micropropagation of aegle marmelos and molecular markers
DESCRIPTION
Standardization of micropropagation protocol in Aegle marmelos corr.TRANSCRIPT
In vitro plant regeneration and genetic assessment among regenerates using molecular
markers in bael (Aegle marmelos Corr.)
Central institute for subtropical Horticulture, Lucknow, India
Rajesh Pati, PhD
e-mail: [email protected]
Introduction
• Bael (Aegle marmelos Corr.) is an important medicinal fruit tree.
• The fruit pulp contains marmelosin, which is a laxative, diuretic, is being used in many patented drugs in India.
• The bael tree can suitably be grown under various wasteland situations. However, its commercial orcharding is not expanding at a faster pace due to severe shortage of planting material.
• Conventional method of bael propagation (Inarching, budding and soft wood grafting) is season bound and slow.
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• Micropropagation technology can be gainfully employed in mass multiplication of improved bael varieties.
• We have developed micropropagation protocol of bael through shoot bud culture. It was imperative to test genetic fidelity of micropropagated plants using molecular markers (RAPD, DAMD and ISSR).
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Advantages • The production of disease free plantlets
• The rapid production of large numbers of genetically identical plantlets
• Introduction of new varieties and or genotypes
• Germplasm conservation
• Production of plantlets from species in which plant development from seed is difficult
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Objectives • Standardization of micropropagation protocol for shoot bud
culture from mature elite tree. • Standardization of acclimatization procedure for
micropropagated plants of bael. • Field testing of micropropagated plants of bael. • Checking of genetic stability of micropropagated plants
through molecular markers.
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• 14 years old fruit bearing tree of Aegle marmelos variety CISH-B1 and CISH-B2 of about was chosen for study.
• 30-45 cm long shoots were excised from the elite donor tree.
• The shoots were defoliated and three different sub-experiments were undertaken to optimize ideal explant.
• Nodal segments were obtained from different nodal position (1-5, 6-10, 11-15, 16-20th nodes), length (1, 2, 3 and 4 cm) and number of buds/explant (1, 2, and 3).
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Effect of Nodal position
(A) 1-5 nodal position, (B) 6-10 nodal position, (C) 11-15 nodal position and (D) 16-20 nodal position.
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Different size of explant (1 cm, 2 cm, 3 cm and 4 cm long).
Effect of Length of shoot
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Effect of Number of buds/explant
(A)1 axillary bud (B) 2 axillary buds and (C) 3 axillary buds
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Pre-sterilization 0.1 % Carbendazime (Bavestin) + 25 mg/l Rifampicin (or 0.1% Streptomycin sulphate), citric acid (100 mg/l) and 2-3 drops of Tween-20 per 100 ml of distilled water and leave for one hour and wash with distilled water. Post-sterilization The explants were treated with different sterilizing agents (70% ethyl alcohol, 0.1% HgCl2 and 4% NaOCl) for different durations (4, 6 and 8 minutes) and washed with sterile distilled water.
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In vitro morphogenesis Nodal explants was inoculated in MS medium fortified with different plant growth regulators like BAP (0, 0 .5, 1, 2, 3 mg/l), Kinetin (0, 0.5, 1, 2, 3 mg/l), IAA (0.5-1.0 mg/l).
In vitro microshoot proliferation Microshoot was inoculated in MS mediun fortified with different plant growth regulators like BAP (0, 0.5, 1, 2, 3 mg/l), Kinetin (0, 0.5, 1, 2, 3 mg/l), IAA (0.5-1.0 mg/l) and AdS (12.5, 25, 50 and 100 mg/l).
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In vitro rooting 3 cm long shoots were inoculated in ½ and full strength MS basal media containing IBA (10.0, 15.0 mg/l) and IAA (0.5, 1.0 and 1.5 mg/l) either alone or in various combinations for rooting.
Acclimatization Carrier substrates containing autoclaved soil, soil + sand + FYM (1:1:1) and coconut husk fortified with All the treatments were supplemented with ½ strength MS nutrients salt solution.
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Biochemical Studies • The chlorophyll (a, b and total) was estimated as per the
method described by Arnon (1949).
• Nitrate reductase activity was estimated as per the method described Srivastava (1975).
• Total soluble protein was estimated as per the method described by Lowery et al. (1951).
• The reducing sugar was estimated as per the method described by Ranganna (1986).
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DNA isolation Total genomic DNA was extracted from leaf tissue of micropropagated bael plants using Qiagen Miniprep DNA isolation kit. Polymerase Chain Reaction (PCR) PCR reactions were carried out on the total genomic DNA in a final volume of 25µl reaction mixture with the 13 RAPD, 3 ISSR and 2 DAMD primers. Agarose Gel Electrophoresis The PCR amplification products were electrophorised on 1.5% agarose gel for three hours at 5V/cm. After completion of electrophoresis, gel was stained with ethidium bromide and visualized on a transilluminator and acquire gel images under Gel Doc System (Alpha Inn. Co.).
