Abstract of a thesis doctor of Philosophy in biology: Atudy 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

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Aims of the study: Assess the composition and distribution of Amorphophallus genus (Amorphophallus spp.) contains glucomannan in some northern provinces in Vietnam; choosing the Amorphophallus species that has high level of glucomannan and developing potential for reproduction and growing in some norther provinces in Vietnam; testing the reproduction and growing of the candidate species in some northern provinces in Vietnam.

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Nội dung Text: Abstract of a thesis doctor of Philosophy in biology: Atudy 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

MINISTRY OF EDUCATION VIETNAM ACADEMY OF SCIENCE AND TRAINING AND TECHNOLOGY GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY ----------------------------- TRAN VAN TIEN 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 Major: Botany Code: 62.42.01.11 ABSTRACT OF A THESIS DOCTOR OF PHILOSOPHY IN BIOLOGY HA NOI - 2017
This thesis was fulfilled at Graduate University of Science and Technology Supervisors: 1: Dr. Nguyen Van Du 2: Assoc. Prof. Dr. Ha Van Huan Reviewer 1: …............................................................................................ Reviewer 2: …............................................................................................ Reviewer 3: …............................................................................................ The thesis will be defended at the University of Science and Technology, 18 Hoang Quoc Viet, Cau Giay, Ha Noi City, 2017 The thesis can be found at: The National Library of Vietnam Library University of Science and Technolog
INTRODUCTION 1. Rationales of study Some of the species in Amorphophallus genus. From Araceae family that contains glucomannan, one of the sugars with large molecular that has chain structure and already being grown in many countries in the world such as Japan. China, India in order to harvest the root for food and functional food production. According to Chua M., Baldwin TC., Hocking TJ., Chan K. (2010), the root of some species in Amorphophallus genus has glucomannan, a water-soluble polysaccharide. Products containing glucomannan in Amorphophallus’s root has glucose control capability in blood, reducing the lipid levels in the blood, and decrease the hungry in obese people. In Vietnam, the root has been used in traditional dishes from a long time ago in local ethnic groups in some northern provinces in the country. However, Amorphophallus has only been harvested in a small scale in some local areas and these dishes are prepared like some kind of tofu, called “Mò gỉ” (Nùng language) or “Cò ký thơ” (Mông language), noodle, fried bread….. (Nguyen Van Du, 2012). Studies about the Amorphophallus in Vietnam show some species in this genus contains glucomannan, their distribution characteristics, and there have been some studies on the reproduction and growing of these plants. In addition, according to the General Statistic Office of Vietnam in 2016, the local people in mountainous area in Northern Vietnam has per capita income of only 337.2 thousand VND/ month. Since their income are low, the poverty rate is very high, at 41.42%, and many houses lack food in 2-6 months/year/. Therefore, in order to make Amorphophallus as an agriculture product for economic development in northern Vietnam, study about this plant and its reproduction and growing method is valuable for the economic and essential. With the reasons above, the author chose the present study “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” as scientific thesis. 2. Aims of the study - Assess the composition and distribution of Amorphophallus genus (Amorphophallus spp.) contains glucomannan in some northern provinces in Vietnam. - Choosing the Amorphophallus species that has high level of glucomannan and developing potential for reproduction and growing in some norther provinces in Vietnam. - Testing the reproduction and growing of the candidate species in some northern provinces in Vietnam. 3. Main contents of the study - Investigate and assess the species, their distribution and local knowledge about Amorphophallus containing glucomannan in northern Vietnam. - Study the characteristics, growing, development and select the species which has high level of glucomannan and developing potential in some provinces in Northern Vietnam. 1
