Auxillary bud proliferation

approach- shoot tip, somatic

embryogenesis-
procedure and requirements for
organogenesis
Axillary and apical shoots contain quiescent or active meristems depending on the
physiological state of the plant. When these shoot tips are cultured on a basal
medium containing no growth regulators, these typically develop into single
seedling like shoots with strong apical dominance

Auxillary bud proliferation approach- shoot tip, somatic embryogenesis- procedure and requirements for organogenesis 1
 On the contrary, when the shoots of the same explant material are grown on
culture media containing cytokinin, axillary shoots develop precociously which
proliferate to form clusters of secondary and tertiary shoots. These clusters when
subdivided and transferred onto fresh medium again these will form similar
clusters.
This subdivision process may be continued indefinitely when provided with basic
nutrients. About
5-10 multiplication rates on 4-8 weeks of micro-propagation cycle may ultimately
lead to extremely impressive clonal propagation range of 0.1-3.0 x 10° within a
year.

(For surface sterilization we incubate 1-1cm long top parts in a solution of 1%

sodium hypochlorite for 10 minutes. Now wash these top parts with sterilized
distilled water 4 times so that the excess chemical present on the surface is
removed.)

Auxillary bud proliferation approach- shoot tip, somatic embryogenesis- procedure and requirements for organogenesis 2
 (Now remove the outer leaves from each apex so that shoot tips make visible.)

(Now 1 mm long shoot tips are cut with the help of a blade or knife, and transfer on
to the surface of the agar medium.)

Auxillary bud proliferation approach- shoot tip, somatic embryogenesis- procedure and requirements for organogenesis 3
 (Now incubate this culture at 25 ° C temperature and 16 hours light period. As a
result the shoot system is developed first and then the root system develops in a
few days. Now establish these plants in pots.)

Auxillary bud proliferation approach- shoot tip, somatic embryogenesis- procedure and requirements for organogenesis 4
 Axillary Bud Method
The axillary buds or lateral buds are formed in the angle between leaf and stem
which lead to the formation of several branches.

The axillary bud method involves the development of the axillary bud. The shoot
tips are isolated from the tip of the axil of leaves that develop axillary bud under
the effect of a high concentration of cytokinins.

Auxillary bud proliferation approach- shoot tip, somatic embryogenesis- procedure and requirements for organogenesis 5
 The role of cytokinin is to suppress apical dominance and promote the
development of axillary buds. The axillary bud method is often combined with the
single node method.

Key points include:

1. The ratio of auxin and cytokinin used in the axillary bud method is 10:1.

2. The developmental stage of explant in culture is directly proportional to the

concentration of cytokinin to be used in the culture media. For example,
mature culture requires a higher concentration of cytokinin compared to the
juvenile culture.

3. The cytokinin requirement and concentration used in axillary bud proliferation

are also dependent on the species or cultivar of the plant you want to culture.

4. Often it is advisable to induce the formation of shoot tip before increasing the
cytokinin concentration to induce axillary bud formation.

5. If the growth and development of the axillary bud are unsatisfactory, you must
remove the apical meristem from the cultured explant.

6. Often, liquid media promotes the axillary shoot formation.

7. It has been observed that, in a wide range of species, several substituted

pyridyl phenyl urea compounds and thidiazuron stimulate axillary branching.

Factors That Impact Axillary Bud Proliferation

Several factors affect the axillary bud proliferation under in vitro conditions, out of
which of them are discussed below. note that the effect of factors varies
depending on the species of the plant you are working on.

Plant growth regulators: Axillary bud growth is highly dependent on the

presence of benzyl adenine (BA-synthetic cytokinin that promotes the growth
and development of the in vitro cultures by stimulating cell division). More
callus and shoots are produced by the explants cultured in the media
containing BA compared to the media without BA. Moreover, rooting in the
explants is induced by keeping them in basal media without any growth
regulators. The presence of 1-Naphthaleneacetic acid (NAA) in the media
induces negative effects on the root formation.

Auxillary bud proliferation approach- shoot tip, somatic embryogenesis- procedure and requirements for organogenesis 6
 Explant size: It has been observed that the explants larger in size showed
more positive responses compared to the smaller ones. It's because the larger
explants contain more nutrient reserve and endogenous plant growth
regulators that support the in vitro development of axillary buds.

Explant density: The number of explants cultured in a jar determines the

multiplication rate and output of the cultures. For example, a jar containing 10
explants will show a lower rate of multiplication and explant survival compared
to a jar containing 5-6 explants.

Somatic Embryogenesis:
The sporophytic generation of a plant is initiated with the zygote, which is the
initial cell (product of gamete fusion) that bears all the genetic information to
construct the adult individual. In angiosperms, the zygote divides transversally,
resulting in two cells. One of them, the apical cell, is small and dense with an
intense activity of DNA synthesis. Further ordinal divisions of this cell give rise to
the embryo head that will be the new plant. The other resultant cell (basal cell) is a
large and highly vacuolated one that will confirm the suspensor complex, which
plays an important role during the early stages of the young embryo. In vivo-
produced somatic embryos generally follow the same pattern; however, they are
not initiated from a zygote, but from a somatic cell as occurs in the polyembryony.

