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Questions and Answers
Propagation can occur through ______ buds.
Propagation can occur through ______ buds.
terminal
Micropropagation can free plants from specific ______ diseases.
Micropropagation can free plants from specific ______ diseases.
virus
Micropropagation allows for a more ______ adjustment of factors influencing vegetative regeneration.
Micropropagation allows for a more ______ adjustment of factors influencing vegetative regeneration.
flexible
Plants may acquire a new temporary ______ through micropropagation.
Plants may acquire a new temporary ______ through micropropagation.
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A specialized and ______ production facility is needed for micropropagation.
A specialized and ______ production facility is needed for micropropagation.
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Explants and cultures must be grown on a medium containing ______ or another carbon source.
Explants and cultures must be grown on a medium containing ______ or another carbon source.
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Micropropagated plantlets are more susceptible to ______ loss in an external environment.
Micropropagated plantlets are more susceptible to ______ loss in an external environment.
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Mother plants must be ______ of the variety or species.
Mother plants must be ______ of the variety or species.
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The customary second step in micropropagation is to obtain an ______ culture.
The customary second step in micropropagation is to obtain an ______ culture.
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Shoot cultures are conventionally started from the apices of ______ or main shoots.
Shoot cultures are conventionally started from the apices of ______ or main shoots.
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Larger explants, while sometimes used with advantage, can be more difficult to ______ from micro-organisms.
Larger explants, while sometimes used with advantage, can be more difficult to ______ from micro-organisms.
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The shoot tip used are usually macerated from shoots originating from ______ tip culture.
The shoot tip used are usually macerated from shoots originating from ______ tip culture.
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According to some researchers, there is a competition between cell proliferation and the formation of ______ particles in the meristem region of a plant.
According to some researchers, there is a competition between cell proliferation and the formation of ______ particles in the meristem region of a plant.
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The growth and proliferation of axillary shoots in shoot cultures is promoted by incorporating ______ into the growth medium.
The growth and proliferation of axillary shoots in shoot cultures is promoted by incorporating ______ into the growth medium.
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In some plants, pinching out the main shoot axis is used as an alternative, or an adjunct, to the use of ______ for decreasing apical dominance.
In some plants, pinching out the main shoot axis is used as an alternative, or an adjunct, to the use of ______ for decreasing apical dominance.
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Conventional shoot culture continues to be the most important method of ______, although node culture is gaining in importance.
Conventional shoot culture continues to be the most important method of ______, although node culture is gaining in importance.
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Single ______ culture is another in vitro technique that can be used for propagating some species of plants from axillary buds.
Single ______ culture is another in vitro technique that can be used for propagating some species of plants from axillary buds.
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In this method, intact individual shoots may be placed on a fresh medium in a ______ position.
In this method, intact individual shoots may be placed on a fresh medium in a ______ position.
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Each shoot may be cut into single-, or several node pieces which are ______.
Each shoot may be cut into single-, or several node pieces which are ______.
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Sucrose increases the osmotic potential when it is hydrolyzed into ______ and fructose.
Sucrose increases the osmotic potential when it is hydrolyzed into ______ and fructose.
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Only thiamine (vitamin B1) is essential in a culture, as it is involved in carbohydrate ______.
Only thiamine (vitamin B1) is essential in a culture, as it is involved in carbohydrate ______.
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Activated charcoal is useful for absorbing brown or black pigments and oxidized ______ compounds.
Activated charcoal is useful for absorbing brown or black pigments and oxidized ______ compounds.
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Agar is used to solidify tissue culture media into a ______.
Agar is used to solidify tissue culture media into a ______.
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Agarose is a purified extract of agar that leaves behind agaropectin and ______ groups.
Agarose is a purified extract of agar that leaves behind agaropectin and ______ groups.
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Murashige and Skoog (MS) medium is the most commonly used basic tissue culture medium for plant ______ from tissues and callus.
Murashige and Skoog (MS) medium is the most commonly used basic tissue culture medium for plant ______ from tissues and callus.
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Agar should be used at a concentration of 0.6 – 0.8% to properly ______ the medium.
Agar should be used at a concentration of 0.6 – 0.8% to properly ______ the medium.
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After fertilization, the new egg cell nucleus is ______.
After fertilization, the new egg cell nucleus is ______.
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The ______ of the embryo pushes it into the developing endosperm for nutrition.
The ______ of the embryo pushes it into the developing endosperm for nutrition.
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Protoplasts isolated from embryogenic suspensions may give rise to ______ embryos directly.
Protoplasts isolated from embryogenic suspensions may give rise to ______ embryos directly.
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Indirect embryogenesis provides an efficient method of ______.
Indirect embryogenesis provides an efficient method of ______.
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To increase somatic embryos on zygotic embryos of sunflowers, the larger embryos were cut into ______ equal pieces.
To increase somatic embryos on zygotic embryos of sunflowers, the larger embryos were cut into ______ equal pieces.
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Stage II focuses on the production of suitable ______.
Stage II focuses on the production of suitable ______.
