Propagation of pomegranate (Punica granatum L.) by tissue culture

1 • Introduction
2 • Different in vitro techniques in micro-propagation
3 • Micro-propagation
5 • Advantages of micro-propagation
6 • Plant parts for regeneration
7 • Stages of micro propagation
8 • Factors affecting in vitro multiplication
9 • Problems encountered during micro-propagation
10 • Review of literatures (Case studies )
11 • Conclusion
Topic Division

Introduction
 Botanical name: Punica granatum L.
 Family: Punicaceae
 Origin: Iran
 India is one of the major pomegranates producing country
 In India: Area - 0.13 m ha
Production - 1.35 m tons
Productivity - 10.3 t/ha (Anon. 2014)
 Salinity and drought hardy fruit crop
 A fully matured fruit contains many of the important nutrients, minerals,
protein, fat, fiber, carbohydrate, etc.
 The fruit are rich in Fe, Ca, and antioxidant component like phenol,
pigments and tannins
(Sepulveda et al., 2000).
1

 The conventional method - hardwood cutting and layering
 However, it has several limitations
- Low success
- Less availability of planting material at a time
 This results is non- availability of planting material through-out the year
 Micro propagation would help in overcoming difficulties of vegetative
propagation, producing true to-type plants, rapid and mass production of
planting materials
 Tissue cultures of edible pomegranate via shoot organogenesis, somatic
embryogenesis and enhanced auxiliary bud proliferation have been reported
2

Different in vitro techniques in micro-propagation
Meristem culture
Shoot tip culture
Callus culture
Cell culture
Protoplast culture
Somatic embryogenesis
Anther culture
3

Micro-propagation
 It refers to the production of plants from very small plant
parts, tissues or cell, grown aseptically in a test tube or
containers under controlled nutritional, environmental and
aseptic condition.
 Suitable for the rapid and large-scale clonal multiplication of
elite germplasm.
 Well known as a means of producing millions of identical
plants (“clones”) under aseptic conditions
 The size of meristem tissue used for micro-propagation is
about 0.1-0.5 mm size having only one or two leaf primordia.
4

Advantages of Micro-propagation
Year-round availability of plants
Fast multiplication of true-to-type planting material
Disease-free plant production
Export and import of germplasm become easy requiring
minimum quarantine checks
Easy transport of propagation material
Conservation of plant diversity
5

Small space is required to maintain and multiply large number of
plants.
Small tissue is required as an explant, hence saves the scion wood
to a great extent.
Micro propagated plants exhibit vigorous growth and higher
yields
It helps in reducing the breeding cycle, through embryo rescue
and somaclonal variation
Production of homozygous plants
6

Basic requirements for Micro-propagation
1. A well equipped laboratory
2. Asepsis
3. A suitable culture medium
4. Controlled environment
7

Plant parts for regeneration
Regeneration from existing meristems.
Regeneration from adventitious meristems.
Regeneration by somatic embryogenesis
Regeneration of whole plant from small excised plant parts
include the following
8
Shoot tip and nodal bud
Leaf, internodes, stems and roots
Originated from somatic or vegetative cells

Stage 0: Selection of mother plant and explant isolation
Stage 1: Explant Establishment
Stage 2: Shoot Multiplication
Stage 3: Rooting of Shoots
Stage4: Hardening and Transfer to Soil/Field
9

Factors Affecting In Vitro Multiplication
Selection of explants
The organ that is to serve as a tissue source
Size of explant and overall quality of parent plant
Physiological state
10
Select the explant from good, healthy, disease free mother plant.
Shoot tip, meristematic tissue, cotelydonary tissue, root tip etc.
Larger explant regenerated earlier and faster
Younger trees or plants provide efficient explant than older one

Starting material for
micro propagation
Leaf
Tip bud
Internode
Axillary
bud
Root
Explant
Cell tissue or organ of a
plant that is used to start
in vitro cultures.
11

