Direct organogenesis, embryogenesis, micro grafting, meristem culture and its importance for fruit crops

2. Pawan Kumar Nagar
M.Sc. (Horti.) Fruit science,
IInd Semester
REG. NO: 04-2690-2015
Direct organogenesis, embryogenesis, micro
grafting, meristem culture and its importance for
fruit crops

3. What is plant tissue culture ??
“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. Different techniques in plant tissue culture may
offer certain advantages over traditional
methods of propagation, including:
 The production of exact copies of plants that produce particularly good
flowers, fruits, or have other desirable traits.
 To quickly produce mature plants.
 The production of multiples of plants in the absence of seeds or
necessary pollinators to produce seeds.
 The regeneration of whole plants from plant cells that have been
genetically modified.
 The production of plants from seeds that otherwise have very low
chances of germinating and growing.

5. Organogenesis
Definition
“The formation of roots, shoots or flower buds from the cells in culture in manner similar to
adventitious root or shoot formation in cuttings is called organogenesis’’
 Organogenesis starts in the callus in response to the stimulation given by the chemicals in the medium.
 Organogenesis takes place in two stages, namely caulogenesis or shoot initiation and rhizogenesis or root
initiation.
 Both types of organogenesis are controlled by the hormones present in the medium. generally a high
auxin:cytokinin ratio induce shoot formation.
 Organogenesis starts with the development of a group of meristematic cells called meristemoids, which
initiate the formation of a primordium.
 Depending on the factors within the system, this primordium develops into shoot, root or embryoid.

8. Two types of organoginasis
1. Direct regeneration
2. Indirect organogenesis
 This two types depend on hormonal
combination of the culture media.
Intermediate ratios around 1:1 favor callus growth. Indirect organogenesis
Auxin/cytokinin 10:1-100:1 induces roots Direct regeneration
Auxin/cytokinin 1:10-1:100 induces shoots.
Rule of thumb:

9. Direct organogenesis
Explant → Meristemoid → Primordium
 In many plants, subculturing of callus results
in undesired variations of clones (somaclonal
variations).
 To avoid this, direct regeneration of the explants
into plantlets can be tried.
 This has been achieved in many plant species
by altering the hormonal combination of the culture media.

11. Indirect organogenesis
Explant → Callus → Meristemoid → Primordium
• In indirect organogenesis, callus is first produced from the explant. Organs can
then be produced from the callus tissue or from a cell suspension produced
from that callus.

12. The major factors affecting the process of regeneration are:
1. Source of Explant
 The organ that is to be served as tissue source
 The physiological and ontogenic age of the organ
 The season in which the explant is obtained
 The size of the explant
 The overall quality of the plant from which explants are taken.
2. Culture Environment
 Physical form of the medium i.e. presence or absence of agar
 The pH of the medium3. Light quality and quantity
 Temperature
 Relative humidity
 The gaseous atmosphere within the vessel.
3. Nutrient Media and Constituents

13. Embryogenesis
• Somatic Embryogenesis
“The process of a single cell or a group of cells initiating the
developmental pathway that leads to reproducible regeneration
of non-zygotic embryos capable of germinating to form
complete plants”
• Under natural conditions, this pathway is not normally
followed, but from tissue cultures somatic embryogenesis
occurs most frequently and as an alternative to organogenesis
for regeneration of whole plants.

14. How Somatic Embryos produced?
• In somatic embryogenesis, embryo-like structures, which can
develop into whole plants in a way analogous to zygotic
embryos, are formed from somatic tissues.
• These somatic embryos (SE) can be produced either directly
or indirectly.
• Two ways of somatic embryogenesis:

15. 1. Direct embryogenesis
 In direct somatic embryogenesis, the embryo is formed directly from a cell or
small group of cells without the production of an intervening callus.
 Direct somatic embryogenesis is generally rare in comparison with indirect
somatic embryogenesis.
2. Indirect embryogenesis
 In indirect somatic embryogenesis, callus is first produced from the explant.
 Embryos can then be produced from the callus tissue or from a cell suspension
produced from that callus.

17. Somatic embryogenesis - Synthetic seeds

18. Importance
 In poly embroyonic crops like citrus, zygotic as well as nucellar embryonic plants are
obtained separately.
 Embryos of big and heavy fruits like coconut can be taken out of the fruits and pre-
serve in tube in sterile distilled water for about two months and then cultured in
media.
 In this process easy international exchange of germplasm is possible.
 In many interspecific and intergeneric crosses the hybrid embryos fail to develop to
maturity. In such cases before the embryo gets damaged can be taken out of the ovule
and cultured in artificial media, which gives rise to complete plantlets.
 One major path of regeneration
 Mass multiplication
 Production of artificial seeds

19. Micrografting (shoot tip grafting)
• Micrografting consists of the placement in aseptic conditions of a maintained
scion onto an in vitro grown rootstock.
• The results of in vitro micrografting and the plant material derived from it can be
further multiplied in tissue culture conditions or acclimatized to outdoor
conditions.
• Micrografting is a technique that potentially can combine the advantages of
rapid in vitro multiplication with increased productivity that results from grafting,
superior rootstock and scion combinations.
• Among various methods of micrografting, slit or wedge grafting has been found
most suitable in case of fruit crops.
• In vitro shoot tips are better as compared to in vivo shoot tips for carrying out
micrografting resulting in higher graft success, less contamination, lower shoot tip
necrosis and good vigour of micrografts.

