Research to identify the scientific name, in vitro propagation process and biological activity of essential oil from indigenous ginger in Bac Kan (gừng đá)

ication method based on genetic sequence 2.4.2.1. Total DNA extraction method 2.4.2.2. Electrophoresis testing total DNA extraction results 2.4.2.3. DNA quality check on agarose gel 2.4.2.4. DNA quality check by spectrophotometer 2.4.2.5. DNA amplification by PCR 2.4.2.6. PCR product sequencing 2.4.2.7. Sequence editing 2.4.2.8. Building a phylogenetic tree 2.4.3. Tissue culture methods 2.4.3.1. Reasearch on clean in vitro specimen creation 2.4.3.2. Reasearch on callus regeneration 2.4.3.3. Reasearch on regeneration of shoot buds from callus 2.4.3.4. Reasearch on quick shoot buds producing 2.4.3.5. Reasearch on complete seedlings creation 2.4.3.6. Reasearch on plant hardening and transfer substrate 2.4.3.7. Reasearch on physiological and biochemical changes of in vitro plant in the transfer stage 2.4.4. Characteristics of growth and development of in vitro native plants 2.4.5. Biochemical analysis method 6 Chưng cất thu tinh dầu bằng phương pháp lôi cuốn hơi nước. Việc phân tích định tính được thực hiện trên hệ thống thiết bị sắc ký khí và phổ ký liên hợp GC/MS của hãng Agilent Technologies HP 6890N. Distillation of essential oil is conducted by steam extraction method. The qualitative analysis was performed on the gas chromatography–mass spectrometry (GC/MS) systems of Agilent Technologies HP 6890N. 2.4.6. Method of analyzing antimicrobial activity Antimicrobial activity was conducted to assess the antibiotic ability of the extracted specimens on 96-well microtitre microplates according to the method of Vander Bergher and Vlietlinck (1991) and McKane & Kandel (1996). 2.5. Research location and duration The study of morphological analysis was conducted at Vietnam National Museum of Nature - 18 Hoang Quoc Viet - Hanoi. The specimens were sent to the Chinese Academy of Sciences, Guangzhou, China. Implementation time: from 2/2016 to 6/2017. The study of specific gene segment sequencing was done at the Institute of Biotechnology - Vietnam Academy of Science and Technology. Implementation time: from 01/2017 - 7/2017. In vitro propagation studies were conducted at the Agricultural Genetics Institute - Vietnam Academy of Agricultural Sciences; Hung Vuong University - Viet Tri City - Phu Tho Province. Implementation time: from 2/2016 to 6/2017. The study of biochemical analysis and antibiotic activity were conducted at the Institute of Natural Products Chemistry - Vietnam Academy of Science and Technology. Duration: from 7/2017 - 8/2018. The study of assesment of the growth and development abilities of tissue culture Bac Kan ginger was conducted in Phu Tho and Na Ri district - Bac Kan province. Implementation time: from 4/2016 - 8/2018. 7 CHAPTER 3 RESULTS AND DISCUSSION 3.1. Taxonomy of Bac Kan ginger plant 3.1.1. Biological traits of Bac Kan ginger plant 3.1.1.1. Some agronomic and morlogical traits Table 3.1: Some typical traits of Bac Kan ginger No Trait Expression level 1 Plant height 80-150 cm 2 Dissection of plant at the base Rounded 3 Smell of stem Yes 4 Length of petioles Short (3-6 mm) 5 Leaf blade shape Elliptic-lanceolate 6 Leaf length From 13 - 16 cm 7 Leaf width From 2,5 – 3,3 cm 8 Ratio of length/width of leaf From 3 – 5 9 Leaf color Dark green 10 Leaf stripe Yes 11 Leaf margin Pubescent 12 Leaf margin color Dark green 13 Leaf smell Yes 14 Flowering frequency Once a year 15 Number of flowers/cincinnus 2 - 4 flowers 16 Flower structure Inflorescence 17 Flower color Sepia, yellow 18 Root shape Branching 19 Root size Small (200g) 20 Root skin color Sepia 21 Root flesh colour in the center Gray 22 Secondary color in the root flesh Yellow 23 Yield of root/cluster (kg) 150 – 300g 24 Number of tubers on cluster 4 – 6 tubers. 