Horticultural Work Experience

HEMVATI NANDAN BAHUGUNA GARHWAL UNIVERSITY,

SRINAGAR GARHWAL UTTARAKHAND

TOPIC: Hands on Training on Micro-propagation of Horticultural Crops

PRESENTED BY:
Dharmesh Ranwa
B.Sc. Horticulture
8th Semester(2020-24)
 Contents

 About the ICAR-Central Institute Of Arid Horticulture, Bikaner

 What is micropropagation/ tissue culture?

 Steps involved in micropropagation/ tissue culture technique in fruit crops

 Methods of micropropagation

 Advantages and disadvantages of micropropagation

 Case study first: Micro-propagation of Cactus Pear

 Case study second: Micro-propagation of Mulberry

 Case study third: Micro-propagation of Pomegranate

 Case study fourth: Micro-propagation of Date palm

 Case study fifth: Micro-propagation of Date palm by inflorescence

About the ICAR-Central Institute Of Arid Horticulture, Bikaner
During V-five year plan an ad-hoc project under AP Cess fund scheme was started by Indian Council of Agricultural
Research in 1976 at 10 centres for conducting research on some selected fruits in semi-arid areas of India.
In April, 1993 the Project Coordinator along with the establishment of AICRP on AZF was shifted from HAU, Hisar
to Bikaner, which actually started functioning at Beechwal, Bikaner from September, 1994. After visualizing the
progress made by NRCAH in short span of time and future needs of the arid region, on 27th September, 2000, the
NRCAH was elevated to full fledged Institute as Central Institute for Arid Horticulture, Bikaner (CIAH) and Central
Horticultural Experiment Station (CHES), Godhra, Gujarat (Earlier under IIHR, Bangalore) was merged as its
Regional Centre on October 1st, 2000. Two division i.e. Division of Crop Production and Division of Crop
Improvement has been created during month of August 2013.

Location
The headquarter of ICAR-Central Institute for Arid Horticulture (CIAH) is located on National Highway No. 15 (Bikaner-Sri Ganganagar Road)
10 km away from the Bikaner city, which lies at 280 N latitude, 730 180E longitude and at altitude of 234.84 m above sea level. The recurrent
drought and extreme aridity are common phenomena. The average rainfall is about 230 mm/ annum. May-June hottest (mean maximum
temperature 42-90C and mean minimum temperature 29.6 0C) and December-January (mean maximum temperature 23.7 0C and mean
minimum temperature 8.9 0C) are coldest months of the year. Occasional frost is also experienced during January and February. The soil of the

area is sandy, desertic, poor in fertility and water holding capacity .

What is micropropagation/ tissue culture?

Micropropagation is a method of plant multiplication

using extremely small pieces of plant tissue known as
explant taken from a carefully chosen and prepared
mother plant, and growing these under laboratory
conditions to produce new plants.

Tissue culture is an art and science of in vitro plant multiplication. Micropropagation means the process by
which complete plant in test tube, bottle, flask etc. is developed taking any part of the plant like root, stem, leaf,
flower etc. by growing in standardized controlled climatic conditions under the laboratory in an appropriate
medium nourished with various nutrients and hormones.
Steps involved in micropropagation/ tissue culture technique in fruit crops

There are various steps involved in tissue culture technique. Each and every step has some specific techniques and care should be taken during
whole process for successful outcomes. These steps are as under-

1. Selection of suitable donor mother plant and preparation

2. Trimming for initiation/stimulation of lateral shoot growth

3. Pre-treatment sprays

4. Providing forcing solution for initiating bud break

5. Appropriate nourishment and watering

6. Establishment of aseptic culture

7. Proliferation of axillary shoots

8. Shoots/ clump of shoots elongation and development

9. Root development in micro-shoots/ shoot clumps

10. Hardening of plantlets under greenhouse/ polyhouse/ shade net house

Methods of micropropagation

1. Meristem culture

2. Callus culture

3. Suspension culture

4. Embryo culture

5. Protoplast culture

Plant tissue culture media

Different media have been standardized and reported in several crops which have their own specifications. Some
major plant tissue culture media used commercially are given here under with their specifications-
1. Murashige& Skoog Medium (MS): used for micropropagation, organ culture, callus culture and cell
suspension culture.
2. Gamborg B5 Medium: used for callus culture and cell suspension culture.
3. Nitsch Medium: used for anther callus culture.

