VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY -*** - GRADUATION THESIS TOPIC: RESEARCH ON IN VITRO PROPAGATION OF SCHISMATOGLOTTIS WALLICHII HANOI – 2022 VIETNAM NATION UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY – – – – – –   – – – – – – GRADUATION THESIS TOPIC: “RESEARCH ON IN VITRO PROPAGATION OF SCHISMATOGLOTTIS WALLICHII” Supervisor : Dr Nguyen Thi Lam Hai Student name : Le Thi Phuong Oanh Student code : 637061 Class : K63CNSHE HA NOI – 2022 DECLARATION OF AUTHORSHIP I hereby declare that this is my research work The data, results and reports mentioned herein are honest and have not been used to be published in previous theses, dissertations, and scientific research works I hereby certify that the citation information used in the thesis is clearly stated in the source, ensuring that the citation is correct as prescribed I take full responsibility for this promise! Hanoi, day month year 2022 Student Le Thi Phuong Oanh i ACKNOWLEDGEMENTS In order to complete this graduation thesis, I would like to thank the Faculty of Biotechnology of Vietnam National University of Agriculture for organizing and facilitating support and assistance in the process of studying and researching the topic With the deepest gratitude, I would like to send to the teachers in the Department of Plant - Faculty of Biotechnology of Vietnam National University of Agriculture, especially Dr Nguyen Thi Lam Hai, who followed me closely during the research and imparted valuable knowledge to us throughout the time at school Thanks to the guidance and teaching of the teachers, my research topic can be completed well I would like to sincerely thank Mr Vu Cong Son and the staff at T.E.N Biotech Biological Joint Stock Company - who directly helped, cared and guided me to successfully complete this report in the past time The final thesis was carried out over a period of nearly months My initial step into practice is still limited and there are still many surprises, so it is inevitable that I will make mistakes, I look forward to receiving valuable comments from teachers so that my knowledge in this field can be improved more complete and at the same time have conditions to supplement and raise their consciousness Thank you sincerely! Hanoi, day month year 2022 Student Le Thi Phuong Oanh ii CONTENTS DECLARATION OF AUTHORSHIP I ACKNOWLEDGEMENTS II LIST OF ABBREVIATIONS VI CONTENT OF FIGURE VII CONTENT OF TABLE VIII ABSTRACT PART INTRODUCTION .2 1.1.Prefaceace 1.2 Objectives 1.3 Requirements PART LITERATURE REVIEW 2.1 General Introduction 2.1.1 General introduction about Schismatoglottis wallichii 2.1.2 Scientific Classification 2.1.3 Botanical characteristics of Schismatoglottis wallichii 2.1.4 Ecological characteristics 2.2 Some studies on tissue culture of species of the family Araceae 2.3 Introduction of Temporary Immersion Bioreactor system (TIB system) 13 2.3.1.Principle of Temporary Immersion Bioreactor system (TIB system) 13 2.3.2 Some micropropagation studies of Temporary Immersion Bioreactor system (TIB system) 15 PART RESEARCH MATERIALS AND CONTENTS AND METHODS 19 3.1.Object, material, location, time of culture 19 3.1.1 Research Materials 19 3.1.2 Location, time of culture 19 3.1.3 Research tools 19 3.2.Research contents 19 iii 3.2.1 Evaluation of the effect of growth hormones on the multiplication stage of Schismatoglottis wallichii 19 3.2.2 Comparison of growth of Schismatoglottis wallichii on the multiplication stage in different culture systems 21 3.2.3 Evaluation of the effect of growth hormones on the rooting stage of Schismatoglottis wallichii 22 3.2.4 Comparison of growth of Schismatoglottis wallichii on the rooting stage in different culture systems 23 3.3 Tracking Indicators 24 3.4 Research Method 24 3.4.1 Method of proceeding 24 PART 4: RESULTS AND DISCUSSION 26 4.1 Effect of growth hormones on the multiplication stage of Schismatoglottis wallichii 26 4.2 Effect of auxins on shoot proliferation and growth of Schismatoglottis wallichii after weeks of culture 27 4.2.1 Effect of IBA (3-Indolebutyric acid) in combination with BA on shoot proliferation and growth of Schismatoglottis wallichii 28 4.2.2 Effect of α-NAA (α-Naphthaleneacetic acid) in combination with BA on shoot proliferation and growth of Schismatoglottis wallichii 30 4.2.3 Comparison of efficiency on shoot proliferation and growth of Schismatoglottis wallichii between IBA and α-NAA in combination with BA supplemented medium after weeks of culture 31 4.3 Comparison of growth of Schismatoglottis wallichii on the multiplication stage in different culture systems 33 4.4 Effect of Auxin on the rooting stage of Schismatoglottis wallichii 35 4.4.1 Effect of IBA (3-Indolebutyric acid) on the rooting stage of Schismatoglottis wallichii 36 iv 4.4.2 Effect of α-NAA (α-Naphthaleneacetic acid) on the rooting stage of Schismatoglottis wallichii 37 4.4.3 Comparison of efficiency on quality of Schismatoglottis wallichii between IBA and α-NAA supplemented medium on the rooting stage after weeks of culture 39 4.5 Comparison of growth of Schismatoglottis wallichii on the rooting stage in different culture systems 40 PART CONCLUSIONS AND RECOMMENDATIONS .43 5.1 Conclustion 43 5.2 Recommendations 43 REFERENCES 44 APPENDICES 46 v LIST OF ABBREVIATIONS Abbreviations Annotate MS Murashige and Skoog medium CV Coefficient of variation LSD5% Least significant differences BA Benzyl Aminopurine TIB Temporary Immersion Bioreactors IBA Indole-3-butyric acid α-NAA α-Naphthaleneacetic acid vi CONTENTS OF FIGURE Figure 1: Schismatoglottis wallichii Figure 2: The Herb of Schismatoglottis wallichii Figure 3: The blade (leaves) of Schismatoglottis wallichii Figure 4: The Inflorescences of Schismatoglottis wallichii Figure 5: Technological design and operational principle of Temporary Immersion Bioreactor system 14 Figure 1: Schismatoglottis wallichii shoots in BA supplemented mediums after weeks of culture …………………………………………………………… 27 Figure 2: Schismatoglottis wallichii shoots in BA supplemented mediums combined with IBA after weeks of culture 29 Figure 3: Schismatoglottis wallichii shoots in BA supplemented mediums combined with α-NAA after weeks of culture 31 Figure 4 Schismatoglottis wallichii shoots between BA supplemented medium combined with IBA and α-NAA after week of culture 32 Figure 5: Schismatoglottis wallichii shoots in vitro culture systems after weeks of culture 35 Figure 6: Schismatoglottis wallichii in IBA (3-Indolebutyric acid) supplemented mediums on the rooting stage after weeks of culture 37 Figure 7: Schismatoglottis wallichii in α-NAA (α-Naphthaleneacetic acid) supplemented mediums on the rooting stage after weeks of culture 39 Figure 8: Schismatoglottis wallichii in α-NAA (α-Naphthaleneacetic acid) and IBA (3-Indolebutyric acid) supplemented mediums on the rooting stage after weeks of culture 40 Figure 9: Schismatoglottis wallichii in vitro culture systems on the rooting stage after weeks of culture 42 vii CONTENTS OF TABLE Table 1: Effect of BA (Benzyl Aminopurine) on shoot proliferation and growth of Schismatoglottis wallichii after weeks of culture 26 Table 2: Effect of IBA (3-Indolebutyric acid) in combination with BA on shoot proliferation and growth of Schismatoglottis wallichii after weeks of culture 28 Table 3: Effect of α-NAA (α-Naphthaleneacetic acid) in combination with BA on shoot proliferation and growth of Schismatoglottis wallichii after weeks of culture 30 Table 4: Efficiency of in vitro culture systems of Schismatoglottis wallichii after week of culture 33 Table 5: Effect of IBA (3-Indolebutyric acid) on the rooting stage of Schismatoglottis wallichii after weeks of culture 36 Table 6: Effect of α-NAA (α-Naphthaleneacetic acid) on the rooting stage of Schismatoglottis wallichii after weeks of culture 38 Table 7: Efficiency of in vitro culture systems of Schismatoglottis wallichii on the rooting stage after week of culture 40 viii ABSTRACT To aid with fresher air and offer beauty to living spaces, the usage of decorative plants has grown in popularity in recent years As a result, decorative plants like Schismatoglottis wallichii are quite popular Because it has a significant economic impact, Schismatoglottis wallichii is one of the most significant ornamental plants The objective of this study was to compare the efficacy of the temporary imersion bioreactor (TIB), a semi-solid culture system, and liquid culture systems for Schismatoglottis wallichii in order to determine the most ideal culture system for in vitro multiplication of Schismatoglottis wallichii The best outcomes for plants were likewise achieved when cytokines and auxins were added during the rapid multiplication and roots stages Using in vitro cultures and disease-free samples, the following outcomes were attained by rotating between several cultures: On MS + mg/l BA + 0.75 mg/l IBA + g/l agar, the maximum shoot multiplication yield (3.56 shoots/plant, 3.24 cm/bud, 5.9 leaves/plant, and 100% rooted) was attained In the rooting stage, MS + 0.75 mg/l α-NAA + g/l agar was the best medium for chimatos plants (average root length: 3.21cm, 8.35 roots/plant, mass plant weight: 10.42 g) the temporary imersion bioreactor (TIB)is the most effective in vitro culture technology for Schismatoglottis wallichii during both rapid multiplication and rooting PART INTRODUCTION 1.1 Prefaceace Schismatoglottis wallichii is a species of Angiosperms (Flowering Seed Plants) (Monocotyledon) in the Araceae family This species was acquired by Hook.f first scientific description in 1893 The leaf surface is wrinkled and swollen, parallel to the leaf veins The primary leaf color is green, often variegated with dark-green and grey strips When the need to love bonsai is more concerned, Schismatoglottis wallichii is also one of the favorite plants in the bonsai collection of people who are passionate about bonsai In recent years in Vietnam, the hobby of ornamental plants is also very developed Schismatoglottis wallichii has also become a sought-after favorite Exploitation in the wild is