Rice Science ›› 2018, Vol. 25 ›› Issue (2): 61-72.DOI: 10.1016/j.rsci.2017.08.005
• Orginal Article • Next Articles
Radhesh Krishnan Subramanian1,2, Muthuramalingam Pandiyan1, Pandian Subramani1, Banupriya Ramachandradoss1, Chithra Gunasekar1, Ramesh Manikandan1()
Received:
2017-04-10
Accepted:
2017-08-14
Online:
2018-03-28
Published:
2017-12-22
Radhesh Krishnan Subramanian, Muthuramalingam Pandiyan, Pandian Subramani, Banupriya Ramachandradoss, Chithra Gunasekar, Ramesh Manikandan. Sprouted Sorghum Extract Elicits Coleoptile Emergence, Enhances Shoot and Root Acclimatization, and Maintains Genetic Fidelity in indica Rice[J]. Rice Science, 2018, 25(2): 61-72.
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Germination medium | Basal medium | Additive | Hormone |
---|---|---|---|
MS | MS | - | - |
HMS | ½ MS | - | - |
MIBA-1 | MS | - | 0.5 mol/L IBA |
MIBA-2 | MS | - | 1.5 mol/L IBA |
MSSE-10 | MS | 10% SE | - |
MSSE-20 | MS | 20% SE | - |
MSSE-30 | MS | 30% SE | - |
MSSE-40 | MS | 40% SE | - |
Table 1 Different types of media for germination and elongation.
Germination medium | Basal medium | Additive | Hormone |
---|---|---|---|
MS | MS | - | - |
HMS | ½ MS | - | - |
MIBA-1 | MS | - | 0.5 mol/L IBA |
MIBA-2 | MS | - | 1.5 mol/L IBA |
MSSE-10 | MS | 10% SE | - |
MSSE-20 | MS | 20% SE | - |
MSSE-30 | MS | 30% SE | - |
MSSE-40 | MS | 40% SE | - |
Fig. 1. Coleoptile emergence on different percentages of sorghum extract in medium.A, Germinated sorghum seeds in distilled water; B, Germinated ADT36 rice seeds on HMS medium; C, Germinated ADT36 rice seeds on MS medium; D, Germinated ADT36 rice seeds on MIBA-1 (1) and MIBA-2 (2) media; E, Coleoptile formation on MSSE-10 (1), MSSE-20 (2), MSSE-30 (3) and MSSE-40 (4) media; F, Dissected germinated coleoptile (1), leaf base (2) and seed (3) media. MS, Murashige and Skoog; HMS, Half of MS; MIBA, MS with indole-3-butyric acid; MSSE, MS with sorghum extract. The lengths of bars for A to F are 1 cm.
Fig. 3. Callus induction.A, Calli from mature seed; B, Calli from leaf base; C, Calli from coleoptile with 0.5 mg/L 2,4-D (2,4-dichlorophenoxyacetic acid); D, Calli from coleoptile with 1.0 mg/L 2,4-D; E, Calli from coleoptile with 1.5 mg/L 2,4-D; F, Calli from coleoptile with 2.0 mg/L 2,4-D; G, Calli from coleoptile with 2.5 mg/L 2,4-D; H, Proliferated calli; I, The closer view of proliferated calli; J, Effect of synthetic kinetin and 6-beniylamino purine (BAP) on callus proliferation. The lengths of bars for A to I are 1 cm. Bars for J represent the standard deviations.
Fig. 4. Callus regeneration.A, Three-day-old regenerated callus (incubated at dark); B, Regenerated callus with SAMs (shoot apical meristems); C, Upper view of regenerated callus with shoot primordium; D, Bright field microscopic-cross sectional view of torpedo shaped callus; E, Globular callus; F, Regenerating globular callus; G, Regenerated callus with SAM. The lengths of scare bars for A to C are 1 cm.
