Plant Tissue Culture History & Techniques PDF

- But why do we even do tissue culture on plants? - it can be done for various purposes - commercial cultivars--- help reduce costs of orchid plants; economic advantage; fast commercial propagation - agricultural purposes--- crop improvement, reduced food...

- But why do we even do tissue culture on plants? - it can be done for various purposes - commercial cultivars--- help reduce costs of orchid plants; economic advantage; fast commercial propagation - agricultural purposes--- crop improvement, reduced food insecurity and shortages, improving nutritional value, pest control, disease resistance, etc. - pharmaceutical or medicinal purposes--- to extract certain substances like that could be medically beneficial - plant conservation--- saving rare or endangered plants by multiplying them to make a large number of the same species in less time or by providing them a safer environment; safer to export - crossing or breeding--- crossing distantly related species by protoplast fusion and regeneration of novel hybrids - for chromosome doubling and induction of polyploids--- helps increase food size and improve taste as well as nutritional value - **History of Plant Tissue Culture** - 1756--- Durhamel du Monceau H. L discovered callus formation from decorticated elm trees - 1839--- Schwann T.H was of the view that that each living cell of a multicellular organism is able to develop into independent organisms if provided proper external conditions (climate, pH, nutrients, growth hormones, etc) (Schwann was right but could not be fully proven until the father of tissue culture made some innovations) - 1893--- Rechinger C tried to isolate stem fragments and root slices of varying thickness from callus formation. He found that the minimum limit of divisibility which is 1.5 mm. He believed that a slice must be thicker than 1.5 mm to be divisible (He was wrong, but sort of right at the time because microscopes and technologies didn't exist at the time) - note: none of these scientists used artificial or synthetic media or nutrients, they did not have microscopes or autoclave machines and any other technologies for successful experimentation - 1902--- Haberlandt G is the father of plant tissue culture due to his experiments on the culture of isolated plant cells. He also stated why some of his experiments failed in his publications. He also found that: - unlike Rechinger, unlimited fragmentation wouldn't influence cell proliferation - culture mediums could grow well using Knops solution, asparagine, peptone (a protein hydrolysate formed by an amino acid with enzymatic method or acidic dilution) and sucrose - He prepared this medium which helped show which components can promote growth - He stated the reasons for failure in his experiments to be the following: 1. Selection of Genera: He selected a monocot which is more challenging to grow than dicots. Even to this day, monocots are hard to grow 2. Selection of Explant: He believed that he selected the "wrong parts" of an explant. He selected mature differentiated mesophyll cells and palisade tissues. He stated that undifferentiated cells should have been selected instead. You could say that he used recalcitrant explants as the growth response was not that good. Even today, although it is harder to grow differentiated cell cultures, it is still possible but not that preferred. We can culture any part of a plant today. 3. Contamination: He was unable to control contamination. Even today, 90% of the time, scientists face contamination issues in their tissue cultures. Only 10% yield is free of contaminants. This is just nature's way of doing things. No amount of technology has helped solve this problem. - 1922--- Robbins W J excised meristematic root and stem tips of maize under sterile conditions. This term did not exist at the time of Haberlandt. As for Robbins, despite sterile conditions, his cultures did not survive for a long time which was a gap of research or a drawback that other scientists later tried to fill. Today, you can keep a culture alive for as long as you want, given that you frequently provide favourable conditions and subculture it. It depends on what you intend to do with that culture. Some cells in the culture will still die though. - 1934--- White P R continuously grew cultures of meristematic cells of tomato using a medium. For the first time in history, someone was able to grow a "continuous" culture using unspecialised meristematic cells. The gap of research was now filled. He used a medium of salt, yeast extract, sucrose and 3 vitamin Bs (pyridoxine, thiamine and nicotinic acid) - 1902---1934 --- **Golden period** of plant tissue culture. This is because satisfactory mediums were successfully formulated which led to easier experimentation. - 1941--- Van Overbeek and his colleagues established the importance of coconut milk for the growth and development of very young Datura embryos. They have medicinal properties for intensinal problems and stomach ulcers. - 1942--- White and his colleagues initiated studies on crown gall and tumor formation in plants. Agrobacterium Tumefaciens is a natural genetic engineer which causes proliferation of undifferentiated cells. We now also have a gene gun method but it is much more expensive which is why A. tumefaciens is preferred. - 1944--- Skoog F started his work on organogenesis in tobacco callus. Tobacco is a model crop for tissue cultures. Organogenesis is the development of organs or the regeneration of organs. - 1951--- Morel G and Wetmore R H got successful cultures from monocots which were once considered recalcitrants to the cultural condition. - 1952--- Steward and his colleagues discovered the synergistic action of 2,4-D (2,4- Dinitrile hydrazine). - Synergistic action definition: - refers to the phenomenon where two or more plant growth regulators (PGRs) interact in a way that produces a **greater effect** than if they were used individually. This enhanced response is often observed when auxins and cytokinins are combined - 1958---1960--- Morel G and Martin C cultured orchid plants using yeast extract, fruit juice, protein hydrolysate and undefined compounds (compounds where you don't know the exact composition of components in a solution like CHON or how many vitamins are in a compound, etc. You just know that it's there). Casein is usually used to make protein hydrolysate - 1959--- Reinert J observed somatic embryo formation from callus cultures of carrot grown on agarified medium. - Molten agar: agar is solid, it is boiled to a 100C to form a hot liquid. It cannot be poured directly on a culture this hot so it is cooled down to around 40C called molten agar, which you then mix with cell suspension color, hence the agarified medium. - Somatic embryos are formed to avoid waiting for season or supply. We can produce embryos in any season in any quantity without depending on low yield cultivars, seasons or suppliers. - 1959--- Melchers and Bergmann were the first to culture haploid tissues (n or 1n). They are used for various reasons but the most important one is to study the effect of recessive mutations. Other reasons include making homozygous plants from n haploid plants. It can also be done to pollinate more plants, to perform anther or egg cell culture, to preserve an old plant or to get desirable traits. - 1960--- Cocking discovered the technique of isolation and culture of protoplasts. He removed the cell wall enzymatically of a tomato fruit locule to form a protoplast. A protoplast has no cell wall. They are plant or bacterial cells without cell walls. They are used for transformation and fusion, regeneration, and isolation. Protoplasts can be used to extract anything. Somatic hybrids have been developed from somatic cells. Protoplasts can be fused together such as the somatic fusion of a tomato and a potato to form a pomato. - 1960--- Bergmann was the first to plate cells from suspension cultures on to a solid medium. This became known as the Bergmann Plating Technique. - 1960--- Morel discovered a technique to produce virus free progenies by meristem culture in Cymbidium.