmCHI gene isolated from soybean cultivars was successfully expressed in the T paniculatum and created two transgenic T paniculatum lines with higher flavonoid content than the wild-type plants 3) Create the hairy root lines from the T paniculatum to serve as a material for selecting the hairy root lines with high pharmaceutical content The scientific and practical significance of the dissertation The results of the dissertation are of scientific and practical significance in the approach to improve flavonoid content by overexpressing the CHI gene that encodes the key enzyme of flavonoid synthesis and creating the hairy root lines in the T paniculatum Scientific significance, the research results of the dissertation will be the basis for applying the technique of creating hairy roots and 4 transgenic plants to improve the pharmaceutical content in T paniculatum and some other medicinal plants Practical significance, the hairy roots and the transgenic T paniculatum lines provide materials for selecting T paniculatum varieties with high flavonoid content The results of the study have opened up the future prospects for applying to create hairy roots and overexpression techniques in improving pharmaceutical content in medicinal plants Dissertation structure The dissertation has 129 pages (including appendix) and is divided into chapters and sections: Introduction (04 pages); Chapter 1: Literature Review (37 pages); Chapter 2: Materials and Research Methods (16 pages); Chapter 3: Results and Discussion (41 pages); Conclusions and Recommendations (02 page); Publications related to the dissertation (03 page); References (14 pages); Appendix (12 pages) The dissertation has 16 tables, 33 figures and 131 references Chapter LITERATURE REVIEW The dissertation has 131 references, including references in Vietnamese; 125 references in English to summarize the relevant content, including: (1) Studies on in vitro culture of Talinum paniculatum plants; (2) Flavonoids and the pathway of flavonoid biosynthesis in plants, (3) CHI and expression of gene encoding CHI Talinum paniculatum (T paniculatum) plants contain flavonoids and saponins, which have strong antioxidant properties and are used in the treatment of numerous diseases, such as inflammation, allergies, and gastric ulcers Currently, there is no published research on the flavonoid content of T paniculatum plants However, it has been determined that the species in the genus Talinum have very low flavonoid content (about 0,897 mg/g fresh leaves) (Afolabi cs, 2014) Flavonoids are synthesised by the phenylpropanoid biosynthesis pathway and chalcone isomerase (CHI) is the key enzyme in flavanone biosynthesis that catalyses the intramolecular cyclization of bicyclic chalcones (i.e., naringenin chalcone) into tricyclic (S)-flavanones (i.e., 5 naringenin) To improve the content of bioactive compounds in T paniculatum (including flavonoids), until now, studies on improving the content of bioactive compounds including flavonoids in T paniculatum have mainly focused towards increasing the biomass of cells and hair roots Zhao et al (2009) proposed to select appropriate materials and concentrations of growth-promoting substances to maximize the formation of calli and buds, the rooting proportion, and the survival rate of seedlings in a greenhouse Muhallilin et al (2013) studied the effect of auxin-type plant growth regulators (IAA, NAA, IBA and 2.4-D) at various concentrations (1 mg/l, mg/l and mg/l) for root induction on leaf explants of T paniculatum While Yosephine et al (2012) studied the effect of aeration and inoculum density on biomass and saponin content of T paniculatum Gaertn hairy roots in a balloon-type bubble bioreactor by transforming a leaf sample of T paniculatum with A rhizogenes In Vietnam, there have been no published reports on creating and cloning hairy root lines in Talinum paniculatum plants Other the effective approach to enhance the flavonoid content in T paniculatum plants is overexpression of the chalcone isomerase gene in the phenylpropanoid biosynthesis pathway In the world, there have been a number of studies on overexpression of CHI genes in tomato plants (Muir et al., 2001), tobacco (Li et al., 2006), peony (Lin et al., 2014) Results of total flavonoid, flavonol and anthocyanin content increased many fold compared to non-transgenic control plants Currently, there is no research work that transferred CHI gene into T paniculatum.Thus, the application of technology to improve flavonoid content of T paniculatum by overexpressing the CHI gene that encodes the key enzyme of flavonoid synthesis should be considered and focused on research Chapter MATERIALS AND RESEARCH METHODS 2.1 RESEARCH MATERIALS Seeds and samples of T paniculatum collected from September 2015 to March 2016 in localities: Tan Yen district, Bac Giang province (BG); Thai