Tissue Culture Application in Fruit Crops

1. WELCOME
IN
CREDIT SEMINAR
1

2. TISSUE CULTURE APPLICATIONS
IN FRUIT CROPS
BY
KAMALPREET SINGH
M.Sc (Horticulture.) Fruit Sci.
ROLL NO. 79083
2

3. INTRODUCTION
 Plant tissue culture is a collection of techniques used
to maintain or grow plant cells, tissues or organs under
sterile conditions on a nutrient culture medium of
known composition. M Razdan (2003)
 Plant tissue culture refers to growing and multiplication
of cells, tissues and organs of plants on solid or liquid
media with nutrients under aseptic and controlled
environment.
 Plant tissue culture is genetic description which
embraces plant protoplast ,plant cell and tissue of
plant. Plant tissue culture comprises a set of in- vitro
techniques, methods, strategies. Tissue culture had
been exploited to create genetic variability, to improve
the health of plant material, increase the number of
desired germplasms.
P. Ponmurugan and K. Suresh Kumar (2012)
3

4.  Totipotency :- The potential or
inherent capacity of a plant cell to
develop into an entire plant . It
implies that all the information
necessary for growth and
reproduction of the organism is
contained in the cell.
 Explant :- The plant tissue or
organ excised and use for invitro
culture is know as explant.
B.D Singh (2010)
Source:- U kumar (2008)
RELATED TERMS
4

5. IMPORTANT CONTRIBUTORS TO PLANT
TISSUE CULTURE
 Haberlandt German botanist
Gottlieb Haberlandt (1902) developed
the concept of invitro cell culture.
 Folke Skoog in 1955 discovered
cytokinins e.g. kinetin as potent cell
division factor. In 1957 ,Skoog and
Miller predicted shoot and root
initiation.
 Toshio Murashige student of skoog
developed standard methods of
propagation of species in vitro in fruit
plants. Name known as Murashige
and Skoog media.
.Ponmurugan and Suresh Kumar (2012)
5

6. A GLANCE IN HISTORY
 In 1902, Mr. Haberlandt; father of plant tissue culture(He
proposed that plant cells could be cultured.)
 In 1930- Mr. White cultured tomato root tip and
subcultured to fresh medium containing salts, yeast extract
and sucrose and vit B.
 During this period ,some plant growth regulators, additives
and vitamins was discovered for the plant micro-
propagation.(discovery of PGR-Indole Acetic Acid, in 1937)
 In 1962, Murashige and Skoog published a recipe for MS
media.
 In 1972, protoplast fusion has been done in tobacco.
U Kumar (2008)
6

7. WHY TISSUE CULTURE
 A single explant can be multiplied into several
thousand plants in less than a year.
 Once established, It can give a continuous
supply of young plants throughout the year.
 Disease free plants ready by the tissue culture
techniques.
 This technique is rapid continuous and efficient.
 Breeding cycle is reduced.
 This technique useful in hard to propagate plant.
 Germplasm preservation also possible.
 Clones through this method are ‘true to type’ as
compared with seedlings, which show greater
variability.
U Kumar (2008)
7

8. MICROPROPAGATION
 Tissue culture is a term used for the growth of
plants or more commonly plant parts in sterile
culture. Micro propagation is a method of
propagating plants using very small parts of plants
that are grown in sterile culture.
 A whole plant can be regenerated from a small
tissue or plant cells in a suitable culture medium
under controlled environment. The plantlet so
produced are called tissue culture raised plant.
M.K Sadhu (2014)
8

9. ADVANTAGES OF TISSUE CULTURE
 Plant production is reliable and consistent.
Multiplication rates also high.
 Plant produced via tissue culture are usually
true-to-type and uniform.
 Produce mature plant quickly.
 Disease resistant plants are produced by tissue
culture.
 High rate of fecundity is obtained.
M.K Sadhu (2014)
9

10. DISADVANTAGE
 the experiment involved in tissue culture are
expensive.
 Handled by highly trained people and careful
observation.
 As all the plants are genetically similar, there is
reduction is genetic diversity.
 if precautions are not taken the whole stock, may
be contaminated or infected.
 The techniques is a complex procedure and it is
has varied procedure requires special and carefull
observation.
M.K Sadhu (2014)
10

11. STEPS INVOLVED IN THE MICROPROPAGATION
Cleaning of glassware
Preparation of nutrient medium
Selection and sterilization of explant
Inoculation of aseptic explant into nutrient medium
Proliferation of shoots on a multiplication medium
Transfer of shoots for Sub-culturing
Rooting and hardening of plantlets
Field Trials
11

