Protocols for Micropropagation of Woody Trees and Fruits

348 B.K. BISWAS AND S.C. GUPTA
(iv) Injection volume — 5 µl
(v) Flow rate — 0.5 ml min –1
(vi)
(vii)
Detector
Detection at
—
—
SPD – 2AS
UV – 215 nm
Reagents. Use the routine reagents for estimation of azadirachtin, such as (i) HPLC
grade methanol, (ii) HPLC grade water, and (iii) azadirachtin of at least 99% purity.
Azadirachtin Estimation. Inject 5 µl of standard azadirachtin solution as well as
sample solutions separately into HPLC unit to get area reproducibility for two consecutive
injections. The percentage of azadirachtin in samples can be calculated, on
the basis of HPL chromatograms by employing the following formula:
Azadirachtin content (% by mass) = A1/A2 × m2/m1 × P. Where, A1 = peak area
of azadirachtin in sample solution injected, A2 = peak area of azadirachtin in
standard injected, m1 = mass in gm, of the sample taken for the test, m2 = mass in g,
of the standard azadirachtin, P = purity of reference azadirachtin. Prepare HPL
chromatograms for all samples and select the tree having highest azadirachtin
content in its seed kernels.
2.2. Micropropagation
2.2.1. Explant Collection
Though micropropagation has been presently accomplished using nodes, rachis, leaf,
apical meristem and cotyledons but protocols only for nodes and apical meristem
culture are described below. Collect young shoots from the selected tree, growing
orthotropically either on the main tree trunk or on the primary and secondary branches
spreading horizontally, as well as those arising on coppiced branches.
2.2.2. Surface Sterilization
Remove the leaves from the twigs and thoroughly wash under running tap water for
45–60 min. Cut the twigs into 10–15 cm long pieces and treat with Polysan detergent
for 30–45 min with continuous shaking on a shaker at 110 rpm. Wash again in running
tap water for 10 min and treat with freshly prepared chlorine water (30–45 min) or
0.1–0.2% HgCl2 (15–30 min). The duration and method of treatment will depend on
the type of twigs, i.e. if very hard and old, then treat for a longer duration (chlorine
water for 45 min and HgCl2 for 30 min). Give a dip in 70% ethanol for 1–3 min.
Finally, wash them 3 or 4 times with sterilized distilled water.
2.2.3. Node
Subsequently, cut the twigs into 1–1.5 cm long pieces each comprising only one
node and inoculate two explants in each culture tube. To minimize cross contamination,
in some cases, it is advised to culture only one explant per test tube (Figure 1a).
Some nodal explants may still develop pink bacterial infection on the medium even