20. This comprises the following steps
• Aseptic condition was maintained throughout all stages of micrografting process.
• Seedlings were removed carefully from the agar medium with sterilized forceps.
• For use as rootstock, seedlings were cut back to 1 cm above the cotyledonary
nodes and leaves were excised using a microscalpel.
• Terminal shoots to be used as scions were excised from the other seedlings at 2
cm below the apex.
• Lower leaves removed from the basal 1 cm of scion.
• Excised scion and rootstocks were stored in sterile de-ionized water until used in
the grafting process.

21. • The shoot tip was inserted at the top of decapitated rootstock by
making an incision.
• The cortex was exposed by the horizontal cut of the incision.
• Grafted plants were cultured in a liquid nutrient medium
containing plant cell culture salt solution of MS, modified White's
vitamins and 75 g/1 sucrose.
• When at least two expanded leaves were formed micrografted
plants were transferred to pots containing a steam sterilized soil
mixture suitable for plant.

24. Importance
• Overcoming graft incompatibility
• Rapid mass propagation of elite scions by grafting onto rootstocks that
have desirable traits like resistance to soil borne pathogens and
diseases
• To allow survival of difficult to root /shoots
• Development of virus free plant

25. Meristem culture
Cultivation of axillary or apical shoot meristems, particularly
of shoot apical meristem, is known as meristem culture.
Meristem culture involves the development of an already
existing shoot meristem and subsequently, the regeneration
of adventitious roots from the developed shoots.
It usually does not involve the regeneration of a new shoot
meristem.

26.  Shoot apical meristem lies in the ‘shoot tip’ beyond the youngest leaf or the
first leaf primordium; it measures upto about 100 µm in diameter and 250 µm
in length.
 Thus a shoot tip of 100- 500 µm would contain 1-3 leaf primordia in addition
to the apical meristem.
 Shoot tip culture is widely used for rapid clonal propagation for which much
larger, e.g., 5-10 mm, explants are used.
 Therefore, most cases of meristem culture are essentially shoot-tip cultures.
Nodal explants of various sizes are also commonly employed for rapid clonal
propagation.

29. • Procedure
 Collect rapidly growing apex of a shoot
 Remove all the leaves except the smallest leaves, wash thoroughly under
running water with one drop of Tween 20.
 Disinfect the working area of the laminar flow cabinet with 70% alcohol
or rectified spirit.
 Dip washed explants in 0.1% mercuric chloride solution for 5-7 min for
surface sterilization, and wash with sterile distilled water thoroughly for 3-
4 times.

30.  Place the shoot tips on sterile filter paper.
 Hold the stem firmly with a forcep and remove the young leaves with a needle.
 Remove the underlying leaf primordia gradually.
 Remove up to the 3rd and 4th leaf primordia and keep intact the 1st and 2nd leaf
primordia carefully without damaging the fragile dome shaped apical meristem.
 With a surgical scalpel remove the apical dome (0.1-0.3 mm).
 Transfer the dome to the culture tubes containing medium.
 The culture tubes maintain at 25 ± 2° C for 12 h light (3000 lux)/12 h dark cycle.

31. Growth and development:
 Within 2-3 weeks of inoculation apical meristem grows and forms shoots.
 Each single shoot undergo proliferation, these shoots are separated and
cultured in rooting media.
 Root initiation occurs within 18-21 days of inoculation.
 Once the plantlet has two to three leaves and strong root, they are
transferred to liquid basal media with the help of Paper Bridge
 After about 8 weeks the plantlets are soaked in 0.2% Bavisiin a fungicide
for 10 min and are potted in vermiculite mixture, till they grow.

32. • Importance:
Production of virus free germplasm.
Mass production of desirable genotypes.
Facilitation of exchange between locations (production of clean material).
Cryopreservation (cold storage) or in vitro conservation of germplasm.
can produce virus-free plantlets for micropropagation and international
exchange of germplasm.
Viruses persist in the vegetative plant parts, which are used for
propagation.

33. • References:
• Methods in plant tissue culture by U. Kumar
• http://www.whatisthebiotechnology.com/blog/wp-
content/uploads/2013/10/ORGANOGENESIS-IN-PLANTS.pdf
• https://www.scribd.com/doc/35044234/Importance-of-Plant-Tissue-Culture
• http://www.biologydiscussion.com/essay/plant-breeding-essay/essay-on-plant-
tissue-culture-history-methods-and-application/17639
• https://en.wikipedia.org/wiki/Plant_tissue_culture

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