12. EXPLANTS SOURCE
 1. Shoot –tip culture
Shoots develop from a small of cells known as
shoot apical meristem, described as the culture of
terminal (0.1-1.0 mm) portion of a shoot.
Ponmurugan and Suresh Kumar (2012)
12

13.  2. Meristem-tip culture
“Meristem-tip culture” is the invitro culture of a
generally shiny special dome-like structure
measuring less then 0.1 mm in length.
Ponmurugan and Suresh Kumar (2012)
13

14.  3. Axillary Bud Culture
The nodal or axillary bud consists of a
piece of stem with axillary bud with or without a
portion of shoot. when only the axillary bud is
cultured it is designated as “Axillary bud” culture.
Ponmurugan and Suresh Kumar (2012)
14

15.  4. Cell suspension and callus cultures
Plant parts such as leaf discs, intercalary meristems,
stem pieces ,immature embryos ,anthers ,pollen
,microspores and ovule have cultured to initiate
callus.
.
Ponmurugan and Suresh Kumar (2012)
15

16. TYPES OF MICRO-PROPAGATION
1. Callus culture
2. Suspension culture
3. Pollen culture
4. Ovule culture
5. Root tip culture
6. Shoot tip culture
7. protoplast culture
8. Leaf primordial culture
16

17. 1.CALLUS CULTURE
Callus culture concerns the initiation and continued
proliferation of undifferentiated parenchyma cells from
parent tissue or clearly defined semi solid media.
John M walker (2009)
17

18. 2.SUSPENSION CULTURE
A suspension culture refers to cell or groups of cells
dispersed and growing in an aerated liquid culture
medium is placed in a liquid medium and shaken
vigorously and balanced dose of hormones.
Cytokinin induced adventitious buds in kiwi fruit in a
suspension culture, sub-culture for about a week.
Robert verpoorte (2011)
18

19. 3.POLLEN CULTURE
 The culture of pollen grains which germinate invitro.
Such cultures may eventually form monoploid callus,
from which shoots embryoids develop into monoploid
plants.
MA Germana (2011)
19

20. 4.OVULE CULTURE
 Female gemetophyte cells are also a source for haploid
production. For haploid production for female gametophyte
it is necessary to know : (1) the events related to the
induction of haploidy in these tissues,
(2) Factors that control invitro development of proembryo
into the fully organized plants, and (3) major differences in
the growth patterns of invitro development of unfertilizes
ovule cells (female gametophyte) and in pollen cells (male
gametophyte).
U Kumar (2008)
20

21. 5.SHOOT TIP CULTURE
 The culture of terminal part of shoot to a plant in-vitro
condition or in lab called shoot-tip culture. Mostly the
shoot-tip culture used for obtain disease free plant without
genetically changes. The shoot-tip plant are more efficient
to cultivation of differentiation in vitro because cells of
them newly generated and healthy comparison to other
parts.
M Nithya Devi (2012)
21

22. 6.ROOT TIP CULTURE
 Isolated root tips of apical produce invitro root systems
with indeterminate growth habits. These were among the
first kinds of plant tissue cultures (white,1934) and remain
important research tools in the study of development
phenomena.
M Nithya Devi (2012)
22

23. 7.PROTOPLAST CULTURE
 The first step in protoplast culture involves the regeneration of the
cell wall around the protoplast membrane. Once the cell wall has
formed, cell division must be induced in the new cell.
U Kumar (2008)
23

24. 8.LEAF PRIMORDIAL CULTURE
 Leaf culture a form of tissue culture in which excised
leaves, leaf material, leaf primordia are grown on a sterile
growth medium. Mature leaves can be kept healthy under
culture conditions for considerable periods. Leaf primordia
have been used to study growth and differentiation
processes.
M.K Sadhu (2014)
24

25. STAGES OF MICRO PROPAGATION
 Micro propagation is now typically divided
into 5 stages. Stages 1-4 were originally
proposed by Murashige; Debergh and
Maene added Stage 0.
 Stage 0:- Donor Plant Selection and
Preparation
Explant quality and responsiveness is
influenced the physiological phytosanitary
condition of the donor plants.
 Stage 1:- Establishment of Explant in
culture
Surface-sterilization- disinfestations: Must
free explant tissues of all contaminating
microorganisms.
M .K Razdan (2003)
25

