Adventitious shoot regeneration.....pptx

ADVENTITIOUSSHOOT
REGENERATION

CONTENTS
 Adventitious Shoot
 Plant Regeneration
 Objectives Of Adventitious Shoot Regeneration
 Plant Tissue Culture
 Organogenesis
 Types Of Organogenesis
 Procedure Of Adventitious Shoot Proliferation
 Factors Affecting Organogenesis
 Application Of Organogenesis
 References
Adventitious Shoot Regeneration…
1

ADVENTITIOUSSHOOT
 Adventitious shoot is a kind of shoot not formed in its main
location (at the end of stem and node).
 It is formed in the internodal part of explants or other parts.
 The reason for adventitious shoot formation is
dedifferentiation and redifferentiation of some cells.
 Adventitious shoots can be generated either directly from
explants containing external parts of phloem and surface
cambium.
 They can be generated indirectly from the surface of callus.
2

PLANTREGENERATION
 Plant regeneration occurs when plants repair or replace damaged structures
based on the totipotency and pluripotency of their cells.
 Tissue culture is one of the most widely used regenerative technologies.
 Regeneration of plants can be through either non-adventitious system or the
adventitious system.
 Regeneration of plant from auxillary bud is an example of non-adventitious
system.
 Adventitious system of regeneration can be through the organogenesis and
embryogenesis.
3

 In the organogenesis, the regeneration of plant is through formation of
adventitious organs from the explants such as root, lower part of stem or trailing
leaves.
 Adventitious shoots initiated directly on organ explants usually develop from a
shoot apex formed superficially from epidermis or hypodermis.
4

OBJECTIVESOFADVENTITIOUSSHOOTREGENERATION
 The establishment of plant adventitious shoot regeneration systems mainly has
two intentions :-
(I) On the one hand, an adventitious shoot regeneration system provides
technology of the rapid reproduction of plants.
(II) On the other hand, they are basis of gene function research in plants.
5

PLANTTISSUECULTURE
 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.
 Plant tissue culture is widely used to produce clones of a plant in a method
known as micropropagation.
 The three common pathways of plant tissue culture regeneration are :-
(i) propagation from pre-existing meristems (shoot culture or nodal culture)
(ii) organogenesis
(iii) non-zygotic (somatic) embryogenesis
6

ORGANOGENESIS
 The development of adventitious organs or primordia from undifferentiated cell
mass in tissue culture by the process of differentiation is called organogenesis.
 Caulogenesis: The type of organogenesis by which only adventitious shoot bud
initiation take place in the callus tissue.
7

 In plant tissue culture, undifferentiated tissue is referred to as callus.
 Although a callus can contain meristematic nodules that may not be obvious to
the naked eye but which never develop further unless suitable conditions are
supplied.
 Development of organized structures can follow one of three pathways :-
1. Shoot regeneration, based on a unipolar structure with a shoot apical
meristem.
2. Root regeneration, essentially a unipolar structure with a root apical
meristem.
3. Somatic embryogenesis in which there is a bipolar structure.
8

TYPESOFORGANOGENESIS
(A)Indirect organogenesis
(B) Direct organogenesis
9

INDIRECTORGANOGENESIS
 Indirect organogenesis indicate the plant organ formation on callus tissues
derived from explants.
 The process of indirect organogenesis is more useful in the development of a
transgenic plant.
 There are two ways that can be used to develop a transgenic plant in the indirect
organogenesis method :
(i) Transformed callus is used to regenerate a new plant that is transgenic.
(ii) A modified explant is used to develop callus in the shoot, transform explant is
initially used.
10

 Effect of plant growth regulators and differentiation :
 The classic observations of Skoog and Miller that the direction of differentiation
could be influenced by the ratio of the exogenously supplied growth regulators
auxin and cytokinin.
 They observed in tobacco stem pith cultures that a high ratio of auxin to
cytokinin led to initiation of roots whereas a low ratio led to development of
shoots.
 Although there are many species for which this simple manipulation will not
work, in general auxins e.g. IAA, NAA will stimulate regeneration of roots, and
cytokinins e.g. BAP will promote regeneration of shoots or embryos.
11