4. CHU (N6) Medium: used to promote for organ culture and cell suspension culture.

5. Schenk &Hidebrandt Medium (SH): used for callus and cell suspension culture.

6. BM Medium: used for seed culture and micropropagation of orchids.

Plant tissue culture media used for in vitro multiplication are composed of following six basic ingredients:

1. Complex mixture of salts (Macro salts & Micro salts): essential plant elements (mineral ions).

2. Organic supplement: vitamins and amino acids.

3. Carbon source: generally sucrose (sugar).

4. Gelling agents: such as agar

5. Plant Growth Regulators: auxins, cytokinins, gibberellins, abscisic acid, ethylene and antibiotics.

6. Antibiotics: They are produced by some microorganisms which suppress the growth of other microorganisms
and/ or even destroy them.
Advantages of micropropagation

1. This is an alternative method for vegetative propagation with enhanced multiplication rate.

2. Large quantities of identical plants can be obtained from a single plant tissue within a very short time period.

3. The germplasm stocks can be maintained for several years using this technique.

4. It helps in the production and maintenance of pathogen-free plant varieties.

5. Genetic uniformity of the propagules can be maintained through this technique.

6. This method is independent of season and can be carried out anytime.

Disadvantages of micropropagation

7. It is essential to have expensive and complex facilities, skilled employees, and specific processes.
8. High production costs are caused by costly facilities and high labour inputs.
9. Contamination or pest and diseases can result in significant losses in a short period of time.
10. Somatic variation at a higher level.
11. Plant growth in the field was poor.
 Case Study First

Micropropagation of cactus pear

Introduction

Botanical Name: Opuntia ficus-indica

Family: Cactaceae

Chromosome no.: 2n = 6x = 66 and 2n = 8x = 88

Origin: Native America

Cactus Pear is a fruit that comes from one of several varieties of opuntia cactus. The cactus pear is a succulent shrub or
tree that ranges on average from 1.5-3 m in height. The branches (cladodes or “paddles”) are flattened and are grey-
green in colour.The cactus pear plants though shallow rooted but have capacity to absorb and store water in its
parenchyma even under unfavourable climatic conditions due to high mucilage production in both cladode and fruits.
Steps involved in sterilization and inoculation of explants made from cactus pear cladode

 Selection of healthy disease free elite mother plant of spineless, vegetable type cactus pear
 Detaching of suitable cladode having budsfrom mother plant
 Treatment of whole cladode by dipping in to soap solution followed by washing with DD water
 Treatment with bavistin solution followed by washing twice with DD water
 Surface sterilization of cladodes with HgCl 2 added with Tween 20 solution
 Washing thrice with DD autoclaved water under laminar
 Preparation of suitable explant size having one bud each
 These explants were immediately inoculated on shoot proliferation medium under laminar
Put in to dark for response
 Process of selection of explant source, preparation of explants and inoculation

Cactus pear cladode Surface cleaning of cladode Sterilization of cladodes

Explant preparation and inoculation under laminar Inoculated explants in shoot proliferation medium
1. Media preparation:

In the case of cactus pear we have used M.S. media for inoculation/shoot proliferation from explants because it is beneficial for
root & shoot development. The M.S. media used which comprised of different harmones and concentrations of harmones with
and without activated charcoal.