- Study about some reproduction technologies for the species which has high level of glucomannan and developing potential in some provinces in Northern Vietnam. - Study about the growing and development of Amorphophallus konjac in some provinces in Northern Vietnam. 4. Scientific and practical meaning of the study - Scientific meaning: the result of the present thesis contribute into the completion of the knowledge about Amorphophallus spp. in Vietnam. In addition, the results can be used for further study in the various arts relating to Amorphophallus. - Practical meaning: it is a scientific base for development some Amorphophallus species containing high level of glucomannan in Vietnam, which can be used in functional food production and other fields. 5. Design of the study Besides the introduction, summary, suggestions, references and appendix, the thesis consists of three parts: Chapter 1: General study – 25 pages, Chapter 2: subject, content, and method of the study – 22 pages, Chapter 3: The result of the study and discussion – 70 pages. CHAPTER 1 OVERVIEW OF RESEARCH 1.1. Research on Amorphophallus 1.1.1. Location and Classification of the genus Amorphophallus According to the dictionary of Vietnamese herbs by Vo Van Chi (2012), Amorphophallus belongs to the family Araceae, its other Vietnamese names and dialects are Khoai Nưa, Khoai ngái, Tò ngủ, Mò gỉ, Cò kí thơ. 1.1.2. Morphological characteristics of Amorphophallus The genus are herbaceous, tuberous, with a height of 10 cm (A. pulsilus) to meters (A. titanum) (Mayo, 1997, Bown, 2000). The tuber of Amorphophallus come in all shapes and sizes, ranging from elongagted shapes, ranging from elongated, radish shapes, spherical to head shapes. Many of them has the upper body nearing the sphere but the bottom branching. Amorphophallus has the root cluster, usually concentrated at the apex of tubers. The roots reach a length of 15cm and fat. The tree has single leaves and 2 - 3 leaves rarely grow together. Leaf stems are usually fat and green, with white spots, or brown with white spots, or with dark spots, smooth outside, covered by scaly leaves at the early stages. The trunk is split into three branches and the leaf consists of a vertical leaf stalk and a horizontal blade, which may consist of a number of small leaflets. The flowers, hermaphroditic and solitary, consist of a fragrant spadix of flowers; the spadix of flowers varied in shape and size. 1.1.3. Composition and Distribution Characteristics of Amorphophallus 1.1.3.1. Composition Characteristics There are about 200 species in the tropical regions such as Africa and Asia. (Hetterscheid và Ittenbach, 1996; Sedayu, 2010). The number of species is up to 25 species in Vietnam, in which five of them have tubers containing glucomannan according to Nguyen Van Du and his colleagues. 2
1.1.3.2. Distribution Characteristics The genera grow mainly in tropical regions such as Africa and Asia. They are endemic plants of rainforest in Southeast Asia and 24 ° C is considered as the ideal average annual temperatures for the genera. (Hetterscheid, 1994; Mayo, 1997; Liu, 2004). Eight of twenty-five species are distributed in the mountainous provinces of North Vietnam, the others are diversified according to various ecological conditions from the North to South of Vietnam. 1.1.4. Ecological Characteristic and Development of Amorphophallus containing glucomanna  Ecological Characteristic The genera grow increasingly in the shaded environment, well-drained soils and rich humus with a pH of 6.5 to 7.5. The species will have high glucomannan content provided that indirect sunlight and the temperatures from 20 to 25 -250C.  Characteristic of Growth and Development Liu et al. (1998) studied the growth and development of some species in China, indicating the ecological conditions of the genera. According to the research team, the species is not a water-intensive plant and waterlogging tolerance. However, they live well in warm and can tolerate temperature change from 5 to 43 0C and the most appropriate temperature is 20 to 250C, when the temperature drops below 00C and up to 480C the tree will die after 5 days. Amorphophallus grows and develop in season, they often shed leaves in winter or dry season. 