Introduction
The first and foremost question is what Plant Tissue Culture (PTC) is? Why do we need culture tissues when the plant can grow on their own? So, PTC is a technique where the totipotency (capability to differentiate from one cell into another cell) of the plants is utilized to generate the clones of the whole plant from cells, tissues or organs on artificial media under the aseptic in-vitro controlled environment. Even though the plants can develop independently, some plants can’t produce viable seeds thus, propagated through PTC. Moreover, there is a list of applications such as large scale multiplication, disease elimination, plant improvement and production of secondary metabolites, crop improvement and production of transgenic plants. It is also a means of understanding the constituents responsible for cell differentiation and organ formation.
History
Although tissue culture began in the early 18th-century, the foundation of plant tissue culture was laid by the father of tissue culture, German botanist Gottlieb Haberlandt. In 1898, he first attempted in vitro to grow palisade tissues from the leaves, pith, epidermis and epidermal hairs; however, cells survived only for a month. Later in 1902, he proposed the concept of in-vitro cell culture. After that, a landmark of discoveries occurred, such as in 1904 embryos of several cruciferous sp. cultured by Hannig; root and stem tips successfully cultured by Kolte and Robbins in 1922; first plant growth hormone Indole acetic acid discovered in 1926 by Went; Vit. B growth supplement in tissue culture introduced by White in 1934 demonstrated in tomato root tip; Gautheret, White and Nobecourt established endless proliferation of callus cultures in 1939; using shoot tip culture whole plant of Lupinus raised by Ball in 1946; Callus tissues were broken into single cells by Muir in 1954; In 1955 and 1957 Skoog and Miler discovered kinetin as cell division hormone and a concept of hormone control of organ formation respectively; in 1960 first time enzymatic degradation of the cell wall and protoplast were generated by Cocking; cell suspension and isolated single cells by plating by Bergmann in 1960; In 1962 Murashige and Skoog developed MS medium with higher salt concentration; In 1964 first haploid plants from pollen grains of Datura (Anther culture) produce by Guha and Maheshwari; Power et al. in 1970 achieved protoplast fusion; in 1974 Reinhard introduced biotransformation in plant tissue cultures; – Ti plasmid DNA from Agrobacterium tumefaciens integrated in plants by Chilton et al. in 1977; somatic hybridization of tomato and potato resulting in pomato in 1978 by Melchers et al.; term somaclonal variation introduced by Larkin and Scowcroft in 1981; in 2005 Rice genome sequenced under International Rice Genome Sequencing Project (Altaf Hussain et al., 2012). Nevertheless, the research and use of plant tissue culture for different applications have continued until the present day, and a lot of advancement will occur in the future.
Culture media
Picture Credit – Innovative Instrument
Inorganic salts/nutrients include,
Macro Nutrients: Nitrogen (NO3, NH4), phosphorus (K2HPO4, KH2PO4 or Na salts), Sulphur (SO4), Magnesium (MgSO4), Calcium (CaCl2, Ca(NO3)2), Potassium (KCl, K2HPO4).
These have structural and functional in protein synthesis (N & S), nucleotide synthesis (P, N & S), cell wall synthesis (Ca), enzyme co-factors (Mg), and membrane integrity (Mg).
Micro Nutrients: Boron (B), Cobalt (Co), Iron (Fe–Usually chelated with NaEDTA), Manganese (Mn), Molybdenum (Mo), Copper (Cu), Zinc (Zn), Iodine (I).
Fe & Mn are essential for photosynthesis, Mo, Zn & Cu are important co-factor for many enzymes, B is crucial for lignin Biosynthesis.
Hormones: The concentration and ratio differ and are standardized as per the particular plant.
Auxin: Used to stimulate cell elongation, cell division, embryogenesis, organogenesis, and embryogenesis. Callus induction takes place at an intermediate level of their ratio. Synthetic counterpart/analog used in culture media IAA (Indol-3-acetic acid), 2,4-D (2,4-Dichlorophenoxyacetic acid), BTOA (2-Benz-thizolyacetic acid), NAA (Alpha-Nephthaleneacetic acid), IBA (3-indol butyric acid).
Cytokinin’s: Used to induce shoot bud and cell division in tissue culture.
These are kinetin (6-furfurylaminopurine), 6-BAP (6, benzylaminopurine), Zeatin, 2iPA (2, isopentenyladenine)
Note: Naturally occurring forms of Auxin and Cytokinin quickly degraded outside the living system; thus, synthetic analogs are used in PTC. High Auxin : Cytokinin ratio is used for root regeneration, whereas low levels give rise to shoot regeneration.
