Protocols for Micropropagation of Woody Trees and Fruits

2.6. Mass Multiplication in Bioreactors
MICROPROPAGATION OF PINEAPPLE
A procedure for the mass propagation of pineapple (cv. ‘Smooth Cayenne’) plants
using a temporary immersion bioreactor has been described by Escalona et al.
(1999). This procedure is automated and involves three distinct phases: shooting,
bud differentiation and elongation. The initial explant used in this protocol is shoots
from previously established cultures in a ‘shooting medium’ formulated by Daquinta
and Benegas (1997).
In this protocol the bioreactor system should consist of two 1000-ml vessels –
one to act as a reservoir for the liquid medium (volume = 1000 ml) and the other
(volume ranging from 25 to 100 ml) for growing the plants. The two vessels were
connected by silicone and glass tubes and the airflow can be sterilized during
passage through 0.2-µm hydrophobic filters (Escalona et al., 1999). Using an air
compressor, sterile medium can be pressured to flow from the reservoir to completely
immerse five explants (2–3 cm shoots) placed in the other vessel (plant vessel). The
medium can be drained back into the first vessel by controlled reversion of air flow.
The frequency and length of immersion can be electronically controlled. Exposure
to the medium for 2 min every 3 h at 25°C under cool-white fluorescent tube (80
µmol · m –2 · s –1
) with a 16 hr photoperiod is necessary. Addition of paclobutrazol
(3.3 µM) in the immersion medium promotes shoot growth and induces axillary bud
proliferation but avoids unnecessary leaf development during the shoot-formation
stage (Escalona et al., 1999). Shoot multiplication period ranged from 4–7 weeks.
After this a change in medium composition to MS supplemented with BAP (2.2 µM)
and GA3 (2.9 µM) for 7 days (step 1) and later to MS supplemented with GA3
(2.9µM) – step 3, can achieve shoot elongation. All shoots produced by this method
can be rooted in the green house ex vitro by transplantation onto a substrate consisting
of compacted red ferralitic salt mixed in a 1:1 ratio with sugarcane mill baggasse.
The temporary immersion system combines the advantages of solid and liquid medium
and exhibit high multiplication rates.
2.7. Micropropagation via Synthetic Seeds
Synthetic seeds can be prepared using tiny shoots (2–5 mm), from in vitro grown
stock cultures, as encapsulation propagules (Soneji et al., 2002c). Isolated shoots
should be mixed in an alginate matrix [3% sodium alginate (Sigma) prepared in MS
basal medium or SMM and autoclaved] for encapsulation. Each shoot thus coated
with alginate can be picked up using a pair of forceps and gently dropped into an
autoclaved solution of CaCl2·2H2O (1.36 g/150 ml). The coated buds should be
allowed to remain in this solution for 30 min for complexation to occur. At the end
of 30 min, the CaCl2·2H2O solution can be carefully decanted off and the encapsulated
shoots washed 3–4 times with sterile tap water and blot dried on sterilized
filter paper to remove the excess solution. Synthetic seeds are ready to be either
cultured on solidified nutrient media for germination or stored in sealed Petri plates
at 4°C in the refrigerator.
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