1INTRODUCTION
1.1Definition:
Plant tissue culture is the technique of growing plant cells, tissues and organs in an artificial prepared nutrient medium under aseptic conditions. It aims to induce plant organs, or portions of them, to proliferate cells, each one of which may be regenerated into whole plants. These plants are replicas of the parent plant.
Gottlieb Haberlandt (28 November 1854) was the first scientist to plan for plant tissue culture and so he was popularly known as the “Father of Plant Tissue Culture.” He pointed out the possibilities of the culture of isolated tissues as Plant tissue culture. He suggested the potentialities of individual cells via tissue culture and also suggested that the reciprocal influence of tissues on one another could be determined by this method.
Plant tissue culture is widely used to produce clones of a plant by a methodknownasmicropropagation.Ithasbenefitedandadvancetheknowledgeoffundamental botany, especially in the field of agriculture, horticulture, plant breeding, forestry, somatic cell hybridization, phytopathology and industrial production of plant metabolites.
1.2 Applications of Plant Tissue Culture:
Micro propagation is widely used in forestry and in flori culture to conserver are or endangered plant species by multiplying them.A plant breeder uses tissue culture to screen cells rather than plants for advantageous characters, e.g., herbicide resistance/ tolerance.
1.3 Micropropagation:
The regeneration of whole plant through tissue culture is popularly called ‘Micropropagation’. This is a technique where a callus mass has been initiated from a single explants taken from any living part of a donor plant and within very short time and space, a large number of plantlets can be produced from such callus tissue. Again, it is possible to make a large number of callus pieces from the original stock culture during sub culturing.
1.3.1. Stages of Micropropagation:
- The process of micro propagation individually divides into five stages.
a) Stage0: Pre-propagation Stage
The pre-propagation stage requires proper maintenance of the mother plants in the greenhouse under disease and insect free conditions with minimal dust. Cleanen closed areas, glasshouses, plastic tunnels and net covered tunnels, provide high quality explant source plants with minimal infection.
Collection of explants for clonal propagation should be done after appropriate pre-treatment of the mother plants with fungicides and pesticides to minimize contamination in the in vitro cultures. This improves growth and multiplication rates of in vitro cultures. The control of contamination begins with the pre-treatment of the donor plants.
The choice of explant depends on the methods of shoot multiplication to be followed. All plant organs viz. bulb segment, inter-bulb segments, shoot tip, root tip. For axillary bud induction, callus culture, somatic embryo genesis explants bulb segments, internodes and leaves are collected.
b) Stage1: Initiation of Aseptic Culture:
In this stage sterilization of explants and establishment of explants were done. The plant organ used to initiate a culture is called explant. The choice of explant depends on the method of shoot multiplication to be followed.
- For micro propagation work the explant of choice is nodes
- For callus culture work the explant of choice is inter nodes and leaves.
- For somatic embryo genesis the explant is inter nodes and leaves.
c) Stage2: Multiplication of Culture:
This is the most important stage and the rate of multiplication determines the largely success of micro propagation system this can be achieved by-
- Enhanced axillary branching
- Adventitious bud formation
- Through callusing
i) Enhanced axillary branching:
The axillary bud present in the axil of each leaf either develops into a single shoot or form a cluster of shoots in the presence of cytokinin (BAP1.0 mg/l) in the medium.
ii) Adventitious Bud Formation:
Buds arising from any part other than the leaf axils or shoot apex are called adventitious buds. It is a standard horticulture practice.
iii) Through Callusing:
In tissue culture, the mass of differentiated cells commonly known as callus.
This either gives rise to shoot bud or bipolar structure resembling embryo (somatic embryo). This method is used when aim is to induce variability especially in self-pollinating species with narrow genetic base.
d) Stage3: In Vitro Rooting of Shoots
In-vitro grown shoots lack root system. For induction of roots, they were transferred to rooting medium. For rooting half strength MS medium supplemented with 1.0mg/l auxin was used.
e) Stage4: Hardening and Acclimatization of Tissue Culture Plantlets
This is the final stage and requires careful handling of plants. The transplantation from completely controlled conditions should be gradual. This process of gradually preparing the plants to survive in the field conditions is called acclimatization.
