Study of in vitro selection and plant regeneration of Indica rice tolerant to iron
Determination of Dry Weight of Callus
Germination of Somatic Embryos
Everyȱfourȱweeks,ȱtheȱcallusȱsamplesȱofȱknownȱfreshȱ Theȱ groupȱ ofȱ bothȱ non-tolerantȱ andȱ tolerantȱ
weightȱ(200ȱmg)ȱwereȱdriedȱtoȱconstantȱweightȱatȱ700ȱ embryogenicȱ callusesȱ wasȱ furtherȱ transferredȱ toȱ
Cȱinȱanȱoven.ȱTheȱrelativeȱgrowthȱrateȱ(RGR)ȱinȱtermsȱ ½ȱ strengthȱ MSȱ mediumȱ supplementedȱ withoutȱ orȱ
ofȱ freshȱ massȱ ofȱ theȱ callusȱ growthȱ wasȱ calculatedȱ withȱ 0.25ȱ -ȱ 0.5ȱ mg/lȱ NAA,ȱ 0.25ȱ –ȱ 1.0ȱ mg/lȱ BA,ȱ 12ȱ
withȱtheȱformulaȱofȱShah et al. (1993).
mMȱ Feȱ andȱ 3%ȱ (w/v)ȱ sucroseȱ forȱ maturationȱ andȱ
germination.ȱTheȱculturesȱwereȱroutinelyȱmaintainedȱ
atȱeveryȱ4-weekȱintervals.ȱInȱanotherȱexperiment,ȱtheȱ
embryogenicȱculturesȱwereȱkeptȱinȱtheȱdarkȱforȱtwoȱ
weeksȱ thatȱ wereȱ thenȱ transferredȱ toȱ theȱ lightȱ (16hȱ
Photoperiod)ȱ forȱ germinationȱ ofȱ somaticȱ embryos.ȱ
Eachȱtreatmentȱconsistedȱofȱ15ȱreplications,ȱandȱtheȱ
experimentsȱwereȱrepeatedȱtwoȱtimes.
Table 1. Effect of Kinetin, BA, NAA and 2,4-D on callus
induction from immature zygotic embryo of O.sativa Var.
Khandagiri and Var. Jagannath after four weeks of culture.
MS + growth regulators
Percent of explants forming
(mg/l)
callus (Mean ± SE)*
Var.
Var.
Kn
BA
NAA 2,4-D
Khandagiri
Jagannath
0
0
0
0
0
0
0.5
0
0
0
0
0
1.0
0
0
0
22.4 ± 0.3 a
24.6 ± 0.7 b
1.5
0
0
0
26.6 ± 0.8 d
32.2 ± 0.8 d, e
0
0.5
0
0
0
0
0
1.0
0
0
12.8 ± 0.4 a
16.2 ± 0.6 a
A &B. Embryogenic callus developed from immature zygotic
0
1.5
0
0
18.4 ± 0.5 b
16.8 ± 0.7 a
embryos of Var. Khandagiri (A) and Var. Jagannath (B) on MS
medium supplemented with 1.0 Kn + 3.0 mg/l 2,4-D + 12 mM
1.0
0
0
2.0
54.6 ± 0.7 h
52.2 ± 0.6 f
Fe after four weeks of subculture.
1.0
0
0
3.0
74.8 ± 0.8 k
68.8 ± 0.6 h
C &D. Proliferation of embryogenic calluses (C) and
1.5
0
0
3.0
72.6 ± 1.2 j
64.6 ± 0.8 g
germination Germination of somatic embryos on MS medium
1.5
0
0
4.0
62.4 ± 0.7 i
52.8 ± 0.7 f
supplemented with 1.0 mg/l BA + 0.25 mg/l NAA + 12 mM Fe
+ 2% surose after three weeks of subculture.
0
1.0
0
3.0
26.4 ± 0.8 d
32.2 ± 0.4 d, e
E . Germination of somatic embryos of Var. Jaganath on 1.0
0
1.5
0
4.0
30.4 ± 0.6 e
30.4 ± 0.6 d
mg/l BA + 0.25 mg/l NAA + 12 mM Fe + 2% surose after three
1.0
0
2.0
0
26.8 ± 0.4 d
24.8 ± 0.5 b
weeks of culture.
1.5
0
3.0
0
30.8 ± 0.8 e
28.6 ± 0.7 c
F. The tolerant regenerated plantlets establish in pot with
panicle formation after two months of transfer
2.0
0
4.0
0
32.2 ± 0.6 e, f
30.2 ± 0.6 d
0
1.0
2.0
0
24.6 ± 0.7 c
26.2 ± 0.8 b, c
RGRȱ=ȱLnȱ(finalȱmass)ȱ–ȱLnȱ(initialȱmass)/ȱweeks
0
1.5
3.0
0
28.4 ± 0.8 d, e
30.6 ± 0.7 d
GrowthȱToleranceȱIndexȱ(GTI)ȱwasȱcalculatedȱusingȱ
0
2.0
3.0
0
32.3 ± 0.5 e, f
32.2 ±0.6 d, e
theȱformula:
0
2.0
4.0
0
35.6 ± 0.4 g
34.2 ± 0.8 e
GTIȱ (%)ȱ =ȱ Mean%ȱ ofȱ callusȱ growthȱ inȱ mediaȱ withȱ *15 replicate per treatment; repeated three times.
a- k Mean having the same letter in a column were not significantly
additionȱofȱFeȱ/ȱMean%ȱofȱcallusȱgrowthȱinȱmediaȱ
different by Post-hoc multiple comparison tests P< 0.05.
withoutȱadditionȱofȱFeȱxȱ100
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