Nguyen city (TN1); Dai Tu district, Thai Nguyên province 6 (TN2); Son Tay town, Ha Noi capital (HT); Hoanh Bo district, Quang Ninh province (QN) The seeds of T paniculatum were cultivated and analyzed on the basis of the morphological characteristics of roots, tubers, stems, leaves, flowers, fruits, seeds, and providing materials for morphological and molecular biology analysis The A rhizogenes ATTC 15834 were provided by the Department of Plant Cell Technology, Vietnam Institute of Biotechnology, Vietnam Academy of Science and Technology The Agrobacterium tumefaciens (A tumefaciens) CV58 carrying the 35S-CHI-cmyc construct in a pCB301 transgenic plasmid were provided by the Department of Modern Biology & Biological Education, Thai Nguyen University of Education 2.2 CHEMICALS, EQUIPMENTS AND RESEARCH LOCATIONS The chemicals used in the study are bought from worldwide famous firms like Fermentas, Bio-Neer, Invitrogen, Trizol Reagents, Maxima® First Strand cDNA Synthesis The experiments were carried out on the modern equipments of the Genetic Engineering Department, Faculty of Biology, College of Education - Thai Nguyen University, and Laboratory of Gene Technology, Department of Applied DNA Technology and Department of Plant Cell Technology, Institute of Biotechnology, Vietnam Academy of Science and Technology 2.3 RESEARCH METHODS 2.3.1 The identification of T paniculatum plants was done according to the method suggested by Pham Hoang Ho (1999), Do Tat Loi (2004), search on website http://www.tropicos.org/Name/26200178, and molecular classification methods based on some DNA barcodes such as ITS region, three partial sequences of matK, rpoC1 and rpoB genes 2.3.2 In vitro culture methods: (1) Sterilization methods seeds; in vitro method of multiple shoot in T paniculatum; (3) Culture method to create hairy roots in T paniculatum plants 2.3.3 Agrobacterium-mediated transformation via T paniculatum cotyledonary nodes and regeneration and selection of transgenic T 7 paniculatum plants were performed followed the method previously described by Olhoft et al (2006) 2.3.4 Methods of analysing transgenic plants: Determine the presence and incorporation of GmCHI gene into the host genomes of transgenic T paniculatum plants using PCR and Southern blot of Southern (1975) Analysis of recombinant CHI protein expression in transgenic plants by Western blot and ELISA was performed according to the method of Sun et al (2006) Determination of total flavonoid content in T paniculatum by absorption spectroscopy method were performed followed the method previously described by Kalita et al (2013) 2.3.5 Methods of analysis and data processing: The data in the study were statistically processed by SPSS software to determine average values, variance, standard deviation, average sample error Chapter RESULTS AND DISCUSSION 3.1 RESULTS OF IDENTIFYING OF T PANICULATUM SAMPLES 3.1.1 Morphological characteristics of T paniculatum samples collected in some localities The results of comparing the five T paniculatum samples collected from the localities (Tan Yen district, Bac Giang province; Thai Nguyen city; Dai Tu district, Thai Nguyên province; Son Tay town, Ha Noi capital; Hoanh Bo district, Quang Ninh province) show that samples of T paniculatum are similar in morphology, including roots, stems, leaves and flowers (Figure 3.1) Tuberous roots of T paniculatum are cylindrical with many small roots (Figure 3.1 B) The stems are upright and divided into several branches (Figure 3.1 A) The leaves are staggered, generally oval, ovate-oblong, or egg back shaped; thick, glossy with wavy veins, without hairs (Figure 3.1 C) The flowers of the plants have five reddish purple wings, two sepals, more than ten stamens, and a spherical ovary (Figure 3.1 D) The fruits are small, and the ripe fruit is ash gray in color (Figure 3.1 E) The seeds are very small, slightly flat, and black (Figure 3.1 F) 8 Figure 3.1 T paniculatum plant A: T paniculatum plant; B: Tuberous roots; C: branch, leaves; D: bud and flowers; E: fruits; F: seeds After comparing the morphological characteristics observed in the samples of T paniculatum with the characteristics of T paniculatum according to the description of Pham Hoang Ho (1999), Do Tat Loi (2004) and also looking up http://www.tropicos.org/Name/26200178 shows samples of BG, TN1, TN2, HT, QN T paniculatum were determined to belong to T paniculatum species, Talinum genus, Portulacaceae family However, if the plant is in its growing stage without flowers, it is easy to confuse it with the same species of T triangulare In addition, the classification of T paniculatum encounters obstacles if the plants have been completely or partially processed As a result, it is necessary to use an extra method and criterion for the classification The DNA barcoding method can be used to accurately identify the T paniculatum