Example:

A. M.S. media of BA (6- Benzyladenine) 5mg/l

B. M.S. media of BA 3mg/l
C. M.S. media of BA 2mg/l
D. M.S. media of NAA(Napthalene acetic acid) + BA (1mg + 2mg)/l

2. Explant selection:
In the case of cactus pear we were taken the bud in form of explant. The selected explant
should be needed free from insect/pest & disease.
The explant should be taken from healthy & disease free mother plant with the help of
secateurs.
3. Sterilization:
i. Dip the explant in soup solutionfor 10 minutes.
ii. Wash with double distilled autoclave water.
iii. Dip the explant in Bavistin + PVP solution for 10 minutes.
iv. Wash with double distilled autoclave water.
v. Dip the explant in HgCl2+ Tween 20 + Antimicrobial supplement.
vi. Wash with double distilled autoclave water thrice.

4. Inoculation:
The small pieces of explant without damaging buds are inoculated in M.S. media.
The inoculation process held on laminar air flow in protected climate for decrease
the contamination. It is done very carefully with sterilized tools (Knife, Scalper,
Spatula, Forceps, Petri-plates etc.) to save explants from contamination.
After the inoculation the inoculated explants placed in controlled climate dark
room.
 Process of in vitro plant formation of spineless cactus pear

5. Observation:
The weekly observation of inoculated cactus pear
were recorded with respect to following points-
 Number of inoculated explants/flasks/bottles/test-
tubes.
 Number of contaminated explants.
 Number of sprouted explants.
 Percentage of sprouted explants. Bud initiation Shoot development

 Shoot proliferation.
 Shoot elongation.

Shoot proliferation Subculturing of micro shoot for elongation Shoot elongation

6. Subculturing:
In the subculturing process the explants are subcultured in new media after some time of inoculation for
better root & shoot development.

In this process the contaminated explants are also subcultured in new media with 70% alcohol treatment to
protect from contamination.
 Case Study Second

Micropropagation of mulberry (MI-572)

Introduction:

Botanical name: Morus spp.

Family: Moraceae
Chromosome number: 2n = 2x = 28
Origin: Asia and North America

The present findings of M. alba demonstrate the possibility for mass propagation of mulberry through nodal and shoot tip
culture. For successful micropropagation axillary buds or shoot tip cultures are preferred as pre- existing meristem easily
develop into shoots while maintaining clonal fidelity.

Mulberries are fast growing, deciduous, woody perennial plant having deep root system. Plants are generally dioecious. The
black mulberry is the smallest over red and white mulberry and sometimes grows up to 30 feet in height, but becomes a bush if
not trained at younger stage.
Steps involved in sterilization and inoculation of explants made from mulberry

 Selection of healthy disease free elite mother plant of mulberry

 Detaching of suitable shoots having budsfrom mother plant
 Preparation of suitable explant size having one or two buds
 Treatment of explants with bavistinsolution followed by washing twice with DD water
 Surface sterilization of explants with HgCl 2 added
 Washing with DD autoclaved water under laminar
 These explants were immediately inoculated in shoot proliferation medium under laminar
 Put in to dark for response
1. Media preparation:

In the case of mulberry we used the M.S. media for inoculation because it is beneficial for root & shoot development.The M.S. media used of
different harmones and their concentrations with and without activated charcoal.

Example:

A. M.S. media of BA (6- Benzyladenine) 5mg/l

B. M.S. media of BA 3mg/l
C. M.S. media of BA 2mg/l
D. M.S. media of NAA(Napthaleneacetic acid) + BA (1mg + 2mg)/l
 Process of in vitro plant formation of mulberry

Mulberry shoots with dormant bud Treatment with bavistin Treatment with HgCl2

Explant preparation and inoculation under laminar Inoculated explants in shoot proliferation medium
2. Explant selection:
 In case of mulberry shoots having the non-sprouted buds used as the explant.
 The explant should be taken from healthy & disease free mother plant with the help of secateurs.
3. Sterilization:
i. Dip the explant in soup solution for 10 minutes.
ii. Wash with double distilled autoclave water.
iii. Dip the explant in Bavistin + PVP solution for 10 minutes.
iv. Wash with double distilled autoclave water.
v. Dip the explant in HgCl2 + Tween 20 + Antimicrobial supplement.
vi. Wash with double distilled autoclave water thrice.

