Phytohormones commonly used in tissue culture and their functions
Product Manager：Harrison Michael
I. Process of plant tissue culture
1. Concept of plant tissue culture
Plant tissue culture is an advanced biotechnology that allows scientists to grow the cells, tissues, or organs of plants under sterile conditions in a laboratory controlled environment. This method not only is helpful to study the biological characteristics of plants, but also for breeding of rare and endangered plants, production of virus-free plants, plant pathology research and development of new crop varieties have important applications.
2. Process of plant tissue culture
1. Selection and preparation of explants
Suitable plant tissues were selected as explants, such as shoot tips, leaves, root tips, or embryoid bodies.
The explants were cut and processed appropriately to facilitate inoculation and culture.
2. Disinfection of the explants
The surface of the explants was disinfected using appropriate disinfectants such as 70% alcohol and 0.1% mercuric chloride.
Disinfection time need to strictly control, so as to avoid harm the organization.
3. The preparation of the culture medium
Essential minerals: including potassium nitrate (KNO3), ammonium sulfate (NH4NO3), potassium dihydrogen phosphate (KH2PO4), etc., provide the main mineral nutrition required by plants.
Trace elements, such as ferric sulfate, manganese sulfate (FeSO4) (MnSO4), zinc sulfate (ZnSO4) and so on, these are necessary for plant normal development and development.
Organic matter: add vitamins (such as vitamin B1 and B6), plant hormones, such as auxin and cytokinin) and sugar (usually sucrose), to provide the carbon source and promote development.
Phytohormones: Added according to culture purposes, such as 2,4-D for dedifferentiation, IAA, IBA, and NAA for rooting, and 6-BA for promoting shoot formation.
AGAR: As a coagulant, it solidifies the medium and facilitates the attachment and development of plant tissues.
pH regulation: The pH of the medium was adjusted to about 5.8 to meet the development requirements of most plants.
4. Sterilization of media
The prepared medium was loaded into culture bottles or Petri dishes and sterilized by pressure steam to ensure a sterile environment.
5. Get vaccinated
The sterilized explants were inoculated into the culture medium under sterile conditions.
Make sure to avoid any possible contamination during vaccination.
6. Control of culture conditions
The inoculated medium was placed in a constant temperature incubator with controlled temperatures usually between 22 and 28 ° C.
Light duration and intensity were adjusted according to the development needs of the plants.
7. Observe and record
The development of the tissues was observed regularly, and data such as development rate and morphological changes were recorded.
Observe whether there is contamination and deal with it in time.
8. Adjustment of the culture stage
According to the organization's development and differentiation, timely adjust the composition of culture medium, such as the hormone replacement type or concentration.
Dedifferentiation and redifferentiation processes are carried out to promote callus formation and bud differentiation.
9. Rooting and transplanting
When the tissues had formed small plants, the medium was adjusted to induce rooting.
After take root of small plants need to gradually adapt to the environment, and then transplanting to sterile soil or other medium.
10. Follow-up management
Transplanted plants require proper management of temperature, humidity, and light.
Plants were observed for adaptation and pruned and fertilized if necessary.
II. Hormones and classification commonly used in plant tissue culture
Phytohormones are indispensable endogenous substances in plant development and development. They play a crucial role in plant tissue culture, playing a regulatory role, affecting cell division, differentiation, rooting, sprouting and other processes. The following are some of the hormones commonly used in plant tissue culture and their classification and function:
Auxins
Auxin is a kind of main promote cell elongation and differentiation and development of plant hormones.
Functions:
·Promotes cell elongation and division
·Induction of root differentiation and development
·Play a role in the development of organs, such as promoting the development of the flower.
Auxins:
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Cytokinins
Cytokinins mainly promote cell division, induce bud formation and inhibit senescence.
Functions:
·It promotes cell division and tissue proliferation.
·Differentiation of buds and lateral bud development were induced.
·Inhibition of senescence in leaves and fruits.
Cytokinins:
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Gibberellins
Gibberellins affect cell elongation and differentiation, break quiescence, and promote germination.
Functions:
·It promotes cell elongation and affects stem development.
·Break dormancy in seeds and shoots.
·Promotes flower development and sex differentiation.
Gibberellins:
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Abscisic Acid (ABA)
Absciinic acid acts in plants in response to environmental stress, promoting dormanness and inhibiting germination.
Functions:
·Promotes dormancy of seeds and shoots.
·It regulates the adaptability of plants to the environment.
