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1. ADVENTITIOUS SHOOT
PROLIFERATION
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M.Phool Badshah

2. Introduction:
 Propagation of selected plant lines through tissue culture is
called micro-propagation.
 There are several defined steps in a typical micro-propagation
system which are given here:
I. The first step is the initiation of a sterile culture of the explant(Stage I).
II. The second step is the multiplication of shoots or other propagules from
the explant (Stage II). Adventitious shoot proliferation is the most
frequently used multiplication technique in micropropagation systems
(Chu 1992).The culture media and growth conditions used in Stage II
systems are optimized for maximum rates of multiplication.
III. The third step is the development of roots on the shoots to produce
plantlets (Stage III). Specialized media mayor may not be required to
induce roots, depending on the species.
IV. The final step is to produce self-sufficient plants (Stage IV), which usually
involves a hardening-off process and acclimation to growing in soil mixes
under greenhouse conditions for later transplanting to the field

4. Objectives and Goals:
• To set up a micropropagation system by obtaining sterile
cultures from explant tissue ofAfrican violet (Stage I).
• To observe multiple shoot formation from leaf cultures of
African violet (Stage II).
• To induce rooting on the micro propagated shoots of African
violet to produce plantlets (Stage III).
• To harden off the African violet plantlets and establish them
in soil (Stage IV).

5. Plant Materials
 Obtain plants growing in pots from a local nursery. At least two different
cultivars showing different patterns of leaf color variegation should be
used, if possible. Plants should be healthy and vigorous with little or no
signs of disease or pest problems.
 Equipment :
 Sterile beakers, 100 ml
 Sterile petri dishes, 100 X 20 mm
 Plastic stakes, 4"
 Plastic wrap or sandwich bags

6. Procedures:
 Preparation of Reagents and Media:
 Ethanol : Prepare 80 ml of a 70% ethanol solution in a
sterile 100-ml beaker.The same solution can be reused for
all leaves being prepared for culture.
 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. (2) 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.

7. a
 Sterile Distilled Water : Place sterile water in
sterile 100-ml beakers. Use three different
beakers of water for the leaves from each
cultivar or plant being prepared with each
surface sterilization protocol.
 Culture Media : Culture medium MS-AV should
be prepared in 100 X 20-mm petri dishes;
alternative culture vessels may be appropriate.
Culture media 1/2- MS and 1I2-MS-IAA should
be prepared in Magenta boxes, but other deep
culture vessels such as baby food jars are
acceptable.

8.  Treatment of Materials:
 African violet cultures should be placed in an incubator set at 25°C with
either continuous light or a 16-h light/8-h dark photoperiod at 15 /-!mol
m -2 S-I.
 Design of Experiment :
 The experiments are designed to develop experience in the basic
operations of micropropagation.
 Culture initiation is a critical first step.The tissues are exposed to two
different surface sterilization procedures.
 Two types of leaf explants are compared for differences in shoot
initiation.The shoot proliferation tests provide the most information
when different variegated cultivars of African violet are compared.
 Two different rooting media are compared for development of
complete plantlets from the shoots.

9. Protocols :
 Stage I. Culture Initiation :
 Observe and record the pattern of leaf colors for each cultivar or plant
provided.
 Excise four leaves from each plant or cultivar provided. Gently wash in
warm water with a mild detergent, and rinse under tap water.
 Using forceps, briefly dip each leaf into 70% ethanol.
 Place one pair of leaves of each cultivar into a solution of 35% commercial
chlorine bleach for 5 min. Place the other pair ofleaves of each cultivar
into a solution of 10% commercial bleach for ISmin. Maintain the identity
of each leaf and its surface sterilization treatment.
 Rinse each pair ofleaves 3 X in sterile distilled water,S min each time.
 Place the leaves in a sterile petri dish. Using sterile forceps and scalpel,
cut each leaf into sections 1 cm square, with and without vein in the
explant.

10. aaa
 Place the leaf sections, labeled with or without vein, on MS-AV medium.
Record the location of cut sides vs. intact leaf margins for each explant.
 Seal each culture and place in the incubator for 4 weeks.
 Observe weekly for signs of contamination. Record from which surface
sterilization treatment contaminated leaves came. 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.
 Keep a record of which plantlets originated from each original leaf
section explanted from each cultivar.

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 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.
 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 1I2-MS-IAA
medium for comparison of root induction frequencies.

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 Seal the cultures and incubate them for 4 weeks.Transfer the shoots to
fresh 1/2-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 :
 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.
 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.

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 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.
 Observations and Measurements :
 Shoot Development :
Observe weekly for signs of contamination and for changes in
the morphology of the cultured tissues. At the end of the first monthly
passage, calculate the contamination rate for each cultivar and surface
sterilization treatment. At the end of each monthly passage, summarize
the shoot production frequency and number of shoot buds formed.
 Root Development :
Observe weekly. At the end of the rooting period, summarize
the frequency of shoots developing roots. Separate results according to
cultivar and rooting medium treatment.

14. Results
 Examples of African violet cultures as they appear at each of the
micropropagation Stages I through IV .
 Adventitious bud induction during Stage II (4 weeks) was prolific, with
an average of about 20-25 buds per explant (1 cm2) for most cultivars
of African violet.
 The cut or wounded portions of the explants responded faster and
more efficiently than the intact leaf margins.
 The buds appeared earlier at the wounded vein sites, then later at
nonvein wound sites.
 Smaller pieces of subcultured tissue gave rise to a greater total
number of propagated shoots compared to the larger pieces of
subcultured tissue.

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 Averaged over several passages, we observed multiplication rates of lOx
to 15X per passage during Stage II.
 Thus, from one well-formed shoot, a total of 10-15 well-formed shoots
was obtained monthly. Root induction during Stage III approached 100%
frequency using 1I2-MSIAA medium and was considerably lower on 1I2-
MS medium.
 The development of an adventitious root on a propagated shoot in a
typical plant regeneration system . More than 85% of the
micropropagated plantlets were established in soil successfully during
Stage IV.