Microtip Tissue Culture Process

As a clean plant center, the CPCNW receives grape varieties or cultivars for admission into our program and these plants undergo a series of pathogen testing in the initial screening phase.  If the plants are found to be infected with pathogens, they will go through the microtip tissue culture process, which is a part of the therapy phase. Tissue culture is the first step in creating clean plants. Shoot tips are collected from the infected grape plants. Under a powerful microscope, using tweezers and micro needles, the tissue culture specialist dissects the shoot tip. The outer leaves of the shoot tips are pulled off to reveal the meristematic dome. A microtip with 0.2 mm long set of cells, including the meristematic tissue, is cut and removed from the shoot tip. The excised microtip is smaller than the point of a sewing needle and is filled with rapidly dividing cells that serve as the primary blueprint for developing the plant’s shoots or roots. On a pathogen-infected plant, it is the one bundle of cells that are disease free.

Once the microtip is excised, it will be placed in a test tube with tissue culture media. The media is a combination of salts, sugars, vitamins, hormones and other nutrients necessary for the microtip to grow. The test tubes are then incubated in an LED-lit growth chamber with controlled light and temperature conditions for growth and development. As the microtip matures, it develops shoots and is moved to an agar-based media to encourage root growth. When the roots are properly formed, plantlets are transferred from tissue culture containers to small plastic pots with sterilized soilless media. They are sealed with a plastic bag in order to maintain moisture. This is the first step of introducing the tissue culture plants to the outer environment. The plants do not leave the LED-lit growth chamber until sufficient rooting and plant growth has occurred.  Gradually, the plants will mature and be ready to be moved to the greenhouse as the acclimatization and the hardening phase progresses.

In the greenhouse, plants will be transferred to larger containers with standardized growing media specially formulated for containerized grape planting and maintenance. Plants are then trained using bamboo sticks and grown for six months. These microtip tissue culture plants will be tested again for all the known pathogens so the selection of mother plans phase can begin. Plants which are free of all the pathogens tested in the final screening are selected as the mother plant. Mother plants are then moved to a foundation screen house and extra care and maintenance are given in order to keep them clean. The mother plants also undergo genotyping to ensure they are true-to-type. From the screen house mother plants, more plants are produced and planted out in the foundation vineyard to generate clean planting material for release and distribution. The CPCNW maintains the clean status of the foundation vineyard through annual testing to verify the pathogen-free status.