1.1.5. Value in Use The Amorphophallus species have long been used in tropical and temperate Asia as a food source and a traditional medicine (Liu, 1998). One of the most well-known species is the Amorphophalus konjac, planted in China more than 2,000 years ago (Liu, 1998; Long, 1998). Its tubers treat asthma, cough, hernia, chest pain, burns and skin disorders (Niwa, 2000; Xu, 2001). Recently, the flour of Amorphophallus species is emphatic in use as food fiber. These nutrients help enzyme-resistant, keeping us full longer (McCleary, 2003). The major bioconversion molecule in konjac is soluble fiber including cellulose-free polysaccharide and glucomannan (Takigami, 2000; Edison, 2010; Parry, 2010). Amorphophallus have been used as a traditional food for a long time. However, food made from the species is used in small areas and remote places by ethnic groups or starvation resistance (famine), not used as common food. According to the ethnobotanical surveys of the Institute of Ecology and Biological Resources of the species in Hue, a small province in Vietnam. Amorphophallus species were planted in Quang Tho, Quang Dien hundreds of years ago, becoming familiar dishes and a specialty vegetable (N.V. Du, 2012). 1.2. Glucomannan – An Overview 1.2.1. Glucomannan Introduction Glucomannan is mainly a straight-chain polysaccharide, with molecular weights of 200 ÷ 2000 kDa, soluble in water, the component sugars are β-(1→4)-linked D- mannose and D-glucose. Glucomannan content in the root depends on the habitat, cultivation conditions, growth time, development and extraction process, from the initial harvest. 3
1.2.2. Research on Glucomannan Content To assess the glucomannan colorimetric methods are the most commonly used, biochemical analysis techniques is typically the method applied for classical plants. Other Quantitative methods are applied such as using 3,5-dinitrosalicylic acid reagent (3,5-DNS), and phenol-sulfuric acid colorimetric method. 1.3. Reseach on plant breeding 1.3.1. Plant Breeding in the World The species can be propagated from the seeds or from the vegetative organs. the plants are multiplied from the vegetative organs and bulbs (tubers) is commonly used in commercial farming (Follett và Douglas, 2002; Edison, 2010). Buds- attached stems were cut at harvest and buried in 60% grains of sand or hummus with 10-15cm in depth to prevent roots growing in the back of tubers and become dried tubers until the next planting season (Follett and Douglas, 2002). seed multiplication is not as common as the others because seeds are extremely difficult to germinate in natural conditions (O'Hair and Asokan, 1986). In southern China, indigenous peoples make it easier to breed by removing the ripe peel and then mixing it with 1: 4 humid sand; then filled with a thick layer of soil (Long, 1998). Propagation by leaf tissue, callus has been studied by foreign biologists for decades. Kobayashi & M. Yonai, S. introduced the method of propagation of Amorphophallus konjac from stems, bulbs, and tubers. According to the researcher, the rate of mosaic virus infection was 92%. disease-free seed tubers were selected from the seedlings. As the results, the researcher multiplied 1,000 seedlings from the issues in 3 months, 1,000 seedlings from scar tissues of petioles weighting 10 mg in 4 months. 1.3.2. Plant Breeding in Vietnam Amorphophallus is not an economic tree and has been included in the crop structure. Therefore, study materials of the tree are limited, especially for research on Amorphophallus species with high glucomannan content. Recently, the findings of Le Xuan Dac, Nguyen Van Du in 2014 showed that the suitable environment for multi-shoot production was AMNA2 (MS + 30 g / l saccharose + 8 g / l agar + 2 mg / l BAP + 0.2 mg / l NAA) with a multiplication factor of 5.7. The best medium is ARIB6 (MS + 30 g / l saccharose + 8 g / l agar + 0.6 mg / l IBA), and the rate of root growth is 100%. The plants grow best when Average shoots/buds growth is 5.8. The suitable containers made by rice husk and soil for the species and the percentage of live seedlings will be 95.3%. These studies show that tissue culture techniques can be guaranteed to produce disease-free plants and multiplied in large numbers. However, the cost of breeding is quite expensive 1.4. Research on Planting, Harvesting and Processing in the World and in Vietnam 1.4.1. Research in the World 1.4.1.1. Research on Plantin Most of the planting methods are the result of experience accumulated by many generations of farmers such as China, Japan, India. Applications based on scientific research have recently introduced (O’Hair và Asokan, 1986). Japanese planting process (Jinenjo process) is used more than 100 years ago, Amorphophallus been planted 4