limited, so the propagation of Schismatoglottis wallichii by plant tissue culture is of more interest The advantage of systems is that they increase multiplication rates, allow greater availability of culture medium components, and reduce production costs Plant tissue culture has been applied to production since the 20th century when economics and science developed, many plant tissue culture systems were applied and achieved high efficiency Temporary immersion bioreactor system, in recent years, this system has begun to be applied to large-scale production of micropropagation in some countries such as Brazil, and the Netherlands is considered to help plants grow quickly and save a lot of labor costs in culture Therefore, this system has great potential for the production of micropropagation In Vietnam, there have been several studies on the application of bioreactor systems for tissue culture, but they are few and the applicability is not high Therefore, to meet the requirements for the production of high-quality Schismatoglottis wallichii in vitro, I conducted a research project: “Research on in vitro propagation of Schismatoglottis wallichii” 1.2 Objectives The aim of the present study is: - To find-out the optimal medium for the morphogenesis of Schismatoglottis wallichii - Comparison of micropropagation performance of Schismatoglottis wallichii on several different culture systems during rapid propagation and rooting stage 1.3 Requirements - Study the effect of growth regulators on shoot proliferation and rooting stage - Compare the efficiency of in vitro culture systems (the temporary immersion bioreactor (TIB), the semi-solid medium culture system and the liquid medium culture system) PART LITERATURE REVIEW 2.1 General Introduction 2.1.1 General introduction about Schismatoglottis wallichii Schismatoglottis wallichii is a plant of the family Araceae It is found in islands around Southeast Asia, In Malesia, from Peninsular Malaysia, Singapore, Sumatra, Java, Borneo, Philippines, and east New Guinea Habitat of terrestrial native Schismatoglottis wallichii (Primary Rainforest, Secondary Rainforest, Freshwater Swamp Forest) The preferred climate zone is Tropical Schismatoglottis wallichii is a light-loving plant, so it is suitable for planting in the garden, terrace, balcony, or in the window that receives light every day with a highlight with a white stripe on the leaves as a decorative accent to create attraction in the bonsai industry Figure 1: Schismatoglottis wallichii 2.1.2 Scientific Classification Science name: Schismatoglottis wallichii Hook.f Common name in Viet Nam: ‘Mường Thái’ Regnum Plantae Unclassified Angiospermae Unclassified Monocots Ordo Alismatales Familia Araceae Genus Schismatoglottis Species S wallichii Now, Schismatoglottis has more 100 recognized species from tropical and subtropical Asian peninsulas, especially it is found in primary rainforests Around the world, many growers and researchers on Schismatoglottis species as well as choose to create new lines and varieties to serve human needs 2.1.3 Botanical characteristics of Schismatoglottis wallichii Herb: Up to 50 cm, but typically 20 cm or so Stem epigeal, upright to decumbent, 1.5–2 cm diam., pleonastic, hapaxanthic, 1-2 cm thick, and bright green modules Petiole 12-48 cm long, glabrous, sheathing in the lower 1/3 to 3/5; wings of sheath fully attached, tapering, persistent to slowly degrading in the marginal distal part; blade (broadly oblanceolate to) oblong-lanceolate to narrowly ovate, usually rather distinctly asymmetric, bright to mid-green, occasionally suffused with purple and/or purple-backed, adaxially sometimes (to very shallowly cordate), midrib slightly prominent abaxially (dry) or not, secondary venation arising mainly from the midrib; tertiary venation obscure Figure 2: The Herb of Schismatoglottis wallichii Blade: Blade is moderately tough, elliptic to ovato-sagittate, semi-glossy mid-green, occasionally variegated with a single grey-green central stripe, or scattered grey green throughout, or (rarely) entirely grey, measuring 10-29 cm long by 3.5–16 cm wide The margins are smooth to conspicuously crispulate, and the base is obtuse to more or less truncate to somewhat cordate with posterior lobes spreading Primary lateral veins 1.5–3 cm apart on either side of the midrib, impressed adaxially and prominent abaxially; interprimary veins irregular but noticeable; secondary venation unimpressive; tertiary venation hardly perceptible Figure 3: The blade (leaves) of Schismatoglottis wallichii Inflorescence: Inflorescences 1–8 together, powerfully esteric-smelling at anthesis, subtended by short cataphylls 2–8 cm long, these often but not always bearing reduced but well-differentiated petiole and blade; peduncle about half length of petiole at anthesis but inflorescences first exposed when peduncle very short, and this elongating further in fruit; spathe 4–7 cm long; lower spathe green, narrowly ovoid, 1–2 cm long, differentiated from spathe limb by an abrupt constriction; spathe limb white, broadly ovate, inflated over the appendix then abruptly acuminate for 1–1.5 cm, caducous Spadix 6– 12 cm long, sessile, or with the dorsal part of pistillate flower zone naked at insertion, distally weakly clavate; pistillate flower zone about half length of the spadix, obliquely inserted on spathe; ovaries more or less flask-shaped, c mm tall; stigma sessile, button-like; interstellar staminodes scattered, occasionally absent, narrowly stalked and clavate-headed, about twice the height of the pistils; sterile zone between pistillate and staminate flower zones abruptly slightly thicker than distal part of the pistillate zone, reduced to a few whorls of columnar, rounded-topped staminodes markedly dissimilar to interstellar ones; staminate flower zone cylindric, c 0.7–1 cm diam.; stamens more or less rectangular, connective reaching another apex, c 0.7 mm across, thecae more or less flat opening via tiny pores; pollen ivory, in short strings; appendix weakly clavate Figure 4: The Inflorescences of Schismatoglottis wallichii Fruit: Fruiting spathe narrowly urceolate, to c cm long, dark green with darker striations, declinate, walls splitting and reflexing at fruit maturity to release fruits; fruits 2–4 mm long × 1–2.5 mm wide, greenish-white; seeds ovoid ellipsoid, ca 0.4 mm diam, encased with whitish green gel 2.1.4 Ecological characteristics Habitat: Schismatoglottis wallichii are found primarily in tropical parts of Southeast Asia, New Guinea, and Melanesia The majority of the species are native to the Island of Borneo So it is a tropical plant ideally grown in 65 to 75 degrees Fahrenheit (18 to 24 degrees Celsius); The optimum humidity for plants is between 80%-90% Ecological: Schismatoglottis wallichii observed growing in cliffs or steep cliffs around rainforests 1700 m above sea level • Soil: Because it is found in tropical rainforests, it prefers moist, well- drained soil Therefore, the tree must be taken care of to avoid drying and always provide enough moisture for the tree • Water: In addition to providing nutrients from the water, the roots of the plant also need oxygen to keep the plant healthy However, water the plant just enough to avoid over-watering, causing root rot and waterlogging Use potted plants with holes to help plants drain excess water out to avoid waterlogging causing root rot • Light: Schismatoglottis wallichii also likes strong indirect light, so it is suitable for planting in the garden, terrace, balcony, or in a window to receive light every day • Fertillizer: The advantage of the plant is that it is easy to take care of, so it does not require much nutrition, you can fertilize once a month in spring and summer because this time the tree needs more nutrients and limiting overfertilizing makes the plant dead 2.2 Some studies on tissue culture of species of the family Araceae Schismatoglottis is a tropical genus of around 100 species, most of which are Malesia, Bornean species The genus extends east to Vanuatu and north to subtropical China, with a very marked decrease in species richness and diversity across and east of Wallacea and north of the Malay peninsula It is absent in India and Australia (Hay & Yuzammi, 2000) Demand and consumption of ornamental plants and trees with beautiful shapes are highly attractive Harvesting wild plants as their primary source of raw materials can lead to genetic diversity and habitat loss (Shalini Arsogah et al.2018) Controlled conditions include an adequate supply of nutrients, appropriate pH, temperature, and gas and liquid media suitable for the growth and propagation of plants at peak efficiency (Hussain, Qarshi, Nazir, & Ullah, 2012) The establishment of in vitro aseptic cultures of the family Araceae is not easy because rhizome cultures are located in the subsoil and are susceptible to pathogens (Shalini Arsogah et al.2018) In the study by Shalini Arsogah et al.