25 Root length 5 – 10cm 26 Root length at the widest part 2 – 3cm 27 Growth time Long >10 months 3.1.2. Classification based on morphological traits 3.1.2.1. Classification to genus level 8 According to the morphological analysis results compared to the classification key of Nguyen Quoc Binh (2011), Bac Kan ginger plant has traits such as inflorescence on the top, stem with leaves, dimpled shaped pistil, spherical fruits, which are the characteristics of the plants of the genus Alpinia (Alpinia) to distinguish them from other genera of the Ginger family (Zingiberaceae) [6]. Based on that, we identified the plant named Limestone ginger in Bac Kan to be in the genus Alpinia (Alpinia), which is one of the new valuable findings because previously it was commonly called "Limestone ginger" and understood as a plant of the genus Ginger (Zingiber). 3.1.2.2. Classification to species level After identifying that the genus source of Bac Kan ginger belongs to Alpinia genus (Alpinia), we used classification key to species of Alpinia genus (Alpinia) in Vietnam [8]. However, the trait description shows that this species did not match with any of the species so far reported from Vietnam. On this basis, we continued to examine neighboring areas (in China), used the plant species list and species identification key of species of China's Alpinia genus (Alpinia) [48] and compared to near species, the results identified Bac Kan ginger with the scientific name Alpinia coriandriodora D. Fang with typical traits: typical aroma and deciduous. This is an additional species to the Vietnamese flora, increasing the total number of known species of the genus Alpinia Roxb. in Vietnam to 34 species [18]. This plant was published by D. Fang in the journal Acta Phytotax. Sine. 16 (4): 79 1978 [57]. 3.1.3. Classification of Bac Kan ginger based on the ITS and matK gene sequences 3.1.3.1. Total DNA extraction Extracted DNA of 6 research specimens. The results showed that the A260/A280 indexes of all specimens ranged from 1.8-2.0, proving that the total DNA obtained is guaranteed for future experiments. 3.1.3.2. Analysis of amplified products 9 The results of electrophoresis showed that the PCR product of the matK primer pair was about 900bp, the size of the PCR product of the ITS primer pair was about 800bp, which is consistent with the theoretical size. 3.1.3.3. Classification of Bac Kan ginger based on the ITS gene sequence 3.1.3.3.1. ITS gene sequencing results Successfully solved 06 new ITS sequences and registered on Genbank with the codes: MN227653, MN227654, MN227655, MN227656, MN227657 and MN227658 (Appendix 3). After the modification and removal of all ITS genomic vacancies, the ginger specimens obtained had nucleotide sequence similarity of ITS genome from 99 - 100% and length of 698bp. There are 02 nucleotide positions with differences between specimens, regarding nucleotide position 597, while specimens of GD01LT, GD02LT and GD01XD are type T nucleotides, the remaining specimens of GD03LT, GD02XD, GD03XD are type C nucleotides; regarding nucleotide position 605, while the GD03LT specimens are type T nucleotides, the remaining specimens are type C. The differences between the nucleotides in the aforementioned positions may indicate a genetic diversity among Bac Kan ginger plants, which is a very important basis for research to select superior lines, crossbreed, and select high-yield breeds. The six ITS gene sequences of the research specimens were compared to the ITS sequences of the genus Alpinia (taxid: 94326) published on NCBI using the Blast nucleotide tool. The results showed that the species with the ITS sequence at the highest level of similarity with the ITS sequence of Bac Kan ginger specimens include: Alpinia chinensis (EU909426.1), Alpinia japonica (EU909427.1), Alpinia officinarum (EU909422 .1), Alpinia pumila, Alpinia nieuwenhuizii. 3.1.3.3.2. Building a phylogenetic tree by ITS gene sequence The phylogenetic tree of Bac Kan ginger specimens by ITS gene indicator (Figure 3.7) was constructed from Maximum Likelihood 10 method with a value of boostrap value 1000, showing that 06 specimens of Bac Kan ginger were classified in the same group as: Alpinia coriacea, A. pumila, A. japonica, A. polyantha, A. intermedia, A. stachyodes, A. maclurei, A. suishaensis, A. guangdongensis. This result is quite appropriate because in fact, the above species have the same distribution area as A. coriandriodora of southern China. 