continuously for many years until the bulbs reach their weight for harvesting and processing. In addition, Japanese cultivation process is modern ("Uedama system"), tubers are divided into age groups and planted separately. (Kurrihara, 1979; Douglas, 2006). Typically, the shoots are cultivated at dense density for a year to create propagation material for subsequent seasons (Douglas, 2005). After harvest, large bulbs will be sold and the rest will be stored by age groups in cooler warehouses during the winter and will be planted next spring (Kurihara, 1979; Douglas, 2006). Although bulb output is higher compared to traditional culture method “Jinenjo”, the method's detrimental effect such as diseases and pests, slow growth and development on tree and soil. The quality of bulbs deteriorated continuously after 3 years cultivation (Kurihara, 1979). 1.4.1.2. Research on haversting and prosessing Harvesting seasons play an important role in output and quality of bulbs. Studies show that the glucomannan content reaches the peak before the deciduous stage and the hibernation period (Liu, 1998; Bown, 2000). Early harvest before becoming mature bulbs can hinder early hibernation period and prevent biomass in foliage separating from the bulbs, thus impeding the final development of the bulb (Bown, 2000). As mentioned above, larger bulbs are harvested for sale at the end of the fall in Japan; The remainder is either kept in the field for continued growth in the next season or stored in age groups in ventilated warehouses throughout the winter (Kurihara, 1979; Follett , Douglas, 2006). 1.4.2. Research in Vietnam 1.4.2.1. Research on Planting According to Nguyễn Thị Ngọc Huệ and Đinh Thế Lộc's studies suggested that Amorphophallus species should be grown in February and April. If it is grown in late May and June causing short growth time, the productivity will be limited. Planting material is tubers so fertilizers for 1 ha in which manure of 1.5 tons and quantity of N: K: P at 13:13:21 with a density of 5 tree per 1m. Research on growing techniques “Nghiên cứu sinh trưởng của cây Nưa chuông (Amorphophallus paeoniifolius) in Thua Thien Hue" by 3 authors Vo Thi Mai Huong, Tran Vu Ngoc Thi, Nguyen Thi Thu Phuong of University of Science - Hue. The findings of research concluded that the first, second and third buds grow in about 20 days, 55 days and 83 days respectively after root cuttings. Growth time of the first, second, third stems is about 65, 87, 78 days respectively. Height, diameter, and leaf length of terms increase rapidly during the first 5-6 weeks. The height is 39.97 - 96.94 cm, the maximum is 3. The diameter reaches 2.48-3.92 cm and the leaf length is 40.82-71.12 cm. Calculation of theoretical productivity will be 149,2, in which the second stem get the highest output (58.8 tons / ha). 1.4.2.2. Reseach on Havesting Amorphophallus applied researches in daily life have not studied in Vietnam. According to Nguyễn Văn Dự and colleagues (2012) only pointed out that Amorphophallus of tubers are used for cooking in ethnic groups such as Mong, Nung or Ha Nhi. 5
When the leaves are completely faded and the weather is in the middle of the dry season in November, Amorphophallus of tubers will get high percentage dry weight compared to harvesting in September. CHAPTER 2 MATERIALS, CONTENTS AND METHODOLOGY OF STUDY 2.1. Materials, locations and time of research 2.1.1. Subjects and scopes of research Specimen of research is Amorphophallus naturally developing in Northern Highlands Provinces of Vietnam. There are total of 159 research specimens in some Northern Highlands Provinces of Vietnam. Specimens are made and preserved in Institute of Ecology and Biological Resources under Vietnam Academy of Science and Technology. 2.1.2. Locations and time of research 2.1.2.1. Locations of research - Research of component investigation, allocation and indigenous knowledge of exploitation and usage of Amorphophallus with glucomannan implemented in Northern Highlands Provinces of Vietnam. - Research of sexual and tissue culture multiplication implemented in the experimental area of Linh Duoc Son Co-operative in street No.10, Hoa Binh city, Hoa Binh province. - Research of asexual reproduction by tuber carried out in Van Ho commune, Van Ho district, Son La province. - Research of planting Amorphophallus carried out in Van Ho commune, Van Ho district, Son La province. - Amorphophallus is experimentally planted in Van Ho commune, Van Ho district, Son La province; Quyet Tien commune, Quan Ba district, Ha Giang province; Ngoc Son commune, Lac Son district, Hoa Binh province. 