Potato is by far the most popular food in the whole world! Very few people may dislike french fries, or crispy potato chips or potato curry - but people who likes potato are ever increasing in numbers! So, the demand is increasing - especially in a developing country like Bangladesh where population is ever increasing - Potato Tissue Culture may play a vital role. I think it will be specially helpful to Potato Researchers and Tissue Culture Researchers.
TISSUE CULTURE OF POTATO
1. INTRODUCTION
Potato plant can be propagated through true potato seed (TPS), tuber, or stem cutting, but only tuber propagation is generally used for commercial potato production.The tuber vegetative propagation has the advantage to guarantee the varietal purity from generation to generation (except for rare mutations). However, tuber propagation has some major disadvantages: tubers are rich in water and other nutrients, which is a favorable medium for the maintenance and development of pests, viruses, bacteria, and fungus. In addition, the storage of tubers is more difficult than the storage of true potato seeds, and tubers must be reproduced every year.
The most frequent contaminations through tuber propagation are viruses (PVX, PVY, PVA, PVS, PVM and PLRV being the most common throughout the world though many other viruses can also be found). Virus contamination symptoms are difficult to identify on tubers, but are more obvious on the aerial part of the plant (stems and foliage). As a result, it is not possible to sort out clean tubers from contaminated ones only on their external aspect. Viruses are circulating in the sap, and multiply in the foliage of the plants. Therefore, quality tuber seeds have to be produced from plants originating from clean plant materials (e.g. from tissue culture).
2. ELABORATORY
2.1. Micro propagation Unit
A micro propagation unit includes a tissue culture laboratory and a propagation green house. When planning a micro propagation unit, we have to consider the following factors: available space, environments, financing, type of work to be developed, and required production capacity. When propagation in large scale, the facilities and the equipments must be designed to actually perform the work and to maintain an optimum production flow.
tissue culture potato Basic process: The basic process normally carried out in a tissue culture laboratory are as follows.
a) Glassware washing;
b) Culture media preparation;
c) Media and equipment sterilization
d) Ex-plants preparation and aseptic transference of cultivated materials;
e) Incubation and growth of cultivated materials up to maturity; and
f) The rooted plant-lets’ transplantation, is accomplished, in part, with the help of laboratory personnel.
Basic organization: The laboratory for plant tissue culture requires a basic organization that comprises three areas:
a) General laboratory (or media preparation area) provided with spaces for common or independent work, and some equipments and materials can be used by several workers at the same time;
b) Area for the aseptic manipulation of plant material (or transference area);
c) Culture maintenance area (or culture rooms) with controlled conditions for light.
There should be two separate rooms at least. One for washing, sterilization, storage and culture media preparation; and the other for culture maintenance (culture room). The transference chamber can be located in the general laboratory or in an area specifically designed as a transference room, according to available conditions.
Washing and media preparation area: The area for washing should have a big washbasin (can be stainless steel, and acid and alkali resistant), tap water, tables that allow stand-up work and shelves to dry and keep the wasted materials. The media preparation area must be equipped with a refrigerator to keep the chemicals and solutions used in the media culture, scales, a potentiometer, a kitchen, a media mixture, a water distiller, and an autoclave or pressure pot. The last two must be located as close as possible to the washbasin. The stove may be used to dry the materials.
Culture area: This is the culture incubation area, where optimum media conditions change according to the species in culture. Following factors should be taken into consideration:
- Temperature variation.
- Light intensity and quality.
- Relative humidity.
- Photo-period.
Temperature is controlled with an air-conditioning equipment or heater. According to the cultivars, the average temperature of an incubation room should be 25oC±2oC. It is recommended to use thermostats, which prevent temperature variations in the room from exceeding the culture requirements. The airflow must be uniform within room to maintain the same temperature in the whole environments.
The air-conditioning equipment directly controls the relative humidity. If the relative humidity drops below 50%, there will be a water loss in the culture media, and an increase the mineral salt concentrations which can damage the cultures. With a high relative humidity (80~100%) contaminants could enter the culture containers. The optimum average is about 60~70%.
The light source is provided by fluorescent lamps and the photoperiod is controlled by an hourly timer. The fluorescent lamps have an advantage over the incandescent lamps because they have better light quality, distribute the light uniformly and produce less heat. However, some cultivars grow better with a combination of both types of light.
