Orientation of mature embryos on culture medium influences callus induction
--Jasbir Kaur Madan, Ashutosh Pandey, Pranab Gayen and K. R. Sarkar

Immature embryo has been used extensively to obtain embryogenic callus and regeneration in maize tissue culture. Utilization of mature embryos for callus induction has also been reported (Wang, Plant Cell Rep. 6:360-362, 1987). For callus induction from immature embryo, the placement fashion of the embryo on the medial slant plays an important role. The plumule-radicle side of the immature embryo, when placed downward, gave a greater frequency of induction and amount of callus induction than the scutellar side placed downward (Green and Phillips, RE, Crop Sci. 15:417-421, 1975).

When mature embryo is placed facing the medium, the plumule starts to germinate quickly, pushing the explant up and disconnecting it from the medial surface, thereby restricting the callus induction. Hence, placement of embryo on the medium plays a critical role in successful cultures. An efficient mode of placement of the embryo to enhance callus induction frequency was identified in our study.

The callus induction medium was MS medium supplemented with 2,4-D 2mg/l, casein hydrolysate 1g/l, inositol 100mg/l, sucrose 3% and solidified with 0.7% agar. The pH was adjusted to 5.8 before autoclaving.

Sixty mature seeds per replication of seven inbreds with two replications were used for this experiment. Five different embryo placements (Table 1) were taken into consideration.

The culture was kept under dark conditions at 26±1 C for 14 days and callus induction frequency was scored visually. The results indicate that the embryo when placed laterally, half inserted into the media (T5), showed better callus induction frequency and callus growth as compared to other treatments for all the inbreds studied. When the data on callus induction frequency over the inbreds for different treatments were subjected to DMRT ranking, it was observed that lateral placement was distinctly superior to the other four orientations.

Table 1. Callus induction frequency of mature embryos from different inbreds with different embryo placements.
 
 
Callus induction frequency (%)*
Inbreds
T1
T2
T3
T4
T5
3783
66.10
66.65
52.78
58.81
87.91
3786
68.36
70.84
59.09
67.39
86.41
4526
38.39
46.64
47.34
29.16
60.41
4603
21.42
15.00
24.94
11.17
47.51
4532
31.73
46.73
33.32
19.20
59.09
4493
16.44
33.51
16.32
32.77
53.56
4604
12.49
23.04
30.00
11.57
38.52
Mean
33.70b
43.20b
37.68b
32.86b
61.91a
*Each entry is the mean of two replications.
T1=Embryo half inserted vertically in the medium; plumule downward
T2=Embryo half inserted vertically in the medium; radicle downward
T3=Embryo flat on the medium; plumule-radicle axis touching the medium
T4=Embryo flat on the medium; plumule-radicle side upward
T5=Embryo placed laterally, half inserted in the medium; plumule-radicle axis touching the medium
a,b=DMRT ranks 


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