2.1.2.2. Time of research From Dec 2013 to Mar 2017 2.2. Contents of research - Investigate of component assessment, allocation and indigenous knowledge of Amorphophallus with glucomannan in Northern Highlands Provinces of Vietnam. - Research into ecological characteristics, growth and selection of Amorphophallus with high glucomannan contents which has prospect of developing in some Northern Highlands Provinces of Vietnam. - Research into some technologies of multiplication of Amorphophallus with high glucomannan contents which has prospect of developing in some Northern Highlands Provinces of Vietnam. - Research into growth of Amorphophallus konjac in some Northern Highlands Provinces of Vietnam. 2.3. Methods of research 2.3.1. Inheritance method 6
During the study, the author inherits research results from evaluation system of subspecies, monograph documents of muiltiplication, planting and processing of Amorphophallus all over the world and in Vietnam. 2.3.2. Method of investigation and collection of specimens Method of ivestigation and collection of specimens based on Plant Research Method in 2007 and handbook of research of plant diversity in 1997 by Nguyen Nghia Thin. The investigation and collection of specimens are carried out in 14 Northern Highlands Provinces of Vietnam. Fourteen investigation trips were implemented in 14 Northern Highlands Provinces of Vietnam such as Hoa Binh province (Tan Lac, Lac Son, Hoa Binh city), Son La province (Van Ho, Thuan Chau, Moc Chau), Phu Tho province (Tan Son), Dien Bien province (Dien Bien), Lai Chau province (Muong Te), Lao Cai province (Bat Xat), Yen Bai province (Van Chan), Tuyen Quang province (Son Duong), Thai Nguyen province (Dinh Hoa), Ha Giang province (Quan Ba, Pho Bang), Cao Bang province (Nguyen Binh, Thach An), Lang Son province (Trang Dinh), Bac Giang province (Luc Ngan) and Bac Can province (Ngan Son). 2.3.3. Method of assessment of species components In order to study the components of Amorphophalluss in Northern Highlands Provinces of Vietnam, the thesis topic applies the comparative morphology based on research documents of Amorphophalluss by Vo Van Chi (2012), Nguyen Van Du (2005), Pham Hong Ho (2003), so on. The analysis of specimens is implemented in Plant Specimen Department under Institute of Ecology and Biological Resources. Besides, the author also refers to some specimens of Amorphophallus of some countries on the Internet such as China, Japan, Thailand, so on in particular standard specimens (Tyrus). 2.3.4. Method of Amorphophallus selection with the prospect of developing in some Northern Highlands Provinces of Vietnam To contribute to economic development in Northern Highlands Provinces of Vietnam, selected Amorphophallus must bring its economic values and good growth ability, suitable to ecological conditions of that area. Therefore, the author chooses on the two following criteria: Criteria 1: Selection on high glucomannan contents Tuber specimens of Amorphophallus collected after defining species names, are analysed their glucomannan contents based on colorimetry by Melinda Chua and a co-worker (2012) through the reagent of 3,5-dinitro salicylic acid at 550nm wavelength at the Institue of Natural Products Chemistry. Criteria 2: Selection of species with the prospect of developing in some Northern Highlands Provinces of Vietnam With found Amorphophallus with high glucomannan contents, the author analyzes and selects species which have the prospect of developing in Northern Highlands Provinces of Vietnam. For agricultural production, selected species must bring its economic values and good growth ability. Thus, the author evaluates the characteristics of growth, easy multiplication ability and their economic values for selection. During the real investigation, according to the research documents of 7