samples without confusion with other herbs 3.1.2 Characteristics of the ITS region and partial sequences of matK 3.1.2.1 Characteristics of the ITS region The results of testing PCR products by electrophoresis on 1% agarose gel with kb DNA ladder are shown in figure 3.2 The results showed that the PCR products of all samples obtained a DNA band of about 600 bp in size, which was similar to the predicted size of the ITS region The results of the sequencing indicated an ITS segment of 643 bp in size Using the BLAST tool in NCBI, the ITS sequences isolated from five T paniculatumin samples (ITS-TN1, ITS-TN2, ITS-BG, ITS-HT, ITS-QN) were 99 % homologous to the three ITS sequences of T paniculatumin GenBank, which had an accession number JF508608, 9 EU410357 and L78094; Thus, it can be concluded that the ITS region isolated from the five T paniculatumin samples is the ITS region of T paniculatum species Figure 3.2 PCR analysis of ITS region M : Marker kb; 1: ITSTN1, 2: ITS-TN2, 3: ITS-BG, 4: ITS-HT, 5: ITS-QN Figure 3.5 PCR analysis of partial sequences of matK M: Marker kb; 1: matK-TN1, 2: matK-TN2, 3: matKBG, 4: matK-HT, 5: matK-QN 3.1.2.2 Characteristics of the partial sequences of matK The results of testing PCR products by electrophoresis on 1% agarose gel with kb DNA ladder are shown in figure 3.5 The results showed that the PCR products of all samples obtained a DNA band of about 800 bp in size, which was similar to the predicted size of the partial sequences of matK The results of the sequencing indicated an matK segment of 808 bp in size Using the BLAST tool in NCBI, the matK sequences isolated from five T paniculatumin samples (matKTN1, matK-TN2, matK-BG, matK-HT, matK-QN) were 99 % homologous to the three matK sequences of T paniculatumin GenBank, which had an accession number AY015274, KY952520, GQ434150; Thus, it can be concluded that the partial sequences of matK isolated from the five T paniculatumin samples is the partial sequences of matK of T paniculatum species In addition to the sequence of ITS regions (inside the nucleus) and the matK gene segment (chloroplast gene), we sequenced two chloroplast gene segments rpoC1 and rpoB Two partial sequences of rpoC1 and rpoB genes isolated from T paniculatum plants are 595 bp and 518 bp in length, respectively Using the BLAST tool in NCBI, the two partial sequences of rpoC1 and rpoB genes isolated from five T 10 10 paniculatumin samples are the partial sequences of rpoC1 and rpoB genes of T paniculatum species 3.1.3 Discuss the results of identifying T paniculatum samples Based on morphological characteristics, T paniculatum samples were identified with characteristics of nutritional organ, reproductive organs and similar to the description characteristics of T paniculatum according to Pham Hoang Ho (1999), Do Tat Loi (2004) However, if the plant is in its growing stage without flowers, it is easy to confuse it with the same species of T triangulare, therefore, it is not possible to identify these T paniculatum samples from the same species or different species As a result, it is necessary to use an extra method and criterion for the classification The DNA barcoding method with the ITS region and three partial sequences of matK, rpoC1, rpoB genes can be used to accurately identify the T paniculatum samples without confusion with other herbs ITS region and three partial sequences of matK, rpoC1 and rpoB genes isolated from T paniculatum plants are 643 bp, 808, 595 and 518 bp in length, respectively Using the BLAST tool in NCBI, the three partial sequences of matK, rpoC1 and rpoB isolated from five T paniculatumin samples were 97 %, 99 %, 99 % homologous to the chloroplast gene sequences of T paniculatum due to Liu et al (2018) solve the sequence which had an accession number Based on the combination of the characteristics of morphology and nucleotide sequences of ITS region, matK, rpoC1 and rpoB genes, the T paniculatum samples collected in some northern provinces of Vietnam were determined to belong to T paniculatum species, Talinum genus, Portulacaceae family 3.2 GENERATE TRANSGENIC T paniculatum LINES GmCHI 3.2.1 The develoment in vitro regeneration system for gene transfer in T paniculatum plants 3.2.1.1 The results of the optimal conditions for sterilization seeds Table 3.3 shows that the seeds sterilized with 70% alcohol for minute and then with 60 % bleach for 10 minutes resulted in the highest efficiency (the rate of uninfected bottles is 92.23 %, the rate of germinated seeds reaches 91.55 %, the buds grow well) 10 11 11 Table 3.3 Effect of 60 % bleach and 0.1 % HgCl2 on seeds germination rate after 10 days of culture (n = 30) Sterilizatio Rate of Rate of Size of Morphological of n time uninfecte germinatin seedling seedling (minutes) d bottles g seeds after 10 (%) (%) days (cm) Effect of 60 % bleach on rate of germinating seeds 10 