In vitro plant regeneration and genetic
assessment among regenerates using molecular
markers in bael (Aegle marmelos Corr....
Introduction
• Bael (Aegle marmelos Corr.) is an important medicinal fruit
tree.
• The fruit pulp contains marmelosin, whi...
• Micropropagation technology can be gainfully employed in
mass multiplication of improved bael varieties.
• We have devel...
Advantages
• The production of disease free plantlets
• The rapid production of large numbers of genetically identical
pla...
Objectives
• Standardization of micropropagation protocol for shoot bud
culture from mature elite tree.
• Standardization ...
Mother plant CISH-B1 (A) Fruit of CISH-B1 (B)
Materials and Methods
A B
e-mail: rajeshpati777@gmail.com
Mother plant CISH-B2 (A) Fruit of CISH-B2 (B)
A B
e-mail: rajeshpati777@gmail.com
• 14 years old fruit bearing tree of Aegle marmelos variety
CISH-B1 and CISH-B2 of about was chosen for study.
• 30-45 cm ...
Effect of Nodal position
(A) 1-5 nodal position, (B) 6-10 nodal position,
(C) 11-15 nodal position and (D) 16-20 nodal
pos...
Different size of explant (1 cm, 2 cm, 3 cm and 4 cm
long).
Effect of Length of shoot
e-mail: rajeshpati777@gmail.com
Effect of Number of buds/explant
(A)1 axillary bud (B) 2 axillary buds and (C) 3 axillary
buds
e-mail: rajeshpati777@gmail...
Pre-sterilization
0.1 % Carbendazime (Bavestin) + 25 mg/l Rifampicin (or 0.1%
Streptomycin sulphate), citric acid (100 mg/...
In vitro morphogenesis
Nodal explants was inoculated in MS medium fortified with
different plant growth regulators like BA...
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) a...
Biochemical Studies
• The chlorophyll (a, b and total) was estimated as per the
method described by Arnon (1949).
• Nitrat...
DNA isolation
Total genomic DNA was extracted from leaf tissue of
micropropagated bael plants using Qiagen Miniprep DNA is...
S.No. Primer name Sequence (5’-3’), length Annealing temperature (0C)
a. ISSR primers
1. MP2 (GA) 8 YC, 18 mer 42
2. MP3 C...
These optimized PCR conditions were used for all PCR based
experiments in the present study.
Steps/Stages Temperature (0C)...
Effect of season on explant collection
September-October was found ideal because 85.7% explant shows in vitro
bud burst wi...
Effect of different sterilants
0
10
20
30
40
50
60
70
80
90
Percentsurvival
Control 70% Ethyl
alcohol
0.1%
HgCl2
4% NaOCl ...
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
Daystaken...
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 ...
Effect of number of buds per explant
Explants having single bud showed maximum numbers of shoots/explant
(5.33 and 4.0 res...
In vitro bud induction
• Quicker bud burst (5.33 and 6.33 days) were recorded when
explants were inoculated in MS+BAP 2.0 ...
(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 s...
In vitro proliferation
MS+BAP 2.0 mg/l + IAA 1.0 mg/l containing proliferating
medium produced 9.67 and 9.33 microshoots/e...
(A) CISH-B1 (B) CISH-B2
In vitro microshoot proliferation after 8 week
A B
e-mail: rajeshpati777@gmail.com
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...
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 CIS...
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
e-mail: rajeshpati777@gmail.com
Acclimatization
Coconut husk supplemented with ½ strength MS plant salt
mixture proved to be ideal substrate regarding max...
Acclimatization of in vitro rooted plants in coconut husk
(A) CISH-B1 (B) CISH-B2
A B
e-mail: rajeshpati777@gmail.com
Accl...
Acclimatization in shade net house
e-mail: rajeshpati777@gmail.com
(A) CISH-B1 (B) CISH-B2
A B
Acclimatized plantlets growing in earthen pots
e-mail: rajeshpati777@gmail.com
A B
(A) CISH-B1 (B) CISH-B2
Field established micropropagated plants
e-mail: rajeshpati777@gmail.com
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 ...
DNA fingerprinting of Bael obtained by DAMD primers 33.6b
Genetic fidelity test of regenerates of bael plants
e-mail: raje...
The results clearly indicated that no genetic variation in mother tree and
micropropagated plants was observed.
Genetic fi...
Conclusions
• A rapid mass multiplication technique using enhanced
axillary branching has been developed for two elite Bae...
• Pati R, Mishra M, Chandra R and Muthukumar M (2013). Histological
and biochemical changes in Aegle marmelos Corr. before...
e-mail: rajeshpati777@gmail.com
Upcoming SlideShare
Loading in...5
×

Micropropagation of Aegle marmelos and molecular markers

702

Published on

Standardization of micropropagation protocol in Aegle marmelos corr.

