Protocols for Micropropagation of Woody Trees and Fruits

IN VITRO CULTURE OF TREE PEONY 485
PHASE: INITIATION MULTIPLICATION ROOTING
WPMP1 WPMP2 WPMP3
Additional
requirement
of Calcium
(mg L –1 ) (mM) (mg L –1 ) (mM) (mg L –1 ) (mM)
CaCl2.2H2O 441.00 3.00 – – – –
Calcium gluconate
monohydrate
– – 1342.5 3.00 1342.5 3.00
Growth
regulators
(mg L –1 ) (µM) (mg L –1 ) (µM) (mg L –1 ) (µM)
BA 1.00 4.44 1.00 4.44 – –
IBA – – – – 1–10
4.92–
49.2
Preparation to make 1 L of medium: Weigh all the ingredients and dissolve in
300–400 mL of bi-distilled water (conductivity 2 ± 0.5 µS cm –1 ). For microelements,
vitamins and growth regulators stock solutions will be used and an adequate
amount of milliliters will be added to the bi-distilled water. Adjust the volume of
the solution to 500 mL. In the meanwhile, add 8 g of agar to 500 mL of bi-distilled
water and heat for 10–12 min, taking care to avoid evaporation. Add the hot agarsolution
to the previously prepared ingredient solution and mix for some seconds.
The pH of all media are adjusted to 5.85 ± 0.01 while the media are liquid (T =
76°C ± 1). Subsequently media, poured into tubes and vessels, are autoclaved at
120°C, 101 kPa, 15 min.
2.1.1. Steps in the Procedure
1. Collect axillary buds from mother plant stock; buds with progressively
expanded leaves will bemore reactive to the in vitro culture (Figure 5A).
2. Remove the bud scales (Figure 5B) and sterilize by a dip in HgCl2 solution
(0.5% w/v; 3 min) followed by a solution of NaOCl (1% available chlorine;
15 min) and a rinse with sterile double distilled water. Before inoculum,
buds are submitted to an additional dip in PPM (Plant Preservative Mixture,
Plant Cell Technology, Inc TM ; 50% v/v; 30 min).
3. For shoot development, place buds on 25 × 150 mm test tubes closed by
plastic cap (Bellco Kaputs ) and containing 10 mL of agar gelled WPMP1
medium (Table 2; Figure 5C). Incubate at 19 ± 1°C in darkness.
4. After 1 day, browning exudates from the explants are visible at different
degree according to the cultivar (Figure 5D). At this time, explants need to
be transferred without any cutting to a fresh WPMP1 medium and incubated
again in darkness at the same temperature.
5. After 2 weeks of culture, explants are incubated at the same temperature
under a 12 h photoperiod with light energy 50 µmol m –2 s –1 provided by
fluorescent lamps (TLD 36W/33 Philips).