Most of the cultivars require an illumination that varies about 500~3000 lux . Some of them need more than 5000 lux, and others just need darkness as in the case of in vitro tube induction.
The arrangement and the number of the shelves, where magenta vessels and tubes with culture are placed, will vary according to the room’s dimensions. Shelves can be metallic or wooden, and should painted white.
The shelves’ dimensions may vary. However, it is recommended to have an incubation platform of 0.45m×0.90 m, with a height of 0.30m among the shelves because it allows good illumination, access to, and control of the incubated materials. The space between the soil and the first platform must be 0.15 m to facilitate soil cleaning. A distance of 0.05~0.10m must be kept between the wall and the shelves to allow the free circulation of air.
For the laboratory walls antifungal-epoxic-paint (used in cool temperature chamber) is recommended as a preventive measure.
It is recommended to put a tray on the floor, with a rug containing an acaricide and a fungicide to impregnate the shoes of those who enter the culture room.
The culture room must be isolated from the external environment to maintain the appropriate temperature and the relative humidity and to avoid the entrance of contaminants. Just in case, the windows should also sealed. Access to the culture room will be allowed only to the people who work there.
A tissue culture laboratory can be located in any geographical area. The internal controlled environmental conditions allow isolation with a minimum of external influence.
To decide where to build the tissue culture laboratory, the following factors must be taken into consideration:
- Environment growing condition for potato;
- Availability of electricity;
- Availability of water and drainage; and
- Good all-around communication.
Production process
The production processes involves:
In vitro culture establishment stage: Take plant for the test-tube from field. The selected plant should be from a clean lot to guarantee the quality and uniformity. Virus-free plantlets can be obtained by the processes of thermo-therapy, and meristem culture. The plant-lets will be used as a source of explants for production process.
Production stage: It consists of taking the massive propagation of explants and plantlets. The time of each propagation cycle depends on the species behavior, the culture medium, and the environmental conditions to which it is subjected, the average is between three or four weeks for each step.
The colorless plastic and /or glass containers are used in propagation. It is considered that 20 explants per container make a good plantlet development possible.
Aseptic condition in the laboratory
Asepsis in the facilities: Four basic environments should meet : an office, a wash and media preparation room, a transference room and and a culture room, which require a minor to major grade of cleaning.
Asepsis in the washing and media preparation room: The wash room is for cleaning the test tube. Covered shelve to avoid dust and cleaned tables are required.
The media preparation room requires good cleaning to avoid media contamination before it is used.
Asepsis in the transference room: The transference room must be kept very clean since it is where the laminar chamber located and it is next to the culture room. Only the material necessary for propagation will be permitted within the chamber.
Asepsis in the culture room: Continuously disinfecting the floors and shelves is required.
2.2. Multiplication Plan for In vitro Plantlets for Green house
The infrastructure of the tissue culture laboratory makes possible the propagation of large quantities of plantlets to provide materials for one or several green house. The scale of the tissues culture laboratory and the quantity of plantlets in vitro depend on the amount of pre-basic seed production according to the plan.
3. STOCK SOLUTIONS
The Murashige & Skoog basal medium (1962) is used a lot in potato plantlet production. However, there are several kinds of media have been developed by CIP, IVF and other institute to reduce the cost of plantlets in vitro production.
3.1. Preparation of the Murashige & Skoog basal Medium (MS)
Preparation of stock solutions:
Stock A – Salts;
Stock B – MgSO4;
Stock C – Na2EDTA, FeSO.7H2O; and
Stock D – Vitamins.
Take different quantities of stock A, B, C, D and make up to 1 liter with distilled water.
3.2. Preparation of solution VGA (vitamins and gibberellic acid) (omitted)
3.3. Preparation of vitamin solution (omitted)
3.4. Preparation of hormone stock solution: GA3, NAA, BAP, IAA, KIN, and 2,4-D (omitted)
3.5. Preparation of antibiotics: rifampicin and sodic cephotaxim (omitted)
3.6. Preparation of calcium hypochlorite (omitted)
3.7. Preparation of an acaricide solution: use the solution while fresh (omitted)
3.8. Preparation of solution for pH adjustment: HCl and KOH (omitted)
4. CULTURE MEDIA
The growth of the plantlets in vitro depends on nutritional and environmental factors, which interact to provide a plantlet with similar characteristics to those grown in the field. The nutritional factors are based on the MS medium composed of organic salts, vitamins, amino acids, carbohydrates, growth regulators and organic supplements.
