micropropagation technique in fruit crops

1. Credit Seminar
on
Micropropagation Technique in
fruit crops
COLLEGE OF HORTICULTURE & FORESTRY,
JHALAWAR(RAJASTHA)
SPEAKER
Priyanka katara
Ph.D. Scholar (Horticulture)
Fruit Science
SEMINAR INCHARGE
Dr. Prerak Bhatnagar
Asstt. Professor
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2. Contents
Introduction
Need of Micropropagation
 Advantages
Constraints
Stages of Micropropagation
Approaches of Micropropagation
Case Study
Conclusion
2

3. Micropropagation involves
production of plants from very
small plant parts, tissues or
cells, grown aseptically in a test
tube or other containers under
controlled environment.

4. In vitro clonal propagation through tissue
culture is referred to as micro propagation.
Use of tissue culture technique for micro
propagation was first started by Morel (1960)
for propagation of orchids, and is now applied
to several plants.
Micro propagation is a handy technique for
rapid multiplication of plants.
Micropropagation are generally used in
banana, strawberry, papaya, date palm, bael
and citrus etc.
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5. Seasonal limitation.
 Long juvenile phase of fruit plants.
Storage problem in recalcitrant seeds eg: mango, citrus.
Parthenogenesis eg. Banana.
Transfer of viruses through planting material.
Large number of planting materials are required.
Some fruits do not response to the vegetative propagation
methods eg. Papaya.
Need of Micro propagation
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6. ADVANTAGES
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7. It ensures true to type plants.
It is independent of seasonal constraint.
A new plant can be regenerated from a miniature
plant part.
Facilitates long distance transport of propagation
materials and long term storage of clonal materials.
Large scale multiplication in lesser time and space.
Production of female plants in dioecious plants is
possible eg. papaya.
Production of virus free plants.
It is the only viable method of regenerating
genetically modified cells.
This helps to save endangered species and the
storage of germplasm.
Rejuvenation of old fruit tree
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8. CONSTRAINTS
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9. Requires skill and manpower.
Slight infection may damage the entire lot of
plant.
The cost involved in setting up and
maintenance of laboratory is very high.
The seedling grown under artificial condition
may not survive when placed under
environment condition.
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10. Techniques of Micro
propagation
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11. Fig 1: Overall technique of micropropagation
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12. Stages of
Micropropagation
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13. Establishment stage
Proliferation
Rooting
Acclimatization
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14. Fig 2: Stages of micropropagation
Establishment
stage
Proliferation
stage
Rooting stage
Acclimatization
stage
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15. Approaches of
Micropropagation
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16. 1. Multiplication by axillary buds/apical shoots.
2. Multiplication by adventitious shoots.
3. Organogenesis: with callus or without callus
4. Somatic embryogenesis: The regeneration of embryos
from somatic cells, tissues or organs.
Micro propagation mostly involves in vitro clonal propagation
by four approaches
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17. 1. Axillary buds/apical shoots
culture
This approach of micropropagtion utilizes
cultures of either shoot tip or nodal explants on a
medium supplemented with, usually, a cytokinine
and often an auxin.
 Development of shoots from explants.
Each leaf on such shoots has an axillary bud
and they are also stimulated to developed axillary
branching.
After some time (4-6 weeks), the axillary
branching in a culture reaches the maximum.
The individual shoots are then excised and sub-
cultured onto fresh medium to initiate a new cycle
of multiplication by axillary branching.
Axillary
branchesSingle shoot
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18. Fig.3: A diagrammatic representation of micropropagation of plants by axillary bud
(or shoot tip) method (A) Rosette plan (B) Elongated plant
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19. 2. Multiplication by Adventitious Shoots
In many species, adventitious shoot buds are used for
micropropagation.
Adventitious shoot buds are produced in vivo in many plant
species eg. stems, bulbs, tubers and rhizomes.
These are often used for conventional propagation of plant like
banana.
When leaf, stem or other explants of plants are cultured in vitro, a
very large number of adventitious shoot buds develop.
In grapes, fragmented shoot tips have been used to induce
adventitious shoot buds.
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20. Organogenesis is the process of morphogenesis involving the
formation of plant organs i.e. shoots, roots, flowers, buds from
explant or cultured plant tissues.
For appropriate organogenesis in culture system, exogenous
addition of growth regulators—auxin and cytokinin is required.
i. Low auxin and low cytokinin concentration will induce
callus formation.
ii. Low auxin and high cytokinin concentration will promote
shoot organogenesis from callus.
iii. High auxin and low cytokinin concentration will induce
root formation.
It is of two types — direct organogenesis
indirect organogenesis
3. Organogenesis
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21. Direct organogenesis : somatic tissue of plants are capable of
regenerating adventitious buds/shoots. These buds are formed
directly from a plant organ or any piece of tissue without
forming any callus structure.
In direct organogenesis: some time plant regeneration from
cultured explants involves the initiation of basal callus formation
and then shoot bud differentiation. Meristems, shoot tip, axillary
buds, immature leaf, immature embryo are good for callus
initiation.
Conti…
21

