Study on the composition, distribution of amorphophallus spp. tubers containing glucomannan and selection of species which has the potential to be grown in some northern provinces in Vietnam

environment conditions, PBQ4: Put inside sand, PBQ5: Put inside ground. 2.3.6.3. Method of multiplication research of Amorphophallus konjac with tissue culture technology To conduct the thesis topic, 7 experiments must be carried out with the following arrangements: 1. Effect of chemicals and time of sterilization to in vitro clean sampling ability Make use of chemicals: 0,1% of HgCl2 within the sterilization time between 4 to 8 minutes and 60% of NaClO (javel) within the sterilization time between 6 to 18 minutes, then clean for 4 to 5 times with sterile distilled water. The experiment is done with 6 formulars including KT1, KT2, KT3, KT4, KT5, KT6. 2. Effect of nutrient environment to reproductive ability of shoots of in vitro Amorphophallus konjac By this experiment, taking shoots of cultivar in vitro to transplant into different fundamental nutrient environments, includes: Environment ½ MS; MS (Murashige and Skoog medium); WPM (Woody Plant Medium); B5 (Gamborg Medium); N6 (Chu medium), these environments are complemented with 0,5 mg/l BAP + 8 g/l agar + 30 g/l of sucrose, to determine the suitable nutrient environment to reproduce shoots of in vitro Amorphophallus konjac. The experiment includes 5 formulas including MT1, MT2, MT3, MT4, MT5. 3. Effect of BAP concentration to reproductive ability of shoots of in vitro Amorphophallus konjac For Amorphophallus konjac, we study the effect of BAP concentration to reproductive ability of shoots in the range of between 0,5 and 3,5 mg/l. The experiment is conducted with 7 formulas in which each formula is carried out on the fundamental nutrient environment defined in the experiment 2, complementing 8 g/l agar + 30 g/l sucrose and growth promoting factors in different concentrations. The experiment includes 7 formulas including CT0, CT1, CT2, CT3, CT4, CT5, CT6. 4. Effect of BAP and Kinetin concentration to reproductive ability of shoots of in vitro Amorphophallus konjac Maximum BAP concentration for reproductive ability of shoots in vitro is used to research the effect of BAP and Kinetin to reproduct shoots quickly. Environment formula MS + 8 g/l agar + 30 g/l sucrose + BAP concentration is suitable in the experiment 3 and kinetin is complemented in different concentrations between 0,2 and 1,0 mg/l. The experiment includes 5 formulas including CT7, CT8, CT9, CT10, CT11. 5. Effect of nutrient environment to rooting ability of in vitro Amorphophallus konjac Shoots which are created with the huge quantity and meet the standard (with the size of between 3 and 5 cm), are cut and transferred to the rooting environment: Environment formula MS (1962) and ½ MS (macro and micro-mineral substances reduce ½) + 8 g/l agar + 0,5 mg/l IBA + 1g/l of activated carbon, complementing sucrose with different contents between 12 and 20g/l. The experiment includes 8 formulas including CR1, CR2, CR3, CR4, CR5, CR6, CR7, CR8. 10 6. Effect of BAP concentration to rooting ability of shoots of in vitro Amorphophallus konjac After determining the proper nutrient environment for rooting of shoots of in vitro Amorphophallus konjac, conduct the research the effect of growth promoting factors IBA to the rooting of shoots. The experiment requested to use the suitable nutrient environment is determined in the experiment 5 and complements IBA with different concentrations between 0 and 1mg/l. The experiment includes 6 formulas including CIR0, CIR1, CIR2, CIR3, CIR4, CIR5. 