The plants produced in tissue culture, although green in color; do not prepare sufficient food for their own survival. Also, inside the culture vessels humidity is very high and thus the natural protective covering of cuticle is not fully developed. Therefore, immediately after transfer plants were maintained under high humidity. Optimum conditions were provided to plants in green house.
1.4. Advantages of micro propagation
Micro propagation has a number of advantages over traditional plant propagation techniques.
- The main advantage of micro-propagation is the rapid multiplication of plants that are clones of each other.
- Micro-propagation can be used to produced is ease-free plants.
- Growth manipulation: Nutrient levels, light, temperature and other factors can be more effectively controlled to manipulate the growth, multiplication and regeneration
- Micro-propagation produces rooted plantlets ready for growth, saving time for the grower when seeds or cuttings are slow to establish or grow.
- Round the year production: Micro-propagation is independent of season. As micro- propagation could be carried out throughout the year; production cycle can be scheduled to meet peak demands
- It can have an extraordinarily high frequency rate, producing thousands of propagules while conventional techniques might only produce afraction of this in number.
- It is the only viable method of regenerating genetically modified cells or cells after protoplast fusion.
- Micropropagation often produces more robust plants, leading to accelerated growth compared to similar plants produced by conventional methods – like seeds or cuttings.
- Some plants with very small seeds, including most orchids, are most reliably grown from seed in sterile culture.
2. OBJECTIVE
To establish a protocol for In vitro micropropagation of Aegle Marmelos , Moringa oleifera
Background
We chose Moringa for plant tissue culture because the lab work on problem based questions. In our case there was no problem but demand for the plants of Moringa using PTC practices. The respective farmer Mr Raghunath Shinde from Pabal ; had feild of Moringa and he observed that in his farm only few plants gave more production than other and he approached Vigyan Ashram for the Moringa saps ; thereafter we decided to practised on Moringa.
Another plant I chose was Aegle marmelos; which is woody medicinal plant for my eight semester READY program project .
3. Materials and Methods
3.1 Materials
Table No.-3.1. Glassware and Instruments
| Glassware | Instruments |
| Culture Bottles | Weighing Balance |
| Conical Flasks | Magnetic Stirrer |
| Petri Plate | pH Meter |
| Beaker | Laminar Air Flow |
| Pipettes | Autoclave |
| Measuring Cylinder | Hot Air Oven |
| Volumetric Flasks | Water Distillation Unit |
| Test Tube | Refrigerator |
Chemicals:
Table No.-3.2. Chemicals
| For MS Media | For Sterilization |
| Macronutrients | Ethanol |
| Micronutrients | Sodium hypochloride |
| Agar powder | Tween20 |
| Vitamin | |
| Growth regulator | |
| Amino acid | |
| HCl | |
| NaOH | |
| KOH | |
| Sucrose |
Others
Table No. -3.3. Others
| Forceps | Sterile Autoclaved Water |
| Spatula | Tissue Paper |
| Scalpel | Filter Paper |
| Pointed Forceps | Cotton Plugs |
| Sterile Surgical Blade | Scissors |
| Distilled water | Autoclave Bags |
3.2 Methodology
3.2.1. Sterilization
The glass wares such as culture bottles, measuring cylinders and the other equipment like forceps, blade holder were washed under running tap water using detergent followed by rinsing with double distilled water and then wrapped in aluminum foil and subsequently autoclaved at 121ºc at 15 lbs. pressure for 20 minutes.
3.2.2. Preparation of stock solutions
Separate stock solutions of macronutrients, micronutrients, potassium iodide, iron, glycine and various vitamins were prepared by dissolving each chemical separately in small quantity of double distilled water and making up the required volume with double distilled water. The stock solutions of growth regulators were prepared by dissolving them in small quantity of appropriate solvents, heating gently and then making up the volume with double distilled water. The glassware’s such as culture bottles, measuring cylinders and the other equipment like forceps and blade holder were washed under running tap water using detergent.
