TO ESTABLISH PROTOCOLS FOR ANDROGENESIS, MICROPROPAGATION & SOMATIC EMBRYOGENESIS IN SELECTED PLANTS

Submitted to: Department of Biotechnology GGDSD College Affliated to Panjab University CHANDIGARH

Submitted by: Swati Sharma B.Sc Biotechnology(Hons.)- G.G.D.S.D college CHANDIGARH

Certificate 1

This is to certify that the project entitled TO ESTABLISH PROTOCOLS FOR ANDROGENESIS, MICROPROPAGATION & SOMATIC EMBRYOGENESIS IN SELECTED PLANTS has been carried out by ______________ under supervision of ______________________ in the Department of Biotechnology, GGDSD College, Chandigarh.

Dr. Navneet Batra Co-ordinator Dept. of Biotechnology G.G.D.S.D College, Chandigarh

Certificate 2

This is to certify that the project entitled TO ESTABLISH PROTOCOLS FOR ANDROGENESIS, MICROPROPAGATION & SOMATIC EMBRYOGENESIS IN SELECTED PLANTS has been carried out by ______________ under supervision of ______________________ in the Department of Biotechnology, GGDSD College, Chandigarh.

Mrs Samriti Dhawan Project supervisor Lecturer Dept. of Biotechnology G.G.D.S.D College, Chandigarh

ACKNOWLEDGEMENTS

I express my heartfelt gratitude to all the people involved in this project, without whom completion of this project would not have been possible. Firstly, I submit my sincere thanks to Department of Biotechnology, New Delhi, for encouraging science students by pursue research by offering such benovalent opportunities in the form of short term Summer Training Projects. I also owe my gratitude to our College principal, Dr A.C. Vaid for initiating efforts, so that such opportunities reach the students. I thank Dr Navneet Batra, Head of Department and Mrs Samriti Dhawan my project supervisor, for their able guidance throughout the project. I am grateful to the laboratory staff for their support, co-operation and patience. At last however not the least, I thank God, my parents and college teachers with whose help I was in a position to grab this opportunity and fulfill my dream.

LIST OF ABBREVIATIONS

vs & mg l lab lb sq
0

versus and milligrams litre laboratory pounds square degree centigrade temperature -Naphthalene acetic acid 2,4 dichloro acetic acid Kinetin Benzyl amino purine hydrogen ion concentration concentration namely species high efficiency particulate air micrometer percentage per square inch

temp NAA 2,4-D Kn BAP pH conc viz sp HEPA m % psi

min hrs ml iP UV EtOH IAA IBA mg NaOH HCl gm

minute hours milli litre iso pentyl adenine ultra violet ethyl alcohol Indle-3-acetic acid Indole-3-butyric acid milli gram sodium hydroxide hydrochloric acid gram

CONTENTS

1. Introduction 2. layout of a Plant Tissue Culture Laboratory 3. Sterile techniques 4. Preparation of Stock Solutions 5. Preparation of Media 6. Culture Techniques: Anther culture (Androgenesis) Micropropagation Embryo culture Somatic Embryogenesis

INTRODUCTION
Green plants owing to there unique ability to perform photosynthesis, are the source of all the energy to all. Plants provide food, feed, fiber, fuel pharmaceuticals, timber, and a variety of other materials. With such divergent uses there is a continuous effort in improving plant varieties. Advent of Plant tissue culture practices has been a great breakthrough. Use of old world Conventional breeding methods for improvement are now of limited use as compared to new world Plant Biotechnology techniques.

CONVENTIONAL BREEDING vs PLANT BREEDING Modification of crop genotypes to make them more useful to humans constitutes plant breeding. It involves the following steps: 1. 2. 3. 4. 5. 6. Introduction creation of variations Selection Evaluation Multiplication Distribution

However there are some limitations to it. One of the chief limitations is limited genetic variability. Plant breeding methods enable the use of genes only from species that are sexually compatible which places a limit on genes accessible for plant breeding. Another technical difficulty is the production of hybrid variety of seeds. Hybrid seed production requires emasculation. Hand emasculation is tedious, and a satisfactory system of genetic emasculation is not possible in all plants. Plant biotechnology is offers useful solutions to all such problems. Plant biotechnology helps to generate plants from isolated cells, tissues and organs on one hand and genetic manipulation on the other. These can be continuously maintained under controlled nutritional and environmental conditions to enable regeneration of complete plantlets. that are finally transferred to the field. Other important areas where plant biotechnology is of utmost importance include production of synthetic seeds, secondary metabolites, rapid clonal multiplication, development of dihaploid homozygous lines etc. Plant biotechnology can be exploited for both crop improvement and crop protection.