Somatic embryos

Somatic embryos are bipolar structures with both apical and basal meristematic
regions, which are capable of forming shoot and root, respectively. A plant
derived from a somatic embryo is sometimes referred to as an "embling"

Auxillary bud proliferation approach- shoot tip, somatic embryogenesis- procedure and requirements for organogenesis 7
 A process where an embryo is derived from a single somatie cell or group of
somatic cells. Somatic embryos (SEs) are formed from plant cells that are not
normally involved in embryo formation.

Embryos formed by somatic embryogenesis are called Embryoids.

The process was discovered for the first time in Daucas carota L. (carrot) by
Steward (1958), Reinert (1959).

Procedure Of Somatic Embryogeneis:

Auxillary bud proliferation approach- shoot tip, somatic embryogenesis- procedure and requirements for organogenesis 8
 1. Petiole explants plants are surface sterilized and cultured on SH medium
(Schenk and Hildebrandt, 1972) containing 2,4-D, kinetin and many other
nutrients. 2,4-D activates the cell cycle of many cells in the petiole - those in
the vascular cambium develop into a callus, whereas some sub-epidermal
cells develop into a somatic embryo.

2. The initial somatic embryos, which are only small dense cell clusters at this
stage, are embedded in a callus mass of non-differentiated cells.

3. To liberate these proembryonic structures, and to stimulate the formation of

more embryos, the callus is dispersed in a liquid medium to form a suspension
culture containing 2,4-D but not kinetin

4. After 7 days, the suspension is sieved and transferred to solid medium lacking
2,4-D On this medium the embryos develop through morphological stages that
appear to be globular, heart and torpedo.

5. Maturation Phase 1Once the majority of embryos reach the torpedo stage (7-
10 days after sieving) they are transferred to an enriched medium containing a
high level of sucrose, nitrogen and sulphur to prevent precocious germination
and to enable deposition of storage reserves. The embryos rapidly accumulate
fresh and dry weight, reaching 1-2 mg dry weight pe

6. Maturation Phase li To induce the acquisition of desiccation tolerance, the

somatic embryos are placed on a modified medium containing abscisic acid
(ABA) for 3 days. Then they are removed from the medium, washed to remove
sugar and other nutrients, and dried.

7. The standard method of drying is to place the somatic embryos in a sealed

chamber over a saturated salt solution designed to give specific relative
humidifies. Daily for one week, the embryos are transferred to a progressively
lower relative humidity chamber and finally are dried at ambient conditions. At
this stage, the embryos have reached approximately 15% moisture and can be
stored for a year or more with good viability.

TYPES OF SOMATIC EMBRYOGENESIS:

1. Direct SE

Auxillary bud proliferation approach- shoot tip, somatic embryogenesis- procedure and requirements for organogenesis 9
 When embryos are formed dircetly from explant tissuc creating an identical
clone without production intervening callus.

2. Indirect SE

when explants produced unditlerentiated mass cells(callus) which is

maintained or differentinted into embryo. Specific growth regulators and
culture conditions are required for callus formation.

Examples of direct somatic embryogenesis:

Auxillary bud proliferation approach- shoot tip, somatic embryogenesis- procedure and requirements for organogenesis 10
 • Somatic embryogenesis has been reported from many plants such as Coffea
arabica, Alfalfa, Daucus carota Ranunculus scleratus, Linum
usitatissimum,Brassica napus,Arachis hypogea etc.
• Leaves,scutellum,hypocotyl,nu cellus and embryo-sac etc are used as explants.

Examples of indirect somatic embryogenesis:

• Secondary phloem of carrot, leaf; tissues of coffee, Petunia, Asparagus etc. In
majority of cases embryogenesis is through indirect method.

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 FACTORS AFFECTING SOMATIC EMBRYOGENESIS

1. Characters of explants:
Even though a variety of explants can be utilized, the correct developmental
stage of the explants are also crucial for the initiation of embryogenic callus.

Young or juvenile explants produced more somatic embryos than older

explants

Various types of explants used in SE

1. Immature zygotic embryos

2. Inflorescence

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2. Plant growth regulators:

i) Auxins

2, 4-D has been the best synthetic auxin used for inducing SEs.
% Continuous supply of auxin causes embryogenic cells to divide
(Proliferation medium) without the appearance of embryos.

Witherell (1971) have suggested that continuous supply of auxin induces

endogenous ethylene production which suppresses embryo development.

So, embryogenic cells after treatment with auxin must be transferred to auxin
free medium that constitute the embryo development medium.

ii) Cytokinins:

Cytokinin produces globular embryo from initial embryos.

Zeatin is promotive when applied to embryogenic cells after days 3-4 transfer
from the proliferation medium to ED medium whereas BAP and kinetin have
inhibitory effect on embryogenesis.

Auxillary bud proliferation approach- shoot tip, somatic embryogenesis- procedure and requirements for organogenesis 12
 High ratio of cytokinin than auxin induces shoot formation and reverse ratio
favours rooting.
others include Gibberllins, inhibits SE.
ABA promote embryo maturation and prevent precocious germination and
secondary embryogenesis.