22. 22
Fig. 4: A. direct Organogenesis, B. Organogenesis through callus formation

23. The process of regeneration of embryos from somatic cells, tissues
or organs is regarded as somatic (or asexual) embryogenesis.
Development of somatic embryos can be done in plant cultures
using somatic cells, particularly epidermis, parenchymatous cells of
petioles or secondary root phloem.
Somatic embryo generally originate from single cells, which divide
to form a group of meristematic cells.
The cells of this meristematic mass continue to divide to give rise
to different stage of SEs i.e. globular, heart -shaped, torpedo-shaped
and cotyledonary stages.
Somatic embryos are bipolar structure.
4. Somatic embryogenesis
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24. Direct Somatic Embryogenesis:
When the somatic embryos develop directly from the excised plant (explants)
without undergoing callus formation, it is referred to as direct somatic
embryogenesis. This is possible due to the presence of pre-embryonic
determined cells (PEDC) found in certain tissues of plants. eg: hypocotyls
tissue.
Indirect Somatic Embryogenesis:
In indirect embryogenesis, the cells from explants (excised plant tissues)
are made to proliferate and form callus, from which cell suspension
cultures can be raised. Certain cells referred to as induced embryo
determined cells (IEDC) from the cell suspension can form somatic
embryos. eg: secondary phloem.
It is of two types — direct somatic embryogenesis
indirect somatic embryogenesis
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25. 25

26. Growth phases of somatic
embryogenesis
1. Induction: cells attain the capacity for embryogenesis, and they may
progress upto the globular stage. It is promoted by auxin, especially in case
of cells in explant are differentiated and in less differentiated cells of somatic
embryos, cytokinin may be enough.
2. Development: development occurs beyond the globular stage. This may
occur on the induction medium or other medium.
3. Maturation: somatic embryo do not grow in size, but they become hardy.
Maturation is promoted by ABA or high sucrose concentration.
4. Germination: Somatic embryo germinate to produce seedlings. This is
promoted by GA3.
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27. The cultivation of axillary or apical meristems, particularly of shoot
apical meristem, is known as meristem culture.
Meristem cultures have been extensively used for quick vegetative
propagation of a large number of plant species.
To produce virus free plant it is essential that the apical meristem
should be excised along with minimum of surrounding tissue.
The shoot tip may be cut into fine pieces to obtain more than one
plant from each shoot tip.
Meristem culture
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28. 28
Fig. 6: Regeneration of plant through meristem culture

29. A callus is mass of undifferentiated parenchymatous cells. When a living plant
tissue is placed in an artificial growing medium with other conditions
favorable, callus is formed. The growth of callus varies with the homogenous
levels of auxin and Cytokinin and can be manipulated by endogenous supply
of these growth regulators in the culture medium. The callus growth can be
referred into three different stages.
•Stage I: Rapid production of callus after placing the explants in culture
medium.
•Stage II: The callus is transferred to other medium containing growth
regulators for the induction of adventitious organs.
•Stage III: The new plantlet is then exposed gradually to the environmental
condition.
Callus culture
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30. A cell suspension culture refers to cells and or groups of cells
dispersed and growing in an aerated liquid culture medium.
It is placed in a liquid medium and shaken vigorously and
balanced dose of hormones.
Suspension culture
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31. 31
Fig. 7: schematic diagram showing the different explants used and the pathway for apical shoot tip
culture, node culture, callus culture and suspension culture.