3.1.3.4. Classification of Bac Kan ginger based on the matK gene sequence 3.1.3.4.1. matK gene sequencing results Successfully solved the sequence for 06 new matK gene sequences and registered on Genbank with the codes: MN335320, MN335321, MN335322, MN335323, MN335324, MN335325 (Appendix 4). After the modification and removal of all vacancies in matK gene region, the ginger specimens obtained (GD01LT, GD02LT, GD03LT, GD01XD, GD02XD, GD03XD) had nucleotide sequence similarity of 100% and length of 700bp. The six matK gene sequences of the study specimens were compared to the matK sequences of the genus Alpinia (taxid: 94326) published on NCBI using the Blast nucleotide tool ( Blast.cgi). The results showed that: the species with matK sequence at the highest level of similarity with matK sequence of Bac Kan ginger specimens include: Alpinia zerumbet, A. mutica, A. kwangsiensis, A. hainanensis, A. uraiensis, A .uraiensis, A. shimadae, A. formosana, A. japonica, A. japonica, A. chinensis, A. calcarata, A. oxyphylla. 3.1.3.4.2. Building a phylogenetic tree by matK gene sequences Based on the matK gene sequence obtained from specimens of Bac Kan ginger and the published database of matK gene sequences of Alpinia genera, we have built a tree diagram of genetic relationships between A. coriandriodora species and species in the genus Alpinia based on the combined data block of matK gene region by the Maximum Likelihood method (Figure 3.9). The tree diagram shows that specimens of Bac Kan ginger are in the same group with the following species: A. guinaensis, A. 11 zerumbet, A. mutica, A. polyantha, A. mutan, A. blepharocalyx, A. rugosa, A.calcarata, A. macrlure, A. guangdongensis and A.japonica. 3.1.3.5. Classification of Bac Kan ginger based on the combination of ITS and matK genes The phylogenetic tree results generated by a single data block such as matK and ITS are similar, but they show that results from a single data block are unclear and support levels are low. Therefore, we used phylogenetic trees from matK and ITS combined data block to identify the phylogenetic relationship of Alpinia and the position of Alpinia coriandriodora (Figure 3.10). The analysis of combined molecular data by the maximum likelihood (ML) and Bayesian Inference (BI) method strongly supported that Alpinia is not a nonmonophyletic group with a very high support index (BS: 100%, PP: 1.0) (Figure 3.10). The phylogenetic tree distinguished six distinct branches of Alpinia, which is completely consistent with the research by Kress et al., (2005) [77]. Among them, Bac Kan ginger (Alpinia coriandriodora) was recorded as a member of Alpinia genus with the phylogenetic position in branch VI (Figure 3.10). The analysis also shows that Bac Kan ginger (Alpinia coriandriodora) has a very close relationship with some Alpinia members in southern China (Guangxi, Yunnan, Quang Dong, Hainan) such as A. japonica , A. coriacea and A. guangdongensis, etc. This result shows that A. coriandriodora has genetic similarities with species in the same distribution area. Moreover, this result is a solid basis to confirm the addition of Alpinia coriandriodora to the Vietnamese flora [51]. 12 Figure 3.10. Tree diagram of the genetic relationship between species of A. coriandriodora and species in the Alpinia genus based on the combined data block of matK and ITS (A), and the phylogenetic position of Alpinia coriandriodora in Alpinia (B) genus. The ML and PP support indexes of BI analysis are presented on the branches. "-" indicates support index of lower than 50%. Red: Position of A. coriandriodora; Green: species belonging to the same taxonomic branch as A. coriandriodora) 13 3.2. Propagating Bac Kan ginger plants by in vitro thin layer culture technology 3.2.1. Creating in vitro clean specimen The effective sterilization formula for specimens of young shoot buds of Bac Kan ginger is dual disinfection with 2.5% NaOCl and 0.5ml Tween20 solution in 5 minutes for the first time, 15 minutes for the second time, between the two periods, rinse well with sterile distilled water at least 3 times. 