Genetic fidelity test of regenerates plants
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S.No. Primer name Sequence (5’-3’), length Annealing temperature (0C)
a. ISSR primers
1. MP2 (GA) 8 YC, 18 mer 42
2. MP3 CT CT CT CT CT CT CT CTRC, 18 mer 42
3. MP7 GGGTGGGGTGGGGTG, 15 mer 42
b. DAMD primers
1. 33.6a AGGGCTGGAGG, 11 mer 55
2. M13b GAGGGTGGCGGTTCT, 15 mer 55
c. RAPD primers
1. OPA1 CAGGCCCTTC, 10 mer 35
2. OPA2 TGCCGAGCTG, 10 mer 35
3. OPA20 GTTGCGATCC, 10 mer 35
4. OPB1 GTTTCGCTCC, 10 mer 35
5. OPB18 CCACAGCAGT, 10 mer 35
6. OPC12 TGTCATCCCC, 10 mer 35
7. OPD1 ACCGCGAAGG, 10 mer 35
8. OPD6 ACCTGAACGG, 10 mer 35
9. OPD7 TTGGCACGGG, 10 mer 35
10. OPE1 CCCAAGGTCC, 10 mer 35
11. OPE2 GGTGCGGGAA, 10 mer 35
12. OPF6 GGGAATTCGG, 10 mer 35
13. OPF12 ACGGTACCAG, 10 mer 35
The SSR (microsattelite) sequences, minisatellite core sequences and the arbitrary sequence decamers used as RAPD primers in amplification reactions.
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These optimized PCR conditions were used for all PCR based experiments in the present study.
Steps/Stages Temperature (0C) Duration (Minutes)
No. of Cycles
Method
Predenaturation 94 2 1 RAPD
Denaturation 94 1
Annealing 35 1 45
Extension 72 1
Final Extension 72 5 1
Predenaturation 94 2 1 ISSR
Denaturation 94 1
Annealing 42-52 2 40
Extension 72 2
Final Extension 72 5 1
Predenaturation 92 2 1 DAMD
Denaturation 92 1
Annealing 55 2 40
Extension 72 2
Final Extension 72 5 1 e-mail: [email protected]
Effect of season on explant collection
September-October was found ideal because 85.7% explant shows in vitro bud burst with least in born contamination
35
45
35
11.67
65
55
17.67
25.75
48.17
57.67
85.23
55.19
0
10
20
30
40
50
60
70
January-February
March-April May-June July-August September-October
November-December
Period of the year
Perc
entag
e
0
10
20
30
40
50
60
70
80
90
Perc
entag
e
% Aseptic culture Bud-burst in infection-free explants (%)
Results
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Effect of different sterilants
0
10
20
30
40
50
60
70
80
90
Perc
ent s
urviv
al
Control 70% Ethylalcohol
0.1%HgCl2
4% NaOCl 0.1%HgCl2+4%
NaOClSurface sterlising agents
4min. 6min 8min
0.1% HgCl2 for 6 minutes shows less contamination high survival of explants (86.67%).
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Effect of nodal position
0
8
6.677.33
0
48.33
87
61.67
0123456789
1-5 6-10 11-15 16 -20
Nodal Position
Days t
aken
fo
r b
ud
b
reak
0102030405060708090100
Bu
d b
reak (
%)
Days taken for bud-break % Bud break
11-15th nodal stem segment proved to the best explants where most of the explants showed bud burst (87%) in just 6.67 days.
0
8.33
77.67
0
46.67
82.67
59
0
1
2
3
4
5
6
7
8
9
1-5 6-10 11-15 16 -20
Nodal Position
Day
s ta
ken
fo
r b
ud
bre
ak
0
10
20
30
40
50
60
70
80
90
Pec
ent
Bu
d b
reak
Days taken for bud-break % Bud break
CISH-B1 CISH-B2
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Effect of size of explant
1.671.33
3.67
2.67
4
3.33
2.332
00.5
11.5
22.5
33.5
4N
o.
of
sh
oo
ts/e
xp
lan
t
1 2 3 4
Size of explants (cm)
CISH B1 CISH B2
3 cm long explant influenced number of shoots (4.0 and 3.33/explant), number of leaves (5.0 and 4.33/explant) and days taken for bud break (6.67-7.0 days) in both the varieties.
8.338.67
7.678
6.677
7.337.67
0
1
2
3
4
5
6
7
8
9
Day
s ta
ken
for
bud
burs
t
1 2 3 4
Size of explants (cm)
CISH B1 CISH B2
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Effect of number of buds per explant
Explants having single bud showed maximum numbers of shoots/explant (5.33 and 4.0 respectively), and leaves (6.33 and 5.0) and days taken for bud break (6.67 and 7.0) was reduced.