4. Inoculation:
The small pieces of explant(with help of blade) without damaging shoot having buds are inoculated in
M.S. media flasks.The inoculation process held on laminar air flow in protected climate for decrease
the contamination.It is done very carefully with sterilized tools (Knife, Scalper,Spatula, Forceps,
Petri-plates, secateursetc.) to save explants from contamination.After the inoculation the inoculated
explants placed in controlled climate dark room.
5. Observation:
The weekly observation of mulberry explant were recorded with respect to following points-
 Number of inoculated explants/flasks/bottles/test-tubes.
 Number of contaminated explants.

 Number of sprouted explants.

 Percentage of sprouted explants.

 Shoot proliferation.

6. Subculturing:
In the subculturing process the explants are subcultured in new
media after some time of inoculation for better root & shoot Shoot proliferation
development.
 Case Study Third

Micropropagation of pomegranate (Bhagwa)

Introduction:

Botanical name: Punica granatum

Family: Punicaceae
Chromosome number: 2n = 2x =18
Origin: Iran

Pomegranate is a fruit-bearing deciduous small tree and belongs to the family of Punicaceae. It is an
economically important plant which is growing tropical and sub-tropical regions of the world due to its
delicious edible traits with pharmaceutical and ornamental usage.The fruit is largely used as a dessert and the seeds
along with the fleshy pulp called aril are dried and also used as a condiment.Pomegranate, (Punicagranatum), bush or
small tree of the family Lythraceae and its fruit. The juicy arils of the fruit are eaten fresh, and the juice is the source
of grenadine syrup, used in flavourings and liqueurs.
Steps involved in sterilization and inoculation of explants made from pomegranate cv. Bhagva

 Selection of healthy disease free mother plant of Bhagva variety of pomegranate

 Detaching of suitable shoots having budsfrom mother plant
 Treatment of shoot pieces by dipping in to soap solution followed by washing with DD water
 Treatment with bavistinsolution followed by washing twice with DD water
 Surface sterilization with HgCl 2 added with Tween 20 solution
 Washing thrice with DD autoclaved water under laminar
 Preparation of suitable explant size having one bud each
 These explants were immediately inoculated on shoot proliferation medium under laminar
 Put in to dark for response

1. Media preparation:

Example:

A. M.S. media of BA (6- Benzyladenine) 5mg/l

B. M.S. media of BA 3mg/l
C. M.S. media of BA 2mg/l
D. M.S. media of NAA(Napthaleneacetic acid) + BA (1mg + 2mg)/l
Media preparation
 Process of in vitro plant formation of pomegranate

Explant selection and collection Treatment with bavistin Treatment with sodium-hypochloride

Explant preparation and inoculation under laminar Inoculated explants in shoot proliferation medium
2. Explant selection:
 In case of pomegranate (Bhagwa) shoots having the buds used as the explant.
 The explant should be taken from healthy & disease free mother plant with the help of
secateurs.
3. Sterilization:
i. Dip the explant in soup solution for 10 minutes.
ii. Wash with double distilled autoclave water.
iii. Dip the explant in Bavistin + PVP solution for 10 minutes.
iv. Wash with double distilled autoclave water.
v. Dip the explant in HgCl2 + Tween 20 + Antimicrobial supplement.
vi. Wash with double distilled autoclave water thrice.

4. Inoculation:

In the case of pomegranate small pieces of explantwithout damaging buds are

inoculated in M.S. media. After the inoculation the inoculated explants placed in
controlled climate dark room.
5. Observation:
The weekly observation of inoculated pomegranate were recorded with respect to following points-
 Number of inoculated explants/flasks/bottles/test-tubes.
 Number of contaminated explants.
 Growth & status of explant.