3.2.2. Regenerating callus from shoot bud cutting Table 3.6. Effect of combination of TDZ and 2.4D on callus regeneration of shoot bud cutting (after 8 weeks of culture). Formula Growth regulators (mg/l) Callus regeneration rate (%) Callus morphology TDZ 2,4D ĐC 0 0 32,22 Some specimens could not regenerate callus and turned black CT8 0,5 1,0 34,44 Callus surface was dry, firm and bright white. CT9 0,5 2,0 44,44 CT10 0,5 3,0 75,56 CT11 0,5 4,0 54,44 Some specimens could not regenerate callus and turned black LSD0,05 5,05 P.value <0,001 To regenerate callus from Bac Kan ginger shoot bud cutting, use a combination of 0.5 mg/l TDZ and 3 mg/l 2.4D, callus regeneration rate was 75.56%, callus had a dry, firm and bright white surface. This research result is consistent with the previous announcement when studying the propagation of Curcuma kwangsiensis Lindl. and Alpinia purpurata by callus regeneration method [80, 114]. 3.2.3. Regenerating shoot buds from callus The research results showed that MS medium supplemented with 2.0 mg/l Vitamin B1 and 3.0 mg/l BAP was suitable for the regeneration 14 of shoot buds from callus, the rate of shoot bud burst reached 78.89%, the rate of shoot bud regeneration reached 9.71 shoot buds/callus. 3.2.4. Multiplication of in vitro shoot buds 3.2.4.1. Effect of BAP and Kin on the ability to regenerate and multiply shoot buds in vitro Research results show that the combination use of 2.0 mg/l BAP; 1.0 mg/l Kin and 0.2 mg/l α-NAA increased shoot bud multiplication rate (reaching 5.98 times) and shoot bud mean height (6.07 cm) compared with the separate use of 3.5mg/l BAP (shoot bud multiplication rate reached 5.03 times, average height of shoot buds reached 4.18cm) according to the study of Trinh Thi Thanh Huong et al (2014). 3.2.4.2. Effect of coconut water on the ability to regenerate and multiply shoot buds in vitro Table 3.9 Effect of coconut water content on shoot multiplication efficiency (after 4 weeks of culture) CT Coconut water (ml/l) Multiplier (times) Shoot height (cm) Number of leaves/buds (leaves) Shoot morphology CT22 0 5,91 5,87 4,67 Fat buds, dark green stems, good growth. CT23 100 6,32 5,90 5,00 Fat buds, dark green stems, good growth. CT24 150 5,69 5,77 4,33 Fat buds, dark green stems CT25 200 4,71 5,27 3,67 Medium bud, light green stem CT26 250 3,71 4,67 3,33 Small buds, light green stems, some yellow leaves. LSD0,05 0,14 0,38 0,81 P- value <0,001 <0,001 0.0142 15 Compared with the results of Trinh Thi Thanh Huong (2014), the use of MS + 30g/l Succrose + 6 g/l agar + 2.0mg/l BAP + 1.0mg/l Kin + 0.2mg/l α-NAA + 100ml/l coconut water improved shoot multiplier, which increased from 5.03 times to 6.32 times, and the shoots obtained were of good quality and fat with large leaves and dark green stems and grew well. Thus, the appropriate content of coconut water to supplement to the medium is 100ml/l and the suitable medium to multiply Bac Kan ginger buds in vitro is: MS + 30g/l Succrose + 6 g/l agar + 2.0mg/l BAP + 1.0mg/l Kin + 0.2mg/l α-NAA + 100ml/l coconut water. 