In the case of potato, the compounds used for the culture media preparation contain the basal medium, vitamins and other substances, according to the explants to be used. Thus, for the introduction in vitro, one of the most important components is GA3, which breaks bud dormancy and accelerates explants growth.
In the conservation medium, Sorbitol is used to induce osmotic stress, which is regarded as nutrient absorption and growth. The tuberization medium contains growth regulators, which induce the micro tubers’ production through stress.
4.1. In vitro Introduction Medium
a) Take MS stock or dissolve MS with distilled water;
b) Add sucrose and vitamin solution, and stir;
c) Add GA3;
d) Bring the volume to a certain ml with distill water;
e) Measure the pH and adjust it to 5.5;
f) Add phytagel;
g) Dissolve the gelling agent with heat. Stir well until it dissolves, and prevent it from boiling;
h) Distribute 2 ml in small tubes;
i) Sterilize at 121oC and 15 pound pressure for 20 minutes; and
j) Take the culture tubes off the autoclave and keep it at 4oC until planting.y
4.2. Potato Propagation Medium
a) Take MS stock or dissolve MS with distilled water;
b) Add sucrose and VGA solution, and stir;
c) Bring the volume to a certain ml with distilled water;
d) Measure the pH and adjust it to 5.5;
e) Add phytagel;
f) Dissolve the gelling agent with heat. Stir well until it dissolves, and prevent it from boiling;
g) Distribute 12 ml in small containers;
h) Sterilize at 121oC and 15 pound pressure for 20 minutes; and
i) Take the culture tubes off the autoclave and keep it at 4oC until planting.
4.3. Potato Meristem Culture Medium (Table 1)
a) Take MS stock or dissolve MS with distilled water;
b) Add sucrose and vitamin solution, and stir;
c) Add putrescine and GA3
d) Fill it up to a certain ml with distill water;
e) Measure the pH and adjust it to 5.5;
f) Add agar;
g) Dissolve the gelling agent with heat. Stir well until it dissolves, and prevent it from boiling;
g) Distribute 12 ml in small tubes;
h) Sterilize at 121oC and 15 pound pressure for 20 minutes; and
i) Take the culture tubes off the autoclave and keep it at 4oC until planting.
4.4. Potato Conservation Medium
More sucrose and sorbitol.
4.5. Potato Tuberization Medium
Much more sucrose, need BAP, CCC.
4.6. Preparation of the Propagation Medium with Activated Carbon
Add activated carbon after dissolving the gelling agent.
Table 1. Preparation of potato culture media (1 litre)
Potato culture media formulation
Reagents Introduction Propagation Meristem Conservation Tuberization Activ. carbon
MS basal salts 1l 1l 1l 1l 1l 1l
VGA solution 5 ml 5 ml
Vitamin. Stock 5 ml 5 ml 5 ml
GA3 (1000 ppm) 1 ml 0.5 ml
Putrescine(10000 ppm) 2 ml
BAP (1000 ppm) 5 ml
CCC 500mg
Sucrose 25 g 25 g 25 g 20 g 80 g 25 g
Sorbitol 40 g
Activated carbon 10 g
Phytagel 3.5 g 3.5 g 4 g
Agar 6.0 g 7.5 g
5. TISSUE CULTURE TECHNIQUES
Fungus and bacteria grow on the plant surface and contaminate the culture media when they are adequately eliminated. The process of explants introduction into in vitro conditions depends mainly on the disinfecting phase. It is recommended to use specific antibiotics and meristems or bud cuttings.
On the other hand, the process of in vitro introduction involves the use of explants in different physiological stage. During the process of in vitro introduction there is a close connection between green house and laboratory, which is the necessary precautions should be taken to avoid the entrance of contaminants into the laboratory.
In the green house: Take cuttings from the mother plant or buds from green house tuber sprouts.