Table No. -3.4. Stock solution of Macronutrients (A)
| Sr. No. | Constituents | Amount (gm/l) | Amount (gm) to be taken for stock solution (20X) for 1000 ml |
| 1. | NH4NO3 | 1.65 | 33 |
| 2. | KNO3 | 1.90 | 38 |
| 3. | MgSo4.7H2O | 0.37 | 7.4 |
| 4. | KH2PO4 | 0.17 | 3.4 |
| 5. | CaCl2.2H2O | 0.44 | 8.8 |
Table No.-3.5. Stock solutions for Micronutrients (B)
| Sr. No. | Constituents | Amount (mg/l) | Amount (mg) (100 x) to be taken for stock solution in 100 ml |
| 1. | H3BO3 | 6.2 | 620 |
| 2. | MnSO4.H2O | 22.3 | 2230 |
| 3. | ZnSO4.7H2O | 8.6 | 860 |
| 4. | CuSO4.5H2O | 0.025 | 2.5 |
| 5. | CoCl2.6H2O | 0.025 | 2.5 |
| 6. | NaMoO4.2H2O | 0.25 | 25 |
Table No. -3.6. Stock solution of Iron (C)
| Sr. No. | Constituents | Amount (mg/l) | Amount (gm.) For 200 ml stock (200X) |
| 1. | Na2EDTA | 37.25 | 7.45 |
| 2. | FeSO4 | 27.85 | 5.57 |
Table No. -3.7. Stock solution of Vitamin (D)
| Sr. No. | Constituents | Amount(mg/l) | Amount (mg)(100X) for 1 L stock |
| 1. | Thiamine HCl | 0.1 | 100 |
| 2. | Pyriodoxine HCl | 0.5 | 500 |
| 3. | Nicotine acid | 0.5 | 500 |
| Sr. No. | Constituents | Amount(mg/l) | Final volume of stock (ml) |
| 1. | KI | 830 | 100 |
Table No. -3.9. Stock solution of Glycine ( (F)
| Sr. No. | Constituents | Amount(mg/l) | Amount (gm/l) stock |
| 1. | Glycine | 2.0 | 2.0 |
Table No. -3.10. Stock solution of Inositol
| Sr. No. | Constituents | Amount(mg/l) | Amount (mg/l) stock |
| 1. | Myo-Inositol | 100 | 100 |
- Media Preparation
According to the available literature on micro propagation of common rue plant, Murashige and Skoog (1962) medium is the most commonly used growing medium for Rutagraveolens L.
The procedure for composition of 1 liter MS medium is given as follows-
- Dissolved 30g of sucrose and 100mg inositol in approximately 200ml in DDH2O.
- After sucrose and inositol dissolution, the stock solutions was added in following order and mixed well.
Table No.-3.11. Media Preparation
| Stock Solution | Name of solution | Quantity(ml/l) |
| A | Macronutrients | 50 |
| B | Micronutrients | 2 |
| C | Iron | 5 |
| D | Vitamin | 1 |
| E | Potassium Iodide | 1 |
| F | Glycine | 1 |
| G | Inositol | 2 |
- pH of liquid medium was adjusted at 5.6-5.8 with the help of 1N NaOH or 0.1N HCl.
- The volume was made up to 400 ml with D.W.
- In another flask, 8 gm agar was added to the 300 ml of D.D.H2O. The agar is then heated at 60ºC to dissolve completely.
- The heated agar solution was added to the stock solutions and mixed thoroughly.
- The total volume is then made up to 1000 ml by addition of D.D.H2O.
- The growth regulators were added in the solution.
- The culture medium was poured into culture bottles (40-50 ml in each bottle approximately) and bottles is then capped and autoclaved at 121ºC at 15 psi.
pressure for 15 to 20 minutes. After sterilization, the medium is allowed to cool to room temperature and then stored in cool and dry place until
Precautions in the preparation of media
- All the stock solutions were stored in glass bottles under refrigeration.
- Iron stock was stored only in an amber coloured bottle.