Methods of disinfection of the explants
 The various types of disinfectants can be used to overcome
the problem of contamination.
 The surface sterilizing agents like sodium hypochlorite,
calcium hypochlorite, and mercury chloride can be used for
sterilization of explants.
 The systemic sterilizing agents like Bavistin (fungicide)
and streptocycline (bactericide) can also be useful for
prevention of contamination.
 The above chemicals may be used alone or in combination to
enhance the survival rate of explants during sterilization
12

Contamination
Release of Phenolic Compounds
Variations in Tissue Culture-Raised Plants
Mortality in Greenhouse
Facilities are costly
Highly technical skills required
13
Problems encountered during micro-propagation

FACTORS INFLUENCING IN VITRO GROWTH AND SHOOT
MULTIPLICATION OF POMEGRANATE
Singh and Patel, 2014, Gujarat, India
15
Case study – 1

Table 1: Effect of adenine sulphate on in vitro shoot
multiplication and growth of pomegranate cv. Ganesh
Adenine
sulphate (mg /L)
Proliferation of
shoot (%)
No. of shoot
/culture
Length of
shoot (cm)
20 17.50 1.67 1.33
30 28.00 2.00 1.68
40 78.25 3.75 3.06
60 61.00 3.92 2.35
80 28.50 1.65 1.00
100 24.45 1.29 0.65
S. Em.± 0.45 0.15 0.10
CD at 5 % 1.34 0.46 0.29
16
Singh and Patel, 2014

Table 2: Effect of different light intensities on in vitro shoot
multiplication and growth of pomegranate cv. Ganesh
Light intensity
(Lux)
Growth rate
of shoot
No. of
shoot/explant
Length of
shoot(cm)
Remark
1000 + 1 0.85 Stunted growth
2000 ++ 2 1.8 Defoliation
occurred
3000 +++ 4.8 3.5 Good growth
4000 +++ 1.8 1.83 Yellowing of shoot
S.Em.± _ 0.13 0.15
CD at 5 % _ 0.4 0.48
+ Little growth, ++ Medium growth, +++ Better growth
17

Figure 1: Effect of different level of
sucrose on in vitro shoot
multiplication of pomegranate
cv. Ganesh
Figure 2: Effect of initial pH of the
medium on in vitro shoot
multiplication of
pomegranate cv. Ganesh
18

Figure 3:
(a) Proliferation of shoots in MS medium containing 1.0 mg/L BAP + 1.0 mg/L Kinetin with
40 mg/L adenine sulphate
(b) Proliferation of shoot at 3000 lux light intensity
(c) Shoot multiplication at 3 % sucrose in medium
(d) Shoot proliferation at 5.8 pH of medium
a
c
19

Conclusion
 Adenine sulphate 40 mg/l maximize proliferation of shoot, no.
of shoot/culture and length of shoot compare to other treatment
combination
 Apart from that light intensity (Lux) 3000 increase no. of
shoot/explant and length of shoot
 Higher no. of shoots/explant and maximum length of shoots
were recorded with pH 5.8
20

African Journal of Biotechnology
Vol. 10(79), pp. 18130-18136, 12 December, 2011
Micro-propagation of pomegranate (Punica granatum L.)
„Bhagava‟ cultivar from nodal explant
Patil et al., 2011, Maharashtra, India
21
Case study – 2

Table 3. Effect of silver nitrate and adenine sulphate in MS medium on the
rates of nodal explants regenerating shoots for establishment stage.
S/N Medium
Concentration
(mg/L)
Number of
shoot/
explant
% of
explants
showing
response
Shoot length
(cm)
1 MS+ Silver nitrate 0.5 4 ± 1 87 ± 1 1.9 ± 0.9
6 MS+ Adenine sulphate 10 3 ± 1 75 ± 1 0.6 ± 0.2
Patil et al., 2011
22

Table 4. Effect of NAA and IBA in MS medium on rooting.
S/N Medium Concentration
(mg/L)
Number of
root/
explant
% of
explants
showing
response
Root length
(cm)
1 MS+ IBA 0.0 1 ± 1 20 ± 1 0.3 ± 0.2
2 MS+ IBA 0.25 4 ± 1 82 ± 1 1.5 ± 0.2
3 MS+ IBA 0.50 6 ± 1 97 ± 1 3.4 ± 0.5
4 MS+ NAA 0.0 1 ± 1 15 ± 1 1.3 ± 0.2
5 MS+ NAA 0.25 4 ± 1 80 ± 1 2.5 ± 0.2
6 MS+ NAA 0.50 6 ± 1 97 ± 1 3.2 ± 0.5
23
Patil et al., 2011