7. Effect of media to survival rate of in viro Amorphophallus konjac in nursery garden To determine the effect of media components to the survival and growth rate of in vitro Amorphophallus konjac during the experiments at the nursery garden, we do test with 4 following formulas: GT1, GT2, GT3, GT4 with 100% of floor B ground; 100% of sand; 50% of ground: 50% of sand; 50% floor B ground: 30% of sand : 20 % of rice husk. 2.3.7. Method of research of some planting technologies of Amorphophallus konjac Methods are applied in the research of planting Amorphophallus konjac based on agricutural experiment allocation method by Nguyen Thi Lan and Pham Tien Dung – Institue of Agriculture and Hanoi Publishing House 2005. The experiments arranged on Randomized Complete Block Design (RCBD) are repeated 3 times in which each time is repeated on 15m 2 and measuring criteria on 40 plants per criterion. Non-critera factors are ensured consistency between experimental formulas. 2.3.7.1. Research the effect of tuber weight to the growth of Amorphophallus konjac The experiment includes 3 following formulas: KL1: Tuber from 10 to 15g, KL2: Tuber from 70 to 100g, KL3: Tuber from 300 to 350g. 2.3.7.2. Research the effect of coverage rate to growth of Amorphophallus The experiment includes 5 following formulas: TC1: Coverage rate of 0%, TC2: Coverage rate of 20%, TC3: Coverage rate of 40 %, TC4: Coverage rate of 60%, TC5: Coverage rate of 80%. 2.3.7.3. Research the effect of seasons to growth of Amorphophallus The experiment includes 4 following formulas: TV1: Cultivate on Feb 05th, TV2: Cultivate on Mar 05th, TV3: Cultivate on Apr 05th, TV4: Cultivate on May 05th. 2.3.7.4. Research of the effect of plantation density to growth of Amorphophallus The experiment includes 3 following formulas: MĐ1: plantation density of 50x70cm = 28571 plants/hectare, MĐ2: plantation density of 50x50cm = 40000 plants/hectare, MĐ3: plantation density of 50x30cm = 66670 plants/hectare 2.3.7.5. Research of the effect of NPK fertilizer to growth of Amorphophallus The experiment includes 5 following formulas: PB1: No fertilizer, PB2: 80 kg N + 30 kg P2O5 + 100 kg K2O/ 1 hectare, PB3: 100 kg N + 40 kg P2O5 + 120 kg K2O/ 1 hectare, PB4: 120 kg N + 50 kg P2O5 + 140 kg K2O/ 1 hectare, PB5: 140 kg N + 60 kg P2O5 + 160 kg K2O/ 1 hectare 2.3.7.6. Research of glucomannan accumulation of Amorphophallus konjac during growth stages 11 The experiment is arranged for 5 stages of analysis of glucomannan contents such as: TL1: glucomannan content during storage, TL2: glucomannan content during shooting stage, TL3: glucomannan content when leaves are maximumly developped, TL4: glucomannan content when 2/3 of leaves are turned into yellow, TL5: glucomannan content when leaves die after 1 month. 2.3.7.7. Trial planting of Amorphophallus konjac in some Northern Highlands Provinces of Vietnam. Evaluate productivity, glucomannan contents and feasibility during planting. Planting model is arranged with 6 following types: MH1: Cultivate in Van Ho commune, Van Ho district, Son La province, MH2: Cultivate under Plum shade in Van Ho commune, Van Ho district, Son La province, MH3: Cultivate in Quyet Tien commune, Quan Ba district, Ha Giang procvince, MH4: Cultivate in the forest in Quyet Tien commune, Quan Ba district, Ha Giang procvince, MH5: Cultivate in bags in Street No.10, Hoa Binh city, MH6: Cultivate in the forest in Ngoc Son commune, Lac Son district, Hoa Binh province. 2.3.8. Tracking data and method of determination 2.3.8.1. Tracking sexual multiplication by seeds - Rate of germinated seeds (%) (the number of germinated seeds/the number of sowed seeds), rate of surviving plants (%) (the number of surviving plants after one month of germination/the number of germinated plants) - Leaf height (cm) (from root to the big split point). - Rate of pestilent insects by naked eyes, taking shots and consulting with the experts 2.3.8.2. Tracking multiplication by nurseling tuber - Height (cm), dimension (cm), weight (g) of Amorphophallus tuber - Rate of shooted tubers (%) (the number of shooted tubers/the number of planted tubers), rate of surviving plants (%) (the number of surviving plants after one month of shooted tubers/the number of shooted tubers) - Leaf height (cm) (from root to the big split point). - Rate of pestilent insects by naked eyes, taking shots and consulting with the experts 2.3.8.3. Tracking multiplication via plant tissue culture 2.3.8.4. Tracking of planting research (1)- Growth and development criteria of Amorphophallus + Time of growth (days): beginning from planting to ½ of leaves on plants is turned into yellow + Height of plants (cm): measuring from the root intersection with body to growth point of highest leaves. Randomly select 40 plants and measure their height every 15 days from ground to the point of highest leaves after plants are arisen for 15 days. - Size of leaves: when leaves are comprehensively developed, measure the length and width of leaves. Follow up 40 marked plants. 