BAP | NAA (mg/L) | Average regeneration (%) | Shoot height | |
---|---|---|---|---|
(mg/L) | (cm) | |||
Initiation of shoot primordium | ||||
0 | - | 5.67 ± 0.58 | - | |
0.1 | - | 3.33 ± 1.53 | - | |
0.2 | - | 11.33 ± 0.58 | - | |
0.3 | - | 12.00 ± 3.61 | - | |
0.4 | - | 15.33 ± 1.53 | - | |
0.5 | - | 13.00 ± 1.00* | - | |
0.6 | - | 14.00 ± 1.73 | - | |
0.7 | - | 14.33 ± 1.53 | - | |
0.8 | - | 16.00 ± 2.00 | - | |
0.9 | - | 20.00 ± 1.00* | - | |
1 | - | 21.67 ± 1.53 | - | |
1.5 | - | 25.00 ± 1.00* | - | |
2 | - | 26.67 ± 1.53 | - | |
2.5 | - | 35.67 ± 1.53 | - | |
3 | - | 33.67 ± 2.52 | - | |
3.5 | - | 38.67 ± 1.53 | - | |
4 | - | 35.67 ± 4.58 | - | |
Development of SAMs | ||||
2.5 | 0.1 | 37.00 ± 1.00* | 8.20 ± 0.09* | |
2.5 | 0.2 | 36.67 ± 1.15* | 8.77 ± 0.66* | |
2.5 | 0.3 | 37.67 ± 1.53 | 10.07 ± 0.47* | |
2.5 | 0.4 | 38.67 ± 1.53 | 10.87 ± 0.36* | |
2.5 | 0.5 | 36.67 ± 2.08 | 10.30 ± 1.25 | |
2.5 | 0.6 | 43.67 ± 1.53 | 13.32 ± 0.36* | |
2.5 | 0.7 | 43.33 ± 1.53 | 13.43 ± 0.43* | |
2.5 | 0.8 | 47.00 ± 2.65 | 13.39 ± 1.02 | |
2.5 | 0.9 | 50.00 ± 1.00* | 14.50 ± 0.42* | |
2.5 | 1 | 49.67 ± 4.16 | 13.45 ± 1.84 | |
2.5 | 1.5 | 56.00 ± 2.65 | 15.58 ± 2.06 | |
2.5 | 2 | 56.00 ± 3.60 | 15.70 ± 0.48* | |
2.5 | 2.5 | 62.33 ± 1.53 | 15.72 ± 0.46* | |
2.5 | 3 | 62.00 ± 1.00 | 16.24 ± 0.64* | |
2.5 | 3.5 | 57.67 ± 1.53 | 14.92 ± 0.70* | |
2.5 | 4 | 53.00 ± 2.65 | 12.55 ± 0.67 |
Table 2 Effect of plant growth hormones on callus regeneration initiation of shoot primordium.
BAP | NAA (mg/L) | Average regeneration (%) | Shoot height | |
---|---|---|---|---|
(mg/L) | (cm) | |||
Initiation of shoot primordium | ||||
0 | - | 5.67 ± 0.58 | - | |
0.1 | - | 3.33 ± 1.53 | - | |
0.2 | - | 11.33 ± 0.58 | - | |
0.3 | - | 12.00 ± 3.61 | - | |
0.4 | - | 15.33 ± 1.53 | - | |
0.5 | - | 13.00 ± 1.00* | - | |
0.6 | - | 14.00 ± 1.73 | - | |
0.7 | - | 14.33 ± 1.53 | - | |
0.8 | - | 16.00 ± 2.00 | - | |
0.9 | - | 20.00 ± 1.00* | - | |
1 | - | 21.67 ± 1.53 | - | |
1.5 | - | 25.00 ± 1.00* | - | |
2 | - | 26.67 ± 1.53 | - | |
2.5 | - | 35.67 ± 1.53 | - | |
3 | - | 33.67 ± 2.52 | - | |
3.5 | - | 38.67 ± 1.53 | - | |
4 | - | 35.67 ± 4.58 | - | |
Development of SAMs | ||||
2.5 | 0.1 | 37.00 ± 1.00* | 8.20 ± 0.09* | |
2.5 | 0.2 | 36.67 ± 1.15* | 8.77 ± 0.66* | |
2.5 | 0.3 | 37.67 ± 1.53 | 10.07 ± 0.47* | |
2.5 | 0.4 | 38.67 ± 1.53 | 10.87 ± 0.36* | |
2.5 | 0.5 | 36.67 ± 2.08 | 10.30 ± 1.25 | |
2.5 | 0.6 | 43.67 ± 1.53 | 13.32 ± 0.36* | |
2.5 | 0.7 | 43.33 ± 1.53 | 13.43 ± 0.43* | |
2.5 | 0.8 | 47.00 ± 2.65 | 13.39 ± 1.02 | |
2.5 | 0.9 | 50.00 ± 1.00* | 14.50 ± 0.42* | |
2.5 | 1 | 49.67 ± 4.16 | 13.45 ± 1.84 | |
2.5 | 1.5 | 56.00 ± 2.65 | 15.58 ± 2.06 | |
2.5 | 2 | 56.00 ± 3.60 | 15.70 ± 0.48* | |
2.5 | 2.5 | 62.33 ± 1.53 | 15.72 ± 0.46* | |
2.5 | 3 | 62.00 ± 1.00 | 16.24 ± 0.64* | |
2.5 | 3.5 | 57.67 ± 1.53 | 14.92 ± 0.70* | |
2.5 | 4 | 53.00 ± 2.65 | 12.55 ± 0.67 |
Fig. 5. Plantlet acclimatization and root and shoot establishment on medium with different plant growth hormones (PGHs) or sprouted sorghum extracts (SSEs).A, HMS; B, MS; C, MIBA-1; D, MIBA-2; E, MSSE-10; F, MSSE-20; G, MSSE-30; H, MSSE-40; I and J, Effect of PGHs or SSEs on seed sprouting and coleoptile induction.MS, Murashige and Skoog; HMS, Half of MS; MIBA, MS with indole-3-butyric acid; MSSE, MS with sorghum extract. Bar for I and J represents standard deviation.
