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Plant tissue culture lab,sedf Document Transcript

  • 1. BASIC INFORMATION ABOUT PLANT TISSUE CULTURE AND ESTABLISHMENT OF PLANT TISSUE CULTURE LAB: REPORT PREPARED BY: IQRA JUNEJO SUPERVISED BY: MEHBOOB UL HAQ , CEO, SEDF PREPARED FOR: SINDH ENTERPRISE DEVELOPMENT FUND 1|Page
  • 2. ACKNOWLEDGEMENT: I am thankful to almighty ALLAH who made me capable enough to get opportunity to work as internee in Sindh Enterprise Development Fund. I would like to thank to MR MEHBOOB UL HAQ, CEO, Sindh Enterprise Development Fund who provided me opportunity to work as summer internee under his super vision and to make report on such an important topic of PLANT TISSUE CULTURE & PLANT TISSUE CULTURE LABORATORY. He cooperated and appreciated me in every step which gave me potential through which i became able to make this report. I would like to thanks MR NIAZ MUHAMMAD NIZAMANI, PLANT TISSUE CULTURE LAB OWNER,TANDO ALLAH YAR, for his cooperation. I would like to thanks PROF DR SAIF ULLAH, INCHARE BIOTECHNOLOGY WING,HEJ RESEARCH INSTITUE,UNIVERSITY OF KARACH for his cooperation. I have tried my level best to keep this report simple yet technically correct .I hope I succeed in my attempt. IQRA JUNEJO MANAGEMENT TRAINEE SEDF 2|Page
  • 3. TABLE OF CONTENT S no CONTENT PAGE # ACKNOWLEDGMENT 2 2 PREFACE 4 3 HISTORY OF PLANT TISSUE CULTURE 5 4 PLANT TISSUE CULTURE 5 5 PROCEDURE OF PLANT TISSUE CULTURE 6 6 PLANT PROPAGATION METHODS 6 7 THE TISSUE CULTURE PROCESS 7 8 CONTAMINANT FREE ENIVRONMENT 7 9 TISSUE CULTURE MEDIA 7 10 PROPAGATION RATE 7 11 ADVANTAGES&DISADVANTAGES 8-9 12 COMMONLY USED TERMS IN TISSUE CULTURE 9-12 13 REQUIRED FACILITIES FOR PLANT TISSUE CULTURE LAB 13 14 WASHING ROOM 13 15 MEDIA PREPARATION ROOM 14 16 GLASS WARES AND CULTURE ROOM 14 17 PRECUATIONS OF PROCESS 15 18 QUESTIONNAIR 16-18 19 QUESTIONNAIR AND MEETING REPORT 19 20 MEETING REPORT 20-22 1 3|Page
  • 4. PREFACE This report is about plant tissue culture and plant tissue culture laboratory. This report provides all the useful and basic information about plant tissue procedure and establishment of plant tissue culture laboratory. 4|Page
  • 5. HISTORY OF PLANT TISSUE CULTURE Plants have a remarkable regenerative power, as evidenced by the relative ease with which they can be rooted, transplanted and grafted. Early attempts at growing plants in vitro from isolated parts were unsuccessful, because knowledge of plant nutrition and physiology was inadequate. With the discovery of essential plant hormones, some progress was made starting in the 1920s and 30s. A major advancement was made by Philip R. White in 1939 with his report of continuous culture of carrot and tobacco done completely in vitro. Further progress was made by Folke Skoog, who discovered new and important properties of the hormone auxin. Skoog, along with Toshio Murashige, went on to develop the still widely used standard plant nutrient solution--Murashige-Skoog (MS) medium. The work begun by Kenneth Vivian Thimann in the late 1950s, which demonstrated that kinetin broke the dormancy of lateral buds, allowing them to develop as if they were at the tips of plants, paved the way for rapid advancements. From then on, new and important results were announced almost every year. Today almost any plant can be grown under laboratory control from a wide range of starting tissues. PLANT TISSUE CULTURE Plant tissue culture is a method or techniques to isolate parts of plants (protoplasm,cells,tissues and organs)and grow them on artificial media in aseptic conditions in a controlled space so that parts of these plants can grow and develop into complete plants. 5|Page
  • 6. PROCEDURE OF PLANT TISSUE CULTURE Plant tissue culture processes were initially developed and practiced in university and government-based laboratories. In recent years, however, the process has moved beyond these research facilities to widespread use among commercial enterprises as a cost-effective tool for plant propagation, new variety introductions and research. Plant tissue culture has revolutionized the flower and nursery markets by making valuable new hybrid clones available in commercial quantities comparatively soon after their first discovery. It is possible to start with a single plantlet and, in the space of 10-12 months, create in excess of 250,000 identical copies or clones. Plant tissue culture