92,23c 91,55c 1,58b Fat, normal green Effect of 0,1 % HgCl2 on rate of germinating seeds 91,25b 82,26c 1,39c Fat, normal green Note: The value in each column with the same accompanying letters shows no difference with p < 0.05 3.2.1.2 The results of in vitro multiple shoot regeneration and rooting in T paniculatum Effect of BAP on the generation and shoot regeneration in cotyledonary explants Table 3.4 shows that the MS medium supplemented with 1,5 mg/l BAP is suitable for shoot emergence and bud growth from the cotyledons, the number of shoots/samples reached 1.68 (after weeks) and 1.78 (after weeks) Bảng 3.4 Effect of BAP on the generation and shoot regeneration in cotyledonary explants (n=30) BAP The % Height The Shoots concentratio number compared of number quality n of shoots/ to the shoot of leaves/ (mg/l) samples control (cm) Shoot After weeks 1,5 1,68a 136,58 0,87a 4,74b F, NG After weeks 1,5 1,78a 132,83 2,88c 6,14a F, NG Note: The value in each column with the same accompanying letters shows no difference with p < 0.05 F, NG: Fat, normal green Effect of BAP, the combination of BAP and IBA on the generation and shoot regeneration in the lateral shoot bud explants Results of analyzing the effect of BAP on the generation and shoot regeneration in the lateral shoot bud explants are shown in table 3.5 11 12 12 Bảng 3.5 Effect of BAP on the generation and shoot regeneration in the lateral shoot bud explants (n=30) Increase The BAP The compared Height numbe Shoots concentratio number of quality to of shoot r of n shoots/ wild-type (cm) leaves/ (mg/l) samples plants (%) shoot After weeks 2,0 3,04d 218,7 0,87a 5,22c F, NG After weeks 2,0 3,24c 216,00 2,88b 6,52a F, NG Note: The value in each column with the same accompanying letters shows no difference with p < 0.05 F, NG: Fat, normal green Table 3.4 shows that the MS medium supplemented with mg/l BAP is suitable for shoot emergence and bud growth from the lateral shoot bud, the number of shoots/samples reached 3.04 (after weeks) and 3.24 (after weeks) Comparison of results in table 3.4 and table 3.5 shows the generation and shoot regeneration in the lateral shoot bud explants more effectively than the generation and shoot regeneration in cotyledonary explants at the same BAP concentration Thus, mg/l BAP is the appropriate growth stimulant to create multiple shoots from the lateral shoot bud T paniculatum plant The effect of IAA and NAA on in vitro rooting ability of T.paniculatum The effect of IAA and NAA on in vitro rooting ability of T paniculatum is shown in table 3.7 Table 3.7 Effect of IAA on in vitro rooting ability of T paniculatum (n=30) The rate of IAA The number shoots creates Root length concentration of roots roots (cm) (mg/l) / shoots (%) After weeks 0,5 80,17d 5,13d 0,92b After weeks 0,5 98,12d 13,23d 3,79c Note: The value in each column with the same accompanying letters shows no difference with p < 0.05 12 13 13 Table 3.7 shows that the MS medium supplemented with 0,5 mg/l IAA is suitable for rooting from the lateral shoot bud, the number of shoots/samples reached 3.04 (after weeks) and 3.24 (after weeks) MS medium supplemented with 0.5 mg/l IAA gives the highest rooting rate of 80.17 %, reflect increases of 2.66 fold (after weeks) and 98.12 % reflect increases of 1.09 fold (after weeks) compared to the control Thus, 0.5 mg/l IAA is the appropriate in vitro rooting ability of T.paniculatum Effect of NAA on in vitro rooting ability of T paniculatum in table 3.8 shows that MS medium supplemented with 0.5 mg/l NAA gives the highest rooting rate of 58.33 %, reflect increases of 1.93 fold (after weeks) and 94.36 % reflect increases of 1.05 fold (after weeks) compared to the control Thus, 0.5 mg/l NAA is the appropriate in vitro rooting ability of T paniculatum Bảng 3.8 Effect of NAA on in vitro rooting ability of T paniculatum (n=30) The rate of The number Root NAA concentration shoots creates of roots length (cm) (mg/l) roots (%) /shoots After weeks 0,5 58,33e 3,21c 0,31a After weeks 0,5 94,36c 10,43c 2,79c Note: The value in each column with the same accompanying letters shows no difference with p < 0.05 Comparison of the rate of shoots creates roots and the number of roots/shoots at the same time of the two optimal concentrations of 0.5 mg/l IAA and 0.5 mg/l NAA showed IAA is more effective than NAA Thus, the optimal root stimulant in T paniculatum is 0.5 mg/l IAA 3.2.2 Transformation of GmCHI gene and regeneration of transgenic T paniculatum 3.2.2.1 The results of survey of transgenic materials via A tumefaciens The results of multiple shoot from cotyledons and lateral shoot bud after A tumefaciens infection are shown in table 3.9 and figure 3.9 13 14 14 Bảng 3.9 The effect of multiple shoot from cotyledons and lateral shoot bud after A tumefaciens infection (n=150) The Height of The number Shoots number of Materials shoot