Dharmesh Micropropagation PP
Dharmesh Micropropagation PP
Dharmesh Micropropagation PP
PRESENTED BY:
Dharmesh Ranwa
B.Sc. Horticulture
8th Semester(2020-24)
Contents
Methods of micropropagation
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
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-
3. Pre-treatment sprays
1. Meristem culture
2. Callus culture
3. Suspension culture
4. Embryo culture
5. Protoplast 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.
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).
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.
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
Introduction
Family: Cactaceae
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
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:
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.
In this process the contaminated explants are also subcultured in new media with 70% alcohol treatment to
protect from contamination.
Case Study Second
Introduction:
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
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:
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.
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
Introduction:
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
1. Media preparation:
Example:
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:
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
Introduction:
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
1. Media preparation:
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-
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
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:
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)
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