Protocols for Micropropagation of Woody Trees and Fruits

IN VITRO CONSERVATION AND MICROPROPAGATION OF BREADFRUIT
3. After potting, plants are watered with 20-8-20 fertilizer (Plant Products Co.
Ltd, Brampton, On, Canada) daily.
4. Place the pots in a growth chamber set for 16 h light at 26°C, 8 h dark at
24°C, and 95% relative humidity. Reduce the relative humidity by 5% every
week for 5 weeks and keep it constant at 70%.
5. Five weeks after transplantation, transfer the transplants into 6-inch pots
and grow them for an additional 12 weeks in the same chamber.
6. At the end of 12 week, transfer the plants to 1-gallon pots and place the potted
plants in the greenhouse. Almost 100% of the transplants survive and grow to
the size of juvenile trees, ready for planting outdoors, within 6 months after
transplantation (Figure 1F).
7. Greenhouse-grown juvenile trees are continuous sources of axillary shoot
tips for micropropagation that are free from most of the biotic contaminants
encountered with the materials grown in tropics.
2.7. Determination of Nuclear DNA Content and Ploidy Stability by Flow
Cytometry Analysis
The flow cytometry protocol presented here has been optimized to determine the
nuclear DNA content, ploidy level, and genetic stability in breadfruit. Carrot (Daucus
carota cv. Red Cored Chantenay) is used as internal reference since co-processing of
carrot and breadfruit tissues does not change the relative position of breadfruit peaks.
Carrot cells at the G0/G1 phase (2C) contain 0.98 pg DNA/nucleus (Arumuganathan
and Earle, 1991a). DNA analysis is performed using nuclei isolated from young leaf
tissues.
1. Place leaf tissues of breadfruit and carrot (about 50 mg tissue from each)
into 65 × 15 mm Petri dishes kept on ice.
2. Add 1.5 ml of ice cold nuclei isolation buffer (NIB) (Table 2) into the Petri
dishes on ice and slice the tissues finely with the help of a sharp razor blade.
3. Filter the homogenate through a 37 µm nylon filter to eliminate the tissue
debris and collect the filtrate in a 1.5 ml eppendorf tube.
4. Centrifuge the tube at 8000 g for 5 s to pellet the nuclei.
5. Decant the supernatant and place the tubes on a rack placed on ice.
6. Re-suspend the nuclei in 300 µl of NIB with 25 µg ml –1 RNase.
7. To stain the nuclei, add 10 µl of propidum iodide (PI) from a 1 mg PI ml –1
stock solution prepared by dissolving 1 mg PI in 1 ml milliQ water.
8. Analyze 10–15,000 nuclei/sample with a flow cytometer (e.g., a Coulter
EPICS Elite ESP Flow cytometer, Coulter Corp., Miami, Florida, USA).
The nuclear DNA content is calculated using the linear relationship between the
ratio of the 2C peak positions of breadfruit/carrot on the histogram of fluorescence
intensities (Arumuganathan & Earle, 1991b) using the equation described below:
285