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During Stage III, steps are taken to grow individual plantlets capable of carrying out ______.
During Stage III, steps are taken to grow individual plantlets capable of carrying out ______.
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In Stage IV, plantlets are transferred from in vitro to the ______ environment.
In Stage IV, plantlets are transferred from in vitro to the ______ environment.
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Micropropagated plantlets kept in low-light conditions are not fully dependent on their own photosynthesis and require ______.
Micropropagated plantlets kept in low-light conditions are not fully dependent on their own photosynthesis and require ______.
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The process of transferring plantlets must be done carefully to avoid loss of propagated ______.
The process of transferring plantlets must be done carefully to avoid loss of propagated ______.
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In the initial stages, shoots developed in culture are produced in high humidity and low light ______.
In the initial stages, shoots developed in culture are produced in high humidity and low light ______.
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A typical change in plantlets occurs after they have spent several days ______.
A typical change in plantlets occurs after they have spent several days ______.
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The term ______ culture is preferred for starting cultures from intact shoot meristems.
The term ______ culture is preferred for starting cultures from intact shoot meristems.
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Somatic embryos, axillary, or adventitious shoots can serve as a basis for further cycles of ______.
Somatic embryos, axillary, or adventitious shoots can serve as a basis for further cycles of ______.
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The stomata of leaves produced in vitro may be atypical and incapable of complete ______ under low humidity.
The stomata of leaves produced in vitro may be atypical and incapable of complete ______ under low humidity.
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Study Notes
Sugar
- Nutrient salts contribute approximately 20-50% to the osmotic potential of the medium, and sucrose is responsible for the remainder.
- Sucrose's contribution to osmotic potential increases as it hydrolyzes into glucose and fructose during autoclaving.
- This is important when performing osmotic-sensitive procedures like protoplast isolation and culture.
Vitamins
- Vitamins are organic substances, acting as parts of enzymes or cofactors for metabolic functions.
- Thiamine (vitamin B1) is crucial for carbohydrate metabolism and the biosynthesis of some amino acids in cultures.
Activated Charcoal
- Activated charcoal effectively absorbs brown/black pigments and oxidized phenolic compounds.
- It also absorbs other organic compounds, including plant growth regulators (PGRs) like auxins and cytokinins, and other materials like vitamins, and iron and zinc chelates.
- Adding activated charcoal minimizes carryover effects of PGRs when transferring explants to media without PGRs.
- It darkens the medium, aiding root formation and growth, and promotes somatic embryogenesis and enhances growth/organogenesis of woody plant species.
Gelling Agents
- Agar is used to solidify tissue culture media into a gel (0.6 – 0.8%).
- It enables precise contact between explants and medium while maintaining aeration.
- Agar is derived from seaweed.
- Lower concentrations (0.4%) or low pH can result in a soft medium that does not gel properly.
- Typical tissue culture agar melts at ~65°C and solidifies at ~45°C.
- Agarose is a purified extract of agar, used in protoplast or single cell cultures. It leaves behind agaropectin and sulphate groups.
- Gellan gums (Gelrite™, Phytagel™) are alternative gelling agents, transparent for easy contamination detection.
- Gelling requires a specific concentration of divalent cations like calcium and magnesium.
Types of Plant Medium Cultures
- Murashige and Skoog (MS) medium (1962) is a suitable and widely used basic tissue culture medium for plant regeneration from tissues and callus.
- It was developed for tobacco using the mineral analysis of tobacco tissue.
- MS medium is a "high salt" medium due to its high content of K and N salts.
- To address salt sensitivity in woody species, Lloyd and McCown (1980) developed the woody plant medium (WPM).
- Gamborg's B-5 medium, designed for soybean callus culture, has lower levels of nitrate and ammonium salts than MS medium, commonly used for callus or suspension cultures.
- Developed for monocots and dicots, the Schenk and Hildebrandt (SH) medium.
- White's medium, suited for tissue culture of tomato roots, has a lower salt concentration than MS medium. (Specific compositions of each medium are given in Table 3.1)
Micropropagation Techniques
- Micropropagation is the mass vegetative production of plants in vitro for commercial goals.
- Propagation can happen through terminal/axillary buds or adventitious shoots/embryos from somatic cells.
Micropropagation Advantages
- Using small plant pieces (explants) requires less space.
- Producing virus-tested plants.
- Greater flexibility in adjusting factors affecting vegetative regeneration.
- Propagating species difficult to reproduce sexually.
- Can result in new temporary characteristics (e.g. bushy habit in ornamentals, increased runner formation in strawberries).
- Continuous year-round production.
- Low attention needed between subcultures.
Micropropagation Disadvantages
- Requires a specialized and costly production facility.
- Cultures rely on sucrose or other carbon sources.
- Young plantlets are vulnerable to water loss in external environments due to high humidity during culture.
Stages of Micropropagation
- Stage 0 (Introduction): Mother plant selection and preparation. Plants must be representative of the intended variety and free of diseases. Treatment may be advantageous for in vitro culture success.