processes: Plant propagation methods The tissue culture process Contaminant-free environment Tissue culture media Propagation rate PLANT PROPAGATION METHODS Plants produced by vegetative propagation including top cutting, root divisions, pseudobulbs, offshoots and plantlets, are genetically identical to the mother plant and thus members of a single clone. Plant tissue culture is a laboratory based extension of these plant propagation techniques. Through tissue culture, very large numbers of identical plantlets can be derived from one mother plantlet. This technology and the resulting plantlets now form the basis of many plant nursery and flower trade industries. 6|Page
  • 7. THE TISSUE CULTURE PROCESS The mother plant selected should be healthy and free from all micro-organisms. A piece of live tissue is removed from a part of the plant and placed into culturing flasks containing appropriate nutrient media under aseptic conditions. In successful culture, these cells will divide, multiply and differentiate into thousands of plantlets having the same characteristics as the parent plants. In tissue culture, the presence of bacteria and fungi will prevent the growth of the plant tissue. It is important that the plant material used is thoroughly sterilized and the procedure is carried out in an aseptic environment .CONTAMINANT-FREE ENVIRONMENT Laboratory techniques and specialized equipment such as a laminar flow cabinet combine to present an area for the manipulation of sterile plant tissues. A working environment that has virtually all of the bacteria and fungal spores removed is a requirement for successful plant tissue culture. A sterile environment is prepared, now we prepare sterile instruments to remove an apical shoot from a plant . TISSUE CULTURE MEDIA The excised bud is transferred into a tube containing a sterile nutrient medium. The success of tissue culture depends very much on the stage of explant selected, the sterilization period and the type of culture media used; different types of plants require different sets of culture media. The rich tissue culture media provides a good food source for bacteria and fungi, therefore precautions against microbial contamination must be taken in all in vitro procedures. PROPAGATION RATE Once a plant has been successfully entered to in vitro culture and is growing, a multiplication program may be carried out. Careful manipulation of the culture media and the plant tissues will yield a 4-10 fold increase in plant numbers every 7|Page
  • 8. 18-40 days. When sufficient plant numbers have been developed, a process to move the plantlets from the confines of the culture vessel to a greenhouse is undertaken. The following table compares a conventional propagation scheme on the left with a plant tissue culture production system on the right. Note the number of days and the number of resulting plants: Cutting Propagation Tissue Culture Propagation Root 50 cuttings Day 1 One plant in tissue culture media Roots first appear Day 40 5 plantlets cut & transplanted in media Good rooting observed Day 80 25 plantlets Cold Storage Day 120 125 plantlets Cold Storage Day 160 625 plantlets Cold Storage Day 200 3,125 plantlets Cold Storage Day 240 15,625 plantlets Transplant 50 to Greenhouse Day 280 Transplant 15,625 plants to Greenhouse Transplant 50 to Field Day 320 Transplant 15,625 plants to Field ADVANTAGES: 1. To produce many copies of the same plants then which may be used to produce plants with better flowers, odors, fruits or any other properties of the plants that are beneficial to the human beings. 8|Page
  • 9. 2. To produce plants anytime we want although the climates are not appropriate to produce a plant. Moreover, if seed is not available, it is possible to produce a plant with this method. 3. If there is plant with partially infected tissue, it is possible to produce a new plant without infection. 4. Very helpful in the genetically modified organism studies. 5. Very useful solution for the prevention of starvation in third world countries since the process is highly efficient, by using only one plant, it is possible to produce more than one thousand of the same plant with higher productive if its genome changed. 6. The equipments are cheaper when compared to the animal cell culture. DISADVANTAGES: 1. If large scale production is being thinking, the costs of the equipments are very expensive. 