1) The document describes a procedure for propagating cactus plants through micropropagation using auxiliary buds. 2) Through this technique, shoots can be stimulated to grow from auxiliary buds and separated, then rooted to produce new plants, allowing for rapid multiplication. 3) The procedure involves surface sterilization of explants, culture initiation on various media, subculture and separation of developing shoots, and rooting of shoots to produce finished plants. Through monthly subculturing, millions of plants can be produced from a single original explant.

1. MICRO PROPAGATION BY
AUXILIARY BUDS
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Badshah

2. INTRODUCTION
 One of the most exciting and important aspects of in vitro
cell and tissue culture is the capability to regenerate and
propa-gate plants from cultured cells and tissues.
 The simplest type of in vitro plant propagation is the
stimulation of auxiliary bud development.
 This technique exploits the normal ontogenetic route for
branch development by lateral (auxiliary) meristems.
 The auxiliary buds are treated with hormones to break
dormancy and produce shoot branches.
 The shoots are then separated and rooted to produce
plants. Alternatively, the shoots are used as propagules for
further propagation.

3. HISTORY:
 Chu (1992) provides an excellent summary of the economic
considerations and market demand for plants propagated by
tissue culture, also known as micropropagation.
 Auxiliary bud proliferation typically results in an average
tenfold increase in shoot number per monthly culture
passage.
 In a period of 6 months, it is feasible to obtain as many as
1000000 propagules or plants, starting from a single
explant.

4. OBJECTIVES AND GOALS:
 To initiate aseptic cultures of cactus.
 To observe the formation of shoot branches from
auxiliary buds of cactus, and to apply the
technology for plant propagation.
 To establish micro-propagated plants of cactus in
the greenhouse.

5. PLANT MATERIALS:
 Obtain ~4 (preferably 8 or 12) vernalized, nondormant
plants or lateral stems of cactus growing in pots from a local
nursery. Alternatively, cactus plants can be purchased from
Carolina Biological Supply Company.
 Plants should be healthy and free of symptoms of disease
or pest problems. At least two different species should be
used, if possible. Dwarf, clustering species respond the best
in culture.
 Recommended species include Mammillaria elongata
(golden stars cactus), M. prolifera (hair-covered pincushion
or grape pincushion cactus), or Chamaecereus sylvestrii
(peanut cactus).

6. EQUIPMENT:
 Laminar flow hood, such as Envirco LF830 .
 Electric bunsen burner, Electrothermal Engineering Ltd. BA6101 with
power regulator MC228.
 Forceps, scalpels.
 Sterile beakers, 250 ml.
 Sterile petri dishes, 100 X 25 mm or 100 X 20 mm.
 Culture sealant, e.g., Para film.
 Incubator with light and temperature control, such as PH Environmental
CEC-38-15-G.
 Dissection microscope with fiber optic lamp, such as Zeiss Stemi SV 6
 Magenta GA-7 boxes, 3’ X 3’ X 4, Sigma
 Plastic pots, 2’

7. PROCEDURES:
 Preparation of Reagents and Media:
 Ethanol. Prepare 200 ml of a 70% ethanol solution in a 250-
ml beaker. The same solution can be reused for all stems
from each species being prepared for culture.
 Bleach. Prepare 200 ml of a 50% solution of commercial
chlorine bleach (or a 2.6% solution of sodium hypochlorite)
in a 250-ml beaker. Prepare a different beaker for each stem
being prepared for culture.
 Sterile Distilled Water. Place sterile water in sterile 250 ml
beakers. Use three different beakers of water for each stem
being prepared for culture.

8. A
 Culture Media. All agar-solidified culture media should be
prepared in deep petri dishes. Dishes 100 X 25 mm are
recommended, but 100 X 20 mm dishes are acceptable in
most cases. Other deep culture vessels, such as baby food
jars, are acceptable alternatives. Magenta boxes can be
used for rooting media to provide more room for plant
development.
 Treatment of Materials :
 Cacti cultures should be placed in an incubator set at 28°C
with continuous light at 150 micro-mol m- 2 s-1.

9. DESIGN OF EXPERIMENT:
 This experiment is designed to test one or more species of
cactus with a potentially unknown response in culture.
 The experiment works best when two or more species are
compared.
 Four culture media are compared, varying in growth
regulator composition such as for cytokinin source.
 The media are designed to induce auxiliary bud
development with minimal callus formation.
 Two different explant types are compared on each medium.
 The first explant consists of the entire shoot apex including
several rings of areoles.

10. S
 The second explant is a transverse section of the stem
obtained just basipetal to the shoot apex explant, containing
one or a few rings of areoles.
 A minimum of two replications is suggested for each
species, requiring a total of eight plants or lateral stems to
initiate the experiment.