2.3.8.5. Tracking pestilent insects Diagnose pestilent insects according to Handbook of plant disease diagnosis in Vietnam by Lester W. Burgess, Timothy E. Knight, Len Tesoriero, Phan Thuy Hien 12 which is published by Australian Centre for International Agricultural Research) in 2009. Evaluate pestilent instects by naked eyes, taking shots and consulting with the experts [80]. 2.3.9. Data processing Method of experiment allocation and date processing (by calculating the average numbers, analyzing Anova and relativeness, so on) are summarized and handled by Statistical Product and Services Solutions SPSS 20.0, Sotware for Agricultural Data Analysis IRRISTAT 5.0 and Excel 2010. CHAPTER 3 RESULTS AND DISCUSSION 3.1.Species composition, distribution, and indigenous knowledge of Amorphophallus species in mountainous area in northern Viet Nam 3.1.1. Species composition 3.1.1.1. Species composition Results of field surveys and collection of specimens for species identification in 14 mountainous provinces of Northern Vietnam, the authors found 8 species including the following species: Amorphophallus konjac K. Koch, Amorphophallus corrugatus NE Br. Amorphophallus krausei Engl. & Gehrm, Amorphophallus yunnanensis Engl. & Gehrm, Amorphophallus yuloensis H. Li, Amorphophallus paeoniifolius (Dennst.) Nicolson, Amorphophallus hayi Hett. __ N-a hay, Amorphophallus coaetaneus S. Y. Liu & S. J. Wei. Of these 8 species, the results of glucanannan analysis by Melinda Chua et al. (2012), with reagents of 3.5-dinitro salicylic acid at 550 nm, are 6 species Contains glucomannan. The results are shown in Table 3.1 as follows: Table 3.1. Composition of Amorphophallus that contains glucomannan in mountainous northern Vietnam No. Common name Science name Glucomannan content in dry flour (%) 1 Amorphophallus konjac Amorphophallus konjac K. Koch 44,97 2 Amorphophallus corrugatus Amorphophallus corrugatus N. E. Br. 28,6 3 Amorphophallus krausei Amorphophallus krausei Engl. & Gehrm. 29,2 4 Amorphophallus yunnanensis Amorphophallus yunnanensis Engl. & Gehrm. 25,97 5 Amorphophallus yuloensis Amorphophallus yuloensis H. Li 30,07 6 Amorphophallus paeoniifolius Amorphophallus paeoniifolius (Dennst.) Nicolson 6,53 3.1.1.2. Key to classify 6 species found in mountainous area in northern Viet Nam From the morphological characteristics of the six species of Amorphophallus that contains glucomannan, A. konjac K. Koch, A. corrugatus N. E. Br., A. krausei 13 Engl. & Gehrm, A. yunnanensis Engl. & Gehrm, A. yuloensis H. Li, A. paeoniifolius (Dennst.) Nicolson. The thesis is designed key to classify as follows: Key to classify 6 species of Amorphophallus that contains glucomannan in mountainous area in northern Viet Nam 1A. Flowers with short stems. 2A Big flowers; extra is brown-red.1.Amorphophallus paeoniifolius 2B Small flowers; extra is light yellow...............2.Amorphophallus yuloensis 1B. Flowers with long stems. 