(2018) in the random sampling method, Schismatoglottis calyptrate was selected to be representative of the wax family and used as inoculum to initiate in vitro Surface sterilization is carried out in a multi-layer airflow chamber The sample was then sterilized with 70% Ethanol, followed by treatment with 10% Sodium hypochlorite (NaOCI) The roots of S calyptrata have 2-3 root hairs while the rhizomes and tubers with side shoots are pruned to the appropriate size (2-3 cm) They were then inoculated into vials filled with MS medium (Murashige and Skoog, 1962) supplemented with 30 g/L sucrose and 7.6 g/L agar The pH of the medium was adjusted to 5.7-5.8 with 0.1N Sodium hydroxide (NaOH) or 0.1 hydrochloric acid (HCI) before being autoclaved The prepared nutrient medium was autoclaved at 121˚C at a pressure of 0.15 kPa for 15 All cultures were placed and maintained in a culture rack at 25 + 2˚C under white fluorescent light and 50-60% relative humidity The experiment was performed to examine the effect of sterilizing the explants in 95% ethanol for 10 s and the immersion and heating method on the in vitro growth of the explants The experiment started with washing the sample with detergent and placing the culture under running water for 30 Then, before disinfecting the surfaces, they were soaked in distilled water for 10 minutes In a laminar flow chamber, the sample was first treated with undiluted NaOCI and stirred for several seconds, then sterilized with 95% ethanol for 10 seconds These steps were repeated three times, whereby for each subsequent treatment the cultures were rinsed with sterile distilled water Then the samples were cut to the appropriate size (2-3 cm) and dried using filter paper Before inoculation, the explants were dipped in 95% ethanol for seconds and heated for 30 seconds with a burner The roots of Schismatoglottis calyptrate, on the surface that was disinfected with 95% ethanol and then dipped in 95% ethanol and boiled for 30 seconds gave satisfactory results The highest percentage of sterile shoots (80%) was obtained using this rapid three-step method of flame surface sterilization (FSS) when it was surface-flame sterilization Large rhizomes 10 present a challenge in surface sterilization, so this FSS method could be an ideal alternative Abou Dahab, A.M et al (2000) studied three species of plants in the Araceae family to study the effects of different nutrient media during the rooting stage This experiment was designed to investigate the effect of MS salt at different concentrations (whole, half, and quarter) combined with different concentrations of sucrose (10, 20, and 30) on the growth and development of Syngonium Podophyllum, Spathpihyllum wallisii, and Philodendron scandens cultured in vitro (rooting stage) Three 1.5 cm long shoots were generated from the propagation stage (MS + 2mg/L BA + 30g/L sucrose) The obtained results indicated that MS root medium at full salt intensity supplemented with 30 g/l sucrose produced the highest shoot length, fresh weight, and root count for Syngonium podophyllum While MS at 1/4 salt intensity supplemented with 10g/l sucrose was suitable for Spathiphyllum wallisii, giving the highest shoot length and number of leaves The best medium for root formation was MS basal medium supplemented with 10 g/l sucrose For Philodendron scandens plants, the results indicated that shoots cultured in MS at 1/4 salinity with 30g/l sucrose gave the highest values in terms of fresh weight, root length, and root count In 2016, M A D Monney et al published research on the influence of BA and IBA or NAA Combinations on the Micropropagation of Cryptolepis sanguinolenta The experiments were designed to be grown on MS basal medium (Murashige and Skoog) supplemented with vitamin MS, 30 g/L sucrose, and BA at concentrations of 0.5, 1.0, 1.5, 2, 0, 2.5, or 3.0 mg/L in combination with 0.1 mg/L IBA or NAA The control consisted of a hormone-free MS medium For rooting induction and subsequent rooting, microscopic shoots, not less than cm, were transferred to full-strength MS medium with vitamins (addition of 0.5 mg/L IBA or 0.1 mg/ L IBA) or half-strength MS medium with vitamins (addition of 0.01 mg/L IBA) or hormone-free half-strength MS medium with vitamins The 11 pH was adjusted to 5.7 ± 0.1 using N NaOH and N H2SO4 M A D Monney et al concluded that auxin plays a very important role in the induction and proliferation of shoots In general, the combination of BA and IBA was found to be more effective in shoot regeneration and multiplication during the rapid multiplication stage of Cryptolepis sanguinolenta than the combination of BA and NAA M A D Monney et al successfully combined BA and IBA with the most optimal concentration of MS medium supplemented with mg/L BA and 0.1 mg/L IBA for the shoot and rooting on 1/2 of MS medium with addition of 0.01 mg/L IBA provided an effective protocol for micropropagation of Cryptolepis sanguinolenta so this optimal combination and concentration were suitable for the semi-hardwood specimen Cryptolepis sanguinolenta C Stanly et al published the study Micropropagation of Homalomena pineodora (family Araceae) The purpose of the study was to the effect of BA on Homalomena pineodora cultured in vitro Plants of H pineodora grown under greenhouse conditions at School of Biological Sciences, University of Sains Malaysia, Penang Rhizome specimen (2 cm) was removed from the mother plant Shoot explants were sterilized with two-stage Clorox ® In vitro propagation of these species was established using shoot tip explants MS medium supplemented with different concentrations of BA from 0, 0.5, 1.0, 1.5, and 2.0 mg/L Samples were inoculated into 350mL glass vials for weeks, weeks, and 12 weeks After the monitoring process, C Stanly et al found the most optimal concentration of BA for plants with MS medium supplemented with BA at a concentration of 0.5 mg/L, the number of shoots was significantly higher than that of other concentrations of BA (1.0-2.0 mg L-1) was added to the culture medium In the next experiment, C Stanly et al combined BA and 3-indole butyric acid (IBA) on shoot growth and rooting With MS medium supplemented with BA 0.5 mg/L and different concentrations of IBA (0.0-2.0 mg/L) After weeks it was found that the addition of IBA was not necessary and the addition of BA was sufficient to 12 promote shoot and root growth because when comparing concentrations of IBA (0.0-2.0 mg/L) there was no significant difference in shoot height, root, and number of leaves Concentrations of IBA (1.0-2.0 mg/L) significantly reduced root length A follow-up experiment C Stanly et al compared the effectiveness of liquid and solid media for shoot proliferation with MS medium supplemented with 0.5 mg/l BA The results of this experiment C Stanly et al showed that with the liquid medium supplemented with 0.5 mg/L BA the number of shoots was significantly higher and the shoots also started to produce longer roots compared with the solid medium 2.3 Introduction of Temporary Immersion Bioreactor system (TIB system) Micropropagation, popularly known for large-scale clonal propagation, is the first major and widely accepted practical application of plant biotechnology For commercial purposes, micropropagation will normally be limited by several costs that raise production costs such as labor costs, low propagation rates or long propagation times, and poor survival rates due to the risk of pollution and in the process of acclimatization This makes it difficult to apply this method In order to reduce the cost of micropropagation, many ideas for automation have been explored The Temporary Immersion Bioreactor system applied by M Escalona et al in the pineapple micropropagation study in 1999 is now starting to be applied in practice in recent years 2.3.1 Principle of Temporary Immersion Bioreactor system (TIB system) The Temporary Immersion Bioreactor system is an automated process that can control the rapid multiplication of plant samples under the right conditions In addition, TIB provides precise control over optimal conditions for the tree such as gaseous exchange, illumination etc required for plant growth, development and survival compared with conventional culture flasks TIB consists of three main stages: Multiplication, elongation, and rooting 13 The set-up consists of two vessels, one for the plantlets and the other one for the liquid culture media coupled together through a perforated rubber tubing that permits the flow of the liquid media from one vessel to the other During each stage of the cycle, the air stream is cleaned by passing through hydrophobic filters with a pore size of 0.22 micrometers The air pressure from the air compressor pushes the medium from one container to another to fully immerse the plant The airflow is reversed to withdraw the medium from the culture vessel The electronic timer controls the frequency and length of the immersion interval The three-way solenoid valve provides on/off operation Figure 5: Technological design and operational principle of Temporary Immersion Bioreactor system • (A) Period of exposure The whole volume of the liquid medium is located in the medium storage tank The air inlet of both containers is closed and the solenoid valves are opened to air • (B) Dislocation of the liquid medium from the medium storage tank to the culture chamber The air inlet of the cultivation chamber is closed, and the air inlet of the medium storage tank is opened The overpressure moves the medium into the cultivation chamber 14 • (C) Period of immersion The propagules are immersed in the liquid medium The medium storage tank is empty The air inlets for both containers are closed and the solenoid valves are opened to the air • (D) Withdraw all the nutrient medium back into the medium container The valve for air into the culture vessel is opened, while the valve for air into the culture vessel is closed Pressure helps to move the medium back into the medium container 2.3.2 Some micropropagation studies of Temporary Immersion Bioreactor system (TIB system) In 1999, M Escalona et al published the first study on the Temporary Immersion Bioreactor system on Pineapple Experiments conducted by the researchers used in vitro shoots obtained from cultured liquid cultures as starting materials Three culture methods (solid, liquid, and temporary immersion) were compared With MS medium +2.1mg/L BA + 0.3 mg/L, α-NAA after weeks supplemented with 1mg/L PB Temporary immersion increases the multiplier and fresh and dry weight after 42 days Conventional micropropagation (liquid media) and temporary immersion cultures were compared in combination with paclobutrazol Paclobutrazol promotes the formation of compact shoot clusters with limited leaf growth The highest multiplier rates A temporary immersion system was used to test two approaches during the extension phase: reduction of the shoot formation period or reduction the of initial explant period The highest number of shoots and uniform plants (191.8 plants/l) were obtained when shoots were cultured for weeks in a shoot multiplication medium supplemented with PB Md