32. Micropropagation in different
fruit plants
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33. Medium Fresh weight
(g)
Dry weight (g) Shoot length
(mm)
MS 0.18836ab 0.03441ab 37.533ab
C2D 0.18308ab 0.02754ab 40.667ab
WPM 0.11658b 0.01753b 28.767b
DKW 0.20080ab 0.03069ab 31.567ab
DKW-G 0.30457a 0.04074a 54.500a
Note: Means marked with the same letter are not significantly
different at 5% probability level
Table:1 Effect of five different media on in vitro shoot
growth of Ziziphus spina-christi
Assareh M.H. and Sardabi, 2005 at Iran
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34. Substrate Plant
height
(cm)
No. of
leaves
/plant
No. of
root/plan
t
Length
of root
(cm)
No. of
shoots/pl
ant
Survival
of plant
(%)
Soil 4.33 3.00 1.50 0.899 1.60 31.66
Soil
+Sand +
FYM
4.63 7.33 1.66 1.40 2.33 63.33
Coconut
husk
5.53 7.66 2.33 1.60 3.00 83.33
SEm ± 0.156 0.430 0.319 0.745 0.396 2.886
CD (0.05) 0.540 1.487 1.102 0.257 1.371 9.976
Table:2 Effect of substrate on acclimatization of micro
propagated plants of Aegle marmelos Corr.
Pati, R. et al., 2008 at CISTH, Lucknow
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35. Growth
regulators (µM)
Shoot no. per callus Shoot length (cm)
KN Solid
medium
Liquid
medium
Solid
medium
Liquid medium
2.32 1.8d 1.1d 1.2d 0.7d
4.64 2.3c 1.8c 2.5c 1.6c
6.96 2.8b 2.1b 4.4a 2.1b
8.12 2.9ab 2.4ab 4.0b 2.8a
9.28 3.3a 2.8a 1.5d 0.8d
BAP
2.24 2.8c 1.0c 2.1d 1.2c
4.48 2.9c 1.2c 4.4b 2.2b
6.72 5.3ab 2.1b 7.5a 3.8a
7.84 5.7a 2.9a 7.3a 3.5a
8.96 5.0b 3.2a 3.9c 2.4b
Table:3 Shoot regeneration of date palm (Phoenix dactylifera L.) in
MS solid and liquid medium supplements with KN and BAP
Aslam and Khan, 2009 at U.A.E
Note: Means marked with the same letter are not significantly
different at p ≤ 0.05
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36. Size of
explant
(cm)
Days taken
for callusing
% of explant
with pro-
embryonic
calli
Days taken
for somatic
embryo
initiation
No. of
embryos after
100 days
2.5 22.5 40.44 49.5 31.66
3.5 18.3 44.44 47.5 158.33
4.5 19.5 33.33 46.5 64.66
SEm ± 0.89 1.75 0.84 7.43
CD (P=0.05) 1.73 3.40 1.63 14.44
Table:4 Effect of size of explant on somatic embryogenesis in
Mango cv. Kurakkan
Mishra , M. et al., 2010 at CISTH, Lucknow
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37. Parameter
BBTV BMV
Size of
Meriste
m (mm)
Survival
(%)
Virus
free
no.
Virus
infected
No.
Virus
free
(%)
Virus
free
no.
Virus
infected
No.
Virus
free
(%)
0.3 mm 75 65 10 86.66 70 5 93.33
0.5 mm 85 45 40 52.94 50 35 58.82
1.0 mm 100 10 90 10 12 88 12.00
Table:5 Production of virus-free banana
seedlings using meristem tip culture in vitro
Shamy, et al., 2011 at Egypt
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38. Growth
regulator
(mg l−1)
Frequency of
shoots
showing
root induction
(%)
Days to
root
induction
Root length
(cm)
Number of
root/explant
(cm)
IBA (1.0) 66.66 13.5±1.24 4.5±0.41 2.5±0.21
IAA (1.0) 46.66 16.0±2.10 2.5±0.31 3.5±0.19
NAA (1.0) – – – –
Control 60.00 17.0±2.43 4.5±0.51 4.5±0.24
Mean 43.33
Error ±15.02
Table:6 Effect of IBA, IAA, or NAA (1 mgl−1) on root induction from
the in vitro regenerated shoots of C. limon cv. Kaghzi Kalan
Goswami, K. et al., 2013 at SKNAU, Bikaner
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39. Treatment Concentration of
growth regulators
(mg/l)
No. of shoots Shoot length (cm)
GHRP* IVDS** GHRP* IVDS**
BAP (Benzyl
amino purine)
0.25 1.90c 3.90a 1.65b 1.57b
0.5 0.50 2.18c 2.92b 3.00a 0.71c
1.0 1.00 2.88c 1.27d 0.76c 0.58cd
2.0 2.00 3.72a 1.03d 0.71c 0.63c
TDZ
(Thidiazuron)
0.10 2.78b 1.00d 0.72c 0.33d
mean 2.70a 2.30b 1.30a 1.00b
Table:7 Response of in vivo and in vitro raised explants to
cytokinin in guava (Psidium guajava L.)
Note: Means marked with the same letter are not significantly different at p ≤ 0.05
*GHRP: Greenhoue raised plant **IVDS: in vitro derived shoot Ali, et al., 2003 at
Pakistan39