·It is involved in the defense response of plants.
Ethylene
Ethylene is a gas hormone that affects fruit ripening and leaf and flower senescence.
Functions:
·It is involved in the defense response of plants.
·Affects plant development and fruit ripening.
Other regulatory hormones
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III. Application of phytohormones in various stages of tissue culture
The role of phytohormones in tissue culture is multifaceted; they not only affect the basic physiological processes of cells, but also participate in the regulation of plant responses to environmental changes. In practice, fine regulation of plant cell behavior can be achieved by precisely controlling the concentrations and ratios of different hormones, thus optimizing the effect of tissue culture.
Plant tissue culture typically consists of the following stages: explant culture, infection with agrobacterium tumefacients, dedifferentiation to form callus, and redifferentiation to generate roots and shoots. Each stage requires a specific medium formulation to meet the needs of plant development.
Stage of explant culture
The explant culture stage is the starting point of tissue culture, and its purpose is to prepare the explant by culture for the next step of agrobacterium infection.
Explant medium components:
•MS medium: Basal medium containing the following specific ingredients:
o Potassium nitrate (KNO3) : 1900 mg/L
o Ammonium sulfate [(NH4) 2SO4]: 1650 mg/L
o Potassium dihydrogen phosphate (KH2PO4) : 170 mg/L
o Magnesium sulfate (MgSO4·7H2O) : 373 mg/L
o Iron salt (ferric sulfate heptahydrate and sodium ethylenediamine tetraacetic acid) : 27.8 mg/L ferric sulfate heptahydrate (FeSO4·7H2O) and 37.3 mg/L sodium ethylenediamine tetraacetic acid (NaFeEDTA)
o Otrace elements, including zinc sulfate, manganese sulfate, boric acid, sodium molybdate, copper and cobalt chloride and so on, each element has a particular concentration of mg/L
o Vitamins such as niacin (0.01 g/L), pyridoxine (0.01 g/L), thiamine (0.01 g/L) and glycine (2 mg/L)
•AGAR: 7 g/L, as a coagulant.
•Sucrose: 30 g/L to provide carbon source and energy
Hormone proportion:
At this stage, no or only lower concentrations of phytohormones are usually added to maintain the original state and viability of the tissue.
Stage of infection by Agrobacterium tumefaciens
The purpose of the agrobacterium infection stage was to co-culture the explants with Agrobacterium to achieve the transformation of genes.
Components of Agrobacterium tumefaciens infection medium:
•MS medium: Basal medium, may need to be supplemented with antibiotics such as rifampicin and K+ to screen for transformed cells.
•AGAR: 7 g/L
•Sucrose: 30 g/L
Hormone ratio:
The use of auxin and cytokinin depends on transformation efficiency and the need for target gene expression.
Dedifferentiation to form callus stage:
At this stage of the goal is to make explantation cells lose differentiation status, the formation of callus.
Dedifferentiation medium components:
•MS medium: Basal medium
•AGAR: 7 g/L
•Sucrose: 30 g/L
Hormone ratio:
•Auxin (e.g., 2,4-D) : 1-2 mg/L to promote callus formation.
•Cytokinins (e.g., 6-BA) : 0.1-0.5 mg/L to aid in the dedifferentiation process.
•The ratio of auxin (e.g., 2,4-D) to cytokinin (e.g., 6-BA) is about 2:1 to 4:1.
Redifferentiation generates the root and shoot stages
The goal of the redifferentiation stage is to promote the differentiation of the callus into roots and shoots, and then to form a complete plant.
Redifferentiation medium components:
•MS medium: Basal medium, which may require adjustment of the proportions of specific elements and hormones.
•AGAR: 7 g/L
•Sucrose: 30 g/L
Hormone ratio:
•Auxin (e.g., IBA or NAA) : 0.1-1 mg/L for root formation
•Cytokinin (e.g., 6-BA) : 1-2 mg/L to promote bud differentiation
•The ratio of auxin (such as IBA or NAA) to cytokinin (such as 6-BA) is about 1:10 to 1:5
Conclusion
Each stage of plant tissue culture requires carefully designed media and hormone ratios to ensure a smooth transition of plant cells from one state to another. By precisely controlling the composition and hormone levels of the medium, the development, differentiation and regeneration of plant tissues can be effectively guided, so as to obtain good transgenic plants, mutants and other plant experimental plants. Aladdin helps plant research by providing comprehensive biochemical reagents with high quality and high purity reagents to help life science.
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