3.2.5. Regenerating roots to create complete plants in vitro The results of the study showed that the formula using MS medium supplemented with 0.6mg/l NAA had the best root regeneration and complete plant formation with rooting time from 19 to 21 days, the average height/plant was 9.63cm, the average number of leaves/plant was 5.33 leaves, the average number of roots was 5.51 roots/shoot and the average length of roots was 4.43cm. This research result is consistent with the previous publication of the author when researching on propagation of ginger family, especially it confirmed the research results of Trinh Thi Thanh Huong et al (2014) when researching on Bac Kan Limestone ginger [19,51,112]. A B Figure 3.15. Complete plant morphology after 6 weeks of culture A: On ½MS medium (supplemented with NAA content from 0.2 -1.0mg/l) B: On MS medium (supplemented with NAA content from 0.2 -1.0mg/l) 1,0 0,2 0,4 1,0 0,8 0,6 0,2 0,4 0,8 0,6 16 3.2.6. Transfer stage 3.2.6.1. Transfer substrate On a 100% fine sand substrate, the quality of the plant was significantly improved. The average plant height, number of leaves and roots/plant after 15 days reached 9.43 cm; 5.33 leaves/plant; 5.33 roots/plant compared with the average plant height, number of leaves and roots/plant initially being 9.2 cm, 5 leaves/plant and 5 roots/plant. By the 30th day, the average height of the plant was 9.67 cm, the average number of leaves was 5.67 leaves/plant and the average number of roots per plant was 6.00 roots/plant. 3.2.6.2. Reasearch on physiological and biochemical changes of Bac Kan ginger in the transfer stage 3.2.6.2.1. Free water, bound water content and dry matter content 3.2.6.2.2. Photosynthetic pigment content 3.2.6.2.3. Chlorophyll fluorescence 3.2.6.2.4. Catalase activity 3.2.7. Process of propagating Bac Kan ginger plants by in vitro thin layer culture technology 3.2.7.1. Process maps 3.2.7.2. Process Description * Step 1: Prepare materials * Step 2: Create callus * Step 3: Regenerate shoots from callus * Step 4: Multiply shoots in vitro * Step 5: Regenerate a complete in vitro plant * Step 6: Transplant plants in nursery 3.3. Agro-biological characteristics of Bac Kan ginger plants derived from tissue culture Bac Kan ginger plants derived from in vitro culture were grown in Viet Tri City - Phu Tho Province under field conditions to assess agro- 17 biological characteristics: Growth patterns of plant height; Leaf growth patterns; Tillering patterns; Some factors constituting the productivity. Planting time: April 2016 3.3.1. Growth patterns of plant height The research results showed that the growth of plant height of Bac Kan ginger during the monitoring period was relatively slow. 20 days after planting, the average height of the plant was 4.39cm. 50 days after planting, the average height of the plant was 8.88cm (increased by 4.49cm/30 days); 80 days after planting, the average height of the plant is 13.60 cm (increased by 4.72 cm/30 days). The growth rate of the plant height was highest at 5.41 cm/30 days (170-200 days after planting) and 4.73 cm/30 days (140 - 170 days after planting). 290 days after planting, the average plant height was 41.85cm; 320 days after planting, the average plant height was 44.26 cm. 3.3.2. Leaf growth patterns The research results showed that, corresponding to the growth speed of the plant height, leaf growth patterns of Bac Kan ginger were also relatively slow. 20 days after planting, the average number of leaves was 3.03/stem; 50 days after planting, the average number of leaves reached 3.59 leaves/setm; Growth rate was 0.56 leaves/30 days. From 50 to 80 days after planting, the growth rate was 0.55 leaves/30 days; 80 - 110 days after planting, the growth rate was 0.48 leaves/30 days. From 110 days after planting onwards, the speed of leaf growth of Bac Kan ginger was faster than the first stage. 320 days after planting, the average number of leaves reached 9.48 leaves/stem. 3.3.3. Tillering patterns The results showed that, 20 days after planting, the tillering rate was low, the average number of branches was 1.10 branches/plant, during this period the plants had to adapt to field environment conditions, the ability to accumulate nutrients was limited. After 50 days, when the plant started to grow its root and turned green, Bac Kan 18 ginger plant was able to start tillering, the number of branches/plant reached 1.57 branches and 80 days after planting, it reached 2.23 branches/plant. After 140 days, Bac Kan ginger plant continued tillering but the tillering rate was lower than the first stage. 