26. Stage 2:- Multiplication
Repeated enhanced axillary shoot production.
Encouraged by cytokinin in the medium ,alone
or with a smaller amount of auxin. Amount of
cytokinin and presence and amount of auxin
may determined empirically.
Stage 3:- Rooting (pre transplant stage)
Adventitious rooting of shoot cluster invitro.
For root initiation in vitro ,IBA are important.
Stage 4:- Acclimatization
Process by which an organism or plant
physiologically and anatomically adjust from in
vitro to ex vitro means transfer to natural
Environment.
M .K Razdan (2003)
26

27. APPLICATIONS OF TISSUE CULTURE
1. Clonal Propagation
2. Somaclonal Variation
3. Production of Virus free plants
4. Production of Synthetic seeds
5. Somatic Hybridization
6. In Vitro Plant Germplasm Conservation
7. Mutation Breeding
8. Molecular farming
9. Genetic Engineering
10. Production of secondary metabolites. 27

28. 1.CLONAL PROPAGATION
 Clonal Propagation refers to the process of
asexual reproduction by multiplication of
genetically identical copies of individual plants.
 The clonal propagation is rapid and has been
adopted for commercialization of important
plants such as
banana, apple, pear ,strawberry, cardamom,
many ornamental plants.
P. Ponmurugan and K. Suresh Kumar(2012)
28

29. BENEFITS
 Rapid multiplication of superior clones can
be carried out through out the year ,irrespective
of seasonal variations.
 Multiplication of disease free plants ,e.g. virus
free plants of apple, strawberry, banana, pear
etc.
 Multiplication of sexually derived sterile hybrids.
 It is cost effective process as it requires
minimum growing space.
29

30. 2.SOMACLONAL VARIATION
 The genetic variation found in the invitro cultured
cells are collectively referred to as somaclonal
variation and the plants derived from such cells are
called as ‘somaclones’
(Krishna et.al 2008)
30

31. Larkin and Scowkraft in 1981 coined a general
term “Somaclonal variation’’
Adventages:- Helps in crop improvement.
 Creates additional genetic variants.
 Plants with resistant and tolerant to toxins,
herbicides, high salt and even mineral toxicity.
31
(Krishna et.al 2008)

32. SOMACLONAL VARIATION IN FRUIT CROPS
Variation Presence Variation Absence
Fruit Explant
Source
Reference Fruit Explant
Source
Reference
Kiwifruit Leaf
blade and
Petiole
Prado et.al
(2008)
Almond Axillary
branching
Martins et.al
(2004)
Oil palm Zygotic
embryo
Rival et.al
(2013)
Banana Shoot tip Ray et.al
(2006)
Papaya Axillary
Shoot tip
Kaity et.al
(2009)
Vitis Sp. Nodal
Segment
Alizadeh
et.al
(2008)
Table 1:- Somaclonal variation in different fruit crops.
32

33. 3.PRODUCTION OF VIRUS FREE PLANTS
 In tissue culture application produced virus free
plants. The Viral diseases in plants transfer
easily and lower the quality and yield of the
plants. It is very difficult to treat and cure the
virus infected plants therefore the plant breeders
are always interested in developing and growing
virus free plant.
 In some crops like ornamental plants, it has
become possible to produce virus free plants
through tissue culture at the commercial level.
Nithya, (2003)
33

34. PROCEDURE TO PRODUCE VIRUS FREE PLANT
- Hot water treatment(40˚C,24 h)
-Transfer surface sterilized
segments (5-7 mm) on MS medium
- Transfer explants to Rooting medium
- Root elongation (4 weeks)
- Hardening
- Transfer into soil
34
(Bhojwani et al 2013)
Take explant
Virus Indexing
Direct
Organogenesis
(3 weeks)
Root initiation
(3 weeks)
Plants in Pots
Virus free Plants (in vitro gene bank ,
production of healthy seeds)
D.U, U.P

35. 4.PRODUCTION OF SYNTHETIC SEEDS
 In Synthetic seeds the somatic embryos are encapsulated
in a suitable matrix (e.g sodium alginate), along with
substances like insecticides, fungicides , and herbicides.
These artificial seeds can be utilized for the rapid and
mass propagation of desired plant species as well as
herbicides varieties .
Nithya, (2003)
35

36. Explant selected from healthy plant
Induced callus in explant
Somatic embryo induced in callus
Somatic embryo proliferated
Maturation of somatic embryo
Encapsulation of somatic embryo
Invitro germination
Acclimatization, induce fruit
Produce Synthetic Seed
36
SYNTHETIC SEED PRODUCTION
Buhara et.al 2015Baysal University, Bolu, Turkey

