Micropropagation of Aegle marmelos and 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: [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.


• 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

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


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%).


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


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


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


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

2.62.78

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.


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: [email protected]

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

A B

C e-mail: [email protected]

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) CISH-B1 (B) CISH-B2

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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Micropropagation of Aegle marmelos and molecular markers