In the laboratory:
a) Immerse the cuttings for 10 minutes in a beaker conveniently labeled with the accession name, and containing a solution of 0.5% acaricide with 3 drops/L of Tween-20.
b) Throw away the acaricide solution and rinse the nodes with tap water.
c) Prepare the laminar flow chamber.
d) Immerse the cuttings in 70% alcohol for 30 seconds and take the beakers immediately to the laminar flow chamber.
In the laminar flow chamber:
a) Eliminate the alcohol and replace it with a 2.5% solution of calcium hypo chlorite. Keep the cuttings immersed during 15 minutes.
b) Throw away the hypochlorite, and rinse the cuttings three timeswith sterile water.
c) Keep the nodes immersed in the sterile water until bud extraction.
d) Dissect the buds on a sterile plate and place them in the culture medium.
5.1. Micropropagation
In vitro, plant lets which are free of pathogens are used as essential materials for potato seed programs. Potato micropropagation are based on the rapid growth of individual node cuttings, or stems with multiple node cuttings.
Node micropropagation
This method is based on the principle that the node of an in vitro plantlet placed in an appropriate culture medium will induce the development of the auxiliary bud, resulting in a new in vitor plantlet. Callus formation and re-generation must be avoided because they tend to affect the genetic stability of the genotype.
Under the room-controlled condition, micropropagation is fast. Each mode planted in propagation medium will produce a plantlet, which will occupy the full length of the test tube, after approximately four weeks. The resultant in vitro plantlet may be transplanted to in vitro conditions and in greenhouse for further propagation by cuttings.
Micropropagation by node cuttings in a liquid medium
This technique is applied to produce a large number of nodes rapidly. Stem cuttings with 5 to 8 node are prepared by removing both the apex and the root of the in vitro plant to be progated. The stems are place in the corresponding propagation liquid medium. It is also possible to use isolated nodes. The node will sprout and new plantlets will develop over a period of 3 to 4 weeks.
Micropropagation procedure
a) Prepare the laminar flow chamber by disinfecting the internal surface with alcohol. Sterilize the tools with an instrument sterilizer and place them into a container with 75% alcohol.
b) Open the tube or container with plantlets, and cut the node in the container.
c) Open a tube or container with fresh sterile medium and take the node directly from the container with plantlet and put the nodes into the new container, trying to plunge it slightly into the medium with the bud up.
d) Close the containers.
e) Label the new transferred container correctly.
Common problems in micropropagation
Phenolization
The explants become brown or blackish shortly after isolation. The young tissues are less susceptible than the more mature ones.
Prevention:
a) Removing the phenolic compounds produced by dispersion and absorpting by means of activated carbon or polyvinilpyrolidone (PVP).
b) MOdifying the redox potential
c) Inactivating the phenolase enzymes.
d) Reducing the phenolasic activity and the availability of substrate.
Absence of rooting
The explants can naturally form roots during propagation without an additional rooting stage. However, some may show root production deficiency. Rooting may be induced by incorporating auxins, such IAA, NAA, and IBA, or activated carbon to the culture medium.
5.2. Virus Eradication through Mersitems Culture and Thermotherapy
If a healthy plat is exposed to infections caused by pathogens such as virus, which have a negative effect on yield. However, not all the plant cells may become infected. A group of cells, which are in a continuous non-differentiated multiplication, are virus free "the mreistem"
The in vitro meristem culture, together with growth at high temperature produces potato plantlets free from viruses in more than 90% of planted meristems.
Virus cleaning procedures
Infected plant
↓
Micropropagation in magentas
↓
Thermotherapy chamber
34° C ~ 16 hours
32° C ~ 8 hours
↓
Meristem culture
10 per accession
↓
Evaluation of PSTVd
Virus evaluation-ELISA
Indicator plants
↓
Genetic stability evalation
Morphological comparison in the field
Infected plant vs. clean plant
↓
Replacement with the clean material
5.3 Cost Reduction in Vitro Plantlet Production
Cost reduction can be achieved through following measures:
Replace sucrose with commercial sugar.
Using another type of gelling agents or supports agar and starch.
Using other sources of non mineral distilled water: boiled water.
Using natural light.
Reducing the time between transferences.
Culture plantlets in a higher temperature (but <30C).
