Introduction Techniques of Plant Tissue Culture Micro propagation Stages involved in micro propagation Rooting Function of Auxin Hardening Conclusion References The term plant tissue culture broadly refers to the in vitro cultivation of plants, seeds, plant parts (tissues, embryos, single cells, protoplasts etc.) on nutrient medium under aseptic conditions. Or 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.

1. Plant Tissue culture
Culturestage III - Rooting
By
KAUSHAL KUMAR SAHU
Assistant Professor (Ad Hoc)
Department of Biotechnology
Govt. Digvijay Autonomous P. G. College
Raj-Nandgaon ( C. G. )

2. CONTENTS
Introduction
Techniques of Plant Tissue Culture
Micro propagation
Stages involved in micro propagation
Rooting
Function of Auxin
Hardening
Conclusion
References

3. Introduction
The term plant tissue culture broadly refers to the in vitro
cultivation of plants, seeds, plant parts (tissues, embryos, single
cells, protoplasts etc.) on nutrient medium under aseptic
conditions.
Or
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.

4. HISTORY
Harberlandt 1902 First attempt of plant tissue culture. “The father of
Plant Tissue Culture.”
Skoog 1944 in-vitro adventitious shoot formation in
Tobacco
Morel and Martin 1952 Use of meristem culture to obtain viruses free
Dhalias
George Morel 1965 was the pioneer in applying shoot-tip Culture
for micro propagation of Orchid cymbidium.
Murashige 1974 gave the concept of developmental stage by
defining establishment, proliferation and rooting and
hardening stages.

5.  EXPLANTS
 CALLUS
 PLANTLET
 DIFFERENTIATION
 REDIFFERENTIATION
 TOTIPOTENCY
TERMS USED IN PLANTTISSUE CULTURE

6. Techniques of Plant Tissue Culture
Shoot tip culture :
“The in-vitro culture of an organ(shoot tip) so as to achieve the
development and/or preservation of the original organ.”
Clonal Propagation :
“Clonal Propagation refers to the process of asexual reproduction by
multiplication of genetically identical copies of individual plants”
Micropropagation :
“in-vitro clonal propagation through tissue culture is referred to as
Micropropagation”

7. OTHER POWERFUL APPLICATIONS OF PTC

8. Micropropagation
Micropropagation is the propagation of a plant in a controlled and
artificial environment under aseptic conditions, using a defined growth
medium.

9. Major stages involved in Micropropagation
Selection of mother plant and its maintenance
Initiation and establishment of culture
Multiplication of shoots or rapid somatic embryo
formation
in vitro germination of somatic embryo and/or rooting
Of shoots
Transfer of plantlets to sterilized soil for hardening
under greenhouse environment.
STAGE 0
STAGE I
STAGE II
STAGE III
STAGE IV

10. CLONAL PROPAGATION

11. CULTURE STAGE III - ROOTING
• It is rooting of shoots or shoot clusters in vitro.
• Roots do not initiate on the media used in Step I. Therefore a
transfer is required (in auxin rich medium)
• Auxins are important for root initiation in vitro .
e.g. IAA ,IBA , NAA etc.
• Auxin can be characterized by their ability to induced cell
elongation in stems.

12. Garland & Stoltz 1981
Zimmerman & Broome 1981
Half or quarter the salt concentration to induce the
rooting.
Driver & Suttel 1987 Lower nitrogen and high sucrose Content beneficial
for in vitro rooting.
Sriskandarajah et.al. 1990 Observe rooting in 3 apple scion cultivators when
9.8M IBA was added & NH4NO3 was eliminated.
Haissig, 1974; Thorpe, 1978,
1982 ,1984; Gaspar and
Coumans,1987
rooting of microcuttings requires continuous
availability of free sugars, which favours the
differentiation of a good vascular system.
Kaneko et al. (1988) investigated the effect of CO2 on adventitious root
formation
George and Sherrington,1984 Most often IAA, IBA and NAA (0.1-1 mg 1-1)have
been used for rooting, but IAA, IBA are more effective
Jarvis, 1986 Riboflavin is reported to improve the quality of the root
system in Eucalyptus ficifolia. Several chemicals, often te
rmed 'auxin synergists' or rooting 'co-factors', have been
found to enhance the rooting response of applied auxins
.