6. Subculturing:
In the subculturing process the explants are subcultured in new
media after some time of inoculation for better growth of
explant. In this process the contaminated explants are also
subcultured in new media with 70% alcohol treatment to protect
from contamination.
 Case Study Fourth

Micropropagation of date palm

Introduction:

Botanical name: Phoenix dactylifera

Family: Arecaceae
Chromosome number: 2n = 36
Origin: North Africa

Micropropagation of date palm can be achieved through either somatic embryogenesis or direct organogenesis.
Somatic embryogenesis is characterized by the development of a somatic cell into an embryo with a bipolar
structure, leading to shoot and root formation. This technique is used in many commercial laboratories for its high
potential for rapid and large-scale plant production. On the other hand, organogenesis is characterized bythedirect
formation ofadventitiousbuds on the explant. This may produce new plants after root regeneration. Organogenesis
has lower multiplication efficiency than somatic embryogenesis but it permits the preservation of true-to-typeness
of multiplied plants.
Steps involved in sterilization and inoculation of explants made from off shoot of date palm

 Selection of healthy disease free elite mother tree

 Detaching of off-shoot from mother tree
 Removal of leaves and debris
 Preparation of shoot tip
 Treatment of shoot tip with bavistin followed by washing twice with DD water
 Surface sterilization of shoot tip with HgCl 2 added with antimicrobial supplement
 Washing with DD autoclaved water
 Treatment of shoot tip with sodium hypochloride+Tween 20 followed by whashing with DD autoclaved water
 Preparation of explants under laminar and immediately inoculation of explants on callus initiation medium
 Put in todark for response

1. Media preparation:

A. M.S. media of NAA(Napthaleneacetic acid) + BA

B. M.S. media of without harmone
C. M.S. media of BA + T.D.Z.
D. M.S. media of ABA
Removal of off-shoot Removed off-shoot Trimming and cutting of off-shoot Preparation of shoot tip from off-shoot

Cutting of undesired leafy portion Shoot tip ready for further sterilization Chemicals/solutions used for sterilization

Sterilizing shoot tip Explant preparation and inoculation under laminar Inoculated explants in callus initiation medium

Process of date palm off-shoot selection & collection, preparation of explant & inoculation in callus initiation medium
2. Explant selection:
 In the explant selection of datepalm healthy & disease free mother plant was selected.
 In case of datepalm the mother plant mostly selected 20-25 year old to take the better off shoot.
 The off shoot detaching carefully from mother plant with help of desirable tools.
 The leaves & debris of the off shoot removed & cleaned very carefully without damaging the off shoot.
 Prepared shoot tip & taken it to media preparation room for further sterilization process.

3. Sterilization:
 Shoot tip immerged in soap solution for 8-10 minutes.
 Wash with double distilled autoclave water.
 Shoot tip dipped in Bavistin + PVP solution for 15-20 minutes.
 Wash with double distilled autoclave water.
 Antioxidant treatment with citric acid + ascorbic acid + anti-microbial supplement/l for 20-25 minutes.
 Wash with double distilled autoclave water.
 Sterilized with HgCl2+ anti-microbial supplement for 10-12 minutes.
 Washed with double distilled autoclave water.
 Treat with Sodium-hypochloride solution + Tween 20 for 20-25 minutes.
 Wash with double distilled autoclave water thrice.
4. Inoculation:
 The inoculation of explant should be done on laminar air flow to reduce the chances of contamination.
 The explant (Off shoot) cut in to small pieces for easy inoculation in media bottles/flasks/test-tubes.
 The inoculation should be done with sterilized tools (Knife, Scalper, Ptri-plates, Forceps etc.).
 After the inoculation, inoculated explants were placed in dark for better response.
5. Observation:

The weekly observation of inoculated explants of date palm were recorded with respect to following points-

 Number of inoculated explants/flasks/bottles/test-tubes.

