Plantbiotech notes done by Miss Lim


What is plant tissue culture?


 Refers to the sterile, in vitro, cultivation of plant parts such as organs, embryos and seeds, as well as single cells on either solidified or liquid nutrient media.

 Tissue culture: Refers to the aseptic growing of excised plant parts in vitro.

 Unique to plants is the ability of non-dividing cells to revert to an undifferentiated state that has meristematic (capable of cell division) activity. These cells have the ability to divide and differentiate into a whole plant (i.e. exhibits totipotency).

o Clones are genetically identical to the parent and to each other.



Micropropagation

 Plant tissue culture – also known as micropropagation if the technique is used to clonally produce thousands of plantlets.

 Is a vegetative reproduction method (a type of asexual reproduction in plants whereby individuals are obtained without the production of seeds or spores)

 Can be combined with genetic engineering to give rise to new types of plants



Stages in micropropagation

Stage 1 - Initiation of sterile explant culture: the selection of explants, sterilization of tissue surface to prevent contamination, and transfer of explants to nutrient media

(1) Selection of explants

 Plant growth is usually initiated from a piece of tissue or an organ (mature and differentiated) that is removed from a plant (called an explant).

 When grown on a specific nutrient medium in the presence of a specific ratio of growth hormones, the non-dividing cells revert back to an undifferentiated, meristematic state whereby they form callus tissue.

 Callus tissue has the ability to differentiate into a whole plant or plant organ in a process called redifferentiation.

 The ability of a plant cell to give rise to a whole plant through the process of dedifferentiation and redifferentiation is called totipotency  an ability unique to plants.





 Pieces of stem tissue with nodes, flower buds, leaves or tiny sections of the shoot tip meristem and even epidermal tissue can be used.

 Young explants are chosen as they contain a higher proportion of actively-dividing cells that are more responsive to callus initiation efforts.

 Storage organs and cotyledons are also good choices of explants e.g. potato tuber, storage roots of carrots etc., and cotyledons of soy bean.

 The plant that supplies the material, cells or tissues, that is cultured is called the parent plant. This donor plant should be free from diseases and is actively growing.

 The donor plants carries desirable characteristics such as higher yield, better quality fruits or carry genes for resistance to diseases.



(2) Establishing aseptic cultures

 The surfaces of explants (e.g. shoot tip, root tip and very small pieces of leaf and stem) are sterilized using dilute sodium hypochlorite (e.g. Clorox) to kill bacterial and fungal pathogens that may live on the surfaces.



(3) Transfer of explants to nutrient media

 Explants are transferred aseptically to culture vessels containing mineral nutrients, vitamins, carbohydrate sources (e.g. sucrose) and growth regulators (auxins, cytokinins etc.).

 The medium is usually solidified using agar to provide a firm matrix for developing tissues.

 The containers holding the explants are then sealed and incubated for 1 – 9 weeks.

 During this period, a mass of undifferentiated tissue called callus develops.



(4) Rapid multiplication phase

 Calli (singular: callus) can be repeatedly subcultured onto new culture media to obtain required multiplication rates.

 The proportion of cytokinins to auxins must be controlled.

 A higher proportion of auxins is required for proliferation of callus.







Formation of callus:

 Plants form callus in nature in response to plant injury with cells around the injury rapidly dividing.

 The stimuli involved in the initiation of wound callus are endogenous auxin and cytokinin that results in cell division. Invasion by microorganisms and feeding by insects also induce callus formation.

What do we want calli (singular = callus) for?

 They are used because they

o are the fastest method for shoot multiplication and cloning of a plant species.

o have the potential to develop normal roots, shoots and embryoids that can form plants.

o can be a source of protoplasts as they have thinner cell walls that can be digested more easily.

o can be used to screen for cells with special characteristics e.g. herbicide resistance

Establishment of plantlet:

Differentiation: a cellular maturation process; involves changes in patterns of gene expression that affect the structure and functions of cells.

 Rapid multiplication is arrested.

 Process to induce plantlet formation: shoot elongation, root formation, formation of storage organs etc.

 The plant tissue is induced to form plantlets by modifications of the culture medium.

 Different ratios of plant growth hormones induce shoot and root formation.

Stage 2 – Shoot initiation: multiplication of shoot tissue from explants on nutrient media

 To stimulate development of adventitious shoots (shoots from callus), a high cytokinin: auxin ratio is usually used

Stage 3 - Root initiation: multiplication of root tissue from explants on nutrient media

 To stimulate root formation, a high auxin: cytokinin is used.

 Usually the lowering of cytokinins or eliminating it totally and with addition of auxin, will promote root development.

 The callus will differentiate and eventually form plantlets.