DIRECTORGANOGENESIS
 Direct organogenesis indicate the formation of organs directly on the surface
of cultured intact explants. The process does not involve callus formation.
 It results in the development of planting material with no genetic variation
therefore cloning.
 Uniformity in the planting material is ensured.
 This process is also useful in propagating plants with a better multiplication rate
(the number of plants per explant is higher).
 Direct organogenesis is more of an industrial process as it provides plants with
better multiplication rates and cloning propagation where the genetic variation
is zero.
 A good example is the formation of somatic embryos.
12

PROCEDUREOF ADVENTITIOUSSHOOTPROLIFERATION
 Preparation of Reagents and Media :-
(i) Ethanol : Prepare 80 ml of a 70% ethanol solution in a sterile 100-ml beaker.
The same solution can be reused for all explants being prepared for culture.
(ii) Bleach : Prepare 80 ml of a 35% solution of commercial chlorine bleach in a
sterile 100 ml beaker. Prepare a different beaker for each cultivar or plant being
prepared for culture.
Prepare 80 ml of a 10% solution of commercial chlorine bleach in a sterile 100 ml
beaker. Prepare a different beaker for each cultivar or plant being prepared for
culture.
13

(iii) Sterile Distilled Water : Place sterile water in sterile 100 ml beakers. Use
different beakers of water for the explants from each cultivar or plant being
prepared with each surface sterilization protocol.
(iv) Culture Media : Culture medium MS-AV should be prepared in 100X 20mm
petri dishes; alternative culture vessels may be appropriate. Culture media ½- MS
and 112-MS-IAA should be prepared in Magenta boxes, but other deep culture
vessels such as baby food jars are acceptable.
 Treatment of Materials :-
The cultures should be placed in an incubator set at 25°C with either continuous
light or a 16h light/8h dark photoperiod at 15/Lmol m2 S1
14

PROTOCOLS
Stage 0. Selection/ Preparation :
 Excise explant from the desired plant or cultivar.
 Gently wash in warm water with a mild detergent, and rinse under tap water.
Stage I. Culture initiation/ establishment :
 Using forceps, briefly dip each explants into 70% ethanol.
 Place explants of each cultivar into a solution of 35% commercial chlorine bleach
for 5 min or into a solution of 10% commercial bleach for 1 min for its surface
sterilization treatment.
 Rinse each pair of explant 3X in sterile distilled water, 5 min each time.
 Place the explant in a sterile petri dish. Using sterile forceps and scalpel, cut each
leaf into sections 1 cm square.
15

 Place the explant on MS-AV medium.
 Seal each culture and place in the incubator for 4 weeks.
 Observe weekly for signs of contamination.
 Observe weekly for signs of shoot formation using the dissection microscope.
 Record the time of shoot bud emergence for each cultivar, the frequency of
shoot bud formation according to cultivar and explant type, and the number of
shoots per explant.
Stage II. Shoot Multiplication :
 At the end of the first monthly passage, identify the contamination-free
cultures for continued shoot multiplication.
 Transfer culture material, one explant tissue mass at a time, to a sterile petri
dish.
16

 With sterile scalpel and forceps, cut the mass of plantlets into smaller pieces
and transfer to fresh MS-AV media as follows:
Half of the tissue masses should be cut into very small pieces of material for
transfer, each including at least one shoot tip or rosette.
The other half of the tissue masses should be cut into larger pieces containing
several shoot buds.
Compare the multiplication rates obtained from larger vs. smaller pieces.
 Seal the cultures and incubate them for 4 weeks.
 Repeat the multiplication cycle as often as time permits or as planned.
 Observe the cultures at the end of each culture passage and record the
number of shoots obtained from each explanted piece.
17

Stage III. Rooting of Shoots :
 Excise and transfer ten or more individual shoots, at least 1 cm in length, to 11 2-
MS medium for continued development and elongation of the shoot.
 Also transfer ten or more individual shoots to 112-MS-IAA medium for
comparison of root induction frequencies.
 Seal the cultures and incubate them for 4 weeks.
 Transfer the shoots to fresh ½-MS medium on a monthly basis until roots appear.
 Observe weekly for root initiation and record the frequency of root formation.
Stage IV. Plant Establishment/Acclimatization :
 Gently remove well-rooted plantlets from the culture vessel, keeping the roots
intact.
 Transfer the plantlet with roots encased in agar-media to a container of warm, but
not hot, water and gently rinse the agar-media off the roots.
18