Thus plant tissue culture techniques form an integral part of plant biotechnology and are of great interest. Keeping this in mind work was initiated in this field. The main stress was to skill ourselves with the various culture techniques. As a part of the project we established protocols for micropropagation of Chlorophytum borivilianum, somatic embryogenesis in Vigna unguiculata, and androgenesis in Citrus sp.

Lay out of a Plant Tissue Culture Laboratory

Any laboratory, in which tissue culture techniques are performed, regardless of the specific purpose, must contain a number of basic facilities. These usually include the following: A washing area A media preparation area An aseptic transfer area Maintenance area An observation/data collection area

Washing Area The washing area should contain large sinks, some lead-lined to resist acids and alkalis, draining boards, and racks, and have access to demineralized or distilled water, and double-distilled water. Space for drying ovens or racks, automated dishwashers, acid baths, pipette washers and driers, and storage cabinets should also be available in the washing area.

Media Preparation Area The media preparation area should have ample storage space for the chemicals, culture vessels and closures, and glassware required for media preparation and dispensing. Equipments like hot plates/stirrers, pH meters, balances, water bath(s), distilled and double-distilled water system, bunsen burners with a gas source, refrigerators and deep freezers, a microwave or a convection oven, and an autoclave for sterilizing media, glassware, and instruments. Transfer Area A laminar flow hood or sterile transfer room should be utilized for making transfers. Within the transfer area there should be a source of electricity, gas, compressed air, and vacuum. The most desirable arrangement is a small dust-free room with an

overhead ultraviolet light and a positive-pressure ventilation unit equipped with a highefficiency particulate air (HEPA) filter (0.3-m) of 99.97-99.99% efficiency. Another type of transfer area is a laminar flow hood. Air is forced into the unit through a dust filter then passed through a HEPA filter. The air is then either directed downward (vertical flow unit) or outward (horizontal flow unit) over the working surface. The constant flow of bacteria-free filtered air prevents non-filtered air and particulate matter from settling on the working surface. Culture maintenance area All types of tissue cultures should be incubated under conditions of well-controlled temperature, humidity, air circulation, and light quality and duration. Culture room for growth of plant tissue cultures should have a temperature between 25 and 30C, with a temperature fluctuation of less than 0.5C.The temperature should be constant throughout the entire culture room (i.e., no hot or cold spots). The culture room should have enough fluorescent lighting to reach the 10,000 lux; the lighting should be adjustable in terms of quantity and photoperiod duration. Both light and temperature should be programmable for a 24-hr period. and a humidity range of 20-98% controllable to 3 percent.

STERILE TECHNIQUES
Aseptic technique is necessary for the successful establishment and maintenance of plant cell, tissue and organ cultures. The in vitro environment in which the plant material is cultured is also ideal for the proliferation of microorganisms. In most cases the microorganisms outgrow the plant tissues, resulting in their death. Contamination can also spread from culture to culture. The purpose of aseptic technique is to minimize the growth of microorganisms in cultures. Effective sterilization involves: Sterilization of media Sterilization of explants Sterilization of working area Sterilization of instruments

Sterilizing tools, media, vessels, etc.

Moist heat sterilization Autoclaving is the method most often used for sterilizing heat-resistant items. In order to be sterilized, the item must be held at 121C, 15 psi, for at least 15 minutes. It is important that items reach this temperature before timing begins. Therefore time in the autoclave will vary, depending on volume in individual vessels and number of vessels in the autoclave. Sterilizing tools, vessels, etc. Empty vessels, beakers, graduated cylinders, etc., should be closed with a cap or aluminum foil and never autoclaved empty. Tools should also be wrapped in foil or paper or put in a covered sterilization tray. It is critical that the steam penetrate the items in order for sterilization to be successful. Sterilizing Media and Other Liquids (moist heat & flame sterilization) Culture media, distilled water, and other heat stable mixtures can be autoclaved in glass containers that are sealed with cotton plugs, aluminum foil, or plastic closures.

However, solutions that contain heat-labile components must be filter-sterilized. For small volumes of liquids (100 ml or less), the time required for autoclaving is 15-20 min, but for larger quantities (2-4 liter), 30-40 min is required to complete the cycle. The pressure should not exceed 20 psi, as higher pressures may lead to the decomposition of carbohydrates and other components of a medium. Too high temperatures or too long cycles can also result in changes in properties of the medium. Organic compounds such as some growth regulators, amino acids, and vitamins may be degraded during autoclaving. These compounds require filter sterilization through a 0.22 m membrane. Filters used may be pre-sterilized or larger ones can be set over a sterile flask and a vacuum is applied to pull the compound dissolved in liquid through the membrane and into the sterile flask (filter assembly). Smaller membranes fit on the end of a sterile syringe and liquid is pushed through by depressing the top of the syringe. The size of the filter selected depends on the volume of the solution to be sterilized and the components of the solution. Nutrient media that contain thermo labile components are typically prepared in several steps. A solution of the heat-stable components is sterilized in the usual way by autoclaving and then cooled to 35-50 C under sterile conditions. Solutions of the thermo labile components are filter-sterilized. The sterilized solutions are then combined under aseptic conditions to give the complete medium. In spite of possible degradation, however, some compounds that are thought to be heat labile are generally autoclaved if results are found to be reliable and reproducible. These compounds include 2,4-D, NAA, kinetin, pyridoxine, 2-ip, thiamine, etc. are usually autoclaved. UV Radiation Use of germicidal lamps to sterilize items in the transfer hood when no one is working there is a routine practice. UV lamps should not be used when people are present because the light is damaging to eyes and skin. Plants left under UV lamps will also show mutations.

