In vitro plant regeneration and genetic assessment of bael micropropagated plants using molecular markers

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: rajeshpati777@gmail.com

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.

• 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).

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

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.

Mother plant CISH-B1 (A) Fruit of CISH-B1 (B)
Materials and Methods
A B

Mother plant CISH-B2 (A) Fruit of CISH-B2 (B)
A B

• 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).

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.

Different size of explant (1 cm, 2 cm, 3 cm and 4 cm
long).
Effect of Length of shoot

Effect of Number of buds/explant
(A)1 axillary bud (B) 2 axillary buds and (C) 3 axillary
buds

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.

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).

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.

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).

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

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.

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
Predenaturation 92 2 1 DAMD
Denaturation 92 1
Annealing 55 2 40
Extension 72 2

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
Percentage
0
10
20
30
40
50
60
70
80
90
Percentage
% Aseptic culture Bud-burst in infection-free explants (%)
Results

Effect of different sterilants
0
10
20
30
40
50
60
70
80
90
Percentsurvival
Control 70% Ethyl
alcohol
0.1%
HgCl2
4% NaOCl 0.1%
HgCl2+4%
NaOCl
Surface sterlising agents
4min. 6min 8min
0.1% HgCl2 for 6 minutes shows less contamination high survival of
explants (86.67%).

Effect of nodal position
0
8
6.67
7.33
0
48.33
87
61.67
0
1
2
3
4
5
6
7
8
9
1-5 6-10 11-15 16 -20
Nodal Position
Daystakenforbud
break
0
10
20
30
40
50
60
70
80
90
100
Budbreak(%)
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
7
7.67
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
Daystakenforbudbreak
0
10
20
30
40
50
60
70
80
90
PecentBudbreak
Days taken for bud-break % Bud break
CISH-B1 CISH-B2

Effect of size of explant
1.67
1.33
3.67
2.67
4
3.33
2.33
2
0
0.5
1
1.5
2
2.5
3
3.5
4
No.ofshoots/explant
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.
8.33
8.67
7.67
8
6.67
7
7.33
7.67
0
1
2
3
4
5
6
7
8
9
Daystakenforbudburst
1 2 3 4
Size of explants (cm)
CISH B1 CISH B2

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.ofaxillaryshoots/explant
1 2 3
No. of buds/explant
CISH B1 CISH B2
6.67
7
7.67
8.33 8.33
8.67
0
1
2
3
4
5
6
7
8
9
Daystakenforbudburst
1 2 3
No. of buds/explant
CISH B1 CISH B2

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.

(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.

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.

(A) CISH-B1 (B) CISH-B2
In vitro microshoot proliferation after 8 week
A B

Effect of Adenine sulphate on shoot proliferation
2.6
2.78
3.92
3.35
3.17
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)
Lengthofmicroshoots/explants(cm)
CISH-B1 CISH-B2
8.67
8.33
9.3
9
11.33
10.67
9.67
9.33
8.67 8.67
0
2
4
6
8
10
12
CISH-B1 CISH-B2
No.ofshoots/explant
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.

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.

In vitro rooting
in 4 weeks old
microshoots
(A) CISH-B1
(B) CISH-B2
A
B e-mail: rajeshpati777@gmail.com

In vitro rooting after 4 weeks (A, B) and 6 weeks
(C)
A B
C

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).

Acclimatization of in vitro rooted plants in coconut husk
A B
Acclimatization

Acclimatization in shade net house

A B
Acclimatized plantlets growing in earthen pots

A B
Field established micropropagated plants

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

DNA fingerprinting of Bael obtained by DAMD primers 33.6b

The results clearly indicated that no genetic variation in mother tree and
micropropagated plants was observed.

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.

• 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].
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