Zamilur Rahman et al have identified and prevented microbial contamination during potato culture in the Temporary Immersion Bioreactor system Preventing microbial contamination is a very important part because it is a challenge in large-scale cultures The bioreactor system has many advantages 15 including better control of culture conditions; optimal supply of nutrients and growth regulators; Monitor nutrient absorption Despite such advantages, bioreactor systems carry a great risk of loss of culture media due to microbial contamination Although techniques have been thoroughly disinfected, contaminants can still be introduced into cultures by explants, during laboratory manipulation, and possibly by endophytic microbes Liquid MS medium was used in the TIB systems For shoot growth 30 g/L sucrose was added to the medium and the pH was adjusted to 5.8 before autoclaving Five fungi (Aspergillus, Penicillium, Mucor, Fusarium, and Rhizopus) and six bacteria (Pseudomonas, Staphylococcus, Klebsiella, Corynebacterium, Proteus, and Bacillus) were also isolated from the TIB system Three antibacterial (Gentamycin, Vancomycin, and Tetracycline) and four antifungal (Mencozeb, Propiconazole, Bavistin, and Copper oxychloride) drugs were studied for their effects on pollutants and potato shoot growth Results indicate that against isolated bacteria (35 mm inhibitory zone) and fungi (100%) respectively, Gentamycin (50 mg/L) and Propiconazole (0.15%) were more effective, whereas Gentamycin in combination with Bavistin performed better on potato shoot and root development The Potential of Temporary Immersion Bioreactors (TIBs) in Meeting Crop Production Demand in Nigeria was examined by Paul Terwase Lyam in 2012 The first significant and well-acknowledged practical application of plant biotechnology is micropropagation, which is more often known as large-scale clonal propagation Conventional micropropagation of significant crop species has limited commercial utility due to the vast numbers and high production costs required each year to establish new farms These are mostly caused by high labor costs, slow or prolonged multiplication before plantlets are transplanted, and low survival rates due to contamination threats and acclimation All of these represent a significant setback for the commercialization of various economic species through micropropagation The volume of the culture media (100 - 250ml for 16 500ml and 300 – 400 for 1-liter capacity vessel) immersions each day with a 3hour interval were observed The type of plant determines this situation Depending on the plant species, the time for cultural immersion in the media should be between and minutes Daviel Gómez et al published in temporary immersion bioreactors, the effects of salinity (NaCl) and the carbon source mannitol (0-200 mM) on the micropropagation of pineapple cv MD2 were examined (TIBs) The plant material's aldehyde, chlorophyll, carotenoids, and phenolic content levels were also measured, along with the shoot multiplication rate, shoot cluster fresh weight, and shoot weight It was also determined how much soluble phenolics were present in the culture medium Above 50 mM, pineapple shoot multiplication and fresh weight were significantly reduced by sodium chloride or mannitol The cluster fresh weight reduced by 40% and the multiplication rate by 71.5% when 200 mM NaCl was added NaCl increased phenolics excreted into the culture medium by 1.4 times, soluble phenolics in shoots by 1.4 times, and levels of other aldehydes by 2.4 times Mannitol, on the other hand, reduced the multiplication rate and fresh weight of the clusters by around 60% Chlorophyll B content was raised by mannitol by 1.4 times, and soluble phenolics by 2.1 times As a result, pineapple cv MD2 was shown to be more sensitive to NaCl than to mannitol A 50% reduction was attained using 37.4 mM NaCl and 66.5 mM mannitol, according to multiplication rates These concentrations can be used to screen for/detect soma clonal variations with an improved salinity or drought tolerance as well as stress shoots during micropropagation in TIBs Studies have revealed that the priceless species of decorative Schismatoglottis continue to pique the curiosity of scientists Additionally, there are benefits to increasing the propagation process' efficiency by utilizing various culture systems, particularly liquid medium and bioreactor systems There is only one current study being done in Vietnam on conventional Schismatoglottis being 17 micropropagated In Vietnam, research on the use of Temporary Immersion Bioreactors (TIBs) systems for micropropagation has not gotten much attention With such factors, the research I conducted has a strong scientific foundation and broad future applicability variations that can withstand drought or increasing salinity 18 PART RESEARCH MATERIALS AND CONTENTS AND METHODS 3.1 Object, material, location, time of culture 3.1.1 Research Materials In vitro Protocorm-like-body (0.5–1.0 cm) of Schismatoglottis wallichii 3.1.2 Location, time of culture Location: T.E.N Biotech Biological Joint Stock Company, address number 56A, Kien Thanh residential group, Trau Quy town, Gia Lam district, Hanoi Time of culture: 07/2022 – 12/2022 3.1.3 Research tools Sterilizer, sterile culture box, triangle flask, pipette, PH calibrator, piston, knife, scissors, cutter, UV lamp, technical scale, analytical balance, oven, rig system lights, … Chemicals: The chemicals used in plant multiplication, MS medium used in research include macronutrients and micronutrients according to Murashige and Skoog Saccharose, Agar, growth stimulants such as BA, IAA, α-NAA … 3.2 Research contents 3.2.1 Evaluation of the effect of growth hormones on the multiplication stage of Schismatoglottis wallichii 19 Experiment 1: Effect of BA (Benzyl Aminopurine) on shoot proliferation and growth of Schismatoglottis wallichii after weeks of culture Formula BA concentration (mg/l) F1 (Control) 0.00 F2 0.50 F3 1.00 F4 1.50 F5 2.00 F6 2.50 • Basic culture medium: MS + 3% sucrose + g/l agar Experiment 2: Effect of IBA (3-Indolebutyric acid) in combination with BA on shoot proliferation and growth of Schismatoglottis wallichii Formula IBA concentration (mg/l) F1 (Control) 0.00 F2 0.75 F3 1.00 F4 1.50 F5 1.75 • Basic culture medium: MS + 3% sucrose + g/l agar • Amount of BA based on the best result of experiment 20 Experiment 3: Effect of α-NAA (α-Naphthaleneacetic acid) in combination with BA on shoot proliferation and growth of Schismatoglottis wallichii Formula α-NAA concentration (mg/l) F1 (Control) 0.00 F2 0.75 F3 1.00 F4 1.50 F5 1.75 • Basic culture medium: MS + 3% succrose + g/l agar • Amount of BA based on the best result of experiment 3.2.2 Comparison of growth of Schismatoglottis wallichii on the multiplication stage in different culture systems Experiment 4: Effect of different culture systems on the multiplication stage of Schismatoglottis wallichii Formula In vitro culture systems F1 Semi-solid F2 Stationary liquid F3 Liquid with aeration F4 Temporary immersion bioreactor (TIB) • F1: 6g/L agar (in semi soid) • F3: Continuously for 15 minutes every hours • F4: The immersion frequency is minutes every hours 21 • Basic culture medium: From experiments and 3, Comparison of efficiency on shoot proliferation and growth of Schismatoglottis wallichi between IBA and α- NAA supplemented medium on the rapid multiplication then supplement medium MS • All formulas were arranged in replicates, 10 samples each time, and plants were cultured in 380 ml cylinders with 40 ml of medium for each flask, samples each 3.2.3 Evaluation of the effect of growth hormones on the rooting stage of Schismatoglottis wallichii Experiment 5: Effect of IBA (3-Indolebutyric acid) on the rooting stage of Schismatoglottis wallichii Formula IBA concentration (mg/l) F1 (Control) 0.00 F2 0.50 F3 0.75 F4 1.00 F5 1.25 F6 1.75 • Basic culture medium: MS + 3% sucrose + 1mg/L BA 22 Experiment 6: Effect of α-NAA (α-Naphthaleneacetic acid) on the rooting stage of Schismatoglottis wallichii Formula α-NAA concentration (mg/l) F1 (Control) 0.00 F2 0.50 F3 0.75 F4 1.00 F5 1.25 F6 1.75 • Basic culture medium: MS + 3% sucrose + 1mg/L BA 3.2.4 Comparison of growth of Schismatoglottis wallichii on the rooting stage in different culture systems Experiment 7: Effect of different culture systems on the rooting stage of Schismatoglottis wallichii Formula In vitro culture systems F1 Semi-solid F2 Stationary liquid F3 Liquid with aeration F4 Temporary immersion bioreactor (TIB) • F1: 6g/L agar (in semi soid) • F3: Continuously for 15 minutes every hours • F4: The immersion frequency is minutes every hours 23 • Basic culture medium: From experiments and 6, Comparison of efficiency on shoot proliferation and growth of Schismatoglottis wallichi between IBA and α- NAA supplemented medium on the rooting stage then supplement medium MS • All formulas were arranged in replicates, 10 samples each time, and plants were cultured in 380 ml cylinders with 40 ml of medium for each flask, samples each 3.3 Tracking Indicators • Monitor the indicators every weeks until the end of the experiment Contamination rate (%) = total number of contaminated explants total number of explants cultured × 100% Multiplication rate (Number of shoots/explant) = Total number of shoots Total number of explants cultured Average length of shoots (cm) = Average leave number (leaf) = Rooting rate (%) = 3.4 Total of shoot length Total number of shoots Total number of leaves 𝑇𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑝𝑙𝑎𝑛𝑡𝑠 Total number of rooted shoots Total number of explants cultured × 100% Research Method 3.4.1 Method of proceeding A, Multiplication stage method Taking the starting material, we cut each protocorm-like-body with a height of about - 1.2 cm and transplant it into the medium for evaluation 24 B, Rooting stage method In vitro shoots of Schismatoglottis wallichii plants in a vegetative, welldeveloped state with a height of about - cm were separated into single shoots with cleaned roots