of leaves/ quality shoots/ (cm) shoot samples After weeks Cotyledons 3,02b 1,34a 5,21b Fat a a Lateral shoot bud 1,40 1,13 3,43a Thin Note: The value in each column with the same accompanying letters shows no difference with p < 0.05 The results of table 3.9 and figure 3.9 show that the effect of multiple shoot from cotyledons after A tumefaciens infection reflect increases of 2.15 fold (after week) compared to the lateral shoot bud At the same time shoots are produced from cotyledons with height, number of leaves, and quality of shoots better than shoots created from lateral shoot bud Thus, cotyledons are suitable materials to create multiple shoot for gene transfer in T paniculatum plants Hình 3.9 The effect of multiple shoot from cotyledons and lateral shoot bud after A tumefaciens infection A, B: The generation and shoot regeneration in cotyledonary explants through A tumefaciens infection after weeks and weeks C, D: The generation and shoot regeneration in lateral shoot bud through A tumefaciens infection after weeks and weeks 3.2.2.2 Transformation of the structure carrying the GmCHI gene and regeneration of transgenic T paniculatum The results of transformation of the structure carrying the GmCHI gene T paniculatum through A tumefaciens infection via cotyledonary nodes are shown in table 3.10 and figure 3.10 Table 3.10 showns that from a total of 730 samples, we obtained 18 CHI transgenic plant lines, 14 15 15 accounting for 2.46% of the initial samples In parallel with the CHI transgenic experiment, we cultivated two control batches called DC0 and DC1 All samples in DC0 died as expected, while in DC1, 35 plants survived in the greenhouse Figure 3.10 Transformation and regeneration of transgenic T paniculatum plants A: seeds of T paniculatum; B: germination in germination medium (GM); C: co-cultivation in co-cultivation medium (CCM) in the dark; D: inducing shoot generation; E: multiple shoot regeneration after weeks; G, H: rooting and fully rooted plantlets in rooting medium (RM); I: seedling substratum; K: plants in greenhouse Table 3.10 The results of transformation of the structure carrying the GmCHI gene through A tumefaciens infection Numbe Number Numb Controls r of Number of plants Total er of and samples of shoots grown in sampl plants experime produci produci the es growi nts ng ng roots greenho ng shoots use ĐC0 40 0 0 ĐC1 40 30 68 40 35 Experime 730 200 63 43 28 nts times 15 16 16 Note: DC0 contained non-transgenic samples cultured in medium supplemented with selection antibiotics, and DC1 contained nontransgenic samples cultured in medium without selection antibiotics 3.2.3 The results of analyse transgenic T paniculatum plants 3.2.3.1 Verifying the integration of the GmCHI gene into T paniculatum genomes in the T0 generation The results of testing the transgenic T paniculatum by PCR The results of PCR analysis with the specific primers CHI-NcoIF/CHI- NotI-R in figure 3.11 obtained a single DNA fragment with a size of about 0.66 kb corresponding to GmCHI gene size transferred to the T paniculatum in plants (T0-2.1, T0-2.2, T0-4, T0-7, T0-10, T0-12, T0-14, and T0-16) The results of testing the transgenic T paniculatum by Southern blot Eight PCR-positive plants (T0- 2.1; T0- 2.2; T0- 4; T0- 7; T0- 10; T0- 12; T0- 14; T0- 16) and wild-type plants were subjected to Southern Blot analysis to determine whether the transgene was integrated into the transgenic plant genomes Figure 3.12 shows that DNA bands occurred in transgenic plants (T0-2.1, T0-2.2, T0-4, T0-7, T0-10, and T0-14), while there was no band in T0-12, T0-16 and wild-type plants T0-7 showed DNA bands corresponding to copies, and the remaining lines, T0-2.1, T0-2.2, T0-4, T0-10, and T0-14, had only copy The transformation frequency of the CHI transgene at this stage was 5/730 = 0.68% The growth and development of the transgenic plants with positive Southern blot hybridization results were evaluated, and their next generations were subjected to protein expression analysis 16 17 17 Figure 3.11 The presence of the CHI gene from transgenic T paniculatum lines in the T0 generation identified by PCR using the specific primers CHINcoI-F/CHI- NotI-R Figure 3.12 The integration of the CHI gene into the T paniculatum genome determined by Southern blot from PCR-positive transgenic T paniculatum plants with probe segment CHI labelled with biotin 3.2.3.2 Analysis of the recombinant CHI protein expression in the transgenic T paniculatum lines in the T1 generation All transgenic plants grew and developed normally and could produce flowers and fruits However, only the seeds of plants germinated and developed into T1 generation plants, namely, T1-2.2, T1-4, T1-10, and T1-14 The leaves from these T1 generation transgenic lines were used to analyse the expression of recombinant CHI protein Total protein extracted from the leaves of transgenic