Gibberellins lead to organogenesis (particularly adventitious root formation) and cell division in cell suspension culture.
Out of more than 70 isolated gibberellin compounds, only GA3 and GA4+7 are often used in PTC.
Abscisic Acid: Utilized for somatic embryogenesis, particularly during maturation and germination.
Vitamins: mesoinositol, nicotinic, pyridoxine, thiamine etc.
2 to 6% Sucrose (osmotic stabilizer)and glycine are the most common carbohydrate and Amino acid resp. Used in culture media.
Apart from all these growth regulators in various tissue cultures, Activated Charcoal reduces toxicity by adsorbing brown-black pigment and oxidized phenolics produced during culture. It promotes growth and differentiation in Orchids, carrots, ivy, and tomato, inhibiting growth in tobacco, soybean, etc.
Based on the constituents, the culture medium is classified as chemically defined and chemically undefined.
Chemically Defined: The medium, composition, and concentration of organic and inorganic constituents are known. e.g., White’s medium
Chemically Undefined: As for some media employ natural products/complex growth regulators thus the composition and concentration of the constituents cannot be defined. Example coconut milk (Diphenylurea), casein hydrolysate (common amino acid), yeast extract (source of organic N and vitamins), malt extract, potato extract (essential nutrients and vitamins), etc.
Preparation of Culture media
To differentiate or regenerate the whole plant from explant in the in-vitro condition, we must supply the media with the required amount of suitable inorganic salts(macro and micro salts), vitamins, carbohydrates, amino acids, and hormones.
For any tissue culture experiment first and critical step is media preparation. For any tissue culture experiment first and crucial step is media preparation. The culture media can be solid or semisolid or liquid prepared aseptically. Primarily solid and semisolid media designed for callus culture and liquid medium for suspension culture. The culture media composition depends upon the type of plant species used and the part or type of plant material you will use (Protoplast, tissues, cells etc.). For example, white’s media has low salts and are suitable for root culture whereas, the MS medium has high salts (high N & P) essential for plant regeneration from tissues and callus. Similarly, B5 and Nitsch’s medium has lesser amounts of nitrate & ammonium salts and Salt concentration intermediate than MS medium, respectively, making B5 suitable for Protoplast culture and Nitsch’s medium for anther culture.
Even though media parameters and composition vary from species to species and type and part of the plant used as explant, specific do’s and don’ts we have to take care of while preparing the media are
Water: only Double distilled (DDW), Treated drinking water (TDW) or Millipore water with neutral pH, free from contaminants and ions
It is advisable to prepare a stock solution of different components and then mix as per the requirement, as the need is in trace amount, which is difficult to weigh and can lead to concentration error.
One should use an accurate amount of each components.
The deficiency or surplus of any component leads to the problems like inhibition of protein synthesis, inactivation of enzymes, delayed growth, osmotic pressure imbalance, decrease in photosynthesis etc.
Preferably, thermolabile organic constituents such as vitamins, growth regulators and amino acids are 0.22 – 0.45 µm filter (syringe or membrane filter unit) sterilized and added to the autoclaved media once it is cooled down to 35-40 °C.
Prior to the addition of solidifying agents and autoclave, the pH of the media should be checked (appx. 5.5-5.8), as it affects the solidification of media.
Antibiotics can be used to avoid the contamination or as a selection medium for identification of transgenic callus/embryo.
For non living articles (nutrient media, glass good, DDW, instruments) a routine sterilization procedure should be followed.
Sterilization of plant material
Sterilization on plant material is to be cultured performed in laminar airflow or inoculation chamber before the inoculation on culture media. This procedure is followed to eliminate the surface born micro-organism.
Thoroughly washed plant material in tap water
Immerse in 5% v/v solution of liquid detergent (Teepol) for 10-15 min followed by a tap water wash
Dip explant in 70% ethanol for 60 sec
Transfer autoclave jaw bottle containing 0.1% v/v mercuric chloride (HgCl2) for 10 min.
After 10 min, remove all traces of HgCl2 with several rounds of sterile distilled water.
The surface-sterilized free from contaminants is used to regenerate clones/transgenic plants.
References
Bhatia, S. (2015). Plant tissue culture. Modern applications of plant biotechnology in pharmaceutical sciences, 31-107.
Hussain, A., Qarshi, I. A., Nazir, H., & Ullah, I. (2012). Plant tissue culture: current status and opportunities. Recent advances in plant in vitro culture, 1-28.
Saad, A. I., & Elshahed, A. M. (2012). Plant tissue culture media. Recent advances in plant in vitro culture, 30-40.