Amorphophallus by Vo Van Chi (2012), Nguyen Van Du (2005), Pham Hoang Ho (2003), so on, the author assesses the growth ability, the reproduction ability, ecological conditions to choose the most suitable to develop in Northern Highlands Provinces of Vietnam. 2.3.5. Method of investigation of indigenous knoweledge of exploitation and usage of Amorphophallus in some Northern Highlands Provinces of Vietnam The thesis applies the method of investigation of indigenous knoweledge based on the method of investigation of national plant by Gary J. Martin (2002), Agriculture Publishing House and Rapid Rural Appraisal method (PRA) with the participation of national community, delivery of investigation sheets and interviews (Questionnaire sheets are shown in Annex 2). 2.3.6. Method of muiltiplication research of Amorphophallus konjac Mutiplication method is based on the module manual of producing cultivar by seeds (Ministry of Agriculture and Rural Development, 2010) and allocation of experiements on the method of agricultural experiment allocation by Nguyen Thi Lan and Pham Tien Dung – Institute of Agriculture, Hanoi Publishing House in 2005. 2.3.6.1. Method of multiplication research by seeds a. Experiment 1: Effect of harvest time to germination rate of Amorphophallus konjac seeds The experiment includes 3 following formulas: CTH1, CTH2, CTH3 based on different harvest time of peel color (ripeness of fruits). CTH1: green peel, CTH2: ripe peel, CTH3: dramatically ripe peel. b. Experiment 2: Effect of method of seed preservation to germination rate of Amorphophallus konjac seeds The expriment includes 2 following formulas: BQ1: Store cool at 5oC, BQ2: Store dry in jars c. Experiment 3: Effect of seed preservation time to germination rate of Amorphophallus konjac The experiment includes 3 folllowing formulas: TGQ1: storage time for 3 months, TGQ2: storage time for 6 months, TGQ3: storage time for 9 months. d. Experiment 4: Effect of method of seed treatment to germination rate of seeds and growth of nurseling in nursery garden The experiment includes 5 following formulas: XLH1: Clean seeds with water and then sow, XLH2: Soak seeds in warm water at 40-50°C within 3 hours, XLH3: Soak seeds in warm water at 40-50°C within 6 hours, XLH4: Soak seeds in warm water at 40-50°C within 9 hours, XLH5: Keep humid for seeds in the cloth bags until they are broken and then sow. 2.3.6.2. Method of multiplication research by tubers a. Experiment 5: Effect of treatment of tuber cuts to shooting rate, infection rate and survival rate of Amorphophallus konjac The experiment includes 4 following formulas: XVC1: Keep cuts dry and store, XVC2: Treat cuts with ash, XVC3: Treat cuts with powdered lime, XVC4: Treat cuts with cement. b. Experiment 6: Effect of preservation method to shooting rate, infection rate and survival rate of Amorphophallus konjac 8
The experiment includes 5 following formulas: PBQ1: Put on the floor of warehouse, PBQ2: Store cool at 100c, PBQ3: Arrange on the tuber shelves under the 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. 9
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 10
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 11
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 Glucomannan No. Common name Science name content in dry flour (%) 1 Amorphophallus konjac Amorphophallus konjac K. Koch 44,97 Amorphophallus Amorphophallus corrugatus N. E. 2 28,6 corrugatus Br. Amorphophallus krausei Engl. & 3 Amorphophallus krausei 29,2 Gehrm. Amorphophallus Amorphophallus yunnanensis Engl. 4 25,97 yunnanensis & Gehrm. Amorphophallus 5 Amorphophallus yuloensis H. Li 30,07 yuloensis Amorphophallus Amorphophallus paeoniifolius 6 6,53 paeoniifolius (Dennst.) Nicolson 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 12
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 13
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 14
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 15
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 Growth Volume of Number Sexual Glucomannan period in 3- year- of 3 No. Name reproduction content in dry 1 year old female year-old (seeds) flour (%) (days) tuber (g) tubers Amorphophallus 1 158-175 600-1000 5-7 0 44,97 konjac Amorphophallus 2 150-160 200-400 0 0 28,6 corrugatus Amorphophallus 3 153-162 600-800 0 0 29,2 krausei Amorphophallus 4 145-162 200-300 0 0 25,97 yunnanensis Amorphophallus 5 147-158 200-300 0 0 30,07 yuloensis Amorphophallus 6 154-169 400-600 5-6 0 6,53 paeoniifolius 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 16