plants was denatured and run on 10% SDS-PAGE and analysed by Western blot (Figure 3.13) As seen in figure 3.13, we detected a band of approximately 25 kDa corresponding to the molecular weight of the recombinant CHI protein in T1-generation lines, T1- 2.2 and T1- 10, while there was no band in the T1-4, T1-14 and wild-type lanes, suggesting that the CHI transgene was inherited from the T0 generation to the T1 generation in transgenic lines, T1-2.2 and T1-10, and translated into recombinant CHI protein Thus, the transformation frequency of the CHI gene in this period was 0.27 % (2/730) Figure 3.13 Western blot analysis for recombinant CHI expression in transgenic T 17 Figure 3.14 ELISA determined the recombinant CHI protein content in the two transgenic T paniculatum 18 18 paniculatum lines in the T1 lines (T1-2.2 and T1-10) and nongeneration and non-transgenic transgenic plants (WT – wild type) plants The contents of the recombinant CHI protein in two transgenic lines, T1-2.2 and T1-10, and wild-type plants were analysed by ELISA (Figure 3.14) The recombinant CHI protein contents in T1-2.2 and T1-10 were 6.14 µg.mg-1 and 4.29 µg.mg-1, respectively This result demonstrated that CHI protein was overexpressed in these two transgenic T paniculatum lines 3.2.3.3 Determination of total flavonoid content in the T1 generation transgenic T paniculatum lines Samples including leaves, stems and roots from the two transgenic plants (T1- 2.2; T1- 10) and wild-type plants were used to analyse the total flavonoid content (Table 3.11) Table 3.11 Total flavonoid content of the two transgenic T paniculatum lines T1-2.2 and T1-10 and non-transgenic plants Increase compared to Total flavonoid Samples non-transgenic plants content (mg/g) (%) Wild-type plants 0,57a 100 T1- 2.2 4,24c 743,86 T1- 10 2,74b 480,70 Note: The value in each column with the same accompanying letters shows no difference with p < 0.05 Table 3.11 show that T1-2.2 had the highest flavonoid content (approximately 4.24 mg/g), an increase of 743.86 % compared to that of wild-type plants (approximately 0.57 mg/g) T1-10 had lower flavonoid content (approximately 2.74 mg/g), an increase of 480.70 % compared to that of wild-type plants These results illustrated that overexpression of the CHI gene in two transgenic T paniculatum lines T1-2.2 and T1-10 effectively increased the flavonoid content in transgenic plants 18 19 19 3.2.4 Discuss the results of transformation and creation of transgenic T paniculatum lines The current research directions are mainly focused on cultivation in vitro to increase biomass, and there is no research work that establishes an effective gene transfer method to improve the content of bioactive compounds in T paniculatum, including flavonoids In this study, we overexpressed chalcone isomerase (CHI), a key enzyme in the flavonoid biosynthesis pathway, in order to increase the total flavonoid content in transgenic T paniculatum We isolated the CHI gene from soybean and transferred it into T paniculatum via A tumefaciens following a method previously described by Olhoft et al (2006) From a total of 730 samples, 28 plants belonging to 18 transgenic lines survived In this study, we demonstrated that overexpression of the CHI gene isolated from soybean remarkably increased the total flavonoid content in transgenic T paniculatum plants We have successfully generated two transgenic lines, T1-2.2 and T1-10, that contain 4.24 mg.g-1 and 2.74 mg.g-1 of flavonoid, respectively, which reflect increases of 7.4-fold and 4.8-fold, respectively, compared to that in wild-type plants Moreover, the CHI transgene was inherited from the T0 to the T1 generation and stably expressed, suggesting that we have obtained two stable transgenic lines in which the transgenes would be passed through generations Previously, Li et al (2006) transferred the CHI gene isolated from Saussurea medusa (Asteraceae) into transgenic tobacco plants, leading to an increase in the total flavonoid content 5-fold greater than that in non-transgenic plants Transferring the Ps-CHI1 gene into tobacco (Nicotiana tabacum L.) via Agrobacterium to obtain transgenic plants in the T1 generation caused a 3-fold increase in the flavonoid and flavone content compared to that in the wild type Overexpression of the CHI gene isolated from petunia in tomato (Muir et al., 2001) has generated transgenic tomatoes with a flavonoid content 78-fold higher than that in wild type… These studies confirmed the efficiency of transferring the CHI gene isolated from one species into another species to increase the contents of flavones and isoflavones in transgenic plants In addition to the approach to improve flavonoid content in T paniculatum plants by overexpression of the CHI gene, the technology for 19 20 20 establismenting of hairy root lines is the research direction to increase in vitro