5.33
4
3.33
2.33 2.33
1.67
0
1
2
3
4
5
6
No. o
f axi
llary
sho
ots/
expl
ant
1 2 3No. of buds/explant
CISH B1 CISH B2
6.677
7.678.33 8.33
8.67
0
1
2
3
4
5
6
7
8
9
Day
s ta
ken
for
bu
d b
urs
t
1 2 3No. of buds/explant
CISH B1 CISH B2
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In vitro bud induction • Quicker bud burst (5.33 and 6.33 days) were recorded when
explants were inoculated in MS+BAP 2.0 mg/l +IAA 1.0 mg/l.
• While higher number of axillary shoot (5.33 and 4.67 shoots/explant) were recorded when explant was incubated on and MS+ Kinetin 3.0 mg/l +IAA 1.0mg/l in both varieties.
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(A) Shoot bud induction in CISH-B1, (B) Shoot bud induction in CISH-B2, (C and D) Culture establishment
A B
C D
In vitro shoot bud induction and culture establishment after 4 weeks.
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In vitro proliferation
MS+BAP 2.0 mg/l + IAA 1.0 mg/l containing proliferating medium produced 9.67 and 9.33 microshoots/explant. This treatment also gave maximum leaves/explant (15.33 and 16.66) and highest shoot length (3.1 cm and 3.06) in both varieties.
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(A) CISH-B1 (B) CISH-B2
In vitro microshoot proliferation after 8 week
A B
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Effect of Adenine sulphate on shoot proliferation
2.62.78
3.92
3.353.17
2.4 2.5
3.77
3.33
2.77
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
0 12.5 25 50 100
ADS (mg/l)
Leng
th o
f mic
rosh
oots
/exp
lant
s (c
m)
CISH-B1 CISH-B2
8.67 8.33
9.3 9
11.3310.67
9.67 9.338.67 8.67
0
2
4
6
8
10
12
CISH-B1 CISH-B2
No. o
f sho
ots/
expl
ant
Control ADS 12.5 mg/l ADS 25 mg/l ADS 50 mg/l ADS 100 mg/l
Adenine sulphate at 25.0 mg/l along with BAP 2.0 mg/l + IAA 1.0 mg/l produced more number of shoots (11.33 and 10.67) and maximum length of shoots (3.92 and 3.77 cm) in both varieties.
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In vitro rooting ½ strength MS+IBA 10.0 mg/l and IAA 1.0 mg/l produced the 100 % rooting in CISH-B1 and 95% rooting in CISH-B2, While more number of roots (2.33 and 2.0), root length (5.0 and 4.73 cm) and root diameter (2.70 and 2.05 mm) were recorded with MS +IBA 10 +IAA 1.0 mg/l.
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Acclimatization
Coconut husk supplemented with ½ strength MS plant salt mixture proved to be ideal substrate regarding maximum plant survival (83.33 %), plant grew taller (5.53 cm), produced more leaves (7.66) and roots (2.33).
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Acclimatization of in vitro rooted plants in coconut husk (A) CISH-B1 (B) CISH-B2
A B
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Acclimatization
Acclimatization in shade net house
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(A) CISH-B1 (B) CISH-B2
A B
Acclimatized plantlets growing in earthen pots
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DNA fingerprinting of Bael obtained by RAPD using OPA2, OPB1, OPF6
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 C M1
Genetic fidelity test of regenerates of bael plants
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DNA fingerprinting of Bael obtained by DAMD primers 33.6b
Genetic fidelity test of regenerates of bael plants
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The results clearly indicated that no genetic variation in mother tree and micropropagated plants was observed.
Genetic fidelity test of regenerates of bael plants
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Conclusions • A rapid mass multiplication technique using enhanced
axillary branching has been developed for two elite Bael varieties (CISH-B1 and CISH-B2).
• Here it’s concluded that, the photoautotrophic mode of nutrition give the maximum plant survival during acclimatization, rather than photoheterotrophic.
• This technique could be utilized for cloning large number of bael plants.
• However for commercialization of bael micropropagation scale up and economics has to be worked out. This can be taken in future studies.
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• Pati R, Mishra M, Chandra R and Muthukumar M (2013). Histological and biochemical changes in Aegle marmelos Corr. before and after acclimatization. Tree Genetics and Molecular Breeding. 3(3): 12-18.
• Pati R, Chandra R, Chauhan UK, Mishra M and Srivastiva N (2008). In vitro clonal propagation of bael (Aegle marmelos Corr.) cv. CISHB1 through enhanced axillary branching. Physiology and Molecular Biology of Plants. 14(4): 337-346.
• Pati R, Chandra R, Chauhan UK and Mishra M (2008). In vitro plant regeneration from mature explant of Aegle marmelos Corr.) CV. CISH-B2. Science and Culture. 74(9-10): 359-367.
• Pati R. and Muthukumar M. (2012). Genetic transformation in Aegle marmrlos Corr. In: Biotechnology of neglected and underutilized crops, edited by S.M. Jain and S. Dutta Gupta. Springer . p.343-365. [ISBN: 978-94-007-5500-0].
Related publication
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