Plant tissue culture

INTRODUCTION- For many centuries, humans are known for their dependence on pharmaceutical drugs and medicines for health compromise situations. Drugs are chemically structured compounds and gradually cause various side effects in human metabolism reactions. But technology is at its peak and has replaced drugs with various alternative techniques. Plant-derived metabolites after tissue engineering and gene mutation have shown a positive impact on disease-related issues. To produce active plant compounds, in-vitro plant tissue culture is to go for the technique. Plant tissue culture eliminates the dependence on wild forms or plants as well as to produce engineered metabolites, vaccines, and other beneficial products.

IN-VITRO TECHNIQUES- Cell culture can be performed on both plants and animal cells. Plant tissue culture and animal cell culture are both different techniques having similar agenda- Health benefits at the genetic level. In-plant tissue in-vitro culture techniques, the aim is to produce disease-free plants, rapid multiplication of rare plant genotypes, plant genome transformation, and production of plant-derived metabolites of important commercial value.

Tissue culture also known as micropropagation is a technique to grow a whole plant from a part of the mother plant. In other explanatory terms, it is a technique in which cells or tissues grow on an artificial nutrient medium in-vitro. The artificial medium can be semi-solid or liquid to produce primary and secondary metabolites and the regeneration of the whole plant. The main concept on which tissue culture relies is that plants possess the capacity to produce a whole plant, a clone of itself, without any root, stem, or leaves. By providing essential macronutrients and micronutrients, cells can grow on their own and this capability of plants is known as totipotency.

TISSUE DEVELOPMENT- The first step in developing plant tissue culture is the pre-treatment to the mother plant with fungicides and insecticides as disease-free and insect-free plants are preferred. The next step is an initiation in which, small plant pieces called explants are taken from the mother plant and the surface of parts is sterilized with chemicals. An appropriate culture media according to the plant is sterilized and explants are placed and incubated for a short time. Explant conversion to callus is known as callogenesis where cells divide to form an undifferentiated mass. The other process in which direct organ is propagated is known as organogenesis (like root or shoot multiplication). Then these organs/calluses are subcultures after every 4-5 weeks to prevent contamination. Finally, the micro propagated plants are hardened to grow individual plants capable of photosynthesis. The hardening is done gradually allowing the plants to acclimate to ex vitro conditions. Typically, the plants are taken from high to low humidity and from low to high light intensity.

INITIATION STAGE-

STERILIZATION OF PLANT MATERIAL- The first step towards plant tissue culture is to select a mother plant. Mother plant consists of microbes on their surface and living inside them, supporting plant growth and health. However, these microbes tend to become a contamination problem in tissue culture. This problem can be solved by sterilizing plant material with chemical disinfectants such as bleach (10%), alcohol (70%), and hydrogen peroxide (3%) in separate washes. This only sterilizes the surface of the plant tissue and contamination can still arise from inside the plant. Isolating and growing the specific tissue culture can help overcome this issue. Cleanliness and sterility play a major role in plant tissue culture as a single bacteria cell or fungi spore will cause contamination, therefore all work should perform under sterile conditions.

ISOLATION OF CELLS- The isolation procedure involves skilled technicians carefully dissecting the specific tissue under a microscope with fine tools, it should be about 20-50 microns in size. It can take 3-8 months to grow a full plant, requiring delicate maintenance and pathogen testing before being transplanted. Although it can be a lengthy and tedious process, tissue culture is a valuable tool that helps provide disease-free starter plants.

MULTIPLICATION STAGE-

CALLUS FORMATION-The culture medium contains several nutrients and growth regulators that stimulate cell division of explant. Darkness in the culture lab and the presence of agar in media gel helps in forming an irregular, un-organized, and undifferentiated mass of dividing cells called callus. It is generally obtained within 2-3 weeks and is a subculture for the maintenance and growth of cells. If cells grow on the same medium for some time, then gradually nutrients and water exhaust while metabolic toxins accumulate. To avoid this situation and further contamination, sub culturing is necessary up to a certain limit and should be repeated after every 4-5 weeks.

MEDIA FOR TISSUE CULTURE- It is essential to know what type of culture medium exists, what are their applications, and which one would be more suitable for your specific culture. Important components which will make a complete essential media are:

• Elements: Elements like Nitrogen, Phosphorus, Potassium, Calcium, Magnesium, and Sulphur are minimum for plant growth, and other elements like Iron, Manganese, Zinc, Boron, Copper, and Molybdenum are also vital components for a plant’s development.

• Carbon source: Sucrose, Galactose, Lactose, Maltose, and starch are a few examples of effective carbon sources to maintain the culture environment and provide basic energy for metabolic processes.

• Nitrogen source and extracts- For nitrogen sources generally amino acids are added which assimilate tissues immediately. Natural substances or extracts include protein hydrolysates, coconut milk, yeast extract, malt extract, ground banana, orange juice, and tomato juice that help in growth enhancement.

• Growth regulators- Auxins, Cytokinin, and Gibberellins are a few growth regulators that play an important role in stem elongation, tropism, and apical dominance.

• Antibiotics- Ampicillin, streptomycin, kanamycin, and cefotaxime are some of the antibiotics that are common for use in tissue culture laboratories.