- The bottle was thoroughly washed by doubled distilled water.
- Details about the date and concentration of all stock solution were labelled with glass marking pen.
PLATE NO.1: MEDIA PREPARATION
3.1.1. Collection of Plant Material
Stem cuttings including the young leaf and bulb bud were excised from Moringa stock plant grown at Sahyadri Nursery, Astagaon. Tal-Rahata, Dist- A.Nagar.
3.1.2. Surface Sterilization of Explant
All inoculations and aseptic manipulations must be carried out in a laminar air flow cabinet. Before use, the working surface of the laminar air flow cabinet need to be cleaned by swabbing with 70% ethanol and switch on the UV light for 20 min. to reduce the chances of contamination. After that the regular white light is switched on.
The instruments like scalpels, forceps are sterilized by an alcoholic dip followed by glass bead sterilization inside the laminar air flow cabinet. Other requirements like bottles, conical flasks, cotton, distilled water etc. are sterilized by steam sterilization in an autoclave.
Leaves and petiole were removed from stem section and defoliated stems with bulbs were surface sterilized by tap water, 0.1% Bavistin, 0.1% Mercuric chloride (HgCl2) solution for 1 min. After pouring off the HgCl2 solution, immersing it in distilled water.
Explants were treated with following chemicals under laminar air flow.
- Washed multiple times with sterilized tap water.
- Treated with 0.1 % Bavistin for 10 min
- Treated with 0.1 % Mercuric Chloride for 1 min.
- Treated with 70 % Alcohol for 3 mins.
3.1.3. Inoculation of Culture & Incubation of culture
A piece of plant tissue explant is cut from the plant, disinfected i.e., removal of surface contaminant and placed on a medium. A medium typically contains Mineral salts, Carbon sources. Five initiation media having different growth hormone combinations were used in the present investigation. The objective of this stage is to achieve an aseptic initiation of culture i.e., without contaminating bacteria or fungi. The inoculated culture bottles were incubated at 25 ± 2 ºC temperature for 16 hours light and 8 hours dark per dayin 2000-3000 lux light intensity under cool fluorescent white light in the culture room.
PLATE NO.2: INOCULATION
3.3 Initiation of culture
Five different combinations and concentrations of BAP & IAA were used for the initiation of Moringa.
Table No-3.12. Media combinations for Initiation
| Sr. No. | Media | BAP(mg/l) | IAA(mg/l) | No. of test tubes Inoculated |
| 1. | MS Media | 0 | 0 | 5 |
| 2. | MS Media | 1.5 | 0.1 | 5 |
| 3. | MS Media | 1.5 | 0.2 | 5 |
| 4. | MS Media | 1.5 | 0.3 | 5 |
| 5. | MS Media | 1.5 | 0.4 | 5 |
PLATE NO.4:INITIATION
4. Results
4.1. Initiation:
The present investigation aims ‘To establish a protocol for in vitro micropropagation of Moringa , and the related work was carried out at Vigyan Ashram Pabal.
Shoot initiation and establishment from shoot tips explants are cultured on MS media supplemented with different concentration of growth hormones. BAP (mg/l)+ IAA(mg/l) (Control, 1.5+0.1, 1.5+0.2, 1.5+ 0.3, 1.5+0.4 ) and was successfully used. The best result was obtained on MS medium supplemented with 1.5mg/l + 0.1 mg/l BAP +IAA.
1) Shoot Initiation:
Table-4.1. : Result of initiation and rate of survival
| No. | Media | No. of Bottles inoculated | Rate of Survival (%) |
| 1. | Control | 5 | 20% |
| 2. | MS Media +1.5 mg/l BAP+ 0.1 mg/l IAA | 5 | 60% |
| 3. | MS Media + 1.5 mg/l BAP + 0.2 mg/l IAA | 5 | 30% |
| 4. | MS Media + 1.5 mg/l BAP + 0.3 mg/l IAA | 5 | 40% |
| 5. | MS Media + 1.5 mg/l BAP + 0.4 mg/l IAA | 5 | 20% |
PLATE NO.5: RESULTS OF CONC;MS Media +1.5 mg/l BAP+ 0.1 mg/l IAA
Transfer on new medium
- The well grown cultures were transferred after a few weeks into a new medium.