Figure 4. MS medium containing 0.5 mg/L
NAA showing the highest rooting
response.
Figure 5. MS medium containing IBA 0.5
mg/L shows highest rooting
response and thick root formation.
24
Patil et al., 2011

Table 5. Effect of BAP, NAA, silver nitrate and adenine sulphate in MS medium and
woody plant medium on the rates of nodal explants regenerating number of
shoots per explant
S/N
Medium
Number of shoot/ explant
BAP
(1.8 mg/L)
NAA
(0.9 mg/L)
Silver
nitrate
(1 mg/L)
Adenine
sulphate
(30 mg/L)
BAP +
NAA +
AgNO3 +
Adenine
sulphate
1 MS Medium 5 ± 1 3 ± 1 6 ± 1 5 ± 1 12 ± 3
2 WPM Medium 3 ± 1 2 ± 1 4 ± 1 2 ± 1 7 ± 1
25
Patil et al., 2011

Figure 6. MS medium containing BAP,
NAA, silver nitrate and adenine
sulphate show highest shoot
regeneration.
Figure 7. WPM containing BAP, NAA,
silver nitrate and adenine
sulphate showing lower shoot
regeneration as compared to MS
medium.
26
Patil et al., 2011

Table 6. Effect of BAP and NAA in MS medium on the rates of nodal explants
regenerating maximum leaves for proliferation stage.
S/N Medium Concentration(
mg/L)
Number of
leaves/
explant
% of explants
showing
response
Shoot length
(cm)
1 MS+ BAP 0.1 11 ± 1 35 ± 1 0.6 ± 0.1
2 MS+ BAP 0.2 14 ± 1 40 ± 1 0.9 ± 0.3
3 MS+ BAP 0.3 15 ± 1 85 ± 1 1.6 ± 0.8
4 MS+ BAP 0.4 19 ± 1 98 ± 1 2.0 ± 0.6
5 MS+ BAP 0.5 17 ± 1 82 ± 1 1.4 ± 0.2
6 MS+ NAA 0.1 12 ± 1 57 ± 1 1.0 ± 0.2
7 MS+ NAA 0.2 15 ± 1 77 ± 1 1.1 ± 0.4
8 MS+ NAA 0.3 18 ± 1 96 ± 1 1.5 ± 1.0
9 MS+ NAA 0.4 16 ± 1 87 ± 1 1.5 ± 0.9
10 MS+ NAA 0.5 14 ± 1 89 ± 1 1.2 ± 0.6
27
Patil et al., 2011

Conclusion
• From the study we can conclude that, the role of silver nitrate
and adenine sulphate alone or in combination with BAP and
NAA in MS medium induced high frequency and adventitious
shoot regeneration
28

African Journal of Biotechnology
Vol. 12(20), pp. 2863-2868, 15 May, 2013
DOI: 10.5897/AJB2013.11962
ISSN 1684-5315 ©2013 Academic Journals
In vitro mass multiplication of pomegranate from cotyledonary
nodal explants cv. Ganesh
Singh et al., 2013, Maharashtra, India
29
Case study - 3

Table 7. Effect of different media on the establishment and growth of
cotyledonary nodal explants of pomegranate cv. Ganesh
Media Explant
establishment (%)
Days to
establishment
Length of
shoot (cm)
No. of
internodes
MS 75.57 10.43 3.06 2.86
B5 52.86 12.29 1.19 1.07
WPM 33.43 16.86 0.91 1.00
S.Em 0.31 0.26 0.07 0.02
CD at 5% 0.94 0.77 0.21 0.08
Singh et al., 2013
30