320 days after planting, the average number of branches/plant was 5.34 branches/plant. 3.3.4. Some factors constituting the productivity of Bac Kan ginger through tissue culture The research results showed that 320 days after planting, the average number of roots/clump of Bac Kan ginger plant reached 4.73 roots/clump, the average root length was 5.41cm and the average root width was 2.26cm. The average individual yield or weight of roots/clump of Bac Kan ginger was 147.7g/clump. 3.4. Chemical composition of essential oil of Bac Kan ginger 3.4.1. Essential oil extraction results The essential oil obtained from the specimens of leaves was light yellow in color with a pleasant aroma. In the Bac Kan natural ginger specimen, from 400g of fresh leaves, 0.440g of essential oil was obtained, which was 0.110% concentration (based on the specimen of fresh material). Regarding the tissue culture Bac Kan ginger specimens, the content of essential oil obtained from leaves of the specimens grown in Bac Kan and grown in Phu Tho had the same proportion of essential oil content, respectively 0.113% and 0.133%, and both were higher than that of Bac Kan natural ginger specimen. This proves that planting tissue specimens in two different locations (Bac Kan and Phu Tho) did not affect the concentration of essential oil (Table 3.17). Essential oil obtained from the specimens of roots was dark yellow in color and also had a pleasant aroma similar to that obtained from the leaves. When compared with the Bac Kan natural ginger specimens, tissue culture ginger specimens showed a higher concentration of essential oil obtained from the leaves than from the roots. In the Bac Kan natural ginger specimens, from 500g of specimens of fresh roots, 19 0.412g of essential oil could be obtained, which was 0.082% concentration (calculated based on the specimen of fresh material). Regarding tissue culture Bac Kan ginger specimens, essential oil obtained from the roots had a much lower concentration than the essential oil obtained from the leaves (0.075% for specimens of roots compared to 0.113% for specimens of leaves). (Table 3.17). 3.4.2. Chemical composition of essential oil By gas chromatography-mass spectrometry analytical method (GC- MS), the essential oil obtained from leaves of the Bac Kan natural ginger (TNBK) specimen was identified to have 14 components (accounting for 94.38%), the tissue culture Bac Kan ginger (CMBK) specimen had 13 components (accounting for 85,16%), the tissue culture Phu Tho ginger (CMPT) specimen had 18 components (accounting for 84.33%). The composition of essential oil obtained from Rhizome of TNBK specimen had 15 components (94.24%), CMBK specimen had 14 components (85.27%), CMPT specimen had 18 components (88.65%) (Table 3.18 ). Essential oil obtained from leaves contained the main compound as decenal (80.78%), followed by decen-1-ol (4.91%), octenal (2.71%), decanal (1.25%) and 2-decenoic acid (1.03%). Decenal is a major component of coriander essential oil Coriandrum sativum L., which is used as a spice and a flavoring agent [84]. Just like the essential oil obtained from leaves, the essential oil obtained from natural ginger root constituted the highest content of decenal (80.48%), followed by octenal (3.01%), 2-decenoic acid (1.69%), decen-1-ol (1.53%) and dodecanal (1.17%). 20 Table 3.18: Chemical composition of essential oil obtained from research specimens No Compound Leaf specimen (%) Root specimen (%) Molecula r formula TNB K CMB K CMP T1 CMP T2 TNB K CMB K CMP T1 CMP T2 1 Camphene - - - - 0,43 - 0,31 2,79 C10H16 2 Octanal 0,36 0,56 0,41 0,59 0,22 1,05 0,32 0,23 C8H16O 3 Cymene 0,67 0,23 0,68 1,18 0,25 0,15 0,38 0,87 C10H14 4 Limonene - - - 0,14 0,12 - - 0,98 C10H16 5 Cineole 1,8 - - 0,27 - 0,39 0,22 0,45 1,78 C10H18O 6 Octenal 2,71 0,71 1,64 3,42 3,01 0,32 1,36 4,47 C8H14O 7 Borneol - - 0,34 - 0,36 - 1,02 0,98 C10H18O 8 Decanal 1,25 0,58 0,76 1,85 0,91 - 0,25 1,00 C10H20O 9 Fenchyl acetate - - 2,54 - 2,25 2,11 3,35 5,25 C12H20O2 10 Decenal 80,78 39,60 47