- The in vitro raised shoots were excised and transferred to multiplication media and the primary explant was placed on a fresh medium for regeneration of new shoot.
Result
Contamination occur to the culture bottles and tubes ; that was bacterial contamination so we decided to treat explant by giving the treatment of stagamycin before inoculation and follow some laboratory practices regularly.
Daily Laboratory Practices
Mopping the floor ,work bench
Washing
Media Preparation
Spraying the respective plant from explant were taken by fungicide and bacteriocide .
Inoculation
January 2024
1 Daily trails of 30 test tubes of Moringa
2 The survival rate of trial is 60%
3 Aegle mermelos plant collected from Sahyadri Farm Nursery Ahmednagar
4 Planting 3 saps of Napier African 5G grass in polyhose collected from Bhanudas sir
5 Visit Raghunath Shindes feild ( Customer of Moringa plants )
6 Omkar has taken inoculation trails under my instruction since 15th of January
Avanti Mam visit on 18/01 and we had discussion on fish bone analysis of contamination in culture room , and decided to purchase foggers for fogging
February 2024
Daily schedule follow as
1 Cleaning(Floor moping , sterilization of LAF , Autoclave )
2 Glassware washing ( test tubes, culture bottles)
3 Media preparation
Contamination occurs due to dust also interruption of Microbiology lab construction
– We decided to practice on other plants like Banana, Lemon ; plant regarding to this discuss with Avanti Mam an Gavande Mam
– Purchase Apron, Soxhelt, Formaldehyde mask , Acetone
Inititaion stage aapears in 2 weeks successfully with combination of 1.0mg/L BAP + 1.5mg/L IAA
- Initiation results
-Discussion on Fogger,Dixit sir ask Siddhant to help us in installation of fogger in culture room on (01/03/2024)
– Leakages in culture room is sealed with silicon gun
– Purchase Nanodrop hand fogger ( Hand fogger for test tubes fogging manually)
March 2024
Daily schedule follow as
1 Cleaning(Floor moping , sterilization of LAF , Autoclave )
2 Glassware washing ( test tubes, culture bottles)
3 Media preparation
- Agro soray is decided for wall fumigation to check contamination level ,after analysis we refuse the use of Agro spray
- Steamer is used for fumigation
- Decide Wednesday for fumigation weekly
- Banana, Napier , Lemon and Moringa decided for PTC practice
- On 24/03/2024 we scheduled PTC laboratory practices
| Sr/No | Day | Particulars |
| 1 | Monday | Media preparation |
| 2 | Tuesday | Inoculation (Banana) for 30 culture bottles |
| 3 | Wednesday | Instrument Cleaning Fumigation |
| 4 | Friday | Inoculation 2 crops Moringa -20 bottels Lemon – 20 bottels |
| 5 | Saturday | Media preparation |
| 6 | Sunday | Sterilization of scalpels, blades in Hypo solution |
April 2024
Plant Tissue Culture of Lilium – We decided to take trials on lilium and Canna indica for comparative study of Banana .( Trials upto 24th of April)
Introduction
The white lily is a member of the genuine lily family of plants. The herbaceous flowering plants of the genus Lilium all have large, noticeable blooms and grow from bulbs which we used as explant.
The goal of the current study was to create cultural environments that would promote better shoot organogenesis from bulb scales. Additionally, the outcomes of micropropagation using segments of bulb scales are The majority of lilies’ natural habitats are located at high elevations, often more than 2000 metres above sea level. They are appropriate garden plants in a wide range of climates from temperate to subarctic latitudes due to their adaptability to extreme annual fluctuations in temperature and humidity
REFERNECE
Patil et.al., (2021) explained a procedure for its proliferation has to be developed because the plantlets’ ability to multiply by bulb is poor and they are more prone to disease. In this study, we looked at the effects of various concentrations of BAP and NAA on the germination of Lilium candidum shoots from bulbs as well as the effects of IBA, IAA, and NAA on these shoots.