Table 8. Effect of plant growth regulators on frequency of shoot multiplication
of cotyledonary nodal explants of pomegranate cv. Ganesh.
BAP (mg/l) NAA (mg/l) IAA (mg/l) Kinetin (mg/l) Shoot/explant (%)
0.5 - - - 13
1 - - - 11.33
1.5 - - - 14.33
2 - - - 20
0.5 0.5 - - 24.67
1 0.5 - - 76.33
1.5 0.5 - - 23
2 0.5 - - 42.67
0.5 - 0.5 - 18.67
1 - 0.5 - 24.67
1.5 - 0.5 - 22
2 - 0.5 - 25
1 - - 0.5 59
1 - - 1 86.33
S.Em ± 0.56
CD at 5% 1.63
Singh et al., 2013 31

Singh et al., 2013
Figure 8. Effect of serial sub culturing and growth regulators on the multiplication of
cotelydonary nodal explants of pomegranate cv. Ganesh.
32

Table 9. Effect of NAA and strength of medium on the rooting response in
pomegranate cv. Ganesh
Medium
strength
NAA + AC
(mg/l)
Rooting
(%)
Days to root
initiation
Length of
root (cm)
No. of
root/shoot
Length of
shoot (cm)
MS ½
0.1 + 200 63.07 12.43 3.07 3.4 4.4
0.2 + 200 45.73 14.07 2.57 1.67 2.37
0.5 + 200 68.8 10.5 3.87 4.17 6
0.8 + 200 50.73 12.67 2 2.27 2.27
MS full
0.1 + 200 32.33 15.37 1.5 2.6 2.43
0.2 + 200 20.33 13.33 2.43 2 2
0.5 + 200 34.4 12.2 1.83 2.93 2.53
0.8 + 200 23.67 15.97 2.43 2.5 2.13
White medium
0.1 + 200 48.33 14.87 1.2 3.1 2.3
0.2 + 200 22.33 14.84 1.7 2 2.07
0.5 + 200 52.83 11 2.87 3.47 4.07
0.8 + 200 28.4 14.67 2.63 3.23 2.5
S.Em ± 0.5 0.33 0.07 0.11 0.07
CD at 5% 1.47 0.97 0.22 0.34 0.21
Singh et al., 2013 33

Table 10. Effect of different potting mixtures on acclimatization of pomegranate
cv. Ganesh.
Potting mixture Survival of
plantlet (%)
Days to
sprouting
Length of
shoot (cm)
Vermicompost 46.25 18.00 4.50
Leaf mould 0.00 0.00 0.00
Soil 37.50 15.50 5.75
Vermicompost + soil (1:1 v/v) 85.50 11.75 7.75
Vermicompost + leaf mould + soil (1:1:1 v/v) 57.25 18.00 5.00
S.Em ± 0.61 0.31 0.20
CD at 5% 1.82 0.93 0.61
Singh et al., 2013
34

Singh et al., 2013
Figure 9. A. Establishment of explants on MS medium + 1.0 mg/l BAP + 0.5 mg/l NAA
B. Shoot multi[placation on MS medium + 1.0 mg/l BAP + 1.0 mg/l kinetin + 200 mg/l
activated charcoal
C. In vitro rooting on half MS medium + 0.5 mg/l NAA + 200 mg/l activated charcoal
D. Hardening of plantlet in potting mixture of vermicompost + soil (1:1 v/v).
35

Conclusion
 In vitro rooting of regenerated shoot was found in half strength MS
medium supplemented with 0.5 mg/l NAA + 200 mg/l activated charcoal,
which recorded the maximum number of root/shoot and root length.
 In vitro grown plantlets were transferred to vermicompost + soil (1:1v/v)
media kept in net house, which showed better survival of plantlet (85.50%)
within 11.75 days .
 It estimated that using the present protocol of in vitro propagation, large
number of plantlets can be produced in a year starting from single
cotyledonary nodal explants.
36

Agricultural University of Tirana
Albanian j. agric. sci. 2013;12 (1): 1-5
In vitro propagation of pomegranate (Punica granatum L.) Cv.
„Males Yazdi‟
Kaji et al., 2013, Iran
37
Case study -4