2. The procedure is very variable and it depends on the type of the species so sometimes it needs trial-and-error type of experiments if there is not any review about that species. 3. The procedure needs special attention and diligently done observation. 4. There may be error in the identity of the organisms after culture. 5. Infection may continue through generations easily if possible precautions are not taken. 6. Decrease genetic variability. COMMONLY USED TERMS IN TISSUE CULTURE Adventitious: Development of organs such as buds, leaves, roots, shoots and somatic embryos from shoot and root tissues and callus. Agar: Natural gelling agent made from algae 9|Page
  • 10. Aseptic technique: Procedures used to prevent the introduction of microorganisms such as fungi, bacteria, virus and phytoplasma into cell, tissue and organ cultures, and cross contamination of cultures. Autoclave: A machine capable of sterilizing by steam under pressure Axenic culture: A culture without foreign or undesired life forms but may include the deliberate co-culture with different types of cells, tissues or organisms. Callus: An unorganized mass of differentiated plant cells. Cell culture: Culture of cells or their maintenance in vitro including the culture of single cells. Chemically defined medium A nutritive solution or substrate for culturing cells in which each component is specified. Clonal propagation: Asexual multiplication of plants from a single individual or explant. Clones: A group of plants propagated from vegetative parts, which have been derived by repeated propagation from a single individual. Clones are considered to be genetically uniform. Contamination: Infected by unwanted microorganisms in controlled environment Cryopreservation: Ultra-low temperature storage of cells, tissues, embryos and seeds. Culture: A plant growing in vitro in a sterile environment Differentiated: Cultured cells that maintain all or much of the specialized structure and function typical of the cell type in vivo. Embryo culture: In vitro culture of isolated mature or immature embryos. Explant: An excised piece or part of a plant used to initiate a tissue culture. 10 | P a g e
  • 11. Ex vitro: Organisms removed from tissue culture and transplanted; generally plants to soil or potting mixture. Hormone: Generally naturally occurring chemicals that strongly affect plant growth In Vitro: To be grown in glass In Vivo: To be grown naturally Laminar Flow Hood: An enclosed work area where the air is cleaned using HEPA filters Medium: A solid or liquid nutritive solution used for culturing cells Meristem: A group of undifferentiated cells situated at the tips of shoots, buds and roots, which divide actively and give rise to tissue and organs. Micropropagation: Multiplication of plants from vegetative parts by using tissue culture nutrient medium. Propagule: A portion of an organism (shoot, leaf, callus, etc.) used for propagation. Somatic embryos: Non-zygotic bipolar embryo-like structures obtained from somatic cells. Subculture: The aseptic division and transfer of a culture or portion of that culture to a fresh synthetic media. Tissue culture: In vitro culture of cells, tissues, organs and plants under aseptic conditions on synthetic media. Totipotency: Capacity of plant cells to regenerate whole plants when cultured on appropriate media. Transgenic: Plants that have a piece of foreign DNA. 11 | P a g e
  • 12. PLANT TISSUE CULTURE LABORATORY 12 | P a g e
  • 13. REQUIRED FACILITIES FOR PLANT TISSUE CULTURE LAB: In each laboratory where plant tissue culture techniques are used must have a number of facilities that include among others. Outdoor media preparation, sterilization and storage Washing room Media preparation room Water(distilled water) Transfer space (laminar air flow) Culture room Glass wares Microscope Shaker Autoclave chamber Tools dissection (spatula, scalpels,(tweezers)forceps, scissors) Shade or green houses Computer for record keeping WASHING ROOM: Washing room should have a sink. A desk made up of material resistant to acids and akalis Drying ranks and channels for deminerallisi or distilled water Space for the oven drying Equipments /washing machines and dryers Storage racks or equipment cabinets Disinfectants Oven 13 | P a g e