Protocol
Media Preparation
According to the available literature on micro propagation of common rue plant, Murashige and Skoog (1962) medium is the most commonly used growing medium for Rutagraveolens L.
The procedure for composition of 1 liter MS medium is given as follows-
- Dissolved 30g of sucrose and 100mg inositol in approximately 200ml in DDH2O.
- After sucrose and inositol dissolution, the stock solutions was added in following order and mixed well.
Table . Media Preparation
| Stock Solution | Name of solution | Quantity(ml/l) |
| A | Macronutrients | 50 |
| B | Micronutrients | 2 |
| C | Iron | 5 |
| D | Vitamin | 1 |
| E | Potassium Iodide | 1 |
| F | Glycine | 1 |
| G | Inositol | 2 |
3. pH of liquid medium was adjusted at 5.6-5.8 with the help of 1N NaOH or 0.1N HCl.
4. The volume was made up to 400 ml with D.W.
5. In another flask, 8 gm agar was added to the 300 ml of D.D.H2O. The agar is then heated at 60ºC to dissolve completely.
6. The heated agar solution was added to the stock solutions and mixed thoroughly.
7. The total volume is then made up to 1000 ml by addition of D.D.H2O.
8. The growth regulators were added in the solution.
9. The culture medium was poured into culture bottles (40-50 ml in each bottle approximately) and bottles is then capped and autoclaved at 121ºC at 15 psi; pressure for 15 to 20 minutes. After sterilization, the medium is allowed to cool to room
Culture Bottles
Precautions in the preparation of media
- All the stock solutions were stored in glass bottles under refrigeration.
- Iron stock was stored only in an amber coloured bottle.
- The bottle was thoroughly washed by doubled distilled water.
- Details about the date and concentration of all stock solution were labelled with glass marking pen
Collection of Plant Material
Stem cuttings including the young leaf and bulb bud were excised from Lilium stock plant grown at Vigyan Ashram Pabal
Mother Plant Lily
Surface Sterilization of Explant
All inoculations and aseptic manipulations must be carried out in a laminar air flow cabinet. Before use, the working surface of the laminar air flow cabinet need to be cleaned by swabbing with 70% ethanol and switch on the UV light for 20 min. to reduce the chances of contamination. After that the regular white light is switched on.
The instruments like scalpels, forceps are sterilized by an alcoholic dip followed by glass bead sterilization inside the laminar air flow cabinet. Other requirements like bottles, conical flasks, cotton, distilled water etc. are sterilized by steam sterilization in an autoclave.
Leaves and petiole were removed from stem section and defoliated stems with bulbs were surface sterilized by tap water, 0.1% Bavistin, 0.1% Mercuric chloride (HgCl2) solution for 1 min. After pouring off the HgCl2 solution, immersing it in distilled water.
Explants were treated with following chemicals under laminar air flow.
- Washed multiple times with sterilized tap water.
- Treated with 0.1 % Bavistin for 10 min
- Treated with 0.1 % Mercuric Chloride for 1 min.
- Treated with 70 % Alcohol for 3 mins.
Inoculation of Culture & Incubation of culture
A piece of plant tissue explant is cut from the plant, disinfected i.e., removal of surface contaminant and placed on a medium. A medium typically contains Mineral salts, Carbon sources. Five initiation media having different growth hormone combinations were used in the present investigation. The objective of this stage is to achieve an aseptic initiation of culture i.e., without contaminating bacteria or fungi. The inoculated culture bottles were incubated at 25 ± 2 ºC temperature for 16 hours light and 8 hours dark per dayin 2000-3000 lux light intensity under cool fluorescent white light in the culture room.
Inoculation
Initiation of culture
Media combinations for Initiation
| Sr. No. | Media | KN(mg/l) | NAA(mg/l) | No. of Bottles Inoculatd |
| 1. | MS Media | 0 | 0 | 5 |
| 2. | MS Media | 1.5 | 0.1 | 5 |
| 3. | MS Media | 1.5 | 0.2 | 5 |
| 4. | MS Media | 1.5 | 0.3 | 5 |
| 5. | MS Media | 1.5 | 0.4 | 5 |
Transfer on new media
- The well grown cultures were transferred after a few weeks into a new medium.
