Callus cultures were established from 10 lines of Zea mays containing cytoplasms with normal and Texas cytoplasmic malesterile factors. Calli from the different lines varied in their growth characteristics. One callus line was treated with Helminthosporium maydis race T toxin extract

Horner, Harry T., Jr.

Pearson, Christine B.

University of Northern Iowa

Proceedings of the Iowa Academy of Science Volume 82 Number 3-4 Article 7 1976 Callus Formation and Differentiation in Tissue Cultures of Normal and Texas Cytoplasmic Male-Sterile Corn Christine B. Pearson Iowa State University Harry T. Horner Jr. Iowa State University Let us know how access to this document benefits you Copyright ©1976 Iowa Academy of Science, Inc. Follow this and additional works at: https://scholarworks.uni.edu/pias Recommended Citation Pearson, Christine B. and Horner, Harry T. Jr. (1976) "Callus Formation and Differentiation in Tissue Cultures of Normal and Texas Cytoplasmic Male-Sterile Corn," Proceedings of the Iowa Academy of Science, 82(3-4), 163-165. Available at: https://scholarworks.uni.edu/pias/vol82/iss3/7 This Research is brought to you for free and open access by the Iowa Academy of Science at UNI ScholarWorks. It has been accepted for inclusion in Proceedings of the Iowa Academy of Science by an authorized editor of UNI ScholarWorks. For more information, please contact scholarworks@uni.edu. 163 Callus Formation and Differentiation in Tissue Cultures of Normal and Texas Cytoplasmic Male-Sterile Corn1 CHRISTINE B. PEARSON and HARRY T. HORNER, JR.2 PEARSON, CHRISTINE B., and HARRY T. HORNER, JR. (Depart-ment of Botany and Plant Pathology, Iowa State University, Ames, Iowa 50011 ). Callus formation and differentiation in tissue cultures of normal and Texas cytoplasmic male-sterile corn. Proc. Iowa Acad. Sci. 82(3-4): 163-165, 1976. Callus cultures were established from 10 lines of Zea mays con-The establishment of tissue cultures from agronomic plants, and more specifically corn (Zea mays L.), has been recently reviewed by Green, Phillips, and Kleese ( 197 4). Sheridan (1972), Burr and Nelson (1972), and Green et al. (1974) have successfully initiated and maintained calli from excised embryos of a variety of normal corn lines using 2,4-dichloro-phenoxyacetic acid (2,4-D) as the auxin. To our knowledge, there have been no reports to date of cJmparable studies on cytoplasmic male-sterile lines of corn. Tissue cultures can provide a large population of similar undifferentiated cells which might be used to study differen-tiation and regeneration or to monitor host-pathogen re-sponses of particular plant cell lines to determine susceptibil-ity or resistance (Hildebrandt, 1965a, b). In the latter case, normal ( N) cytoplasm an :1 Texas cytoplasmic male-sterile (Terns) corn could be studied by using Helm1nthosporium maydis race T toxin; H. maydis race T was the causative agent of the southern corn leaf blight epidemics of 1970-71 in the U.S. (Tatum, 1971; Ullstrup, 1972). Because of these possibilities, our objectives in this study were to establish and maintain callus cultures from both N and Terns lines of corn, as attempted in an earlier study (Rogers, Gal, and Horner, 197 4), to assess the response of the calli to toxin extracts from H. maydis race T, and to attempt to differentiate the calli intD whole flowering plants. MATERIALS AND METHODS Seeds from the following inbred N and Terns lines of Zea mays L. were used: Oh43, Wf9, CI31A, Bl4, B37, Mol7, A632, A635, A619, and Cl03. Initiation and Maintenance of N and Tcms Cultures Callus cultures were initiated by following procedures modified from Burr and Nelson (1972) and Sheridan (1972). The steps of our procedure were: 1. Corn kernels were sur-1 Journal Paper No. J-8103 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project No. 1914. Research supported by U.S.D.A., C.S.R.S., special grant 177-15-06. 