and then transplanted into the medium to create complete plants C, Experimental methods Using current plant tissue culture methods Each recipe consisted of 10 samples and was repeated times The followup time in the rapid multiplication experiment was weeks, the complete plant formation was weeks, and the nursery acclimatization experiment was weeks The medium was autoclaved at 121ºC and atm pressure for 20 minutes Light intensity: 2000 lux Lighting time: 16h/day Humidity: 60 - 70% Temperature: 25 ± 2ºC D, Data processing methods Data are processed with MICROSOFT EXCEL and IRRISTAT 5.0 Comparative formulas were conducted by the method of checking the difference of means by estimation, using LSD standard with 95% confidence 25 PART 4: RESULTS AND DISCUSSION 4.1 Effect of growth hormones on the multiplication stage of Schismatoglottis wallichii Growth hormones are extremely important factors controlling morphogenesis and complete regeneration of plants Among them, BA is a growth promoter that belongs to cytokinin The most typical physiological roles of this group in plants are cell division stimulants, as well as cell division activators Cytokines have the ability to activate protein synthesis In this experiment, BA was added to the culture medium with a concentration range from 0.5 mg/L to 2.5 mg/L to investigate the rapid multiplication of Schismatoglottis wallichii shoots The results after weeks of culture are presented in Table 4.1 and Figure 4.1 Table 1: Effect of BA (Benzyl Aminopurine) on shoot proliferation and growth of Schismatoglottis wallichii after weeks of culture Formula F1 (control) F2 F3 F4 F5 F6 LSD0,05 CV% BA Shoots (mg/L) (buds/explant) Shoot Leaves Fresh Plant length (Leaves/explant) weight quality (cm/shoot) of shoot (g) 0.0 2.26 1.60 1.86 1.82 ++ 0.5 1.0 1.5 2.0 2.5 3.33 4.03 3.63 3.40 3.93 0.53 2.1 1.86 2.12 1.95 1.87 1.91 0.73 3.6 2.56 2.86 2.46 2.70 2.15 0.37 1.8 2.58 2.70 2.08 2.23 2.04 0.57 1.7 +++ +++ +++ ++ + • +++: Good plant quality: plant is big, strong, stout, uniform, green • ++ : Plants are of good quality: plants may be small, evenly not tall, green, power • +: Low quality of plant: small, weak, stunted, steady plants In some cases, leaves are not droopy, curly leaves, or bunded 26 The results shown in Table 4.1 and Figure 4.1 indicated that MS + mg/L BA + 30 g/L sucrose + g/L agar medium provided the highest shoot proliferation and growth of 4.03 shoots/explant with a median length of 2.12 cm/bud, 2.86 leaves/explant When supplementing with high levels of BA (2.0-2.5mg/L), the number of shoots produced increased (3.93 buds/explant) but the quality of shoots was not good with reduced shoot weight (1.91 cm/bud) and reduced number of leaves (2.15 leaves/explant) compared with the addition of 1mg/L BA Thus, the addition of BA to the growth medium stimulated the shoot proliferation capacity of the explants However, if higher concentrations of BA were added, it inhibited the plant's ability to proliferate shoots BA has excellent qualities in shoot propagation and growth Figure 1: Schismatoglottis wallichii shoots in BA supplemented mediums after weeks of culture 4.2 Effect of auxins on shoot proliferation and growth of Schismatoglottis wallichii after weeks of culture Auxins are phytohormones that affect cell enlargement, root germination, and random shoot formation They induce apical dominance and inhibit lateral bud formation Auxins are commonly used in tissue culture media with or without 27 cytokinins during the propagation phase Combined with the cytokinin and auxin group of growth regulators helps to generate new shoots and improves the ability to regenerate shoots α-NAA and IBA are two popular auxins used in the rooting phase Therefore, at this stage, they are used for experiments In these experiments, the basal medium was MS + mg/L BA + 30 g/L sucrose + g/L agar This experiment was sought to establish the optimum concentration of BA and auxin combination required for shoot production from stem explants of Schismatoglottis wallichii and developed a rooting medium for regenerated shoots 4.2.1 Effect of IBA (3-Indolebutyric acid) in combination with BA on shoot proliferation and growth of Schismatoglottis wallichii In this experiment, IBA was added to the culture medium with a concentration range from 0.75 mg/L to 1.75 mg/L to investigate the rapid multiplication of Schismatoglottis wallichii shoots The results after weeks of culture are presented in Table 4.2 and Figure 4.2 Table 2: The effect of IBA (3-Indolebutyric acid) in combination with BA on shoot proliferation and growth of Schismatoglottis wallichii after weeks of culture Formula IBA (mg/L) Shoots (buds/explant) Shoot length (cm/shoot) Leaves (Leaves/explant) Plant quality F1 (control) F2 F3 F4 F5 LSD0,05 CV% 0.00 3.93 2.31 3.56 +++ 0.75 1.00 1.25 1.75 4.56 3.83 3.23 3.53 0.62 2.5 3.24 3.16 2.16 3.45 0.21 1.8 5.90 5.40 5.10 4.30 0.72 2.4 +++ +++ +++ +++ • +++: Good plant quality: plant is big, strong, stout, uniform, green 28 The results shown in Table 4.2 and Figure 4.2 show that when BA was used in combination with IBA, shoot reproduction was quite high, shoot length was considerable and plant quality was at good peformance In the medium supplemented with 0.75mg/l IBA the best branch reproduction rate is 3.56 buds/explant, the branch length is 3.24cm/plant, the number of leaves is 5.9, and the plant quality is good In this experiment, the plant samples started to produce roots due to the present of auxin IBA in the culture medium However, when the IBA concentration was high, the shoot reproduction rate decreased The optimal medium for shoot proliferation and growth in this experiment is: MS + 0.75 mg/l IBA + 30 g/l sucrose + g/l agar Homalomena pineodora (family Araceae) (C Stanly et al in 2012) studied that adding 0.5mg/L BA and 0.5mg/L IBA gave optimal results consistent with Homalomena pineodora but for Schismatoglottis wallichii in the experiment In this experiment, the optimal concentration was 1mg/l BA and 0.75mg/l IBA, so the supplement was more than that of Homalomena pinodora Figure 2: Schismatoglottis wallichii shoots in BA supplemented mediums combined with IBA after weeks of culture 29 4.2.2 Effect of α-NAA (α-Naphthaleneacetic acid) in combination with BA on shoot proliferation and growth of Schismatoglottis wallichii In this experiment, α-NAA was added to the culture medium with a concentration range from 0.75 mg/L to 1.75 mg/L to investigate the rapid multiplication of Schismatoglottis wallichii shoots The results after weeks of culture are presented in Table 4.3 and Figure 4.3 Table 3: Effect of α-NAA (α-Naphthaleneacetic acid) in combination with BA on shoot proliferation and growth of Schismatoglottis wallichii after weeks of culture Formula α-NAA (mg/L) Shoots (buds/explant) F1 0.00 F2 Leaves (Leaves/explant) Plant quality 3.60 Shoot length (cm/shoot) 2.24 3.46 +++ 0.75 3.96 3.12 3.83 +++ F3 1.00 3.43 2.88 3.00 +++ F4 1.25 2.93 2.72 2.76 +++ F5 1.75 3.16 2.62 2.83 +++ LSD0,05 0.57 0.64 0.59 CV% 4.5 1.6 3.2 (control) • +++: Good plant quality: plant is big, strong, stout, uniform, green The data shown in Table 4.3 and Figure 4.3 indicate that the medium with the maximum shoot proliferation and growth 3.96 buds/plant, a mean length of 3.12cm/shoot, 3.83 leaves/plant was MS + mg/l BA + 0.75 mg/l α-NAA + 30 g/l sucrose + g/l agar Plant quality is also excellent α-NAA had a negligible impact on shoot growth and proliferation 30 When adding α-NAA to the culture medium, the number of multiplying shoots may not be much different than when adding each BA to the medium (4.03 buds/ explant) In addition, it is possible that at the stage of rapid multiplication, the plant has appeared roots So it can be seen that α-NAA has the ability to divide cells and form roots Figure 3: Schismatoglottis wallichii shoots in BA supplemented mediums combined with α-NAA after weeks of culture 4.2.3 Comparison of efficiency on shoot proliferation and growth of Schismatoglottis wallichii between IBA and α-NAA in combination with BA supplemented medium after weeks of culture The auxin group's growth regulators include IBA and α-NAA Based on the findings of Tables 4.2 and 4.3, it was determined that the outcomes of the shoot multiplication rate were maximum when the medium was supplied with the same concentration of 0.75 mg/l In general, it was discovered that BA and IBA combinations were more effective than BA and NAA combinations in the regeneration of shoots and the multiplication of nodal segments of Schismatoglottis wallichii In contrast to α-NAA (3.96 buds/plant), IBA application demonstrated a greater proliferation rate of 4.56 buds/plant (Table 31 4.3) IBA provided strong shoot proliferation and growth but yet for good plant quality at a dosage of 0.75 mg/l Therefor, it can be concluded that MS + mg/l BA + 0.75 mg/l IBA + 30 g/l sucrose medium is best for of Schismatoglottis wallichii shoot proliferation and growth (used as the basic medium for the following experiments) 0.75 mg/L α-NAA 0.75mg/L IBA Figure 4: Schismatoglottis wallichii shoots between BA supplemented medium combined with IBA and α-NAA after week of culture Study on the effect of BA and IBA or NAA combination on the micropropagation of Cryptolepis sanguinolenta (M A D Monney et al 2016) After the research process, the author also reported that the combination of BA with IBA proved to be more effective in regeneration and shoot multiplication during the rapid multiplication stage of Cryptolepis sanguinolenta than the combination of BA and NAA with the concentration of MS medium supplemented with mg/L BA and 0.1 mg/L IBA was the most optimal for shoot and rooting on 1/2 of the MS medium supplemented with 0.01 mg/L IBA providing an efficient protocol for the micropropagation of Cryptolepis sanguinolenta, this optimal concentration and combination was suitable for the semi-hardwood specimen Cryptolepis sanguinolenta whereas for 32 Schismatoglottis wallichii the optimal concentration suitable for this plant is 1mg/L BA and 0.75mgL IBA 