- The in vitro raised shoots were excised and transferred to multiplication media and the primary explant was placed on a fresh medium for regeneration of new shoot.
Multiplication
After a successful shoot formation from bulb segments of Lilium candidum, they were further sub cultured for in vitro shoot multiplication. The in vitro raised shoots were excised and transferred to multiplication media and the primary explant was placed on a fresh medium for regeneration of new shoot. For shoot multiplication, the excised sprouts were transferred to MS medium containing BAP and NAA for its efficiency for shoot multiplication. Maximum of 12 shoots per explant were obtained per explant
Media combination for Multiplication
| Sr. No. | Media | BAP(mg/l) | NAA(mg/l) | No. of Bottles Inoculated |
| 1. | MS Media Control | – | – | 5 |
| 2. | MS Media | 0.5 | 0.1 | 5 |
| 3. | MS Media | 1.0 | 0.2 | 5 |
| 4. | MS Media | 1.5 | 0.3 | 5 |
| 5. | MS Media | 2.0 | 0.4 | 5 |
Results and Discussion
Initiation
The present investigation aims ‘To establish a protocol for in vitro micropropagation of Lilium , and the related work was carried out at Vigyan Ashram Pabal
Shoot initiation and establishment from shoot tips explants are cultured on MS media supplemented with different concentration of growth hormones. KN (mg/l)+ NAA(mg/l) (Control, 1.5+0.1, 1.5+0.2, 1.5+ 0.3, 1.5+0.4 ) and was successfully used. The best result was obtained on MS mediumsupplemented with 1.5mg/l + 0.1 mg/l KN+NAA.
Inoculation of Lily
After a Day
After 2 Days
After 4 Days
After 10 Days
After 16 Days
1) Shoot Initiation : Result of initiation and rate of survival
| No. | Media | No. of Bottles inoculated | Rate of Survival (%) |
| 1. | Control | 5 | 20% |
| 2. | MS Media +1.5 mg/l KN + 0.1 mg/l NAA | 5 | 60% |
| 3. | MS Media + 1.5 mg/l KN + 0.2 mg/l NAA | 5 | 30% |
| 4. | MS Media + 1.5 mg/l KN + 0.3 mg/l NAA | 5 | 40% |
| 5. | MS Media + 1.5 mg/l KN + 0.4 mg/l NAA | 5 | 20% |
Multiplication
Result of Multiplication and Rate of Survival
| No. | Media | Number of Bottles Inoculated | Rate of Survival (%) |
| 1. | Control | 5 | 10% |
| 2. | MS Media + 0.5 BAP mg/l + 0.1 KN mg/l | 5 | 40% |
| 3. | MS Media + 1.0 BAP mg/l + 0.2 NAA mg/l | 5 | 60% |
| 4. | MS Media + 1.5 BAP mg/l + 0.3 NAA mg/l | 5 | 30% |
| 5. | MS Media + 2.0 BAP mg/l + 0.4 NAA mg/l | 5 | 50% |
After 5 days of Subculture
Contamination
Contamination after Subculture (after 6 days)
Discussion
- The aim of the present investigation was to optimize the culture conditions for In vitro micropropagation of Lilium
- For In vitro micropropagation of Lilium were efficiently used as explant for rapid propagation. Treatment of 0.1% Bavistin (10 min) + 0.1% HgCl2 (1 min) + 70% Alcohol gives the best results for sterilization of explant.
- The bulb segment of Lilium was inoculated on MS media containing 1.5 mg/l KN and 0.1 mg/l NAA for initiation showed best results of shoot proliferation. The similar observation were recorded by Patil et.al..
