76 TISSUE CULTURE II Organogenesis PlSc 300 LAB 11 REFERENCE: Text: 663 666; 706 712; 717 718. OBJECTIVES: 1. Learn tissue culture techniques that promote organ formation. 2. Practice making leaf and shoot explants. INTRODUCTION: Plants are propagated in vitro through either enhancement of axillary branching, adventitious shoot formation (organogenesis) with subsequent rooting, or asexual embryogenesis. Caution: As some of you may have discovered with your bean embryos, sterile conditions are absolutely essential. Correct procedures for use of laminar flow hoods will be demonstrated again if needed. Please follow instructions carefully. Organogenesis. For this method of propagation, the explant is used to obtain organs (e.g., shoots, roots, or both). Organ differentiation is controlled by the ratio of cytokinins to auxins in the medium. If a callus stage is involved, minimum concentrations of both are necessary for callusing. Usually, if cytokinin levels are higher than auxin levels, shoot formation is favored. Often only shoots (high cytokinin concentration in the medium) are obtained, and these shoots can often be easily rooted as small softwood or herbaceous stem cuttings.
77 Biologically, this tissue culture technique is analogous to shoot and root differentiation by stem cuttings (except stem cuttings with pre-formed root initials or if pre-existing buds make the new shoot). For cuttings, do you remember which organs (shoots or roots) are promoted by cytokinins and which are promoted by auxins? As with cuttings from certain species, ontogenetic age (degree of juvenility) is an important factor for organogenesis. Shoot Formation Inducing shoot formation from leaf explants is one method of plant organogenesis or regeneration that is widely used with woody and herbaceous plants. Obtaining large numbers of shoots from plant tissue is essential for genetic improvement programs and production efficiency of clones. Leaf explants fulfill this requirement. Callus cultures may also produce many shoots, but this regeneration method may present a problem because of the potential for genetic instability of calluses. Somaclonal variation or genetic mutations appear more frequently for callus-regenerated shoots, especially from long-term cultures of callus. Therefore, a regeneration system with shoots arising directly from the leaf tissue and bypassing or considerably shortening the callus phase is desired. Even though plant regeneration from leaves is an efficient way to reproduce plants, this technique is used only infrequently in commercial propagation. Root Formation An important part of micropropagation is to induce roots to form on microshoots. Root formation on microcuttings is typically the limiting factor in micropropagation rather than shoot proliferation (from axillary buds). Microshoots can be rooted either in vitro or in the regular environment (as microcuttings). In vitro rooted cuttings can be placed on a medium supplemented with auxin or one that lacks a
78 cytokinin. Basal mineral concentration is sometimes reduced to half strength to promote rooting. Addition of activated charcoal is sometimes helpful for root formation on microshoots. Microcuttings rooted in the normal environment are placed under high humidity conditions. Before being placed in a greenhouse, the microcuttings can be treated with an auxin quick-dip before sticking them in a potting/rooting medium. Regardless of where shoots are rooted, indole-3-butyric acid (IBA) is often used to induce root formation on microshoots/cuttings. Other synthetic auxins, such as naphthaleneacetic acid (NAA), can be used in place of IBA to induce rooting. PROCEDURES: A. Shoot Organogenesis with Chrysanthemum 1. Each group should obtain two baby food jars that contains shoot cultures of Iridon mum. Also take two jars each of treatment M1, M2, and M3. 2. All work for this laboratory will be completed in the laminar hood. a. Be sure to spray the jars with a 70% alcohol before putting them into the hood. b. Be sure to sterilize forceps and scalpels between the shoots being removed from the jar. 3. Once a shoot is removed from a jar you MUST WORK QUICKLY so the leaves avoid drying out (become limp). Therefore, be organized and prepared to work quickly. 4. Remove a mum shoot from a jar and place it on a sterile paper towel. 5. Use leaves that are about 1 cm 2 or larger. a. Moving quickly but carefully, cut one leaf off from the shoot so that the petiole is still attached to the leaf blade.
b. Turn the leaf upside down on the paper towel and cut off the 5 79 leaf tip and side leaf margins as shown in class. c. Make one or two slices on the midrib (large vein), but 4 1 do not cut the leaf in half. The idea here is to wound 2 the midrib. Finally, cut off the petiole from the leaf blade. d. After removing the petiole, put the leaf explant in a baby food jar with the adaxial 3 surface (top side) of the leaf in contact with the medium. e. Place four leaf explants in each baby food jar; try to be consistent in the location of leaves used for explants. For instance, make your explants from leaves on the upper halves of the shoots. 6. Baby food jars contain 25 ml of modified MS medium. Treatment M1 lacks plant growth regulators; treatment M2 contains 0.3 M BA and 11.5 M IAA; and treatment M3 contains 0.9 M BA and 3.8 M IAA. 7. Observe explants weekly for organ (shoot) formation. 8. After three weeks from when the explants were taken, record the number of shoots formed on the explants. Remove the cap and count the shoots and/or clearly defined buds formed on the explant surface. Use a dissecting microscope if necessary. 9. Record the following data. a. The amount of contamination in your baby food jars, if any. b. The number of explants forming shoots for each treatment. Be sure to record a proportion (for example, 4 leaves out of 8 explants formed shoots). c. The mean number of shoots formed per regenerating explant for each treatment.