Figure 10. Effect of culture medium type on in vitro shoot proliferation of the pomegranate
cultivar, „Malas Yazdi‟. Columns with different letters are significantly different from
each other at P ≤ 0.05 (Duncan‟s multiple range test). Bars represent SE values
Kaji et al., 2013
38

Figure 11. Effect of different concentration of Kinetin on in vitro shoot proliferation of the
pomegranate cultivar, „Malas Yazdi‟. Columns with different letters are significantly
different from each other at P ≤ 0.05 (Duncan‟s multiple range test). Bars represent SE
values
39Kaji et al., 2013

Figure 12. Effect of different concentration of IBA and NAA on rooting of „Malas Yazdi‟. Columns
with different letters are significantly different from each other at P ≤ 0.05 (Duncan‟s
multiple range test). Bars represent SE values
Kaji et al., 2013 40

Conclusion
 From the study we can conclude that, WPM proved to be more
efficient medium compared to MS.
 The best concentration of kinetin was 9.2 µm, resulting in the
highest number of nodes, shoot length and leaf number.
 Half strength WPM medium supplemented with 5.4 µm NAA
was most effective for rooting of shoots.
 Rooted plantlets were successfully acclimatized and
transferred into field.
41

International Journal of Advanced Research (2015),
Volume 3, Issue 5, 162-165
In vitro Callus induction and Root regeneration through the
mediation of Agrobacterium rhizogenes in (Punica granatum)
Patil and Borkar, 2015, Ahmed nagar, India
42
Case study -5

Table 11. In vitro performance of Agrobacterium rhizogenes on juvenile
segments of pomegranate.
Explants Treatment No. of
explants*
Survival
( %) in
plant
% of callus
developed
Juvenile segments A. rhizogenes 5 100 90%
With Hormone 5 80 82%
Control (without
Hormone)
5 0 _
Patil and Borkar, 2015
43

Table 12. Performance of A. rhizogenes on Pomegranate callus for root
induction.
Explants Treatment Days for
induction of
roots
No. of roots
after six weeks
of infection
% increase in
root no. over
hormone
Treatment
Callus A. rhizogenes 35 – 40 16 128.5
With Hormone 55 – 60 7 _
44

Fig 13: (a) Rooting response of Pomegranate callus for root induction through A.
rhizogenes (b) with hormone
45

Conclusion
 The result indicated that the use of A. rhizogenes strain can be
a successful approach for callus and root induction in
pomegranate
 When this callus was inoculated with A. rhizogenes strain
RPB 13 without application of Auxin cultures in ½ MS
rooting medium these significantly had higher number of roots
as compared to control.
46

Scientia Horticulturae
Scientia Horticulturae 136 (2012) 122-127
Arbuscular mycorrhizal fungi (AMF) induced hardening of micro
propagated pomegranate (Punica granatum L.) plantlets
Singh et al., 2012, New Delhi India
47
Case study – 6

Treatment details
• Pure cultures of arbuscular mycorrhizal fungi (AMF) were procured from
the Division of Microbiology,(IARI) New Delhi.
• Four AMF strains namely
• T0 - control (no strain)
• T1 - Glomus mosseae
• T2 - Acaulospora laevis
• T3 - Glomus manihotis
• T4 - Mixed AMF strain
• The soil based AMF cultures were maintained under glasshouse conditions
in plastic pots, potting mixture (soil:sand:FYM, 2:2:1; FYM is well
decomposed cow dung manure).
48

Table 13: Effect of AMF inoculation on survival and growth attributes of micro
propagated pomegranate plants.
Treatment
Plantlet
survival (%)
Root
colonization
(%)
Plant height
(cm)
Root length
(cm)
Plant fresh
wt./dry wt.
60 DAI 90 DAI 60 DAI 90 DAI 60 DAI 90 DAI 60 DAI 90 DAI 60 DAI 90 DAI
T0
44.00a 37.20a 2.80a 7.00a 17.56a 25.06a 14.76a 19.08a 5.96a 4.76a
T1
90.40b 88.00b 47.40b 87.60b 24.96b 30.50b 23.42b 27.68b 7.86b 5.18ba
T2
88.00b 86.80b 36.80c 84.20cb 21.56c 27.00c 21.06c 27.60b 7.60b 5.72bd
T3
88.20b 86.80b 38.80cb 81.80ac 23.48d 27.08c 21.10c 23.62c 7.48b 5.92d
T4
86.80b 85.40b 44.00cb 84.80cb 23.12d 28.00c 20.76c 23.74c 7.72b 5.32bda
CD at 5% 5.59 5.57 3.58 3.25 1.15 1.23 1.42 1.03 3.49 2.70
49
(Singh et al., 2012)

