LIST OF UNITS
Chapter 3 Results
3.1. ANTHER CULTURE
3.1.3. Regeneration from anther callus
Since the origin of callus often affects the frequency of regeneration, the calli from various multiplication media (Table 3.11) were utilized for regeneration experiments.
Table 3.11: Callus growth index (CGI) of cultivars on various multiplication media. Growth Period: 8 weeks Sl.
No.
Callus proliferation media TV1 TV18 TV19 TV21 317/1 14/100/1
1 MS (3 % Sucrose) + 2,4-D (5 µM) + Kn (5 µM) + L- Glutamine (800 mg/l) + L- Serine (200 mg/l)
0.19±0.01c 0.07±0.02d 0.26 ± 0.02d 2.35±0.05a 0.59±0.01c 0.37±0.02b
2 MS (3 % Glucose) + 2,4-D (5 µM) + Kn (5 µM) + L- Glutamine (800 mg/l) + L- Serine (200 mg/l)
- - - 1.98±0.06b 0.37±0.02d 0.41±0.023b
3 MS (3 % Sucrose) + 2,4-D (3 µM) + TDZ (18 µM)
0.95±0.05b 0.65±0.03c 0.81 ±0.03c 1.59±0.10c 1.52±0.05a 1.64±0.02a
4 MS (3 % Sucrose) + 2,4-D (1 µM) + NAA (1 µM) + BAP (5 µM)
1.61±0.05a 0.97±0.04b 1.85 ±0.05b 1.20±0.03d 0.62±0.04b 0.58±0.03b
5 MS (3 % Sucrose) + NAA (5 µM) + BAP (10 µM)
1.03±0.04b 1.28±0.05a 2.21 ± 0.07a - - 0.05±0.00c
Values are mean ± standard deviation
Among individual cultivar, same letter after the values are not significantly different (p<0.05) according to Duncan’s multiple range test.
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Table 3.12: Effect of various growth regulators and their combinations on regeneration from calli of TV 19 and TV 21 cultivars. Growth period:
10 weeks.
Sl.
No.
Regeneration Treatments TV19 cultivar TV21 cultivar
Observations % cultures showing regeneration
Observations % cultures showing regeneration
1 Control (MS basal medium) Nil Nil Nil Nil
2 BAP (10 µM) Bright green, hard and compact calli Nil Bright green, hard and compact calli Nil
3 Kn (10 µM) Light green, compact calli Nil Light green, compact calli Nil
4 2-iP (10 µM) Light green, compact calli Nil Nil Nil
5 TDZ (10 µM) Dark green, granular, friable calli Nil Dark green, granular, friable calli Nil
6 L-Glutamine (800 mg/l) + L-Serine(200 mg/l) Nil Nil Nil Nil
7 2,4-D(3 µM) + TDZ(18 µM) Moderately hard, bright green callus with massive growth showing
differentiation of shoot-buds
4 (5)* Bright green, granular, friable calli Nil
8 2,4-D (5 µM) + Kn (5µM) Fresh, dark green, granular, friable
calli Nil Fresh, dark green, granular, friable
calli Nil
9 2,4-D (5 µM) + Kn (5 µM) + L-Glutamine (800 mg/l) + L-Serine(200 mg/l)
Fresh, dark green, granular, friable calli
Nil Fresh green, friable callus with green patches
Nil 10 NAA(5 µM) + BAP(10 µM) Dark green, nodulated callus with
green patches (Figure 4 H)
Nil Dark green, nodulated callus with green patches (Figure 4 F)
Nil 11 BAP (10 µM) + L-Glutamine (800 mg/l) + L-
Serine (200 mg/l) Dark green, granular, friable calli Nil Dark green, granular, friable calli Nil 12 BAP (10 µM) + GA3 (1µM) + L-Glutamine
(800 mg/l) + L-Serine (200 mg/l) Dark green, nodulated, friable calli Nil Dark green, nodulated, friable calli Nil 13 BAP (10 µM) + GA3 (3 µM) + L-Glutamine
(800 mg/l) + L-Serine (200 mg/l) Dark green, nodulated callus with numerous asynchronous embryos
75 (18)* Dark green, nodulated callus with numerous asynchronous embryos
85 (20)*
14 BAP (10 µM) + GA3 (6 µM) + L-Glutamine (800 mg/l) + L-Serine (200 mg/l)
Bright green, granular, hard calli Nil Bright green, granular, hard calli Nil
15 BAP (10 µM) + GA3 (1µM) + CH (250 mg/l) Light green, compact calli Nil Nil Nil
16 BAP (10 µM) + IBA (1 µM) Nil Nil Green callus with brown spots Nil
17 BAP (2.5 µM) + IAA(5 µM) + CH(500 mg/l) Light green, dry calli Nil Light green, dry calli Nil
18 Zeatin (10 µM) + Adenine sulphate(20 mg/l)
+ CH (10 mg/l) Green callus with brown spot Nil Green callus with brown spot Nil
*Values in parentheses represent average number of embryos/shoot buds developed per explants.