biomass to increase the contents of flavonoids in T paniculatum plants 3.3 ESTABLISMENT OF HAIRY ROOT LINES IN T paniculatum PLANTS 3.3.1 The results of establisment of hairy root lines in T paniculatum plants 3.3.1.1 Investigate suitable materials to create hairy roots in T paniculatum plants The results of investigate suitable materials to create hairy roots in T paniculatum plants are shown in table 3.12 and figure 3.16 Table 3.12 The results of investigate suitable materials to create hairy roots in T paniculatum plants (n=150, after weeks) Materials The rate of samples The number Root length creates roots of (cm) (%) roots/samples Leaves 65,9c 3,45c 3,25c Lateral shoot 55,6a 1,89a 1,59a bud Cotyledons 58,2b 2,32b 1,82b Note: The value in each column with the same accompanying letters shows no difference with p < 0.05 Hình 3.16 Investigate suitable materials to create hairy roots in T paniculatum plants after weeks A rhizogenes infection The results of table 3.12 and figure 3.16 shown that in the three types of materials that infect by A rhizogenes (cotyledon, lateral shoot bud, leaf tissue), leaf tissue is a suitable material for the highest rate of hairy roots 65.9 % (after weeks), the lowest is the lateral shoot bud for the rate of hairy roots of 55.6% (after weeks) Thus, leaf tissue is a 20 21 21 suitable material for transforming and inducing hairy roots in T paniculatum plants 3.3.1.2 Effect of density of bacteria, concentration AS, infection time, coculture time on the effect of creating hairy roots from leaf tissue in T paniculatum plants Density of bacteria corresponding to OD600=0.6; concentration AS 100 μmol/l; infection time of 10 minutes; days of co-culture; cefotaxime concentrations of 500 mg/l are suitable conditions for inducing hairy roots from leaf tissue (65,9 %) (Table 3.13) Table 3.13 Effect of density of bacteria, concentration AS, infection time, co-culture time on the effect of creating hairy roots from leaf tissue in T paniculatum plants (n=150, after weeks) Effect of density Effect of of bacteria concentration AS The The rate of Concen rate of sample tration sample OD600 s AS s creates (μmol/l creates roots ) roots (%) (%) 0,2 23,42a 50 43,23b 0,4 34,56c 75 47,32c e 0,6 65,9 100 65,9d 0,8 43,24d 125 45,14c 1,0 29,43b 150 40,10a Effect of infection time The rate of Infection sample time s (minute) creates roots (%) 45,23d 10 65,9e 15 40,07c 20 34,12b 25 12,51a Effect of co-culture time Coculture time (day) The rate of samples creates roots (%) 36,12d 65,9e 23,34c 14,12b 4,12a Note: The value in each column with the same accompanying letters shows no difference with p < 0.05 3.3.1.3 Study to determine the bactericidal threshold of cefotaxime The results of determining the bactericidal threshold of cefotaxime in table 3.14 show that the optimal cefotaxime concentration of bactericidal is 500 mg/l for the rate of uninfected implant disk is 93.76 % and the rate of creating hairy roots is 65,9 % This result is consistent with the study of Yosephine et al (2015) Table 3.14 Determine the bactericidal threshold of cefotaxime after weeks Concentration The rate of The rate of samples cefotaxime (mg/l) uninfected implant creates roots (%) disk (%) 21 22 22 500 93,76d 65,9d Note: The value in each column with the same accompanying letters shows no difference with p ≤ 0.05 3.3.1.4 Analysis of hairy roots carried rolC gene by PCR Results of electrophoresis of PCR products of two pairs primers of rolC and VirD2 genes showed that two partial sequences of rolC and VirD2 are 0.5 kb and 0.3 kb in positive control wells, respectively (pRi plasmid 15834); the wells running PCR products of the hairy root lines (lines 2, 3, 6, 7, 8) (Figure 3.17 A) all have the presence of a single DNA band with clear clarity at position 520 bp (same position as the rolC positive control) and no DNA band at position 338 bp of VirD2 gene (Figure 3.17 B); in contrast to positive control wells, negative control and non-transgenic root control (unidentified) wells had no bands in locations 338 bp and 520 bp A B Figure 3.17 Electrophoresis image for PCR product of rolC gene (A) and virD2 gene segment (B) 3.3.1.5 Effect of state of the MS medium on hairy roots growth of T.paniculatum In the three types of state of the liquid MS medium solid, semi-solid, liquid, hairy roots on liquid culture for shaking with the highest growth rate (4.11 g fresh weight), followed by semi-solid medium (3,02 g fresh weight) and finally solid medium (2.12 g fresh weight) increased 7.47; 5.49 and 3.85 fold compared to that in original root volume after weeks of culture (Table 3.15) Thus, in state of the liquid MS medium without growth regulator, shaking culture conditions are suitable for hairy roots growth The image showing the results of cultivating hairy roots in T.paniculatum is shown in figure 3.18 Table 3.15 Effect of state