Published in: Education, Technology
0 Comments
0 Likes
Statistics
Notes
  • Be the first to comment

  • Be the first to like this

No Downloads
Views
Total Views
702
On Slideshare
0
From Embeds
0
Number of Embeds
0
Actions
Shares
0
Downloads
24
Comments
0
Likes
0
Embeds 0
No embeds

No notes for slide

Micropropagation of Aegle marmelos and molecular markers

  1. 1. 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
  2. 2. 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. e-mail: rajeshpati777@gmail.com
  3. 3. • 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). e-mail: rajeshpati777@gmail.com
  4. 4. 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 e-mail: rajeshpati777@gmail.com
  5. 5. 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. e-mail: rajeshpati777@gmail.com
  6. 6. Mother plant CISH-B1 (A) Fruit of CISH-B1 (B) Materials and Methods A B e-mail: rajeshpati777@gmail.com
  7. 7. Mother plant CISH-B2 (A) Fruit of CISH-B2 (B) A B e-mail: rajeshpati777@gmail.com
  8. 8. • 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). e-mail: rajeshpati777@gmail.com
  9. 9. 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. e-mail: rajeshpati777@gmail.com
  10. 10. Different size of explant (1 cm, 2 cm, 3 cm and 4 cm long). Effect of Length of shoot e-mail: rajeshpati777@gmail.com
  11. 11. Effect of Number of buds/explant (A)1 axillary bud (B) 2 axillary buds and (C) 3 axillary buds e-mail: rajeshpati777@gmail.com
  12. 12. 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. e-mail: rajeshpati777@gmail.com
  13. 13. 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). e-mail: rajeshpati777@gmail.com
  14. 14. 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. e-mail: rajeshpati777@gmail.com
  15. 15. 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). e-mail: rajeshpati777@gmail.com
  16. 16. 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 e-mail: rajeshpati777@gmail.com
  17. 17. 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. e-mail: rajeshpati777@gmail.com
  18. 18. 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: rajeshpati777@gmail.com
  19. 19. 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 e-mail: rajeshpati777@gmail.com
  20. 20. 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%). e-mail: rajeshpati777@gmail.com
  21. 21. 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 e-mail: rajeshpati777@gmail.com
  22. 22. 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 e-mail: rajeshpati777@gmail.com
  23. 23. 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 e-mail: rajeshpati777@gmail.com
  24. 24. 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. e-mail: rajeshpati777@gmail.com
  25. 25. (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. e-mail: rajeshpati777@gmail.com
  26. 26. 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. e-mail: rajeshpati777@gmail.com
  27. 27. (A) CISH-B1 (B) CISH-B2 In vitro microshoot proliferation after 8 week A B e-mail: rajeshpati777@gmail.com
  28. 28. 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. e-mail: rajeshpati777@gmail.com
  29. 29. 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. e-mail: rajeshpati777@gmail.com
  30. 30. In vitro rooting in 4 weeks old microshoots (A) CISH-B1 (B) CISH-B2 A B e-mail: rajeshpati777@gmail.com
  31. 31. In vitro rooting after 4 weeks (A, B) and 6 weeks (C) A B C e-mail: rajeshpati777@gmail.com
  32. 32. 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). e-mail: rajeshpati777@gmail.com
  33. 33. Acclimatization of in vitro rooted plants in coconut husk (A) CISH-B1 (B) CISH-B2 A B e-mail: rajeshpati777@gmail.com Acclimatization
  34. 34. Acclimatization in shade net house e-mail: rajeshpati777@gmail.com
  35. 35. (A) CISH-B1 (B) CISH-B2 A B Acclimatized plantlets growing in earthen pots e-mail: rajeshpati777@gmail.com
  36. 36. A B (A) CISH-B1 (B) CISH-B2 Field established micropropagated plants e-mail: rajeshpati777@gmail.com
  37. 37. 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 e-mail: rajeshpati777@gmail.com
  38. 38. DNA fingerprinting of Bael obtained by DAMD primers 33.6b Genetic fidelity test of regenerates of bael plants e-mail: rajeshpati777@gmail.com
  39. 39. The results clearly indicated that no genetic variation in mother tree and micropropagated plants was observed. Genetic fidelity test of regenerates of bael plants e-mail: rajeshpati777@gmail.com
  40. 40. 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. e-mail: rajeshpati777@gmail.com
  41. 41. • 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 e-mail: rajeshpati777@gmail.com
  42. 42. e-mail: rajeshpati777@gmail.com
  1. A particular slide catching your eye?

    Clipping is a handy way to collect important slides you want to go back to later.

×