 Number of contaminated explants.
 Number of explants initiated callus
 Callus multiplication & maturation
 Somatic embryogenesis
 Somatic embryo maturation
 Somatic embryo inoculation for plantlets development
 Somatic embryo germination
 Shoot development
 Shoot elongation
 Root & Shoot growth & development
 Process of in vitro plant formation of date palm

Inoculated explant Callus initiation and multiplication Somatic embryogenesis

Shoot initiation Shoot elongation Plant formation Plantlets with root and shoot
6. Subculturing:
In the subculturing process the explants are subcultured in new media after some time of inoculation for better
availability of nutrients and hormones.
In case of datepalm the callus was subcultured in new media after sometime because the callus multiplication takes
place & the nutrients were absorbed by callus.
 Case Study Fifth
Micropropagation of date palm by Inflorescence
Steps involved in sterilization and inoculation of explants made from immature inflorescence of date palm

1. Selection of healthy disease free elite mother tree

2. Detaching of immature inflorescence from mother tree
3. Removal of spathe and debris by cutting vertically through sterilized scalpel blade
4. Preparation of explants
5. Treatment of explants with bavistin followed by washing twice with DD water
6. Surface sterilization of explants with HgCl 2 added with Tween 20
7. Washing thrice with DD autoclaved water under laminar
8. Immediately inoculation of explants on shoot proliferation medium
9. Put in to dark for response
Process of explant preparation from immature inflorescence of date palm, sterilization and inoculation in shoot proliferation medium

Immature inflorescence/spathe Sterilization of immature inflorescence Explant preparation under laminar

Inoculation under laminar Inoculated explants in shoot proliferation medium Inoculated explants under culture
room
1. Media preparation:

In the case of datepalm we use the M.S. media for inoculation because it is beneficial for callus initiation & multiplication. The M.S. media used of
different harmones& their different concentrations with & without activated charcoal.

Examples:

i. M.S. media of BA + PVP

ii. M.S. media of T.D.Z. + PVP
iii. M.S. media of IAA + PVP
iv. M.S. media of NAA + PVP

2. Observation:
The weekly observation of inoculated explants of date palm were recorded with respect to following points-
 Number of inoculated explants/flasks/bottles/test-tubes.
 Number of contaminated explants.
 Number of proliferated explant/Number of explants initiated callus.
 Callus multiplication/Shoot proliferation.
3. Subculturing:
In the sub-culturing process, the explants are subcultured in new media after some time of inoculation for better availability of nutrients and
hormones.
Conclusion
We have learned and acquainted with various instruments and machineries used in micropropagation/tissue
culture and knew the principle of working of these. Basic aspects and science of micropropagation techniques of
several horticultural crops of arid region learned theoretically and obtained practical exposure inform of hands
on training on date palm, cactus pear, mulberry and pomegranate in detail. We have practically gone through
almost all aspects of micropropagation such as preparation of different hormonal media and composition for
explant inoculation, and also did several practicals on explant preparation, sterilization procedure, inoculation in
suitable callus initiation/shoot proliferation medium and observations of inoculated explants in the given
horticultural crops. Date palm callus cultures (already initiated) have been subcultured periodically for callus
multiplication, callus maturation & somatic embryogenesis, somatic embryo inoculation, shoot proliferation,
plantlets development in RS media. The learned tools and techniques of micropropagation of horticultural crops
will be useful in my future career/ degree programme.
References:
 Micropropagation in Horticulture", by S Gunn ”The Plantsman” (new series) Vol. 5 (2006) pp238–243

 Plant Tissue Culture, Techniques and Experiments 3rd Edition by R H Smith, Academic Press, (2012)

 Advance technology in horticulture crops (pp.221-229)Publisher: AkiNik Publications

 Cactus pear: cultivation and uses by Kamlesh Kumar, Dhurendra Singh, Rama Shanker Singh, 2018/10/1, Publisher: ICAR-
CIAH

 Jain AK, Dandin SB and Serigupta K (1990) In vitro propagation through axillary bud multiplication in different mulberry
genotypes. Plant Cell Rep. 8 : 737-740

 Raj D and Kamlesh K (2010) In vitro regeneration of Punica granatum L. Plants from different juvenile explants. Journal of
Fruit and Ornamental Plant Research. 18(1): 5-22.

 Callus induction, somatic embryogenesis, in vitro plantlet development and ex vitro transplantation of two date palm
(Phoenix dactylifera L.) cultivars by Kamlesh Kumar, Dhurendra Singh and PL Saroj, Publisher: ICAR-CIAH