Numerous growth regulator combinations were tested (Table 3.12) to obtain regeneration from calli. MS basal medium was kept as control. Except TV19 and TV21 cultivars, other cultivars did not show any sign of regeneration on any of the tested media. Both, TV19 and TV21 cultivars did not show regeneration responses on MS basal medium or basal medium supported with either auxin or cytokinin. However, a combination of one auxin and one cytokinin favored shoot-bud differentiation in small percentage of cultures of TV 19. On MS + 2,4-D (3 µM) + TDZ (18 µM) medium, bright green, hard, massively growing calli of TV19, showed differentiation of tiny shoot-buds (5 shoot buds/culture) only in 4 % of the cultures, after 4 weeks of culture initiation. Histology of the regenerating calli showed the presence of distinct shoot-tip with two leaf-primordia (Figures 7 A). Apparently, these shoot-buds did not grow further.
Regeneration via embryogenesis was achieved only when the nodulated calli from callus multiplication media, MS + NAA (5 µM) + BAP (10 µM) and MS + 2,4-D (5 µM) + Kn (5 µM) + L-Glutamine (800 mg/l) + L-Serine (200 mg/l), respectively, of the cultivars TV 19 and TV 21, were transferred to the regeneration medium consisted of MS + BAP (10 µM) + GA3 (3 µM) + L-Glutamine (800 mg/l) + L-Serine (200 mg/l). Asynchronous embryogenesis was observed on regeneration medium after two subcultures, each of 6 weeks duration. An average of 18 embryos per culture were developed, in a single passage, in 75 % of the cultures of TV19 (Figures 6 A, B), while in TV21 cultivar, 85 % of the cultures showed embryogenesis with an average of 20 embryos per culture (Figure 6 C, D). These embryos remained attached to the callus in the form of clusters. All the stages of embryo development, globular, heart, torpedo and dicotyledonous, were observed in single culture. Globular shape, heart-shape and early torpedo shape embryos were fresh, shiny, white or cream in color while late torpedo and dicotyledonous embryos were green and fresh (Figure 6 A, B, D).
In histological sections, distinct stages of embryos at various stages of development were also observed, like globular (Figure 7 B), heart-shape (Figure 7 C), torpedo-shape (Figure 7 D, E) and early dicot (Figure 7 F) and late dicot stages (Figure 7 G). These embryos, on maturity, developed complete vascular strand properly connected from radicular to the plumular end, as revealed from the histological sections (Figure 7 H, I).
SEM images also confirmed first the occurrence of the nodulated calli in the
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multiplication medium of TV 19 cultivar (Figure 8 A) and TV 21 (Figure 8 B, C) which later on showed asynchronous embryogenesis in the regeneration medium. Later, these nodules developed into distinct globular and heart-shaped embryos (Figure 8 D, E).
3.1.4. Embryo maturation, germination and plantlets development