These slides contain authentic material.

1. z
Adventitious Shoot Proliferation
M.PHOOL BADSHAH

2.  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.
Introduction

4.  To set up a micropropagation system by obtaining sterile
cultures from explant tissue of African 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).
Objectives and Goals:

5.  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.
 Equipments:
 Sterile beakers, 100 ml
 Sterile Petri dishes, 100 X 20 mm
 Plastic stakes, 4"
 Plastic wrap or sandwich bags
Plant Materials:

6.  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.
Procedures:

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.  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.
Treatment of Materials:

9.  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 of leaves of each cultivar
into a solution of 10% commercial bleach for I5 min. Maintain the identity
of each leaf and its surface sterilization treatment.
 Rinse each pair of leaves 3 X in sterile distilled water, 5 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.
Protocols:

10.  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.

11.  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.
 With sterile scalpel and forceps, cut the mass of plantlets into smaller pieces and
transfer to fresh MS-A V 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.

12.  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.
 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.

13.  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.
 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.

14.  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.
Observations and Measurements:

15.  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 non
vein wound sites.
 Smaller pieces of sub-cultured tissue gave rise to a greater total number
of propagated shoots compared to the larger pieces of sub-cultured
tissue.
Results:

16.  Averaged over several passages, we observed multiplication rates of 10X 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.