Fig. 6. Fourier-transform infrared spectroscopy spectrum from seeds of direct germinated (A), in vitro germinated (B), regenerated (C) and hardened plants (D).Dashed box represents the cellular component.
Fig. 7. Representative gel picture depicting genetic profile of random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR), and R-ISSR (combination of RAPD and ISSR) primers of direct germinated (lanes 2, 6 and 10), in vitro germinated (lanes 3, 7 and 11), regenerated (lanes 4, 8 and 12), and hardened plants (lanes 5, 9 and 13).The arrow represents monomorphic band.
No. | Primer | Annealing temperature | No. | Primer | Annealing temperature | No. | Primer | Annealing temperature | |||
---|---|---|---|---|---|---|---|---|---|---|---|
37 ºC | 55 ºC | 37 ºC | 55 ºC | 37 ºC | 55 ºC | ||||||
1 | OPD3 | + | + | 10 | ISSR46 | - | + | 18 | OPD3-ISSR35 | - | + |
2 | OPD8 | + | + | 11 | ISSR67 | + | + | 19 | OPD3-ISSR42 | + | - |
3 | OPD18 | + | - | 12 | ISSRA8 | - | + | 20 | OPD3-ISSR67 | - | - |
4 | OPF4 | + | - | 13 | ISSRA10 | - | + | 21 | OPD3-ISSRA16 | - | - |
5 | OPG5 | + | - | 14 | ISSRA16 | + | + | 22 | OPD8-ISSR35 | + | - |
6 | OPH3 | + | - | 15 | ISSRA33 | - | + | 23 | OPD8-ISSR42 | + | + |
7 | ISSR15 | - | + | 16 | ISSRY5 | - | + | 24 | OPD8-ISSR67 | - | + |
8 | ISSR35 | + | + | 17 | ISSRY11 | - | + | 25 | OPD8-ISSRA16 | + | - |
9 | ISSR42 | + | + |
Table 3 Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) primers used to check genetic profile.
No. | Primer | Annealing temperature | No. | Primer | Annealing temperature | No. | Primer | Annealing temperature | |||
---|---|---|---|---|---|---|---|---|---|---|---|
37 ºC | 55 ºC | 37 ºC | 55 ºC | 37 ºC | 55 ºC | ||||||
1 | OPD3 | + | + | 10 | ISSR46 | - | + | 18 | OPD3-ISSR35 | - | + |
2 | OPD8 | + | + | 11 | ISSR67 | + | + | 19 | OPD3-ISSR42 | + | - |
3 | OPD18 | + | - | 12 | ISSRA8 | - | + | 20 | OPD3-ISSR67 | - | - |
4 | OPF4 | + | - | 13 | ISSRA10 | - | + | 21 | OPD3-ISSRA16 | - | - |
5 | OPG5 | + | - | 14 | ISSRA16 | + | + | 22 | OPD8-ISSR35 | + | - |
6 | OPH3 | + | - | 15 | ISSRA33 | - | + | 23 | OPD8-ISSR42 | + | + |
7 | ISSR15 | - | + | 16 | ISSRY5 | - | + | 24 | OPD8-ISSR67 | - | + |
8 | ISSR35 | + | + | 17 | ISSRY11 | - | + | 25 | OPD8-ISSRA16 | + | - |
9 | ISSR42 | + | + |
Fig. 9. T-DNA gene transfer and validation in different explants.A, Checking hygromycin resistance by coleoptile derived callus. 1 to 6 means control, 10, 20, 30, 40 and 50 μg/L hygromycin; B, GUS expression in callus; C, GUS expression in mature seed; D, GUS expression in root; E, GUS expression in leaf blade; F, GUS expression in leaf base; G, GUS expression in coleoptile; H, Cross-section of GUS expressed in leaf base; I-J, Cross-section of GUS expressed in coleoptile; K, DNA blot hybridization of hpt gene integration in different explants (1, Control-wild type leaf; 2, Callus; 3, Mature seed; 4, Root; 5, Leaf blade; 6, Leaf base; 7, Coleoptile).The lengths of bars for A to G are 1 cm.
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