13.  Many commercial laboratories prefer to treat the in vitro multiplied shoots as
microcutting and root them in vivo (ex vitro),if possible.
 For ex vitro rooting, the size of microcuttings may be critical.Treatment of
the microcuttings with root inducing growth regulators (auxins) or a
commercial rooting powder may be necessary for their rooting in vivo.
 An intermediate approach between in vitro and in vivo rooting is to apply
the rooting treatment in vitro and transfer cuttings out of culture before
roots appear.
 During auxin treatment the shoots were placed under higher light intensity
(66~mol m -2 s-i), shorter photoperiod (17 h) and lower temperature (19C)
than during shoot multiplication to promote lignification.
ex-vitro Rooting

14. Advantages
 In vitro rooting is labour intensive and expensive, accounting for 35-75% of the
total cost of micropropagation .
 In vivo rooting combines the rooting and acclimatization stages and, thus, reduces
aseptic handling.
 In vivo formed roots are structurally and functionally of better quality than those
developed in vitro.
 The in vitro formed roots are often thick and lack root hairs and good vasculature.
 These roots frequently die or collapse after the plantlets are removed from cultures
and new, functional lateral and adventitious roots are formed during
acclimatization.
 Under in vivo conditions, callusing at the base of the shoot occurs only rarely
ensuring a continuous vascular connection between root and shoot.
 The in vitro developed roots may get damaged during transplantation.
 For difficult-to-root species it is easier and cheaper to create good rooting
conditions in vivo than in vitro.

15. AUXINS
Auxins induce cell division ,cell elongation, and formation of callus in
cultures.
At low concentration, auxin promote root formation while at a high
concentration callus formation occurs.
Ratio of auxins to cytokinins is high, embryogenesis, calllus initiation
and root initiation occur.
2, 4-D 2, 4-Dichlorophenoxy acetic acid
2, 4 ,5-T 2, 4, 5-Trichloro acetic acid
4-CPA 4-Chlorophenoxy acetic acid
NOA 2-Naothyloxy acetic acid
MCPA 2-Methoxy 4-Chlorophenoxy acetic acid
Dicamba 2-Methoxy 3, 6-dichlorobenzoic acid
Picloram 4-Amino 2, 5, 6-trichloropicolinic acid

16. Functions of Auxin
• Stimulates cell elongation
• Stimulates cell division in the cambium and, in combination with
cytokinins in tissue culture
• Stimulates differentiation of phloem and xylem
• Mediates the tropistic response of bending in response to gravity and
light
• The auxin supply from the apical bud suppresses growth of lateral
buds
• Delays leaf senescence
• Can inhibit or promote (via ethylene stimulation) leaf and fruit
abscission
• Involved in assimilate movement toward auxin possibly by an effect
on phloem transport
• Stimulates growth of flower parts
• Stimulates the production of ethylene at high concentrations

17. HARDENING
 This stage involve the establishment of is done by plantlets in soil.
 This done by transferring the stage III plantlets from the laboratory to
greenhouse.
 Acclimatization: a process of physiologically and anatomically
adjustment from in vitro to ex vitro conditions.
 Relatively slow process, may take weeks.
 Must adjust from high to lower relative humidity (e.g. from 98-99% to
20 - 60%): development of sufficient defenses to control water loss.
 Must adjust from low light to high light: from low photosynthetic
competence to photosynthetic competence.

18. PRIMARY HARDENING
SECONDARY HARDENING

21. CONCLUSIONS

22. REFERENCES
Name of
author
Edition Year Name of
book
H.S.Chawla 3rd edition 2009 Introduction to plant
Biotechnology
U. Satayanarayan reprint 2008 Biotechnology
S.S.Bhojwani
and
M.K.Razdan
reprint 2004 Plant tissue culture
Kalyan kumar De
reprint 1999 An introduction to plant
tissue culture

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QUERIES