Micro Propagation by auxiliary buds.pptx

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1. Micro Propagation by auxiliary buds f M,Phool 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 .