37. 5.SOMATIC HYBRIDIZATION
 Somatic hybridization broadly involves in vitro fusion of
isolated protoplasts to form a hybrid cell and its
subsequent development to form a hybrid plant.
 Development of hybrid plants through the fusion of
somatic protoplasts of two different plant
species/varieties is called somatic hybridization.
M Raj Ahuja (1997)
37

38. SOMATIC HYBRIDIZATION TECHNIQUES
Isolation of protoplast
Fusion of the protoplasts of desired species/varieties
Identification and Selection of Somatic hybrid cells
Culture of the hybrid cells
Regenration of hybrid plants Tomar et.al 2010
38
R.I, jodhpur

39. 6.IN VITRO GERMPLASM APPLICATION
 Germplasm refers to the sum total of genes present in a crop and its
related species.
 The conservation of germplasm involves the preservation of the
genetic diversity of a particular plant.
 This will ensure the availability of valuable germplasm to breeder to
develop new and improved varieties.
 Germplasm conservation depending upon the crop species and
method of preservation of genetic resources from 1 to 15 years.
 Important method of conservation of germplasm is Cryopreservation .
Angelika Filova (2014)
39

40. CRYOPRESERVATION
 The germplasm is stored as a very low temperature
using solid carbon dioxide ( at -79˚C)
 Using low temperature deep freezers (at -80˚C)
 Using Vapour nitrogen (at -150˚C)
 Using Liquid nitrogen (at-180˚C)
 Any tissue from a plant can be used for
cryopreservation e.g, meristems, embryos,
endosperms, ovules, seeds, cultured plant cells,
protoplasts, calluses.
Ponmurugan and Suresh Kumar(2012)

41. 7.MUTATION BREEDING
 Mutagenic agents, such as radiation and certain
chemicals, then can be used to induce mutations
and generate genetic variations from which
desired mutants may be selected.
 Mutation induction has become a proven way of
creating variation within a crop variety.
FJ Novak & H Bruner (1992)
41

42. MUTATION BREEDING CHEMICALLY
1. Take shoot-tip area of explant (0.2 mm size)
2. Cultured on shoot induction medium
3. Stem segments incubated in growth chamber for 2 days
4. Activate the lateral vegetative buds
5. Transferred into 50 ml plastic tubes
6. 35–40 ml EMS(Ethyl MethaneSulphonate) solution and placed on
a shaker .
7. 60–90 RPM for the desired time.
8. Explants were washed with sterile water 4–5 times
9. Shaken in sterile water for 1 h at 60–90 RPM
10. Treated and washed stem segments were cut into small pieces
about 4–5 mm in length
11. Transferred to fresh SIM for incubation in the growth chamber set
at 25 C (±1), 16/8 light/dark with light intensity 1500–2500 LUX for
3–4 weeks.
42
Elhiti et.al (2015)Food Research Centre , Canada

43. 8.MOLECULAR FARMING
 Molecular farming is the use of whole plants or plant
cells/tissues cultured in vitro for the production of valuable
recombinant proteins.
Schillberg et.al (2013)
43

44.  The advantages of plant-based systems can be
summarized as follows.
 Plants are less expensive to set up and maintain than
cultured cells.
 Plant-based systems are extremely versatile.
 which has been established as an economically viable
alternative to mainstream production system and cells
cultivated in large-scale bioreactors.
44
Rosaria et.al 2007

45. 9.GENETIC ENGINEERING
 Although genetic engineering and hybridization by
conventional breeding can augment genetic variation in
plants.
 In terms of quick returns, the time needed to produce a
new genotype can be a critical factor for its commercial
exploitation.
Ahuja et.al (2007)
45

46. PRODUCTION OF GENE
The production of DNA fragments to be cloned
Insertion of the DNA fragments into a suitable vector
Introduction of the recombinant DNA into a suitable host
Selection of the host cell or clones carrying the desired DNA
Using the DNA insert (gene) from recombinant DNA for Propagation 46
B.D Singh (2010)

47. 10.PRODUCTION OF SECONDRY METABOLITES
 Secondary metabolities can be produced by using different
biotechnological approaches, such as callus cultures, cell
suspension cultures and/or organ cultures.
 It was observed, that production of secondary metabolites
is generally higher in differentiated plant tissues, there
were attempts to cultivate whole plant organs, i.e. shoots
or roots in invitro conditions with the aim to produce
medicinally important compounds (Biondi et al., 2002).
Angelika Filova (2014)
47

