Tissue Culture

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Micropagation by micrografting results in offspring that are genetically identical to the parent plant. This is useful for replicating superior cultivars of fruit or flowering trees. Micrografting involves joining a piece of a mature tree (scion) to a seedling (rootstock). The scion will become the new trunk and branches of the tree and the rootstock will become the root system. The cambium is a key component in a successful graft as cambium layers from both the scion and the rootstock must be matched for a successful graft. The cambium is an active layer of cells located between the bark and woody portion of a stem or branch.  Applications of Micrografting techniques

1. Elimination of viruses
The in vitro micrografting technique has proved to be very useful in the regeneration of whole orchards of citrus fruits infected by viruses. This is because it is difficult to obtain rooting of the in vitro isolated apex in trees. However, grafting the apex onto a suitable rootstock would eliminate this problem. 2. Grafted plants bypass the juvenile stage that is associated with sexual seedlings. 3. Production of plants resistant to pest, diseases, cold, water stress, waterlogging, salinity and etc. For the shoot-tip micrografting technique, it is necessary to:

1. Obtain rootstock seed and germinate seedlings in vitro * Make sure the seeds used for rootstock production are not infected by any seed-borne pathogen. * Wrap groups of 10 peeled (shelled) seeds in a gauze. Disinfect the seeds in a laminar flow hood, immersing them in 10% sodium hypochlorite (commercial formulation) plus 1% Tween 20 for 10 minutes. Wash three times with sterile distilled water and, using aseptic techniques, plant two seeds in each sterile culture tubes containing the seed germination medium. Cover with kaputs. * Keep the tubes at 27 ºC in the dark, until the seedlings reach grafting size, approximately in 2-3 weeks. 2. Obtain young shoots

* Budwood approximately 10 cm long with 4-6 buds are preferable. * Wash with ethanol 95% and rinse with water to eliminate dust. * In a laminar flow hood, disinfect them by immersion in 10% sodium hypochlorite and 1% Tween 20 for 10 minutes, wash three times with sterile distilled water. Make a fresh angled cut on one end of a budstick and place the end in a sterile culture tube. * Keep the tubes containing the budwood at a temperature between 25 – 30 ºC, and illuminated at 1000 lux/16 hours. * Most of the buds will sprout between 7 – 14 days. It is important to use shoots measuring less than 5 cm long to avoid apices that are in abscission stage.

3. Micrograft
* Place the apex on the triangular cut or T cut. The base of the apex should overlap with the base of the triangle. If the T cut is used, the tip should be flat on the surface of the cut and the edges of the cut are closed over the tip. * Pick up the grafted seedling with forceps and place the root tip into the hole of the Heller support. Lower the support into the culture tube so that it is level with the medium. Cap the tube and place it in the rack. * Keep the micrografts in the culture room or chamber at a temperature of 26 – 27 ºC and illuminated at 1000 lux/16 hours, for 5-8 weeks or until the plants show at least two to three expanded leaves from the apex. 4. Plant the micrografted plant directly into soil

Examples of micrografting
In Vitro Micrografting of Mature Carob Tree (Ceratonia siliqua L.) A micrografting of carob between seedling and explants from mature female tree. Below showed different methods of wedge grafts to determine the micrografting optimum union. The micrografting on epicotyle method involves: * the seedlings were decapitated 1.5 cm above the cotyledonary joint, * a downward central incision of 0.5 cm was made

* and the wedge of microscion was inserted gently and firmly into the vertical split on the detopped seedlings. Whereas...
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