80 d. Location of shoot origin (cut surface, leaf tissue) and origin of shoots (from callus [a little callus or a large amount] or directly from the leaf tissue). e. The dates when adventitious shoots or roots began to form. Record the number of days from making the explant. B. Root Organogenesis on European Birch Microcuttings 1. Each group should obtain five jars of European birch microshoots. Also take two jars each of treatment B1, B2, B3, and B4. Woody Plant Medium is the basal salt used. Treatment B1 lacks plant growth regulators; treatment B2 contains only 0.1 M NAA; treatment B3 contains only 1.0 M NAA; and treatment B4 contains 5.0 M NAA. 2. Spray all jars and place them in the hood. 3. Get out a sterile paper and then remove one birch shoot from one jar. Use a sterile scalpel to remove any roots and callus from the bases of the stem. 4. Remove a second shoot from the jar, and remove any roots and callus from its base. Next, cut the two shoots to be more or less equal in length. 5. Place the first shoot in one of the jars containing a root induction treatment (B1, B2, B3, and B4). Make sure the base of the stem is about 0.5 cm (~ ¼ inch) deep in the agar. Take a third shoot from a jar, cut off any roots and callus and then cut the shoot to length using the second shoot as a guide. 6. Continue to remove shoots, remove roots and callus from the shoots, and then cut them to length before placing them in a rooting treatment jar. Be sure to work quickly to prevent wilting of the microshoots. Evenly divide up the shoots between the four treatments so that you have three shoots per jar. 7. Place the jars on a shelf in the culture room.
81 8. Observe the microshoots weekly for root formation. 9. After three weeks, record the following data. a. The amount of contamination in your baby food jars, if any. b. The number of microcuttings forming roots in each treatment. Be sure to record the proportion (e.g., 6 shoots out of 10 shoots formed roots). c. The mean number of roots formed per rooted microcutting for each treatment. d. Location of root origin (e.g., from cut surface, callus, internode area, etc.). e. The dates when the adventitious roots began to form for each treatment. Record the number of days from making the microcuttings. The data in sections A9 (a. through e.) and B9 (a. through e.) are required in the laboratory report. Questions: 1. What was the effect of the different concentrations of BA and IAA on shoot regeneration from chrysanthemum leaves? 2. Did callus form before shoots on the leaf explants? Explain your answer. 3. What was the effect of the different NAA concentrations on root initiation? 4. Did appearance of the roots vary with auxin concentration? If so, briefly describe root appearance and speculate why they appear different.
82 COMPOUND Murashige Skoog Woody Plant Medium Anderson Gamborg B5 -------------------------------------------- concentration (mg/liter) --------------------------------------- Macronutrients NH 4 NO 3 1650.0 400.0 400.0 KNO 3 1900.0 480.0 2500.0 Ca(NO 3 ) 2 4H 2 O 386.0 (NH 4 ) 2 SO 4 134.0 CaCl 2 332.2 72.5 332.2 113.24 KH 2 PO 4 170.0 170.0 330.6 130.5 K 2 SO 4 990.0 MgSO 4 180.7 180.7 180.7 122.09 Micronutrients FeSO 4 7H 2 O 27.8 27.8 55.7 27.8 Na 2 EDTA 2H 2 O 37.26 37.3 74.5 37.3 MnSO 4 H 2 O 16.9 22.3 16.9 10.0 ZnSO 4 7H 2 O 8.6 8.6 8.6 2.0 H 3 BO 3 6.2 6.2 6.2 3.0 KI 0.83 0.3 0.75 Na 2 MoO 4 2H 2 O 0.25 0.25 0.25 0.25 CuSO 4 5H 2 O 0.025 0.25 0.025 0.025 CoCl 6H 2 O 0.025 0.025 0.025 Organics myo-inositol 100.0 100.0 100.0 100.0 glycine 2.0 2.0 Vitamins nicotinic acid 0.5 0.5 1.0 pyridoxine HCl 0.5 0.5 1.0 thiamine HCl 1.0 1.0 10.0 c:\plsc300\lab11 organogenesis tc-12.doc