of the MS medium on hairy roots growth of T paniculatum 22 23 23 State of the medium Original root weight (g) Liquid Semi-solid Solid 0,55 0,55 0,55 Fresh root weight after weeks of culture (g) 4,11c 3,02 b 2,12a Increases in root weight (fold) Dry root weight (g) 7,47 5,49 3,85 0,34b 0,23a 0,18a Note: The value in each column with the same accompanying letters shows no difference with p ≤ 0.05 Figure 3.18 Image of adventitious hairy roots induction and culture of T paniculatum roots 3.3.2 Discuss the results of creation of hairy roots lines in T paniculatum The culture of hairy root biomass by A rhizogenes to acquire secondary compounds with biological activity is an effective solution that can overcome the limitations of traditional breeding methods and methods of growing cell biomass For T.paniculatum, study on hairy roots and application of techniques for increasing hairy root biomass were published by Yosephine et al (2012) In Vietnam, the study of creating the roots of hairy roots from plants and especially in the T paniculatum plant is very new This study showed the results of production / establishment of hairy root lines in vitro of T paniculatum through Agrobacterium rhizogenes Of the three types of materials that infect by A rhizogenes (cotyledon, stem, leaf tissue), leaf tissue is a suitable material for transforming and inducing hairy roots Density of bacteria corresponding to OD600 value=0.6; concentration AS 100 μmol/l; Infection time of 10 minutes; days of coculture; cefotaxime concentrations of 500 mg/l are suitable conditions for inducing hairy roots from leaf tissue In state of the liquid MS medium 23 24 24 without growth regulator, shaking culture conditions are suitable for hairy roots growth The obtained hairy root lines (2, 3, 6, 7, 8) were confirmed by the presence of rolC gene and absence of virD2 gene through PCR However, in order to use these T paniculatum roots in producing flavonoids in particular and secondary metabolites in general, it is necessary to continue the study, comparing the content of pharmaceutical substances between the roots of hairy roots with The roots of natural ginseng plants CONCLUSION AND RECOMMENDATIONS Conclusion 1.1 The collected T paniculatum samples in some localities were determined to belong to T paniculatum species, Talinum genus, Portulacaceae family by comparative morphology method combined with DNA barcode analysis 1.2 The cotyledonary and the lateral shoot bud explants are suitable material to create multiple shoots in T paniculatum The MS medium supplemented with 50 ml/l coconut water + 1,5 mg/l BAP is the suitable for shoot emergence and growth from axillary cotyledons The MS medium supplemented with 50 ml/l coconut water + 2.0 mg/l BAP is the suitable for shoot emergence and growth from the lateral shoot bud explants The cotyledons are suitable materials to create multiple shoot for gene transfer in T paniculatum plants From a total of 730 samples, 28 GmCHI transgenic plants were survived in the greenhouse Recombinant CHI protein was expressed successfully in two transgenic T paniculatum lines of T1-2.2 and T1-10 in the T1 generation with contents of 6.14 µg/mg and 4.29 µg/mg, respectively Two transgenic T paniculatum lines of T1-2.2 and T1-10, that contain 4.24 mg/g and 2.74 mg/g of flavonoid, respectively, which reflect increases of 7.4-fold and 4.8-fold, respectively, compared to that in wildtype plants 1.3 Leaf tissue is a suitable material for transforming and inducing hairy roots in T paniculatum Density of bacteria corresponding to OD600 value=0.6; concentration AS 100 μmol/l; Infection time of 10 minutes; days of co-culture; cefotaxime concentrations of 500 mg/l are suitable conditions for inducing hairy roots from leaf tissue In state of the liquid MS 24 25 25 medium without growth regulator, shaking culture conditions are suitable for hairy roots growth in T paniculatum Recommendations 2.1 Continue to analyse and evaluate the two transgenic T paniculatum lines (T1-2.2 and T1-10) in T2 and T3 generations to select the transgenic T paniculatum lines with high and stable flavonoid content 2.2 Continue to analyse and compare the flavonoid content between the hairy roots lines and non-transgenic roots T paniculatum 25 ... its flavonoid content by overexpressing the CHI gene that encodes the key enzyme of flavonoid synthesis, and the GmCHI gene encoding chalcone isomerase, a key enzyme in metabolizing synthetic flavonoid. .. Transformation of the structure carrying the GmCHI gene and regeneration of transgenic T paniculatum The results of transformation of the structure carrying the GmCHI gene T paniculatum through A tumefaciens... of analyse transgenic T paniculatum plants 3.2.3.1 Verifying the integration of the GmCHI gene into T paniculatum genomes in the T0 generation The results of testing the transgenic T paniculatum