11. PROTOCOL:
 Surface Sterilization and Explant
Preparation:
 If plants exhibit lateral branches or multiple stems, excise
them for explant preparation. If no lateral branches are
present, the entin plant, if small, can be removed from the
pot. If the plant is large, excise the pper 2-3 cm of the shoot
apex including several rings of areoles.
 Soak each plant or excised lateral branch in a solution of
liquid detergent for 10 min, then rinse in running tap water
for 5 min.
 Carefully remove any roots and trim any spines present
using scissors or a scalpel. Be careful not to damage the
areoles. Place each stem into 70% ethanol for 1 min.

12. A
 Transfer each stem into a solution of 50% commercial
bleach for 7 min.
 Rinse each stem 3 X in sterile distilled water,S min each
time. Prepare the two explants from each stem: the entire
shoot apex including several rings of areoles compared to a
transverse section of the stem obtained just basipetal to the
shoot apex explant.
 Culture Initiation and Maintenance:
 Place prepared explants of the shoot apex or a transverse
section of stem, one explant per culture vessel, onto each of
four media: MS-Cl, MS-C2, MS-C3, and MS-C4.
 Seal the culture vessels and incubate them for 4 weeks.

13. A
 Observe the cultures weekly with the aid of a dissection
microscope. Note any morphological changes and growth
responses in the cultured tissues. Record the following data:
(1) the time of emergence of shoots from axillary buds, (2)
the frequency of axillary branch development, (3) the
number of axillary branches per responding culture, and (4)
the frequency and amount of callus formation, for each
explant type and culture media used.
 At the end of each monthly culture passage, separate or
excise the developing clusters of axillary shoots and transfer
them to fresh medium of the same composition for further
proliferation of axillary buds. Transfer up to four shoots per
culture vessel.

14. ROOTING AND ESTABLISHMENT OF PLANTS:
 Isolated shoots are placed on MS medium to encourage
shoot elongation and/ or spontaneous rooting. Up to four
shoots can be transferred to each culture vessel.
 Observe the cultures weekly for root initiation. Record the
following data: (1) the time of root emergence, (2) the
frequency of shoots developing roots derived from each of
the explants and shooting media used, and (3) the number
of roots per shoot.
 If after two monthly passages on MS medium rooting is
not observed, use media MS-Cs, MS-C6 and MS-C7
for 1 month to induce root initiation, then transfer back
to MS medium for 1 month to develop roots. Observe
cultures as indicated in step 2, except note the
frequency of rooting according to rooting media used.

15. A
 Well-rooted plantlets of cactus are carefully removed from
the agar-solidified media. Be careful not to damage the
roots. The agar medium is gently rinsed from the roots using
warm, but not hot, water.
 Plantlets are placed in soil mixes in plastic pots. Make sure
that the root tips are pointed downward in the soil mix.
Transfer the potted plantlets to a greenhouse. If possible,
the conditions in the greenhouse should be generally suited
to the given species of cactus.

16. SCHEDULE OF OBSERVATIONS AND
MEASUREMENTS:
 Axillary Shoot Development : Observe weekly. At the end
of each monthly passage, summarize the axillary shoot
response by frequency of cultures responding and number
of shoots developed. Separate results according to species,
explant type, and culture medium treatment. Also note the
undesirable occurrence of callus. Calculate the average
multiplication rate for each species on its optimum
combination of explant and medium over the course of the
experiment.
 Root Development : Observe weekly. At the end of the MS
medium rooting treatment, summarize the response by
frequency of shoots developing roots and number of roots
per shoot Separate results by species and according to
shooting media used. If media MS-Cs, MS-C6 and MS-C7
were used for rooting, separate results according to rooting
media used.

17. RESULTS:
 There was a difference between the explant types for timing of the
axillary bud response. Generally the intact shoot apices responded
faster than the transverse sections. The development of axillary shoots
from areoles, and a typical example of shoot multiplication in an
established axillary bud culture is provided. The development of axillary
branches was easy to observe in 88% of cacti surveyed in our lab.
Multiplication rates varied with the species. We observed 3 X to 10 X
increases in shoot number per monthly passage. The dwarf, clustering
cacti exhibited multiplication rates at the high end of this range. There
was a difference between the culture media for axillary shoot response
among and within species. More than 1/3 of the cactus species we
tested preferred MSCl medium and another 1/3 preferred MS-C2
medium. Of the remainder, about 10% of the species preferred MS-C3
medium, another 10% preferred MS-C4 medium, and the last 10%
responded poorly to any of these media.

18. A
 An example of in vitro-rooted shoots of cactus is shown in. Some of the
shoots developed roots spontaneously on MS medium. About half of the
cactus species we tested developed roots spontaneously on MS
medium, while the remainder required treatment with MS-Cs, MS-C6,
MS-C7 or other auxincontaining media to induce rooting. Well-rooted
shoots grown under high light intensity established in soil readily.