4.3 Comparison of growth of Schismatoglottis wallichii on the multiplication stage in different culture systems Different culture methods have been studied and created with the following goals in mind: to decrease the labor cost associated with micropropagation while boosting the efficiency of the process by raising the shoot multiplication coefficient and the quality of the shoots, among other things Micropropagation may be partially automated or reduced in steps This article will compare and evaluate the impact of several culture systems on the in vitro rapid multiplication stage of Schismatoglottis wallichii In this experiment, the basic medium is MS + mg/l BA + 0.75 IBA + 30 g/l sucrose Shoots were inoculated in semi-solid, Stationary liquid, Liquid with aeration and TIB media to find the optimal culture mode for rapid multiplication of Schismatoglottis wallichii shoots Culture results after weeks are presented in Table 4.4 and Figure 4.5 Table 4: Efficiency of in vitro culture systems of Schismatoglottis wallichii after week of culture Shoot quality 3.90 3.87 3.18 Fresh weight of shoot (mg) 9.66 8.71 9.11 6.17 4.18 10.71 +++ LSD0,05 0.55 0.33 0.70 CV% 2.2 1.6 1.5 F1 F2 F3 F4 In vitro culture systems Survival rate (%) shoots (buds/plant) Shoot length (cm/shoot) Semi-solid Stationary liquid Liquid with aeration Temporary immersion bioreactor (TIB) 90% 85% 80% 5.60 5.46 4.10 97% +++ ++ ++ • +++: Good plant quality: plant is big, strong, stout, uniform, green • ++: Plants are of good quality: plants may be small, evenly not tall, green, power 33 The multiplier of Schismatoglottis wallichii in the Temporary immersion bioreactor system (TIB) with an average multiplier of 6.17 shoots/explant is the best, according to Table 4.4's findings This outcome was improved in liquid with aeration with 4.1 shoots per sample and normal culture on semi-solid system with a multiplier of 5.6 shoots/explant The multiplier, which reached 5.46 shoots/explant in the stationary liquid culture method In different culture systems, static liquid culture has the disadvantage that if the explant is small and grows slowly when submerged in the culture solution, growth will be poor, only if the explant is partially submerged will help plants grow faster, which affects the multiplier and shoot growth When the weak shoots are damaged, they will be underdeveloped or not grow at all The Temporary immersion bioreactor system (TIB) system overcomes the disadvantages of the other culture systems because the exchange of medium between the two flasks exposes the entire culture to the culture solution for a period of time the explants are exposed to air for easy gas exchange and growth, new gas is actively introduced making the concentrations of gases balanced and the plants grow better than in a semi-solid gas culture system with passive exchange and very little Those advantages and disadvantages are reflected in the quality of shoots when shoots in the Temporary immersion bioreactor system (TIB) system develop very well, and the shoots are sturdy; when growing static liquid, shoots develop but there is succulent and vitrification phenomenon; shoots in the semi-solid system grew more slowly than in the other systems Additionally, the results showed that liquid cultures are effective at increasing biomass and the number of shoots The present temporary immersion bioreactor is quite effective for the large-scale spread of Schismatoglottis wallichii 34 Semi-solid Stationary liquid Temporary immersion bioreactor (TIB) Liquid with aeration Figure 5: Schismatoglottis wallichii shoots in vitro culture systems after weeks of culture 4.4 Effect of Auxin on the rooting stage of Schismatoglottis wallichii Single-leaf Schismatoglottis wallichii shoots with leaves were transplanted into a 350 ml flask with MS basal medium supplemented with + 30 g/l sucrose + g/l agar to produce complete Schismatoglottis wallichii plants 35 4.4.1 Effect of IBA (3-Indolebutyric acid) on the rooting stage of Schismatoglottis wallichii In this experiment, IBA was added to the optimal culture medium with a concentration range from 0.5 mg/L to 1.75 mg/L for investigation during the rooting stage of Schismatoglottis wallichii plants The results after weeks of culture are presented in Table 4.5 and illustrated in Figure 4.6 Table 5: Effect of IBA (3-Indolebutyric acid) on the rooting stage of Schismatoglottis wallichii after weeks of culture IBA Rooting Average Root (Root/ Fresh Plant (%) root explant) weight quality length (g) (cm) F1 0.00 100 2.08 6.10 8.24 +++ F2 0.50 100 2.51 5.73 9.08 +++ F3 0.75 100 3.89 7.23 10.84 +++ F4 1.00 100 3.49 6.10 10.12 +++ F5 1.25 100 3.50 5.60 9.69 +++ F6 1.75 100 3.38 6.26 9.40 +++ 2.6 3.2 1.1 0.30 0.92 0.68 CV% LSD0,05 • +++: Good plant quality: plant is big, strong, stout, uniform, green The results from Table 4.5 and Figure 4.5 showed that at all concentrations of IBA addition, the rooting rate was 100% But at different concentrations, the quality of roots and plants will also be different At the basic MS medium supplemented with 0.75mg/L IBA gave the best plant quality, with the highest average root length of 3.89 (cm), plant weight reaching 10.8(g) The number of 36 root 7.23 root/ explant The results showed that with increasing IBA concentration (1mg/L-1.75mgL), the average root length decreased (from 3.49 (cm) to 3.38 (cm)) and the plant weight also decreased (from 10.12(g) to 9.4(g)) Figure 6: Schismatoglottis wallichii in IBA (3-Indolebutyric acid) supplemented mediums on the rooting stage after weeks of culture 4.4.2 Effect of α-NAA (α-Naphthaleneacetic acid) on the rooting stage of Schismatoglottis wallichii In this experiment, α-NAA was added to the optimal culture medium with a concentration range from 0.5 mg/L to 1.75 mg/L for investigation during the rooting stage of Schismatoglottis wallichii plants The results after weeks of culture are presented in Table 4.6 and illustrated in Figure 4.7 37 Table 6: Effect of α-NAA (α-Naphthaleneacetic acid) on the rooting stage of Schismatoglottis wallichii after weeks of culture Fomular α-NAA Rooting Average (%) Root Fresh Plant root (Root/ weight quality length explant) (g) (cm) F1 0.00 100 2.61 6.43 7.96 +++ F2 0.50 100 2.77 7.53 9.06 +++ F3 0.75 100 3.21 8.53 10.42 +++ F4 1.00 100 2.24 8.06 9.41 +++ F5 1.25 100 1.79 7.30 9.89 +++ F6 1.75 100 2.33 7.33 9.37 +++ CV% 5.1 3.8 4.3 LSD0,05 0.19 1.67 0.67 • +++: Good plant quality: plant is big, strong, stout, uniform, green The results presented in Table 4.6 and Figure 4.6 show that MS + 0.75mg/l α-NAA + 30 g/l sucrose + g/l agar medium gave the best quality plant This indicates that the plant is affected by rooting by NAA with average root length, the number of roots/explant increased significantly more than formula without NAA In addition, it is also affected by concentration In formula 5.6, a large amount of NAA was added, but the average length of roots was lower than that of formula 3, the number of roots/explant was also reduced 38 Figure 7: Schismatoglottis wallichii in α-NAA (α-Naphthaleneacetic acid) supplemented mediums on the rooting stage after weeks of culture 4.4.3 Comparison of efficiency on quality of Schismatoglottis wallichii between IBA and α-NAA supplemented medium on the rooting stage after weeks of culture Growth regulators IBA and α-NAA, often known as auxins, exert a variety of physiological impacts on plants, including stimulating effects on brittle roots Auxin supplementation is essential for the plants throughout the rooting process to assist prevent harm to the roots and increase the effectiveness of the nursery acclimation process The plants were 100% rooted when IBA and α-NAA were added, however the quality of the roots was different The root length was longer with the media added with IBA than it was with the medium supplied with αNAA, but there were less roots overall Additionally, α-NAA roots are regarded as having superior, stronger, and thicker hairy roots at the roots At a dose of 0.75 mg/l, α-NAA developed robust roots while still ensuring good plant quality Schismatoglottis wallichii grows best on MS + mg/l BA + 0.75 mg/l α-NAA + 30 g/l sucrose medium during the roots stage (used as the basic medium for the following experiments) 39 0.75mg/L α-NAA 0.75mg/L IBA Figure 8: Schismatoglottis wallichii in α-NAA (α-Naphthaleneacetic acid) and IBA (3-Indolebutyric acid) supplemented mediums on the rooting stage after weeks of culture 4.5 Comparison of growth of Schismatoglottis wallichii on the rooting stage in different culture systems Plants were inoculated into semi-solid, Stationary liquid, Liquid with aeration and TIB media to find the optimal culture during the Schismatoglottis wallichii rooting stage Culture results after weeks are presented in Table 4.7 and Figure 4.9 Table 7: Efficiency of in vitro culture systems of Schismatoglottis wallichii on the rooting stage after week of culture Fomular In vitro culture systems Rooting (%) F1 F2 F3 Semi-solid Stationary liquid Liquid with aeration Temporary immersion bioreactor (TIB) F4 CV% LSD0,05 Root (Root/ explant) Fresh weight (g) 100 100 100 Average root length (cm) 2.91 2.72 1.65 Plant quality 4.06 3.93 3.46 10.40 9.26 9.57 +++ +++ +++ 100 2.89 5.80 11.50 +++ 2.7 0.15 1.5 0.51 1.9 0.93 40 • +++: Good plant quality: plant is big, strong, stout, uniform, green The rooting rate of Schismatoglottis wallichii shoots approached 100% in several culture methods after weeks With an average of 2.89 roots per plant in the cultured TIB system, the maximum number of roots was produced; 5.8 roots per plant were produced in the semi-solid system The semi-solid culture system produced the best results in terms of root length, with an average of 2.91 cm; the other systems, the static liquid system, the aeration liquid system, and the temporary immersion bioreactor system (TIB), reached 2.72 cm, 1.65 cm, and 2.89 cm, respectively The Temporary Immersion Bioreactor System (TIB) system was followed by semi-solid, stationary liquid, aerated liquid, and fresh