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WELCOME

IN

CREDIT SEMINAR

1

TISSUE CULTURE APPLICATIONS

IN FRUIT CROPS

BY

KAMALPREET SINGH

M.Sc (Horticulture.) Fruit Sci.

ROLL NO. 79083

2

INTRODUCTION

Plant tissue culture is a collection of techniques usedto maintain or grow plant cells, tissues or organs understerile conditions on a nutrient culture medium ofknown composition. M Razdan (2003)

Plant tissue culture refers to growing and multiplicationof cells, tissues and organs of plants on solid or liquidmedia with nutrients under aseptic and controlledenvironment.

Plant tissue culture is genetic description whichembraces plant protoplast ,plant cell and tissue ofplant. Plant tissue culture comprises a set of in- vitrotechniques, methods, strategies. Tissue culture hadbeen exploited to create genetic variability, to improvethe health of plant material, increase the number ofdesired germplasms.

P. Ponmurugan and K. Suresh Kumar (2012)

3

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

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)

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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)

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WHY TISSUE CULTURE

A single explant can be multiplied into severalthousand plants in less than a year.

Once established, It can give a continuoussupply of young plants throughout the year.

Disease free plants ready by the tissue culturetechniques.

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’ ascompared with seedlings, which show greatervariability.

U Kumar (2008)

7

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)

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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)

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DISADVANTAGE

the experiment involved in tissue culture areexpensive.

Handled by highly trained people and carefulobservation.

As all the plants are genetically similar, there isreduction is genetic diversity.

if precautions are not taken the whole stock, maybe contaminated or infected.

The techniques is a complex procedure and it ishas varied procedure requires special and carefullobservation.

M.K Sadhu (2014)

10

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 Trials11

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)

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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.


13

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.


14

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.

.


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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

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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)

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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)

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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)

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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)

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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)

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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)

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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)

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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)

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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)

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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)

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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

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

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.

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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

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.

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(Krishna et.al 2008)

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.

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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)

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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

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(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

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

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

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SYNTHETIC SEED PRODUCTION

Buhara et.al 2015Baysal University, Bolu, Turkey

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)

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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 plantsTomar et.al 2010

38

R.I, jodhpur

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

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)

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)

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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 ona 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 piecesabout 4–5 mm in length

11. Transferred to fresh SIM for incubation in the growth chamber setat 25 C (±1), 16/8 light/dark with light intensity 1500–2500 LUX for3–4 weeks.

42

Elhiti et.al (2015)Food Research Centre , Canada

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

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

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

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)

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

ASPECTS OF SECONDARY METABOLITES

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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

RESEARCH FINDING

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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)

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BARI joydebpur ,Gazipur

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)

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Islamic Azad University, Rasht, Iran

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 )

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Dr.Y.S.P Nauni,Solan

-(without) + with PG

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

Table 6 :- Effect of BAP and NAA and strength of medium on

the rooting response in pomegranate cv. Ganesh.

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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

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

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