The process of plant tissue culture varies depending on the goal and circumstance of the researcher. Modern technologies often forego selection and use of embryos for such advanced methods that include utilization of plant tumor cells from any tissue type induced by Agrobacterium tumefaciens. However in poor and under-equipped areas, traditional knowledge still persists as to the cultivation of plant species in the laboratory.
The following scheme is an example of a system used in many developing countries for specific plants that are carefully chosen from manually inspected seeds before extraction of the embryo.
Before explantation, seeds that are at the studied age for picking are harvested. The age in months of the plant is crucial in order to take advantage of stigmatic receptivity of embryos to laboratory conditions, a fact well known by plant biologists.
After harvest the embryo is detached from the rest of the seeds using different modes of extraction. A cork borer can be used to take out the embryo with its endosperm cylinder, in tandem with safety measures that keep the young plant intact. A round rubber pad attached to a stirring rod can help push the embryo out of the seed body.
The embryo with meat cylinder is cleaned with soap and tap water, before rinsing with 95% ethanol and washing three times using distilled water. Around 5% of commercial bleach solution is prepared for final sterilization.
After the steps mentioned, inoculation and incubation proceeds with elimination of microorganisms in the glassware and instruments to be used; the culture media and the explants are sterilized as well. All materials to be used are set in a sterilized laminar hood. An alcohol lamp is prepared with a fireproof block covering to protect it from the blower of the laminar hood.
The cultivator's hands are disinfected with 80% ethanol, while the tools for manipulation are sterilized by immersing in 95% ethanol and passing through the alcohol lamp's flames. An alternative is dipping the instruments in beads heated at 300˚F for 15 seconds.
The endosperm cylinder is transferred to a sterile Petri dish lined with absorbent paper, with the use of a forcep. The embryo is removed carefully with a scalpel, and relocated to a similar Petri dish for drying. A particular culture medium is used in test tubes for inoculation of the embryos, which are positioned with the flat surface on top. Each container is then properly labelled for easy reference.
The tubes are put in a test tube rack and allowed to stay at room temperature in a dark chamber for three to eight weeks. The course of the plant's development is monitored periodically to ensure viability.
The germinating embryos are then transferred to culture shelves having sufficient light. Root growth is anticipated following three to four weeks, where the embryos are evacuated to a new liquid culture medium after the roots have reached 1.5 inches in length. Another 3-4 weeks are allotted for secondary roots and root hairs to manifest.
Bottles with new liquid culture medium are used to house the embryos with whole roots. The setup is monitored for three to five months until seedlings with extensive root systems appear.
After the young plants are incubated in vitro, seedlings with three photosynthetic leaves are taken to a screenhouse. These plants must also have shoots with lengths not lower than 17.5 cm. The seedlings are then hardened for one to two months before potting.
Potting plants with suitable characteristics comes next. Benlate solution is used to soak and disinfect the young plants before being removed from the individual bottles. The uprooted organisms are rinsed meticulously with distilled water, then soaked in benlate solution for 5 minutes.
The plantlets are then planted in a clay pot with sterile soil and properly labelled. Distilled water is used to provide adequate moisture three times a week after a clear plastic bag enclosed with rubber band is positioned on the upper portion of the pot. The plastic is provided with a slit at its base big enough for a wash bottle's nozzle to push through during watering.
The plants are constantly watched for any signs of infection or contamination, and 2-cm holes are punched on the side of the plastic to dispel excess water vapour. At the start of one month, another set of holes are made on the side. Benlate solution is sprayed over the setup and the top portion of the plastic cover is cut off after a week. These steps are then followed by bagging and planting on the field.
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