3A. Stalk shorter than flower, tube and blade are separated....3.Amorphophallus konjac 3B. Stalk shorter than flower, tube and blade are not separated 4A. Flowers with no stem...........................................4. Amorphophallus krausei 4B. Flowers with stem. 5A. Extra is wizen...............................................5. Amorphophallus corrugatus 5B. Extra is smooth.....................................................6. Amorphophallus yunnanensis 3.1.2. Distribution features 3.1.2.1. Characteristics of species distribution of Amorphophallus that contains glucomannan by height Results of the survey on the distribution of 6 species of Amorphophallus that contains glucomannan in mountainous areas in Northern Vietnam are shown in Table 3.2 as follows: Table 3.2. Distribution of 6 species of Amorphophallus that contains glucomannan in mountainous areas in Northern Vietnam No. Name Altitude above sea level (m) 1 Amorphophallus konjac 800-1600 2 Amorphophallus corrugatus 400-1000 3 Amorphophallus krausei 600-1000 4 Amorphophallus yunnanensis 400-800 5 Amorphophallus yuloensis 800-1000 6 Amorphophallus paeoniifolius 0-300 As a result of the fieldwork, high- glucomannan content Amorphophallus species are distributed at higher elevations than those of low glucomannan content. Five species distributed widely in elevation from 400-1600m. Namely, Amorphophallus konjac is distributed at the height of 800-1600m, Amorphophallus yuloensis is distributed at the height of 800-1000m, Amorphophallus krausei is distributed at the height of 600-1000m, Amorphophallus yunnanensis lives at the height of 400-800m, Amorphophallus corrugatus lives in the height of 400-1000m, Amorphophallus paeoniifolius is distributed at the lowest altitude from 0-300m above sea level. As such, it is possible to see that there are differences in climate, terrain, ecology, etc. at different elevations. It greatly influences the existence of species with glucomannan. 3.1.2.2. Species distribution of Amorphophallus that contains glucomannan by height by habitat 14 From the results of the survey on the distribution of 6 species of Amorphophallus by habitat, we can see that the species are widely distributed in different habitats; they occur in the forest, roadside, upland fields and home garden These species mainly live in the canopy cover, cool climate and no flooded land. Of the six species, the Amorphophallus konjac lives the canopy cover in the forest, along the road, in the garden, etc. with different cover ranges usually from 20-70% depending on the temperature of each region. Two species of Amorphophallus yuloensis, Amorphophallus krausei are found only in areas where the canopy has a large coverage of 40-70% in average forest and dense forest. 3 species of Amorphophallus paeoniifolius, Amorphophallus corrugatus, Amorphophallus yunnanensis live under the thin canopy and especially for the species of Amorphophallus paeoniifolius grows well in areas where there have canopy or no canopy cover. 3.1.2.3. Distribution of Amorphophallus that contains glucomannan by the trend to receive sunlight The results of the study show that the Amorphophallus paeoniifolius is distributed in various trend to receive sunlight, indicating that it can adapt well to changes in temperature and intensity of sunlight. The other five species have a more characteristic orientation, mainly in the trend of exposure to less sunlight. Amorphophallus corrugates and Amorphophallus yunnanensis has trend to receive sunlight in 3 trends: east- south, east- north and west- south that receive less sunlight than North-East, South-East, South-West, North-West. 3 species of Amorphophallus konjac, Amorphophallus yuloensis, Amorphophallus krausei have only 2 trend to receive sunlight: East-south, East- North that receive the least sunlight, so the average temperature in these areas is also the lowest. 3.1.3. Distribution chart of Amorphophallus that contains glucomannan 3.1.3.1. Distribution chart of Amorphophallus konjac In the northern mountainous regions of Vietnam, the species is distributed in Y Ty, Bat Xat - Lao Cai and Quan Ba, Pho Bang- Ha Giang. They are frequently seen in the forest, roadside, upland fields, or home gardens, where there is cover. This distribution may be due to the ecological characteristics of the species suitable for cool climates, at elevations between 800 and 1600m. 3.1.3.2. Distribution chart of Amorphophallus paeoniifolius Amorphophallus paeoniifolius is a common in the mountainous region of northern Vietnam, provinces of Hoa Binh (Tan Lac, Lac Son, Hoa Binh city), Son La (Van Ho, Thuan Chau, Moc Chau), Phu Tho (Tan Son), Yen Bai (Van Chan), Tuyen Quang (Son Duong), Thai Nguyen (Dinh Hoa), Cao Bang (Nguyen Binh, Thach An), Lang Son (Trang Dinh), Bac Giang (Luc Ngan), Bac Kan (Ngan Son). They have wide distribution in the area with many habitats and different trends to receive sunlights, at 0-600m altitude compared to sea level. This shows that the ecological characteristics of the species are consistent with different habitats. On the survey line, species is seen with large frequency. 