Fig. 14. Effect of AMF inoculation on in vitro raised pomegranate plantlets at 90 DAI. T0 = control;
T1 = Glomus mosseae; T2 = Acaulospora laevis; T3 = Glomus manihotis; T4 = mixed (IARI)
strain.
50(Singh et al., 2012)

Table 14: Effect of AMF inoculation on physiological parameters of in vitro
raised pomegranate plants..
Treatment
Relative water
content (%)
Net photosynthetic
rate(µmol CO2/m2/s)
Net respiration rate
(µmol CO2/m2/s)
60 DAI 90 DAI 60 DAI 90 DAI 60 DAI 90 DAI
T0 83.80a 88.00a 2.20a 2.35a 7.09a 7.51a
T1 92.80b 96.00b 4.21b 5.25b 3.84b 3.77bd
T2 90.80b 93.40b 3.79c 4..18c 3.99bc 3.68b
T3 91.20b 94.00b 3.70c 4.05d 3.79b 3.22c
T4 90.40b 93.20b 3.53d 3.77e 4.17c 3.87d
CD at 5% 3.20 2.68 0.11 0.07 0.22 0.12
51

Table 15: Effect of AMF inoculation on biochemical enzymatic attributes of in
vitro raised pomegranate plants.Treatment
Total phenols
(µg/g)
Total
chlorophyll
(mg/g)
Reducing
sugars (%)
Catecholase
(Δ400/g/m)
Cresolase
(Δ400/g/m)
60 DAI 90 DAI 60 DAI 90 DAI 60 DAI 90 DAI 60 DAI 90 DAI 60 DAI 90 DAI
T0
16.20a 19.60a 2.95a 3.08a 0.85a 0.91a 364.83a 401.49a 336.05a 385.10a
T1
22.18b 24.48b 3.33b 3.83b 2.26b 1.93b 434.90b 429.00be 362.00b 407.33bd
T2
22.58b 25.67c 3.06a 3.17a 2.13b 1.81b 431.00c 426.33c 390.67c 441.33c
T3
20.80c 23.44d 2.98a 3.18a 2.10b 1.77b 504.44d 522.38d 363.83b 409.00d
T4
24.94d 28.62e 3.70c 3.96b 1.89b 1.59c 419.00e 429.00e 353.33d 396.00e
CD at 5% 0.51 0.42 0.09 0.09 1.03 0.27 2.35 2.36 2.19 8.53
52

Conclusion
 Effect of all the AMF strains was significantly superior over non-
mycorrhizal control with regard to physical, physiological and
biochemical attributes.
 Among all mycorrhizal strains Glomus mosseae followed by G.
manihotis were found superior suggesting their high suitability as
biohardening agents for tissue culture raised pomegranate
plantlets
53

Overall Conclusion
54
From the over all study we can conclude that,
 The role of silver nitrate and adenine sulphate alone or in combination with
BAP and NAA in MS medium induced high frequency and adventitious
shoot regeneration
 In vitro rooting of regenerated shoot was found in half strength MS
medium which recorded the maximum number of root/shoot and root
length.
 In vitro grown plantlets were transferred to vermicompost + soil media
showed better survival of plantlet respectively .
 The use of A. rhizogenes strain can be a successful approach for callus
and root induction in pomegranate
 Among all mycorrhizal strains Glomus mosseae followed by G. manihotis
were found superior suggesting their high suitability as biohardening agents
for tissue culture raised pomegranate plantlets

Propagation of pomegranate (Punica granatum L.) by tissue culture

Propagation of pomegranate (Punica granatum L.) by tissue culture

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