- Stage I: Establishing an aseptic culture. Transferring explants to a sterile environment, fostering shoot tip or callus growth. Discarding contaminated material.
- Stage II: Producing suitable propagules (new plants). Growing out axillary and adventitious shoots or somatic embryos that can be perpetuated.
- Stage III: Preparing for natural growth. Preparing plantlets for eventual transfer to the natural environment, enabling photosynthesis. Includes in vitro rooting of shoots.
- Stage IV: Transfer to the natural environment. Acclimating plantlets to external conditions. Techniques can alter developed shoots/leaves, especially regarding humidity tolerance.
- Additional Stage: Cultures need adjustment after transferring them from culture to the external environment. Appropriate nutrients and suitable proportion of auxin and cytokinin in the culture medium can control the changes occurring to the cells.
Shoot (or shoot tip) culture
- Shoot culture is the preferred naming for cultures originating from intact shoot meristems, allowing for further axillary branch formation (a rapid protocol).
- Shoot cultures can also be initialized from actively growing shoots or dormant buds. Larger explants allow for faster in vitro adaptation.
- The shoot tip is macerated from the shoot originating from the meristem tip. Researchers found that there is a competition issue between cell proliferation/virus particle formation in the meristem region.
- Shoot growth promotion in vitro is usually done by adding cytokinins (growth hormones) to the medium.
- Some cultures will benefit from removing the main shoot axis (pinching). This method reduces apical dominance.
Node culture
- Node culture is an in vitro technique for propagating plant species using axillary buds.
- Shoots can be separated and each cut into single- or multiple units.
- Leaves are usually trimmed to focus on the stem and lateral bud parts.
- In vitro layering is not an exact equal method to usual shoot cuttings.
- Media and methods for rooting are similar to those used in shoot culture.
- Node culture is valuable for species producing elongated shoots, such as potatoes or Alstroemeria, with a benefit towards commercial stability and less callus development.
Multiple shoots from seeds (MSS)
- MSS initiates shoot cultures directly from sterilized seeds placed on media containing cytokinin.
- Multiple shoots develop as germination occurs, allowing for serial subcultures.
- This method works for diverse species, including dicots (soybean, sugar beet, and almonds).
Somatic embryogenesis
- Somatic embryos are directly initialized from explanted tissues, often from the female gametophyte (ovaries, ovules).
- Somatic embryos have a similar structure to true seed embryos.
- These embryos, uniquely different from shoot/root buds, have recognizable shoot and root poles.
- Somatic embryos can be produced from many plant sources (nucellus tissue, adventitious embryos).
- Protoplasts from embryogenic suspensions may directly form somatic embryos without preceding callus formation.
Embryogenesis
- Includes descriptions of plant parts/stages in the embryo/seed (e.g., ovule, ovary, etc.).
Current Applications
- Sugar: Important for osmotic-sensitive procedures.
- Vitamins: Essential for crucial metabolic functions in cultures.
- Activated Charcoal: Helpful in minimizing PGR carryovers, improving root growth, and promoting somatic embryogenesis.
- Gelling Agents: For solidifying tissue cultures to support propagation procedures efficiently, especially in protoplast and single cell cultures.
- Micropropagation: A vital technique for mass production of plants, facilitating commercial plant production, virus removal, and accommodating specific plants difficult to propagate sexually.
- Shoot/node culture: Enables mass propagation, and it is increasingly popular for generating new plant parts.
- MSS (Multiple Shoots from Seeds): Can provide a quicker way to produce multiple shoots.
- Somatic embryogenesis: An important method for producing plantlets and is useful for plant conservation efforts.
- Callus culture: Used in various applications, like initiating cell suspensions, studying plant nutrition, and investigating cell/organ differentiation, morphogenesis, and somaclonal variation. It also enables genetic transformation and secondary metabolite production.
Stages of callus culture
- Induction Phase: Cells in the explant dedifferentiate and divide.
- Division Phase: There is rapid cell division.
- Differentiation Phase: Differentiation and organized structure formation. Includes organogenesis/somatic embryogenesis.
Notes on callus culture
- Explants in agar media with nutrients and appropriate auxin/cytokinin ratios display callusing that gradually covers the tissue surface.
- Callus cultures should be subcultured every 3-5 weeks. Repeated subculture improves callus friability.
- Subculture is the transfer of cultures to fresh media, considered a "passage."
- Callus cultures may display changes like variation.
Current Applications
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Manipulation of auxin/cytokinin ratios in medium influences shoot/root/embryo formation and subsequent whole plant development.
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Calluses can also develop into cell suspensions.
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Use in studying plant nutrition needs.
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Used for further study of cell/organ differentiation and morphogenesis.
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Study of somaclonal variation (natural genetic differences arising)
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Genetic transformation (modification)
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Production of secondary metabolites(useful compounds).
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Description
Test your knowledge on the basics of micropropagation techniques, including the role of explants, culture mediums, and the importance of disease-free propagation. This quiz will cover various aspects from the initial stages to the environmental considerations for micropropagated plantlets.