Stage 4 – Transfer of plants to sterile soil or other substrate under controlled conditions (acclimatization)

 The plantlets are usually incapable of growing outside of the culture vessel.

 Acclimatization involves slowly weaning the plantlets from a high humidity, low light, warm environment (of the in vitro culture) to what would be considered a normal growth environment of the particular species grown.

 It is grown for 4-8 weeks in a green house as a weaning process.

 The plant is taken out from a culture vessel, the agar washed and the plant soaked in fungicide and then grown in sterile soil.

 Rooting hormones may be used to stimulate root development.

 Plants are eventually transplanted to a uniform medium that adequately supports the plant, and is sufficiently porous to allow adequate drainage and aeration.










Advantages of cloning plants by plant tissue culture (micropropagation)


(1) Rapid multiplication: Plants with desired traits can be multiplied rapidly than can be done using conventional plant breeding techniques, which rely on sexual reproduction.

(2) Genetic uniformity: Since plants produced are genetically identical, they all possess the desirable features of the stock plants. It is difficult to produce plants that breed true (homozygous for the desired traits) when sexual reproduction is used.

(3) Production of disease-free plants: There is a possibility of eliminating viral, bacterial and fungal contamination. Pathogens do not normally penetrate to the tips of meristems usually used for explants.

(4) Takes up little space compared with plants growing in the fields.

(5) Production of rooted plantlets ready for growth (faster), rather than seeds or cuttings.

(6) Production of often, more robust plants, leading to accelerated growth compared to similar plants produced by conventional methods.

(7) Propagation is independent of climatic changes, since tissue culture is carried out in a controlled environment.

(8) Effective means of asexual reproduction in some plant species. Some plants like bananas are sterile. Seeds of some plants such as orchids are difficult to germinate.

(9) Ability to air-freight large quantities of plantlets quickly and efficiently.

(10) Genetic engineering: tissue culture is an important tool for genetic engineering. It is the only viable method for regenerating genetically modified cells or cells after protoplast fusion.



Disadvantages of cloning plants by plant tissue culture (micropropagation)

(1) Contamination: a problem faced by commercial tissue culture laboratories is contamination. Contamination can cause very high losses in a short time. An infected plant sample can produce infected progeny. Stocks are usually checked carefully to prevent this.

(2) High production costs: micropropagation requires sophisticated facilities, sterile laboratory conditions and special nutrient media. It also requires trained personnel (i.e. skilled labour) with specialized skills, and a lot of effort to transfer the plantlets from the laboratory to the soil. Mechanization of the process would eliminate most of the labour cost associated, but this has proven difficult so far despite active attempts to develop this technology.

(3) Losses incurred during transfer of plant material from in vitro conditions to the acclimatization stage.

(4) Higher than acceptable levels of somatic variation / somaclonal variation. Callus cultures sometimes undergo genetic changes.

 Taken from plant biotechnology extra reading materials

Somaclonal variation: the genetic variability produced by plant tissue culture.

 Variability can be exploited to improve characteristics of crop and ornamental plants.

 Examples of somaclonal variants and their improved characteristics obtained in different crop species include:

 Corn – herbicide resistance

 Wheat – grain colour and height

 Sugarcane – sugar content, yield and disease resistance

 A number of different factors influence the presence of somaclonal variation during plant tissue culture. Explant sources, the length of time cells are in culture, the culture conditions such as growth hormone types and concentrations, and selective agents such as herbicides or other toxins that are introduced in low doses influence the degree of success of finding plant variants in culture.



 This variability is caused by changes in the chromosome number and structure of cells of explants being cultured.

 Examples of changes that influence genetic variability include:

1. Chromosome rearrangements

2. Single-gene mutations

3. Gene amplification (increase in gene copy number)

4. The activation of transposable elements.

 The resulting plants differ from the original parent plant (Note: Can be a disadvantage if you want genetically identical plants).

 These mutations occur randomly so that the regenerated mature plant is not genetically identical to the parent tissue.



Plant tissue culture (micropropagation) is a broad term used to define different types of in vitro plant culture. Six different types of in vitro cultures are recognized. Each type can result in a whole plant.

1. Callus culture – culture of differentiated tissue from an explant that dedifferentiates. (Pg. 1-4 of this set of notes)

2. Cell culture – culture of cells or cell aggregates (small clumps of cells) in liquid medium – need not know (need not know)

3. Protoplast culture – culture of plant cells with their cell walls removed (refer to extra readings / plant biotechnology reading)

4. Embryo culture – culture of isolated embryos (refer to extra readings

5. Seed culture – culture of seeds to generate plants (need not know)

6. Organ culture – culture of isolated plant organs such as anthers, roots, buds and shoots (refer to extra reading)