40. Concentration of
growth regulators
(mg/l)
No. of roots Root length (cm)
IBA GHRP* IVDS** GHRP* IVDS**
0.5 1.40c 3.10b 0.56cd 1.41b
1.0 1.50c 5.40a 0.68c 2.03a
2.0 1.70c 1.70c 0.25d 1.51b
mean 1.53b 3.34a 0.51b 1.65a
Table:8 Rooting of in vivo and in vitro explants
raised on IBA in guava (Psidium guajava L.)
Note: Means marked with the same letter are not
significantly different at p ≤ 0.05
*GHRP: Greenhoue raised plant **IVDS: in vitro derived shoot
Ali, et al., 2003 at Pakistan
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41. Concentration
of CPPU
(mg.L-1)
Number of shoots Shoot length
Liquid
media
Semi-solid
/double
layer
media
Liquid Semi-solid
/double layer
0 2±0.20b 2±0.22c 0.667±0.06b 0.778±0.05c
0.05 2±0.20b 4±0.36c 0.656±0.04b 0.592±0.04c
0.1 2±0.35b 6±0.67b 0.767±0.07b 1.069±0.10b
0.15 3±0.32a 10±1.36a 1.567±0.11a 1.667±0.19a
0.2 2±0.25b 2±0.32c 0.711±0.06b 0.667±0.08bc
Table:9 Effect of CPPU on number of shoot and Shoot length of
papaya (C. papaya L.) using liquid and semi-solid/liquid double
layer media approaches after eight weeks of culture
Mean values within a column followed by same
letters are not statistically different at P ≤ 0.05
Gatambia et al., 2016 at Kenya
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42. Conclusion
At present micropropagation finds extensive application in horticulture in
several countries. It is used to provide a sufficient number of plantlets from
a stock plant which does not produce seeds, or does not respond well to
vegetative reproduction. It is also used to produce virus free plant at rapid
rate .The major limitation in the use of micropropagation for many plants is
the cost of production. For this reason, many plant breeders do not utilize
micropropagation because the cost is prohibitive.
In India, micropropagation technique is in initial phase and did not
fully exploited as compared to countries. However, it is gradually increasing
because India has great potential to used micropropagation technique
commercially in fruit plant propagation.
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43. THANK YOU
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