  • 14. MEDIA PREPARATION ROOM: In the media preparation room should be available space for storage of chemicals, glass culture and the lid Sturdy table or bench for storage of hot plate magnet stirrer Ph meter, scales and dispenser must be available Vacum devices Distilling units Bunsen burner Refrigerator Freezer for storage of stocks, solutions and chemicals ,microwave ,gas stove Glass ware and other equipments All the chemicals utilized in the manufacturing of culture media Split AC GLASS WARES: Glass ware used in lab of tissue culture generally made up of pyrex Erlenmeyer of various sizes(50,125,250,500,1000,2000 ml) Glass tubes Petri dishes Bottle of jam jam Glass cup Measuring pipettes CULTURE ROOM: Suspension cells, protoplast cells, anther cells aRe the most sensitive to environment Room temperature for the growth of culture generally range between 15c to30c 14 | P a g e
  • 15. The range of temperature greater may be required for the purpose of the experiment Temperature and light must be programmed for 24hrs. Culture room should be well ventilated with 20-98% humidity range. PRECUATIONS: Distilled water should be used. Glass wares should be washed with hot water(.>70c)+soap. Glass ware should be dried in an oven at 150c and wrapped in aluminum foil, then stored in closed cabinet. Culture room should be cleaned once a week with disinfectants and soap. Dissection equipments should be sterilized by sterilization combustion techniques, because these equipments may be responsible for blast of autoclave chamber as equipments are made of metals... Several compounds belonging to the group of proteins, vitamins, amino acids , extracts ,carbohydrates that are thermo labile, there may be decomposition when sterilized by autoclave ,so should be sterilized by filter. Millipore filter with porosity +-0.2 micron (um) is one of the filters that should be used for sterilization of materials that are thermo labile. Parts of plants should be soaked with dis infectant. Don’t wash herbaceous plant. 15 | P a g e
  • 16. QUESTIONNAIR Q1: What kind of soil is ideal for tissue culture planlets? ANS: Ideal media must have 5.75 PH. Q2: we sterilize equipments , tissue of plants is there any need to sterilize soil as well? When we plant tissue culture plantlets in the field? ANS: No. Q3: We wash ex plant with detergent before performing the practical, are plantlets produced as a result of this practical immune to disease, virus after planting in the field? ANS: NO. Q4: What kind of manure or fertilizer will be ideal for tissue culture plantlets after planting in the field? ANS: NPK. Q5: What kind of effect will be on plant tissue culture if we couldn’t sterilize soil before planting in the field? ANS: Nothing. Q6: How long I can keep my plantlets under shade in case of no buyers? ANS: One or two months 16 | P a g e
  • 17. Q7: What are major challenges associated with performance of practical? ANS: Electrical power Unskilled labor Improper weighing of plant materials Improper measuring of chemicals utilized in manufacturing of media Improper operation of Equipments Q8: What are the major challenges associated with transportation of tissue culture plantlets? ANS: High transportation cost Q9: What will be the effect of improper transportation on plant tissue plantlets? ANS: Plants may be damaged Q10: Why the price of tissue plantlets is so high as compared to ordinary plantlets? ANS: Conventional plants are free and production cost of the TC plant is high. Q11: Do tissue culture plantlets need extra attention or special management after planting in the field? ANS: Yes initial care is required that is a disadvantage of TC plant. Q12: What will be the effect of improper or poor management on growth rate of tissue culture plantlets after planting in field? ANS: Proper management shall give uniform crop and early yield. Q13: What are the sources of contamination during performance of practical? ANS: Improper handling and autoclaving, frequent visits of people. Q14: What are the chances of wilting, because of improper management under shade house? And after planting in the field? 17 | P a g e
  • 18. ANS: Over irrigation. Q15: Are there any special techniques applied to harvest fruit produced by tissue culture plantlets? ANS: No. 18 | P a g e