 Plant the regenerant in a small pot or in a plastic sandwich bag with sterile soil
mix.
 Make sure the soil is moist with water, but is not sodden.
 Wrap the pot and plant in plastic wrap, and use a plastic stake to allow the plastic
wrap to form a tent over the plant.
 Plants should not need to be watered for the first few days.
 Place the pots in diffuse light.
 Open the tents to allow air exchange briefly every day.
 After 1 week, let some air in the tent for 1 h each day.
 After another week, increase gradually to several hours per day.
 After a total of 2-3 weeks, remove the wrap and allow the plants to adjust to
ambient conditions.
19

FACTORSAFFECTING ORGANOGENESIS
In vitro organogenesis is controlled by a number of factors other than
phytohormones, such factors are :-
 Size of Explant : Organogenesis is generally dependent upon size of explant.
The large explant consisting parenchyma, vascular tissues and cambium have
greater regenerative ability than the smaller explant.
 Source of Explant : The most suitable part of the plant for starting culture will
depend on species. Leaves and leaf fragment of many plant species like Begonia,
Solanum, Nicotina, Crepis, etc. have shown capacity to regenerate shoot buds.
 Age of the Explant : Physiological age of explant is important for in vitro
organogenesis. In Nicotiana species, regeneration of adventitious shoot is only
noted if the leaf explant is collected from vegetative stage i.e. before flowering.
20

 Seasonal Variation : Bulb scales of Lilium speciosum regenerate bulblets
freely in vitro when explant is taken during spring and autumn period of growth
but same explants collected from summer or winter season does not produce any
bulblets.
 Oxygen Gradient : In some cultures, shoot bud formation takes place when
the gradient of available oxygen inside the culture vessel is reduced. But rooting
requires a high oxygen gradient.
 Quality and Intensity of Light : The blue region of spectrum promotes shoot
formation and red light induce rooting.
 Temperature : Most tissue culture are grown successfully at temperature
around 25°C. In number of bulbous species optimum temperature may be much
lower of about 15-18°C.
21

 Culture Medium : Medium solidified with agar favors bud formation although
there are some reports about the development of leaf shoot buds on culture
grown in a liquid medium.
 pH of the Medium : The pH of the culture medium is generally adjusted
between 5.6 and 5.8 before sterilization. The pH may have a determining role in
organogenesis.
 Ploidy Level : Variation in chromosome number i.e. aneuploidy, polyploidy,
etc. of plant cell in culture has been well documented. With the increase in
chromosome instability there is a general decline in morphogenetic potentiality
of callus tissue.
 Age of Culture : A young culture frequently produces organs. But the
organogenic potential may decrease and ultimately disappear in old culture.
22

APPLICATIONSOFORGANOGENESIS
 Plant tissue culture or organogenesis now has direct commercial applications as
well as value in basic research into cell biology, genetics and biochemistry.
 Micropropagation using meristem and shoot culture to produce large numbers
of identical individuals.
 Screening programmes of cells, rather than plants for advantageous characters.
 Large-scale growth of plant cells in liquid culture as a source of secondary
products.
 Removal of viruses by propagation from meristematic tissues.
23

REFERENCE
 Bhojwani, S. S., & Razdan, M. K. (1986). Plant tissue culture: theory and
practice. Elsevier.
 Long, Y., Yang, Y., Pan, G., & Shen, Y. (2022). New insights into tissue Culture
Plant-Regeneration Mechanisms. Frontiers in Plant Science, 13.
https://doi.org/10.3389/fpls.2022.926752
 Moshtaghi, N. (2020, January 1). Tissue and cell culture of saffron. Elsevier
eBooks. https://doi.org/10.1016/b978-0-12-818638-1.00014-9
 Razdan, M. K. (2002). An introduction to plant tissue culture. Oxford and IBH
publishing.
 https://www.slideshare.net/6263234147/organogenesis-in-plant-tissue-culture
 https://www.slideshare.net/mphoolbadshah/adventitious-shoot-
proliferationpptx
24

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