Autoclave

Filter assembly

Laminar air flow

Glass bead sterilizer

Additional Guidelines for Working in the Transfer Hood The hood should remain on continuously. If for some reason it has been turned off, turn it on and let it run for at least 15 minutes before using. Make sure that everything needed for the work is in the hood and all unnecessary things are removed Check the bottom of the hood to make sure there is no paper or other debris blocking air intake. Spray or wipe the inside of the transfer hood (bottom and sides) with 70% EtOH. Spray everything placed in the sterile area with 70% ethanol. Remove watches, etc., roll up long sleeves, and wash hands thoroughly with soap and water before working. Wipe hands and lower arms with 70% EtOH Make sure that materials in use are to the side of your work area, so that airflow from the hood is not blocked. Dont touch any surface that is supposed to remain sterile with your hands. Use forceps, etc. Instruments (scalpels, forceps) can be sterilized by flaming - dipping them in 95% EtOH and then immediately placing them in the flame of an alcohol lamp or gas burner.

Sterilize your instruments often, especially in between individual petri plates, flasks, etc. The tools should be placed on a holder in the hood to cool or should be cooled by dipping in sterile water or medium before handling plant tissues. Wipe up any spills quickly; use 70% EtOH for cleaning. Clean hood surface periodically while working. Sterilize culture tubes with lids or caps on. When you open a sterile tube, touch only the outside of the cap, and do not set the cap on any laboratory surface. Instead, hold the cap with one or two fingers while you complete the operation, and then replace it on the tube. After you remove the cap from the test tube, pass the mouth of the tube through a flame. Put only sterile objects into the tube. Complete the operation as quickly as you reasonably can, and then flame the mouth of the tube again. Replace the lid. Remove items from the hood as soon as they are no longer needed. All cultures must be sealed before leaving the hood. When finished in the hood, clean up after yourself. Remove all unnecessary materials and wipe the hood down with 70% EtOH. Be sure when you are finished that you turn off the gas to the burner!

Surface-sterilizing Plant Material

Preparation and use of Stock Plants (Cultures) Use of stock plants to obtain starting material may lessen the amount of contamination that is present in field obtained explants. Plants grown in the field are typically more dirty than those grown in a greenhouse or growth chamber, particularly in humid areas. Treatment of stock plants with fungicides and/or bactericides is sometimes helpful initially but may be inhibitory at a later stage. To avoid this, the best solution is to use pre-conditioned plants as source of explants. Seeds may be sterilized and germinated in vitro to provide clean material. In case of field grown material, explants from which starting material will be cut can be washed in running water for 1 to 2 hours, followed by sterilization with a suitable sterilant and a mild detergent added to it. This is followed by thorough washing with sterile distilled water. Ethanol (or Isopropyl Alcohol) Ethanol is a powerful sterilizing agent but also extremely phytotoxic. Therefore, plant material is typically exposed to it for only seconds or minutes. The more tender the tissue, the more it will be damaged by alcohol. Tissues such as dormant buds, seeds, or unopened flower buds can be treated for longer periods of time since the tissue that will be explanted or that will develop is actually within the structure that is being surface-

sterilized. Generally, 70% ethanol is used prior to treatment with other compounds. Sodium Hypochlorite Sodium hypochlorite is the most frequent choice for surface sterilization. It is readily available and can be diluted to proper concentrations. Commercially available bleach is 5.25% sodium hypochlorite. It is usually diluted to 10% - 20% of the original concentration, resulting in a final concentration of 0.5 - 1.0% sodium hypochlorite. Plant material is usually immersed in this solution for 10 - 20 minutes. A balance between concentration and time must be determined empirically for each type of explant, because of phytotoxicity. Calcium Hypochlorite Calcium hypochlorite is obtained as a powder and must be dissolved in water. The concentration that is generally used is 3.25 %. The solution must be filtered prior to use since not all the compound goes into solution. Calcium hypochlorite may be less injurious to plant tissues than sodium hypochlorite.