  • 19. QUESTIONNAIR Q1: WHAT IS PLANT TISSUE CULTURE??WHAT IS THE IMPORTANCE OF IT?? Q2: HOW MUCH AMOUNT OR FUND IS REQUIRED TO SET UP A LAB? Q3 : HOW MUCH MAN POWER IS REQUIRED TO SET UP A LAB?? Q4: WHERE IN PAKISTAN WORK ON PALNT TISSUE CULTURE IS BEING DONE ON PLANT TISSUE CULTURE?? Q5: WHAT BASIC EQUIPMENTS ARE REQUIRED FOR PLANT TISSUE CULTURE LAB? Q6: HOW MUCH TIME IT WILL APPROXIMATELY TAKE TO PROPERLY SET UP PLANT TISSUE CUTURE LAB? Q7: WHAT ARE THE IDEAL LAB CONDITIONS FOR PLANT TISSU CULTURE?? Q8: WHAT IS PROCEDURE OF PLANT TSSUE CULTURE?? Q9: HOW PLANT TISUE CULTURE IS BENEFICIAL FOR OUR BUSINESS?(SMALL AND LARGE SCALE) , WE ARE GETTING GENETICAL IDENTICAL OFF SPRINGS OR PLANTS FROM THIS TECHNIQUE Q10: CAN WE GET DISEASE RESISTANCE VARIETIES FROM PLANT TISSUE CULTURE?? Q11: WHICH VARIETIES OF BANANA ARE BEING DEVELOPED FROM PLANT TISSUE CULTURE??? IN PAKISTAN 19 | P a g e
  • 20. Q12: FROM WHERE I CAN GET PROPER TRAINING ABOUT PLANT TISSUE CULTURE IN PAKISTAN?? Q13: HARMFULL EFECTS OF PLANT TISSUE CULTURE ?IF ANY ?? Q14: BASIC REQUIREMENTS FOR INFRA STRUCTURE OF PLANT TISSUE CULTURE LAB?? Q15: WHICH PARTS OF PLANTS ARE BEING USED FOR PLANT TISSUE CULTURE?? Q16: ARE THERE ANY SPECIFIC RULES AND REGULATIONS FOR PLACE WHERE LAB HAS BEEN SET UP??OR ABOUT SIZE OF LAND, SIZE OF LABORATORY?? (NATIONALOR INTERNATIONAL) Q17: WHAT IS THE LEGAL PROCESS REQUIRED TO SET UP A PLANT TISSUE CULTURE LABORATORY? (IF ANY) MEANS NOC FROM ANY GOVERNMENT OR PRIVATE ORGANIZATIONS, AND OTHER SPECIFIC DOCUMENTS)? Q18: CAN PLANT TISSUE CULTURE CREATE PROBLEM?? IF YES THEN HOW?? 20 | P a g e
  • 21. MEETING REPORT WITH DR SAIF ULLAH KHAN MEETING DATE: Friday, June 28th MEETING TIME: 11:00 am to 12:00 am 12:45 pm to 1:00 pm ATTENDEE: IQRA JUNEJO PLACES OF MEETINGS : (1) Biotechnology wing ,Room#1 H.E.J Research Institute, University Of Karachi (2) Centralized Science Lab, University Of Karachi PURPOSE OF MEETINGS: Research in Establishment of Plant Tissue Culture Lab THINGS DISCUSSED WITH DR SAIF ULLAH KHAN: Sir dr saif is doing incredibly nice work in plant tissue culture, he appreciated me and wished me good luck for my project, Sir as you know that we had already sent the questionnaire to him, He answered all of my questions in a very polite way. Following things were discussed: Only 2-3 persons are required to run the lab , But they must be technically well informed and trained they must be capable enough to handle all the situations. Work on Plant Tissue Culture is being done in National Agriculture Research Centre (NARC) (ISL), NIAZ Tissue culture lab tando allahyar, Commercial Lab (isl), H.E.J (k.u), Botonical Garden(k.u),DHA Lab(khi), Ayub Agriculture (faisalabad). 21 | P a g e
  • 22. 2-3 years are required for technical back up from lab. Matric pass student can also work in lab, No special or specific qualification is required to run the lab. There are no specific rules and regulations are required to run the LAB, Only concerned or authorized persons should be, Allowed to enter in the lab in order to maintain the aseptic environment of lab., but lab should be near to road in order to minimize the transport expenses. Yes plant produced by plant tissue culture is of good quality but some time variations come after one year, otherwise it is genetically identical to mother plant. NO , we can’t produce disease resistance varieties, Only virus and disease free varieties are produced.. NO varieties of different plant are not developed, Only varieties are being introduced in Pakistan, People take small parts of plants from foreign countries and practice tissue culture techniques on these parts.. You OR anybody can take training from NARC (isl) and HEJ research institute, university of karachi, but HEJ is expensive then NARC. NO, There is no harmful effect of this technique only the plant produced by it expensive. NO there are no legal requirements for establisment of lab. NO, plant tissue culture products do not create any problem. No specific rules and regulations are required about size of lab,its depends how much amount you want to invest on it ,or on what scale or capacity you want to establish a lab.. HE provided me list of chemicals and equipments require to establish a lab. Which is attached with this report. Another fruitful meeting was held with MR WAJID ,who works at centralized science lab university of karachi, He assured me that he will give me necessary assistance for my this research project...he also asked me to meet with DR EHTASHAM at Botanical Garden ,university of karachi.. for further assistance as DR EHTASHAM is also working on plant tissue culture.. 22 | P a g e