3. Nitrogen source:
reduced form of nitrogen is the sole source of embryo formation.

4) Others:
Light generally promotes embryogenesis

High temperature usually favorable for embryogenesis

Parameters:

1. pH: 5.6 to 5.8

2. Temperature: 26 °C to 28 °C

3. Humidity: 40 - 80%

4. a) light intensity: 5000 to 8000 lux

b) light duration: 16hrs light and 8hrs darkness

Organogenesis
In plant tissue culture, organogenesis means genesis of organs like shoots,
roots, leaves, flowers, etc. The earliest report on induction of shoot
organogenesis in vitro was by White (1939) using a tobacco hybrid; and the
first observation of root formation was reported by Nobecourt (1939) using
carrot callus. Till late 1950s, the basic regulatory mechanism underlying in
organogenesis was not identified. Skoog and Miller (1957) were responsible to
recognize the regulatory mechanism as a balance between auxin and
cytokinin. As per their finding, a relatively high level of aux in to cytokinin
favoured root formation and the reverse favoured shoot formation. Using this
concept, it has now become possible to achieve organogenesis in a large
number of plant species by culturing explants, calli and cell suspension in a
defined medium.

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 In organogenesis, the shoot or root may form first depending upon the nature
of growth hormones in the basal medium. The genesis of shoot and root from
the explants or calli is termed as caulogenesis (caulm = stem) and
rhizogenesis (rhizo = root) respectively.

Procedure for Organogenesis (in Plants):

1.⁠⁠Selection of Explant:
Tissue source like leaf, stem, root, or meristem is selected.

Young, actively growing tissues are preferred.

2.⁠⁠Surface Sterilization:
Explant is sterilized using disinfectants (e.g., 70% ethanol, sodium hypochlorite) to
prevent microbial contamination.
3.⁠⁠Inoculation:

Sterilized explant is placed on a nutrient medium under aseptic conditions.

4.⁠⁠Culture Medium:
Typically Murashige and Skoog (MS) medium.
Contains essential macro- and micronutrients, vitamins, sugar (carbon source),
and plant growth regulators.

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 5.⁠⁠Plant Growth Regulators (PGRs):
Cytokinins (e.g., BAP, kinetin): Promote shoot formation.
Auxins (e.g., IAA, NAA, 2,4-D): Promote root formation or callus induction.

Ratio of auxins to cytokinins determines the organ type formed.

6.⁠⁠Incubation Conditions:
Culture kept in controlled environment (light, temperature, and humidity).
Typically 16-hour photoperiod, 23–27°C.
7.⁠⁠Organ Formation:
Direct or indirect organogenesis occurs:

Direct: Organs form directly from explant.

Indirect: Organs form from a callus phase.
8.⁠⁠Subculture and Regeneration:
Shoots and roots are regenerated, often requiring separate media.
Plantlets are grown to a certain size.

9.⁠⁠Acclimatization:
Regenerated plantlets are transferred to soil or potting mix.
Gradual adaptation to external conditions in a greenhouse.

Basic Requirements for Organogenesis in Plants:

1. Explants (Source Tissue)

Any part of the plant can be used: leaf, stem, root, node, meristem,
cotyledon, hypocotyl, etc.

Should be young, healthy, and disease-free.

The capacity for organ formation depends on the totipotency of the explant.

2. Sterile Environment

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 A completely aseptic condition is necessary to avoid microbial contamination.

Use of laminar airflow hood, autoclaved instruments, and sterile culture

media.

3. Culture Media
The most common: Murashige and Skoog (MS) medium.

May also use B5, N6, White’s, or other specialized media.

Must include:

Macronutrients and micronutrients

Vitamins (like thiamine, pyridoxine)

Carbon source (usually sucrose)

Gelling agent (like agar)

4. Plant Growth Regulators (PGRs)

Crucial for determining organ formation:

Organ Hormone Requirement

Shoots High cytokinin : low auxin ratio (e.g., BAP, kinetin)

Roots High auxin : low cytokinin ratio (e.g., IAA, NAA, IBA)

Callus (undifferentiated tissue) Balanced auxin and cytokinin

5. Environmental Conditions
Temperature: Usually 24–28°C.

Photoperiod: Typically 16 hours light / 8 hours dark.

Light Intensity: Around 40–60 µmol m⁻² s⁻¹ (fluorescent lights are commonly
used).

Humidity: Maintained to avoid desiccation.

6. Genotype of the Plant

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 Some species or varieties respond better than others.

Genetic makeup can influence the organogenic potential.

7. Stage of Development of Explant

Juvenile tissues have higher organogenic potential compared to mature
tissues.

8. Subculture and Maintenance

Regular subculturing is required to provide fresh nutrients and maintain
organogenic activity.

submitted by:

Mahdiya 164

Naveeda 184

Rutba 192

Anshara 200

Sadiya 29

Auxillary bud proliferation approach- shoot tip, somatic embryogenesis- procedure and requirements for organogenesis 17