48. ASPECTS OF SECONDARY METABOLITES
48
Selection of cell lines
Large scale cultivation of plant cells
Medium and nutrient composition
Elicitor helps to production of SM
Effect of environmental factors
Biotransformation using plant cell culture
Secondry Metabolite Release and Analysis
Jha et.al 2014

49. RESEARCH FINDING
49

50. Treatment Number of shoots
BAP (mg/l) NAA(mg/l) 10 DAI 20DAI 30 DAI
7.5 0 0.75 1.75 2.50
7.5 0.5 0.75 2.75 6.25
7.5 1.0 0.75 2.75 5.25
7.5 1.5 1.0 1.75 4.25
7.5 2.0 1.0 1.75 2.25
LSD (0.01) 0.61 1.05 1.26
Table 2. :- Effect of different concentration of BAP and NAA on
shoot multiplication of banana.
(AMIN et al 2009)
50
BARI joydebpur ,Gazipur

51. Table 3 :- Response of strawberry explant to different
BAP concentrations supplemented in MS medium on
shoot proliferation in strawberry.
BAP
(Mg/ L)
NAA
(Mg/L)
Shoot
Number
Shoot
length
Root
Number
Root Length
0.5 0.5 1.67 4.82 3.33 3.26
1.0 0.5 2.33 3.76 2.33 3.56
1.5 0.5 2.00 3.80 2.00 2.20
2.0 0.5 1.87 3.63 1.57 1.83
2.5 0.5 1.68 3.48 1.33 1.30
CV% 29.50 5.27 23.28 4.28
.(Rahim.et.al 2013)
51
Islamic Azad University, Rasht, Iran

52.  Table 4:- Effect of various concentration and combination of growth
regulators on shoot proliferation in apple.
Growth Regulators
Number/
Shoots
Conc (mg/L) PG (100
mg/L)
No. of usable
shoots
propagule
Average length
of shoots(cm)
Average Leaf
No./Shoot
BAP+
IBA +
GA3
0.5
0.1
1.0
-
+
2.66
6.66
1.63
2.73
12.67
16.00
BAP+
Kn
0.5
0.5
-
+
1.33
2.33
0.667
2.10
6.00
12.67
BAP+
IBA+
GA3
0.1
0.1
1.0
-
+
1.33
1.66
0.933
1.06
6.66
6.66
BAP+
IBA+
GA3
0.1
0.1
0.5
-
+
-
1.00
2.00
1.33
1.20
1.83
0.96
7.00
12.00
8.00
BAP+
GA3
0.1
5.0
+
-
1.66
1.00
1.50
1.00
8.00
4.66
BAP+
NAA
0.1
0.1
+
-
1.66
1.00
1.56
0.633
8.66
6.00
BAP 2.0 + 1.33 1.00 7.66
CD (0.05) 0.6169 0.1723 1.7666
(Sharma.et.al 2010 )
52
Dr.Y.S.P Nauni,Solan
-(without) + with PG

53. Table 5:- Percent response when use BAP and NAA growth
regulators supplemented with MS medium on shoot
multiplication in Kinnow.
Sr no. BAP (mg/L) NAA (mg/L) Percent
Response
1. 0.5 0.5 10
2. 1.0 0.5 16
3. 1.5 0.5 25
4. 2.0 0.5 39
5. 2.5 0.5 56
6. 3.0 0.5 60
7. 3.5 0.5 49
8. 4.0 0.5 22
9. 4.5 0.5 12
(Sharma et.al 2012)
53
DU University Allahabad, U.P

54.  Table 6 :- Effect of BAP and NAA and strength of medium on
the rooting response in pomegranate cv. Ganesh.
54
BAP
(mg)
NAA
(mg)
Shoot
/explant%
Length of
shoot
(cm)
No. of
root/
shoot
Length of
root (cm)
Rooting
(%)
0.5 0.5 24.67 2.43 2.6 1.5 32.33
1.0 0.5 76.33 2.00 2.00 2.43 20.33
1.5 0.5 23.00 2.53 2.93 1.83 34.4
2.0 0.5 42.67 2.13 2.50 2.43 23.67
CD at
5%
1.63 0.21 0.34 0.22 1.47
Singh.et.al 2013Navsari Agri Uni Navsari ,Gujrat

55. CONCLUSION
 Tissue culture is one of the most important part
of applied biotechnology.
 In the coming decades the world’s population
will increase more and accommodation space,
agricultural lands will decrease significantly.
 Increase per capita availability of food by tissue
culture process easily.
 Keeping these in mind we have to ensure a
peaceful, healthy and hunger free greener world
for our next generation. For doing this there is no
alternate of plant tissue culture. 55

56. 56