- The growing shoots were transferred in multiplication stage containing MS Media with 1.0 mg/l BAP and 0.2 mg/l NAA showed best results for multiplication. The similar observation were recorded by Patil et.al
Summary
The bulb segments were used as an explant for micropropagation of Lilium. Treatment of 0.1% bavistin for 10 min, along with 0.1 % HgCl2 for 1 min, 70% alcohol for 3 min and gives best result for sterilization of explants. These surface sterilized explants were inoculated on different hormone concentration for further regeneration and multiplication. The different media concentrations having KN, NAA (Control, 1.5 KN (mg/l) and Control 0.1, 0.2, 0.3, 0.4 NAA (mg/l) ) were used for the initiation of Lilium. Well grown newly formed shoots were transferred onto fresh media and primary explant was allowed to initiate newshoot growth. The media containing 1.0 mg/l BAP+ 0.2 mg/l NAA was proved efficient for the multiplication stage
Conclusion
- The bulb segment Lilium plant can be effectively used for the micropropagation.
- The Lilium is an important medicinal herb with various pharmacological actions.
- Present study shows that after two weeks of inoculation, maximum number of shoot were obtained in medium supplemented with MS Media +1.5 mg/l KN + 0.1 mg/l NAA
- The NAA along with BAP is proved effectively for multiplication, the best results were obtain on the 1.0 mg/l BAP+ 0.2 mg/l NAA.
- From these study it was concluded that 0.5 mg/l BAP + 0.1 mg/l NAA for initiation and.5 mg/l KN + 0.1 mg/l NAA for multiplication was observed
Fogger Installation on 24th of April by help of Mahesh Sir
Duration of Fogging is set at Each Fogg for 1 min after each 150 minutes
The chemical use for fogging is bacteriosin
Neem
Media for Woody Plant Neem
Inoculation
Inoculation of Neem
Initiation
After 14 days
Bubble formation from media leads contamination
May 2024
Banana Micropropagation
Plant tissue culture is a collection of experimental methods of growing large number of isolated cells or tissues under sterile and controlled conditions.
Banana is one of the world’s most important fruit crop. It is grown in all type of tropical agriculture systems. The yield depends not only on the quality of soil and fertilization, but largely upon the control of the diseases.
An important objective of micro propagation of banana is to produce large number of disease free planting material and to satisfy the large need of banana planting materials
Stages of micro propagation of banana
1. Selection of mother plant
2. Preparation of explants
3. Fresh inoculation
4. Multiplication
5. Rooting
6. Planting out
7. Primary hardening
8. Secondary hardening
9. Field planting
Selection of Mother plant – we take sucker as an explnat , I take suckers in feild of vigyan ashram with help of Bhanudas sir
- Trails on Banana for 15 culture bottles – Grow well upto 14 days , after that culture bottles get contaminate
- Trail on Napier for 10 culture bottles
- Subculturing of Lilium
Culture bottles get contaminitated after 6 days , further I discussed with Shanbagh Sir about that and communicate with Lokhande Sir about contamination.
Weekly Sheduled for Month (May)
| Sr/No | Day | Particulars |
| 1 | Monday | Media preparation |
| 2 | Tuesday | Inoculation |
| 3 | Wednesday | Instrument Cleaning Fumigation |
| 4 | Friday | Inoculation |
| 5 | Saturday | Media preparation |
| 6 | Sunday | Sterilization of scalpels, blades in Hypo solution |
Conclusion –
1) In case of Neem I got results upto initiation
2) Tissue culture protocol reported in this study is an alternative mean of propagation of drumstick plantlets with uniform genotypes for breeding selection, field experiments and to make availability of disease free seedlings to farmers for commercial cultivation and to acheive grater profits
Outcome
- In present project work protocol for “In–vitro micropropagation of Moringa oleifera by using axillary branching ” was successfully standardized to study the effects of hormonal concentration on Semi hard plant tissue culture techniques.
- Good laboratory practices
- Expertise in the procedure of preparing chemicals, MS media, and stock solutions.
- Sterilization of glassware and media
- Proper instrument, equipment handling, and aseptic inoculation.
- Stages of in vitro propagation techniques studied i.e Initiation, Multiplication and Subculturing