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 50C (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 50C 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 of seed treatment on seed germination and growth of seedlings in nursery The method “no treatment” gives the worst results. The measures: increasing the seed treatment time to 9 hours, reducing the seed treatment time to 3 hours, or no treatment, just brewing and sowing seeding in the sand for a worse result than to drenching seeds in warm water 40-50 ° C for 6 hours. Brew and wash then sow the seed into the sand when the tree is about 5 to 10 cm high to transfer to the soil for germination rate, the number of live and growing plants in the nursery is best. 3.3.2. Propagating species of Amorphophallus konjac 3.3.2.1. Effect of root cuttings on tubers growth rate Treatment of cement cuttings for optimal treatment (XVC4), with high blooming rate, low number of infected plants and higher survival rate compared to methods of treatment with ash, and lime powder without treating the cut to self-dry. Due to high glucomannan content, if the root cuttings are not treated, they are susceptible to tuberculosis and affecting on tubers growth and survival rate of tubers. For the ash method, the results are higher with no treatment but lower than with the use of lime and bamboo shoots, as the ash is biocide, but the ability to quickly dry the cut by covering the cut is lower than that of lime powder and cement with the higher 17
rate of tubers, leading to low survival rate. For the method of treating cut roots with lime powder, the number of tubers infected was as low as for the treatment of tubers with cement, but this method had higher rate of tubers emergence and lower root rate than treated with cement, due to the treatment of lime, the tubers are susceptible to dry and causing the death. 3.3.2.2. The effect of tubers preservation on incubation rate, infection rate, survival rate The results showed that in the preservation methods, the cold storage method gave the best tubers quality, followed by storage by burying in clean sand, clean soil and trimming to preserve tubers. 3.3.3. Research on plant breeding of Amorphophallus konjac by tissue culture technique 3.3.3.1. Chemical influence and time of sterilization on in vitro clean-forming ability The best sterilization method for the embryo was 60% by Javen for 12 minutes for a clean sample rate and a 100% recovery rate for the regeneration sample, after 15 days of tissue culture. 3.3.3.2. Effect of nutrient environment on tubers regeneration in vitro The results showed that in medium MS, the sample was best reproducible (reaching 91.85%), average number of shoots / samples was 2.45 and good shoot quality (shoots, healthy, dark green leaf, fast growing). 3.3.3.3. Influence of BAP concentration on in vitro shoot regeneration Results showed that the CT4 environmental formula gave the highest rate of regeneration of shoots of 2 mg / l BAP (100%), the lowest rate of regeneration was CT 0 (0 mg / l BAP), reaching 54.81%. Formulas CT1, CT2, CT3, CT5 and CT6 both gave the regeneration rate of over 74%. As a result, it can be asserted that BAP at different concentrations has a strong influence on tubers growth. When the BAP content was higher than 2 mg / l BAP in the following experiments, the number of shoot regeneration and height of shoots decreased, while at the same time, there appeared very large scar tissue. 3.3.3.4. Influence of BAP and Kinetin concentration on tubers regeneration performance of Amorphophallus konjac in vitro The results show that the difference between the combined effects of Kinetin and BAP on the ability to regenerate tubers compared to the individual effects of BAP. After culture for 3 weeks, CT7, with medium MS + 2 mg / l BAP + 0.2 mg / l Kinetin gave the highest percentage of tubers regeneration (100%), average number of tubers / sample, the average height of the tubers was 3.21 cm. 3.3.3.5. Effect of nutrient environment on rooting ability of tubers in vitro Results showed that after 4 weeks of root culture, supplementation of nutrients, growth regulators and different sugar content resulted in different proportion. 1/2 MS + 8 g/l Agar + 0.5 mg / l IBA + 14 g / l Surcrose formula for the highest efficiency (100%), average number of tubers rooting / tubers achieved at 4.98, the average root length was 2.67 cm and the rooting time was 7 days, the root quality was good (fat and healthy roots), and the penetration was strong. 3.3.3.6. Influence of IBA concentration on rooting ability of tubers in vitro Results showed that environmental formula stimulates the best roots is 1 / 2MS + 8g / l Agar + 14g / l Sucrose + 0.4mg / l IBA. 18