• Gelling agents- There is a need for gelling agents to transform the liquid media into a semi-solid state and if not used, explants would submerge and die due to anaerobic conditions. The commonly used gelling agents are agar, agarose, gellan gum, and isubgol.

• There are some commonly used media in plant tissue culture mentioned below that are specific for only certain plants that majorly differ in salt concentrations, extracts, growth regulators, and nitrogen sources: MS MEDIA, LS MEDIA, B5 MEDIA, NN MEDIA, WHITE MEDIA, QL MEDIA, N6 MEDIA

STERILIZATION OF MEDIA- Plant tissue culture media is generally sterilized by autoclaving at 121 °C at 15-20 psi. The time required for sterilization depends upon the volume of medium in the vessel. Several medium components are considered thermolabile and should not be autoclaved. Stock solutions of the heat-labile components are prepared, and filter sterilized through a 0.22 µm filter into a sterile container. The filtered solution is aseptically added to the culture medium, which has been autoclaved and allowed to cool to approximately 35-45 °C. The medium is then dispensed under sterile conditions.

FORMATION OF ROOT, SHOOT, AND ACCLIMATIZATION- After media is dispended organ/callus is placed over media and the differentiation of organs (root and shoot) occurs from callus. This differentiation depends upon the auxins/cytokinin ratio. A high ratio favors root formation and low ratio favors shoot formation, while an appropriate proportion of the two favors root and shoot formation together. In this phase, the entire plant is obtained from the callus, it is also called the regeneration phase. Embryoids may also develop in this phase if the medium concentration is modified. The embryo is an independent structure devoid of vascular supply - it cannot be regarded as an organ. Plants are kept at low minimal salt medium for hours and are transferred to pots containing autoclaved sterilized mixture of clay and core soil leaf molds in equal proportions. Plantlets of the pots are covered with transparent polythene to maintain humidity for days. Plantlets then are transferred to the greenhouse for a week and then to the field. In the field, agricultural techniques are applied like other crops/ plants.

TYPES OF PLANT TISSUE CULTURE-

ROOT TIP CULTURE- Tips of the roots are sterilized, excised, and transferred to a fresh medium. They continue to grow and provide several roots after 7-8 days in-vitro. By this method nutritional requirements of roots, nodules, and root growth conditions can be studied.

SHOOT TIP CULTURE- Shoot tip also known as shoot apex is a simplified form of shoot culture from a pre-existing meristem. It is simple than other cultures as it is associated with the least genetic variation. Soyabean, sweet potato, and sugarcane crops have been freed of viruses due to this special technique.

LEAF CULTURE- The growth of leaves on the culture medium depends upon the stage of the leaves during excision. It is observed that explants from immature young leaves grow better than explants from older leaves. Leaves (800 µm long) may be detached from shoots, surface sterilized, and placed on a solidified medium where they will remain in a healthy condition for a long period. The shoot forming potentials differ in the leaf cultures as per the derivation of the explant and the hormonal factors involved.

FLOWER CULTURE- This culture system is useful in studying microclimates or nutritional effects on the vegetative and reproductive processes of the plant. Flowers two days after pollination are excised, sterilized by immersion in 5% calcium hypochlorite, repeatedly washed with sterilized water, and transferred to culture tubes containing an agar medium. When cultured, such flowers produce fruits. Larger fruits are obtained on medium supplemented with growth hormones. Flowers excised before pollination does not produce fruits.

ANTHER AND POLLEN CULTURE- Immature stage of anther or late stage of anther containing starch-filled pollen usually grow abnormally or the development is generally ineffective and hence for better response always select mature anther or pollen. Anther or pollen grains of different species have been successfully cultured to obtain many haploid plants. Pollen grains are removed from the anther either mechanically or by natural dehiscence. Anthers placed in 5 ml of the liquid medium in a petri dish containing pollen grains in the culture media are sealed with parafilm and incubated. After incubation haploid plantlets are developed.

ADVANTAGES AND APPLICATIONS OF PLANT TISSUE CULTURE- 1990 was the era of transgenic plants and means of important pharmaceutical protein production. From the past 20 years, plant tissue culture offers transgenic systems of decreased costs, easy delivery, and decreased risk of contamination in protein processing. There are many useful plants made human recombinant therapeutic proteins that have overcome several hurdles in disease and health. Few plants derived biological active compounds are as follows:

PLANT MADE PHARMACEUTICAL	PLANT	USE
NDV Vaccine	tobacco suspension culture	against new castle disease virus
VEN150	rice seeds	for HIV
Moss- GAA	moss	pompe disease
Moss-GBA	Moss Nicotiana benthmiana, Alfalfa	Gaucher's disease
Moss-AGLAL		Fabry disease
Vaccine		Influenza Rabies Rotavirus
Antibody	Duckweed leafy biomass	Non- hodgkin's lymphoma
Serum albumin	Rice seed
CaroRX	Tobacco leaves	for dental caries
PBI-220	Tobacco leaves	Antibody for anthrax

REFERENCE: In-vitro plant tissue culture: means for production of biological active compounds (2018)., Plant science laboratories., Plant cell technology., clinisciences