3.1.3.3. Distribution chart of Amorphophallus corrugatus 15 The Amorphophallus corrugatus is distributed in the provinces of Hoa Binh (Tan Lac, Lac Son, Hoa Binh), Son La (Van Ho, Thuan Chau, Moc Chau), Phu Tho (Tan Son), Tuyen Quang (Son Duong), Lang Son (Trang Dinh). They are widely distributed in the area with many habitats and different trends to receive sunlights. This shows that the ecological characteristics of the species are consistent with different habitats. On the survey line, the frequency of encountering species is small, due to the limited reproductive capacity of the species. 3.1.3.4. Distribution chart of Amorphophallus krausei The Amorphophallus krausei is distributed only in Son La (Van Ho, Thuan Chau, Moc Chau), Cao Bang (Nguyen Binh, Thach An). On the survey line, the frequency of encountering species is small. They are clustered in some locations in the survey area. This may be due to the ecological characteristics of the species only suitable for habitats of thick canopy cover. 3.1.3.5. Distribution chart of Amorphophallus yunnanensis The Amorphophallus yunnanensis is distributed in the provinces of Hoa Binh (Tan Lac, Lac Son, Hoa Binh), Son La (Van Ho, Thuan Chau, Moc Chau), Cao Bang (Nguyen Binh, Thach An), Bac Kan (Ngan Son). They are widely distributed in the area with many habitats and different trends to receive sunlights. On the survey line, the frequency of encountering species is not high, it can be seen in the thin forest, bush forest, roadside, hillside or on the kaingin. 3.1.3.6. Distribution chart of Amorphophallus yuloensis The Amorphophallus yuloensis is distributed only in Son La (Van Ho, Thuan Chau, Moc Chau), Dien Bien (Tuan Giao). On the survey line, the frequency of encounters with species is relatively high. They clustered at some locations in the survey area. This may be due to the ecological characteristics of the species only suitable for habitats of thick canopy cover. 3.1.4. Indigenous knowledge on the exploitation and use of Amorphophallus in some mountainous provinces of northern Vietnam 3.1.4.1. Exploiting Amorphophallus in some mountainous provinces of Northern Vietnam - Harvest time (exploitation time) of Amorphophallus With planted Amorphophallus, when the leaves are yellow and near the end of the growing season, it can be exploited, the period from September to November every year. - Tool With a small scale, the terrain is difficult to access, people can use the buffalo, cow, plows and hoes with tools to hold such as basket for transport. - Harvesting techniques With the exploitation of tubers in the nature, Amorphophallus usually grows under the forest canopy, on the slope, it should be used spades, hoes to dig each tree one. Just take the big tubers, small tubers are buried for exploitation in the next year. - Preserve Có hai phương pháp thường được nông dân sử dụng là vùi trong đất ẩm mát ngay ở trên nương hoặc bảo quản trên giàn thoáng mát. Sau khi khai thác những củ đủ tiêu chuẩn làm giống, được chọn lựa, r sạch đất mang củ giống về nhà, xếp nơi 16 thoáng, để vài ngày cho Nưa khô vỏ thì để lên giàn che có mái che trong điều kiện ít ánh sáng. Trong thời gian bảo quản thường xuyên kiểm tra và loại bỏ những củ bị thối. - Pre- processing After harvesting, soil and roots are removed and stored for storage. Before processing, tubers are prewashed to remove soil, dirt and microorganisms. 