weight indicator, which climbed steadily in that order, reaching 11.5g, 10.4g, 9.26g, and 9.57g, respectively The quality of the complete plants is clearly differentiated, when in the semi-solid and TIB systems, the complete plants are sturdy, well developed, without succulent and vitrification; while in the stationary liquid system and in the shaking liquid system, the plants grow less well, showing signs of succulent and vitrification Semi-solid Stationary liquid 41 Liquid with aeration TIB systems Figure 9: Schismatoglottis wallichii in vitro culture systems on the rooting stage after weeks of culture 42 PART CONCLUSIONS AND RECOMMENDATIONS 5.1 Conclustion - The optimal medium used in the rapid multiplication stage of Schismatoglottis wallichii is MS + mg/l BA + 0.75 IBA + 30 g/l sucrose + g/l agar - The optimal medium used in the rooting stage of Schismatoglottis wallichii is MS + 0.75 α- NAA + 30 g/l sucrose + g/l agar - The optimal in vitro culture system for Schismatoglottis wallichii is a temporary immersion bioreactor 5.2 Recommendations - Due to the limited time available for research, additional studies are still needed for some assessment trials, such as those involving the temporary immersion bioreactor TIB system control parameters being adjusted for the stage of complete plant growth and testing analyzing the potential to speed up the stage of complete plant creation while changing the culture medium, changing the medium without transplanting to save labor during the micropropagation process - The results demonstrate the viability and potential of using temporary immersion bioreactor TIB system technique for micropropagation, which is the foundation for Schismatoglottis wallichii perform practical studies on Vietnamese plants with development potential 43 REFERENCES Abou Dahab, A., Arafa*, A M., & and, I I (2000) EFFECT OF DIFFERENT NUTRIENT MEDIA ON THE Agriculture Development Systems Project , 10-15 Altaf Hussain, I U (2012) Plant Tissue Culture: Current Status and Opportunities intech, 1-5 Berthouly, H E (2002) Temporary immersion systems in plant micropropagation Plant Cell Tissue and Organ Culture, 69(3):215-231 Escalona, M S (2003) Physiology of Effects of Temporary Immersion Bioreactors on Micropropagated Pineapple Plantlets In Vitro Cell Developmental Biology of Plan, (39): 651-656 González, M E (1999) Pineapple (Ananas comosus L Merr) Plant Cell Reports, 3-14 Jiakang Zhou 1, Y L (2021) Effects of Plant Growth Regulators on the Rapid Propagation Forests, 13-15 Maame Adjoa Dwumawa Monney, N A (2016) Influence of BA and IBA or NAA American Journal of Plant Sciences, 7-13 Md Zamilur Rahman1, S (2011) Identification and prevention of microbial contaminants of potato culture in Published by Malaysian Society for Microbiology Peter C Boyce1, a (2015) Studies on Schismatoglottideae (Araceae) of Borneo L – Journal of Plant Systematics, 440-450 PIERIK, R (1997) IN VITRO CULTURE OF HIGHER PLANTS Department of Horticulture,, 45-76 S Roels1, * M (2004) Optimization of plantain ( Musa AAB) Plant Cell Tissue and Organ Culture, 58-64 S Roels1, * M (2018) FSS -NOVEL TECHNIQUE FOR IN VITRO PROPAGATION OF ARACEAE AND Suganthi Appalasamy 44 Schismatoglottis wallichii Hook f (2021, october 14) Retrieved from NParks Flora & Fauna Web: https://www.nparks.gov.sg/florafaunaweb/flora/2/4/2442 Stanly, C., Bhatt*, A., Sulaiman, B., & Keng, C L (2012) Micropropagation of Homalomena pineodora Sulaiman & Boyce (Araceae): A Horticultura Brasileira · , 39-42 Thorpe, T A (2017) History of Plant Tissue Culture History of Plant Tissue Culture, 9-15 Wong Sin Yeng, H Y (2016) Studies on Schismatoglottideae (Araceae) of 7790 Wong Sin Yeng, P B (2018) An Annotated Check-list for Schismatoglottis Journal of the International Aroid Society, 138-157 Wong Sin Yeng, P C (2017) Studies on Schismatoglottideae (Araceae) of 2936 Y, K M (2004) Improvement of micropropagation of Japanese yam using liquid and gelled medium culture, Scientia Horticulturae, 102: 461-466 Yuzammi, A H (2000) Schismatoglottideae (Araceae) in Malesia I — Telopea , 14-18 45 APPENDICES The effect of BA (Benzyl Aminopurine) on shoot proliferation and growth of Schismatoglottis wallichii after weeks of culture BALANCED ANOVA FOR VARIATE CHOI FILE OANH 22/11/22 9:25 :PAGE thi nghiem ba VARIATE V003 CHOI LN ER SOURCE OF VARIATION DF SUMS OF MEAN SQUARES SQUARES F RATIO PROB LN =========================================================================== == CT 27.3111 5.46222 2.45 0.035 NL 13.5111 6.75555 3.04 0.049 * RESIDUAL 172 382.756 2.22532 -* TOTAL (CORRECTED) 179 423.578 2.36636 -BALANCED ANOVA FOR VARIATE CCCHOI FILE OANH 22/11/22 9:25 :PAGE thi nghiem ba VARIATE V004 CCCHOI LN ER SOURCE OF VARIATION DF SUMS OF MEAN SQUARES SQUARES F RATIO PROB LN =========================================================================== == CT 1.81304 362608 8.71 0.000 NL 427551 213776 5.14 0.007 * RESIDUAL 172 7.15648 416075E-01 -* TOTAL (CORRECTED) 179 9.39707 524976E-01 -BALANCED ANOVA FOR VARIATE SOLA FILE OANH 22/11/22 9:25 :PAGE thi nghiem ba VARIATE V005 SOLA 46 LN ER SOURCE OF VARIATION DF SUMS OF MEAN SQUARES SQUARES F RATIO PROB LN =========================================================================== == CT 10.9778 2.19556 2.04 0.075 NL 211111 105556 0.10 0.906 * RESIDUAL 172 185.456 1.07823 -* TOTAL (CORRECTED) 179 196.644 1.09857 -BALANCED ANOVA FOR VARIATE KLCHOI FILE OANH 22/11/22 9:25 :PAGE thi nghiem ba VARIATE V006 KLCHOI LN ER SOURCE OF VARIATION DF SUMS OF MEAN SQUARES SQUARES F RATIO PROB LN =========================================================================== == CT 851203 170241 6.71 0.000 NL 2.21723 1.10862 43.70 0.000 * RESIDUAL 172 4.36387 253713E-01 -* TOTAL (CORRECTED) 179 7.43230 415212E-01 -TABLE OF MEANS FOR FACTORIAL EFFECTS FILE OANH 22/11/22 9:25 :PAGE thi nghiem ba MEANS FOR EFFECT CT -CT NOS 30 30 30 30 30 30 CHOI 2.26667 3.33333 4.30000 3.93333 3.80333 3.93333 CCCHOI 1.60600 1.88633 2.12167 1.95100 1.87133 1.91933 SOLA 1.86667 2.56667 2.86667 2.46667 2.70000 2.15000 KLCHOI 1.82200 2.58267 2.70000 2.08067 2.23133 2.04967 47 SE(N= 30) 0.272355 0.372413E-01 0.189581 0.290811E-01 5%LSD 172DF 0.760178 0.103945 0.529145 0.811690E-01 -MEANS FOR EFFECT NL -NL NOS 60 60 60 CHOI 3.50000 4.16667 3.76667 CCCHOI 1.99117 1.89450 1.88217 SOLA 2.50000 2.58333 2.55000 KLCHOI 2.21650 1.96450 2.00217 SE(N= 60) 0.192584 0.263336E-01 0.134054 0.205634E-01 5%LSD 172DF 0.537527 0.735003E-01 0.374162 0.573951E-01 -ANALYSIS OF VARIANCE SUMMARY TABLE FILE OANH 22/11/22 9:25 :PAGE thi nghiem ba F-PROBABLIITY VALUES FOR EACH EFFECT IN THE MODEL SECTION - VARIATE GRAND MEAN STANDARD DEVIATION C OF V |CT (N= SD/MEAN | |NL | 180) | | NO BASED ON BASED ON OBS TOTAL SS RESID SS 1.5383 0.22912 1.0481 0.20377 1.4918 0.20398 1.0384 0.15928 % | | | | 0.0351 0.0000 0.0751 0.0000 0.0493 0.0069 0.9064 0.0000 | | CHOI CCCHOI SOLA KLCHOI 180 180 180 180 3.8111 1.9226 2.5444 2.0611 2.1 3.6 1.8 1.7 The effect of IBA (3-Indolebutyric acid) in combination with BA on shoot proliferation and growth of Schismatoglottis wallichii BALANCED ANOVA FOR VARIATE CHOI FILE TN2 22/11/22 9:45 :PAGE tn anh huong IBA VARIATE V003 CHOI LN ER SOURCE OF VARIATION DF SUMS OF MEAN SQUARES SQUARES F RATIO PROB LN 48 =========================================================================== == CT 34.4666 8.61666 5.72 0.000 NL 23.5600 11.7800 7.82 0.001 * RESIDUAL 143 215.473 1.50681 -* TOTAL (CORRECTED) 149 273.500 1.83557 -BALANCED ANOVA FOR VARIATE CCCHOI FILE TN2 22/11/22 9:45 :PAGE tn anh huong IBA VARIATE V004 CCCHOI LN ER SOURCE OF VARIATION DF SUMS OF MEAN SQUARES SQUARES F RATIO PROB LN =========================================================================== == CT 16.0648 4.01620 21.90 0.000 NL 4.08765 2.04382 11.14 0.000 * RESIDUAL 143 26.2268 183404 -* TOTAL (CORRECTED) 149 46.3793 311270 -BALANCED ANOVA FOR VARIATE SO LA FILE TN2 22/11/22 9:45 :PAGE tn anh huong IBA VARIATE V005 SO LA LA LN ER SOURCE OF VARIATION DF SUMS OF MEAN SQUARES SQUARES F RATIO PROB LN =========================================================================== == CT 101.427 25.3567 12.44 0.000 NL 61.2400 30.6200 15.03 0.000 * RESIDUAL 143 291.393 2.03772 -* TOTAL (CORRECTED) 149 454.060 3.04738 49 -TABLE OF MEANS FOR FACTORIAL EFFECTS FILE TN2 22/11/22 9:45 :PAGE tn anh huong IBA MEANS FOR EFFECT CT -CT NOS 30 30 30 30 30 CHOI 3.93333 4.56667 3.83333 3.23333 3.53333 CCCHOI 2.31900 3.24200 3.16367 2.16367 3.45400 SO LA 3.56667 5.90000 5.40000 5.10000 4.33333 SE(N= 30) 0.224114 0.781887E-01 0.260622 5%LSD 143DF 0.626431 0.218549 0.728478 -MEANS FOR EFFECT NL -NL NOS 50 50 50 CHOI 3.26000 2.40000 2.44000 CCCHOI 3.11020 2.70680 2.88440 SO LA 5.76000 4.34000 4.48000 SE(N= 50) 0.173598 0.605647E-01 0.201877 5%LSD 143DF 0.485231 0.169288 0.564277 -ANALYSIS OF VARIANCE SUMMARY TABLE FILE TN2 22/11/22 9:45 :PAGE tn anh huong IBA F-PROBABLIITY VALUES FOR EACH EFFECT IN THE MODEL SECTION - VARIATE GRAND MEAN STANDARD DEVIATION C OF V |CT (N= SD/MEAN | |NL | 150) | | NO BASED ON BASED ON OBS TOTAL SS RESID SS 1.3548 0.55792 1.7457 1.2275 0.42826 1.4275 % | | | | | | CHOI CCCHOI SO LA 150 150 150 2.7000 2.9005 4.8600 2.5 0.0003 1.8 0.0000 2.4 0.0000 0.0007 0.0001 0.0000 50 The effect of α-NAA (α-Naphthaleneacetic acid) in combination with BA on shoot proliferation and growth of Schismatoglottis wallichii BALANCED ANOVA FOR VARIATE CHOI FILE TN3 22/11/22 9:46 :PAGE tn anh huong NAA choi VARIATE V003 CHOI LN ER SOURCE OF VARIATION DF SUMS OF MEAN SQUARES SQUARES F RATIO PROB LN =========================================================================== == CT 42.2267 10.5567 8.52 0.000 NL 373334 186667 0.15 0.861 * RESIDUAL 143 177.193 1.23911 -* TOTAL (CORRECTED) 149 219.793 1.47512 -BALANCED ANOVA FOR VARIATE CCHOI FILE TN3 22/11/22 9:46 :PAGE tn anh huong NAA choi VARIATE V004 CCHOI LN ER SOURCE OF VARIATION DF SUMS OF MEAN SQUARES SQUARES F RATIO PROB LN =========================================================================== == CT 2.60500 651249 6.01 0.000 NL 3.93905 1.96953 18.16 0.000 * RESIDUAL 143 15.5061 108434 -* TOTAL (CORRECTED) 149 22.0501 147987 -BALANCED ANOVA FOR VARIATE SOLA FILE TN3 22/11/22 9:46 :PAGE tn anh huong NAA choi VARIATE V005 SOLA 51 LN ER SOURCE OF VARIATION DF SUMS OF MEAN SQUARES SQUARES F RATIO PROB LN =========================================================================== == CT 7.24000 1.81000 2.19 0.072 NL 101.080 50.5400 61.13 0.000 * RESIDUAL 143 118.220 826713 -* TOTAL (CORRECTED) 149 226.540 1.52040 -TABLE OF MEANS FOR FACTORIAL EFFECTS FILE TN3 22/11/22 9:46 :PAGE tn anh huong NAA choi MEANS FOR EFFECT CT -CT NOS 30 30 30 30 30 CHOI 3.60000 3.96667 3.43333 2.93333 3.16667 CCHOI 2.24700 3.12367 2.88767 2.72667 2.62500 SOLA 3.46667 3.83333 3.00000 2.76667 2.83333 SE(N= 30) 0.206134 0.590012E-01 0.211139 5%LSD 143DF 0.576174 0.164917 0.590165 -MEANS FOR EFFECT NL -NL NOS 50 50 50 CHOI 2.34000 2.38000 2.46000 CCHOI 3.07320 2.75320 2.70980 SOLA 3.74000 2.04000 1.96000 SE(N= 50) 0.157424 0.465691E-01 0.128586 5%LSD 143DF 0.440023 0.130168 0.359416 52 ANALYSIS OF VARIANCE SUMMARY TABLE FILE TN3 22/11/22 9:46 :PAGE tn anh huong NAA choi F-PROBABLIITY VALUES FOR EACH EFFECT IN THE MODEL SECTION - VARIATE GRAND MEAN STANDARD DEVIATION C OF V |CT (N= SD/MEAN | |NL | 150) | | NO BASED ON BASED ON OBS TOTAL SS RESID SS 1.2145 0.38469 1.2330 1.1132 0.32929 0.90924 % | | | | | | CHOI CCHOI SOLA 150 150 150 2.3933 2.8454 2.5800 4.5 0.0000 1.6 0.0002 3.2 0.0722 0.8610 0.0000 0.0000 The effect of different culture systems on the multiplication stage of Schismatoglottis wallichii BALANCED ANOVA FOR VARIATE CHOI FILE TN4 23/11/22 20:16 :PAGE tn4 VARIATE V003 CHOI LN ER SOURCE OF VARIATION DF SUMS OF MEAN SQUARES SQUARES F RATIO PROB LN =========================================================================== == CT 55.6334 18.5445 11.76 0.000 NL 22.5167 11.2583 7.14 0.001 * RESIDUAL 114 179.817 1.57734 -* TOTAL (CORRECTED) 119 257.967 2.16779 -BALANCED ANOVA FOR VARIATE CCCHOI FILE TN4 23/11/22 20:16 :PAGE tn4 VARIATE V004 CCCHOI LN ER SOURCE OF VARIATION DF SUMS OF MEAN SQUARES SQUARES F RATIO PROB LN =========================================================================== == 53 CT 10.2920 3.43066 6.16 0.001 NL 2.67367 1.33684 2.40 0.093 3 * RESIDUAL 114 63.4465 556549 -* TOTAL (CORRECTED) 119 76.4122 642119 -BALANCED ANOVA FOR VARIATE KL CHOI FILE TN4 23/11/22 20:16 :PAGE tn4 VARIATE V005 KL CHOI CHOI LN ER SOURCE OF VARIATION DF SUMS OF MEAN SQUARES SQUARES F RATIO PROB LN =========================================================================== == CT 68.7564 22.9188 12.21 0.000 NL 1.66880 834401 0.44 0.648 * RESIDUAL 114 213.999 1.87719 -* TOTAL (CORRECTED) 119 284.424 2.39012 -TABLE OF MEANS FOR FACTORIAL EFFECTS FILE TN4 23/11/22 20:16 :PAGE tn4 MEANS FOR EFFECT CT -CT NOS 30 30 30 30 CHOI 5.60000 5.46667 4.10000 6.16667 CCCHOI 3.90567 3.87433 3.18667 4.18800 KL CHOI 9.66267 8.71067 9.11967 10.7153 SE(N= 30) 0.229299 0.136204 0.250146 5%LSD 114DF 0.642322 0.381542 0.700719 -MEANS FOR EFFECT NL -NL NOS CHOI CCCHOI KL CHOI 54 40 40 40 4.57500 5.42500 5.55000 3.81300 4.17075 4.05725 9.28600 9.54825 9.52200 SE(N= 40) 0.198579 0.117956 0.216633 5%LSD 114DF 0.556267 0.330425 0.606841 -ANALYSIS OF VARIANCE SUMMARY TABLE FILE TN4 23/11/22 20:16 :PAGE tn4 F-PROBABLIITY VALUES FOR EACH EFFECT IN THE MODEL SECTION - VARIATE GRAND MEAN STANDARD DEVIATION C OF V |CT (N= SD/MEAN | |NL | 120) | | NO BASED ON BASED ON OBS TOTAL SS RESID SS 1.4723 0.80132 1.5460 1.2559 0.74602 1.3701 % | | | | | | CHOI CCCHOI KL CHOI 120 120 120 5.1833 4.0137 9.4521 2.2 1.6 1.5 0.0000 0.0007 0.0000 0.0013 0.0930 0.6479 The effect of IBA (3-Indolebutyric acid) on the rooting stage of Schismatoglottis wallichii BALANCED ANOVA FOR VARIATE CD RE FILE TN5 22/11/22 9:50 :PAGE tn5 VARIATE V003 CD RE LN ER SOURCE OF VARIATION DF SUMS OF MEAN SQUARES SQUARES F RATIO PROB LN =========================================================================== == CT 10.0611 2.01222 5.73 0.000 NL 484547 242274 0.69 0.508 * RESIDUAL 172 60.4000 351163 -* TOTAL (CORRECTED) 179 70.9457 396344 -BALANCED ANOVA FOR VARIATE SOLA FILE TN5 22/11/22 9:50 :PAGE tn5 55 VARIATE V004 SOLA LN ER SOURCE OF VARIATION DF SUMS OF MEAN SQUARES SQUARES F RATIO PROB LN =========================================================================== == CT 53.3778 10.6756 7.04 0.000 NL 51.4112 25.7056 16.95 0.000 * RESIDUAL 172 260.855 1.51660 -* TOTAL (CORRECTED) 179 365.644 2.04271 -BALANCED ANOVA FOR VARIATE KL CAY FILE TN5 22/11/22 9:50 :PAGE tn5 VARIATE V005 KL CAY LN ER SOURCE OF VARIATION DF SUMS OF MEAN SQUARES SQUARES F RATIO PROB LN =========================================================================== == CT 119.790 23.9580 13.25 0.000 NL 6.23523 3.11761 1.72 0.179 * RESIDUAL 172 311.010 1.80820 -* TOTAL (CORRECTED) 179 437.035 2.44154 -TABLE OF MEANS FOR FACTORIAL EFFECTS FILE TN5 22/11/22 9:50 :PAGE tn5 MEANS FOR EFFECT CT -CT NOS 30 30 30 30 30 30 CD RE 2.08300 2.51533 3.88833 3.48867 3.50067 3.38333 SOLA 6.10000 5.73333 7.23333 6.10000 5.60000 6.26667 KL CAY 8.23667 9.07667 10.8440 10.1213 9.69733 9.40000 56 SE(N= 30) 0.108192 0.224841 0.245506 5%LSD 172DF 0.301976 0.927559 0.685239 -MEANS FOR EFFECT NL -NL NOS 60 60 60 CD RE 3.41683 3.54333 3.46950 SOLA 4.60000 3.45000 3.48333 KL CAY 9.30267 9.72817 9.65717 SE(N= 60) 0.765031E-01 0.158986 0.173599 5%LSD 172DF 0.213530 0.443751 0.484537 -ANALYSIS OF VARIANCE SUMMARY TABLE FILE TN5 22/11/22 9:50 :PAGE tn5 F-PROBABLIITY VALUES FOR EACH EFFECT IN THE MODEL SECTION - VARIATE GRAND MEAN STANDARD DEVIATION C OF V |CT |NL | (N= 180) SD/MEAN | | | NO BASED ON BASED ON OBS TOTAL SS RESID SS 0.62956 1.4292 1.5625 0.59259 1.2315 1.3447 % | | | | | | CD RE SOLA KL CAY 180 180 180 3.4766 3.8444 9.5627 2.6 3.2 1.1 0.0001 0.0000 0.0000 0.5076 0.0000 0.1792 The effect of α-NAA (α-Naphthaleneacetic acid) on the rooting stage of Schismatoglottis wallichii - :PAGE tn6 VARIATE V003 CD RE LN ER SOURCE OF VARIATION DF SUMS OF MEAN SQUARES SQUARES F RATIO PROB LN =========================================================================== == CT 9.45634 1.89127 12.63 0.000 NL 1.24502 622509 4.16 0.017 * RESIDUAL 172 25.7661 149803 57 -* TOTAL (CORRECTED) 179 36.4675 203729 -BALANCED ANOVA FOR VARIATE SOLA FILE TN6 22/11/22 9:51 :PAGE tn6 VARIATE V004 SOLA LN ER SOURCE OF VARIATION DF SUMS OF MEAN SQUARES SQUARES F RATIO PROB LN =========================================================================== == CT 24.5833 4.91667 3.25 0.008 NL 62.6333 31.3167 20.67 0.000 * RESIDUAL 172 260.533 1.51473 -* TOTAL (CORRECTED) 179 347.750 1.94274 -BALANCED ANOVA FOR VARIATE KL CAY FILE TN6 22/11/22 9:51 :PAGE tn6 VARIATE V005 KL CAY LN ER SOURCE OF VARIATION DF SUMS OF MEAN SQUARES SQUARES F RATIO PROB LN =========================================================================== == CT 139.751 27.9501 15.80 0.000 NL 5.36834 2.68417 1.52 0.221 * RESIDUAL 172 304.328 1.76935 -* TOTAL (CORRECTED) 179 449.447 2.51088 -TABLE OF MEANS FOR FACTORIAL EFFECTS FILE TN6 22/11/22 9:51 :PAGE tn6 MEANS FOR EFFECT CT 58 -CT NOS 30 30 30 30 30 30 CD RE 2.61300 2.77500 3.20700 2.24633 1.79567 2.32633 SOLA 6.43333 7.53333 8.53333 8.06667 7.30000 7.33333 KL CAY 7.96000 9.06033 10.4230 9.41200 9.89700 9.37400 SE(N= 30) 0.706642E-01 0.224702 0.242855 5%LSD 172DF 0.197233 1.067171 0.677838 -MEANS FOR EFFECT NL -NL NOS 60 60 60 CD RE 3.06133 2.89067 2.87967 SOLA 4.58333 3.36667 3.30000 KL CAY 9.26017 9.62817 9.62483 SE(N= 60) 0.499672E-01 0.158888 0.171724 5%LSD 172DF 0.139465 0.443477 0.479304 -ANALYSIS OF VARIANCE SUMMARY TABLE FILE TN6 22/11/22 9:51 :PAGE tn6 F-PROBABLIITY VALUES FOR EACH EFFECT IN THE MODEL SECTION - VARIATE GRAND MEAN STANDARD DEVIATION C OF V |CT (N= SD/MEAN | |NL | 180) | | NO BASED ON BASED ON OBS TOTAL SS RESID SS % | | | | | | CD RE 180 SOLA 180 KL CAY 0.2206 2.9439 0.45136 0.38704 5.1 0.0000 0.0171 3.7500 1.3938 1.2307 3.8 0.0081 0.0000 180 9.5044 1.5846 1.3302 4.3 0.0000 The effect of different culture systems on the rooting stage of Schismatoglottis wallichii BALANCED ANOVA FOR VARIATE SOLA FILE TN7 23/11/22 20:19 :PAGE tn7 VARIATE V003 SOLA 59 LN ER SOURCE OF VARIATION DF SUMS OF MEAN SQUARES SQUARES F RATIO PROB LN =========================================================================== == CT 6.66667 2.22222 2.25 0.085 NL 70.6166 35.3083 35.75 0.000 * RESIDUAL 114 112.583 987573 -* TOTAL (CORRECTED) 119 189.867 1.59552 -BALANCED ANOVA FOR VARIATE CC RE FILE TN7 23/11/22 20:19 :PAGE tn7 VARIATE V004 CC RE LN ER SOURCE OF VARIATION DF SUMS OF MEAN SQUARES SQUARES F RATIO PROB LN =========================================================================== == CT 2.19261 730870 7.63 0.000 NL 478165E-02 239082E-02 0.02 0.976 * RESIDUAL 114 10.9178 957701E-01 -* TOTAL (CORRECTED) 119 13.1152 110212 -BALANCED ANOVA FOR VARIATE KL CAY FILE TN7 23/11/22 20:19 :PAGE tn7 VARIATE V005 KL CAY LN ER SOURCE OF VARIATION DF SUMS OF MEAN SQUARES SQUARES F RATIO PROB LN =========================================================================== == CT 88.4138 29.4713 8.86 0.000 NL 55.7907 27.8954 8.39 0.000 60 * RESIDUAL 114 379.117 3.32559 -* TOTAL (CORRECTED) 119 523.322 4.39766 -TABLE OF MEANS FOR FACTORIAL EFFECTS FILE TN7 23/11/22 20:19 :PAGE tn7 MEANS FOR EFFECT CT -CT NOS 30 30 30 30 SOLA 4.06667 3.93333 3.46667 5.80000 CC RE 2.91567 2.72233 1.65000 2.89033 KL CAY 10.4627 9.26300 9.57067 11.4667 SE(N= 30) 0.181436 0.565008E-01 0.332946 5%LSD 114DF 0.508247 0.158272 0.932663 -MEANS FOR EFFECT NL -NL NOS 40 40 40 SOLA 4.95000 3.27500 3.37500 CC RE 2.68975 2.69050 2.70350 KL CAY 11.1550 9.71575 9.70150 SE(N= 40) 0.157128 0.489311E-01 0.288340 5%LSD 114DF 0.440155 0.137068 0.807710 -ANALYSIS OF VARIANCE SUMMARY TABLE FILE TN7 23/11/22 20:19 :PAGE tn7 F-PROBABLIITY VALUES FOR EACH EFFECT IN THE MODEL SECTION - VARIATE GRAND MEAN STANDARD DEVIATION C OF V |CT (N= SD/MEAN | |NL | 120) | | NO BASED ON BASED ON OBS TOTAL SS RESID SS 1.2631 0.33198 2.0971 0.99377 0.30947 1.8236 % | | | | | | SOLA CC RE KL CAY 120 120 120 3.8667 2.6946 10.191 2.7 1.5 1.9 0.0849 0.0001 0.0000 0.0000 0.9758 0.0005 61