3.1.4.2. Amorphophallus processing in some mountainous provinces of northern Vietnam In Quan Ba, Ha Giang Province; Y Ty, Lao Cai province; Nguyen Binh, Cao Bang province, Amorphophallus is processed into dishes like tofu, fried noodles, mixing rice. The process of making is very simple and manual. From the slices of the dried Amorphophallus tubers, the people crushed the flour by machine or mill. The flour is then drenched and filtered with ash. After filtering with the ash, depending on the food is beans or noodles, it it continues to be processed. 3.2. Amorphophallus with glucomannan have potential for growth in some northern mountainous provinces of Vietnam From the field survey, studying the literature on growth characteristics and glucomannan content of Amorphophallus in the mountainous region of North Vietnam, some basic characteristics are summarized in below Table 3.11: Bảng 3.11. Characteristics of growth and development of 6 Amorphophallus species contain Glucomannan in northern mountainous region of Vietnam No. Name Growth period in 1 year (days) Volume of 3- year- old female tuber (g) Number of 3 year-old tubers Sexual reproduction (seeds) Glucomannan content in dry flour (%) 1 Amorphophallus konjac 158-175 600-1000 5-7 0 44,97 2 Amorphophallus corrugatus 150-160 200-400 0 0 28,6 3 Amorphophallus krausei 153-162 600-800 0 0 29,2 4 Amorphophallus yunnanensis 145-162 200-300 0 0 25,97 5 Amorphophallus yuloensis 147-158 200-300 0 0 30,07 6 Amorphophallus paeoniifolius 154-169 400-600 5-6 0 6,53 From the results of Table 3.11, the Amorphophallus Konjac has the best growth and development ability. After 3 years of growing the tubers, volume of female tubers is 600-1000 g, the tubers produced from this female tubers are 5 -7 tubers. So, Amorphophallus Konjac species is a promising plant growing, with volume of female tubers after 3 years growing is 600-1000g, tubers born from female tubers at this age of 5-7 tubers and content 44.97% glucomannan. 3.3. Research on breeding species of Amorphophallus Konjac in Vietnam 3.3.1. Native breeding species of Amorphophallus Konjac 17 3.3.1.1. Effect of harvesting time on seed germination rate The results show that there is a clear difference between the rates of germination at different harvesting times from when the fruit is green, the fruit is ripe and the fruit is very ripe.The results showed that at the time of harvesting ripe fruit, with CTH2 fomula, it gave highest seed germination rate of 64.67%, CTH3 formula of very ripe fruit, it gave second rate of germination germination of 49.33%, in the CTH1 formula the green pods, it gave the lowest germination rate of 17.33%. 3.3.1.2. Effect of seed preservation on grain germination The results showed that there was a significant difference in germination rate of Amorphophallus Konjac in two cold storage methods at 5 0 C (BQ1) and dry preservation by jar. Specifically, with the number of seeds germinated in the cold preservation method, the number of seed germination was 33.33 seeds at the rate of 66.67%, in addition to the method of dry preservation with jar, the rate of germination only reached 34% with 17 seeds germinated. From this data can be seen with the cold storage method at 5 0 C for better efficiency with preservation method of Amorphophallus konjac seeds in jar condition. The cause may be due to the origin of the Amorphophallus konjac lives in tropical climates, through cold weather, the seed preserved under jar conditions is vulnerable to low humidity or extreme cold. Influence the development of seed embryos. 3.3.1.3. Effect of seed preservation time on germination rate There were significant differences in germination rates with different seed preservation time, indicating that preservation time of Amorphophallus Konjac influenced the germination rate of the seed. In the table, three months of storage for the highest germination rate was 72.77%, the storage time of 6 months was reduced to 68.33%, after 9 months of preservation seed germination is reduced rapidly to 45%. The longer the storage time, the quality seed will be less and especially after 6 months of preservation. 3.3.1.4. Effects o