2 Department of Botany and Plant Pathology, Iowa State Uni-versity, Ames, Iowa 500ll. taining cytoplasms with normal and Texas cytoplasmic male-sterile factors. Calli from the different lines varied in their growth characteristics. One callus line was treated with H elminthosporium maydis race T toxin extract. INDEX DESCRIPTORS: Callus Formation, Callus Differentiation. face sterilized for 10 minutes in a solution consisting of 1 g mercuric chloride and one drop of 'Tween 80 detergent in 100 ml water. 2. Kernels were rinsed four times in sterile water and left to soak overnight in the last rinse to soften the kernel for embryo dissect:on. 3. Embryos were aseptically removed from the kernels with forceps and surface sterilized for seven minutes in a rnlut:on of 10 ml Chlorox and one drop of 'Tween 80 detergent in 90 ml water. 4. Whole em-bryos were rinsed four times in sterile water, placed on po-tato dextrose agar (Difeo) in petri plates, and incubated for two weeks at 28° C in the dark, during which time they ex-panded and grew, producing roots and elongated shoots. 5. MesocJtyl regions were aseptically excised with forceps and placed on 50 ml of modified Murashige and Skoog ( 1962) medium (sucrose concentration was lowered to 25 g per liter, agar was reduced to 6 g per Lter, and kinetin and 2,4-D levels were adjusted to 1 mg per liter and 10 mg per liter respectively) for initiation of callus. 2,4-D levels of 1, 3, 5, 10, 15, 20, and 25 mg per liter were tested for initiation of callus. 6. For maintenance of calli the 2,4-D concentrations of 0, 1, 2, 3, 5, and 10 mg per liter were tested, and the calli were incubated at 28° C in the dark, with subculturing every 30 days. Differentiat'on of N and Tcms Cultures The modified Murashige and Skoog medium was used, but with the 2,4-D and indoleacetic acid replaced by 0-5 mg cx-naphthaleneacetic acid per liter and 0-5 mg kinetin per liter. Differentiating calli were placed in a growth chamber with a 14 hour photoperiod (1,100 footcandles, fluorescent lights) at 27° c during the day and 16° cat night. After two weeks the calli were either subcultured on the same medium or transferred to sterile vermiculite in the greenhouse and watered with Hoagland's solution (Hoagland and Amon, 1938). Liquid Cultures L;quid culturing was attempted by using calli in:t:ate:i on the solid me:lium. The modified Murashige and Sko1g med-ium was used without agar and w~th 0-10 mg 2,4-D per liter. After inoculation with 112 cm3 chunks of calli, cultures were shaken continuously on a 60 cycle per minute reciprocal shaker under fluorescent lights. 1Pearson and Horner: Callus Formation and Differentiation in Tissue Cultures of NormalPublished by UNI ScholarWorks, 1975164 PRoc. low A AcAD. Sc1. 82 ( 1976) Figures 1-7. Stages depicting initiation, maintenance, and differentiation of Bl4 Terns corn callus. Figure 1. Plucked embryo on potato dextrose agar medium. X2.3. Figure 2. Enlarged embryo after two weeks on potato dextrose agar medium. X2.4. Figure 3. Excised mesocotyl region of embryo. X2.4. Figure 4. Compact round clumps of callus on maintenance medium. X2.4. Figure 5. Callus with roots and root hairs. X2.2. Figure 6. Fluffy white callus growth. X2.2. Figure 7. Callus on differentiating medium shows prolific root growth and a few green stubby protuberances. X2.4. Toxin Treatment Healthy calli from N and Terns Bl4 corn were placed on the modified Murashige and Skoog medium containing 2 ml per liter H. maydis race T toxin extract and incubated as previously described for 30 days. The diluted toxin extract caused a 50 percent reduction in primary root elongation of germinated B37 Terns seedlings after 24 hours' exposure. RESULTS AND DISCUSSION In early experiments, whole embryos or portions of embryos were placed directly on modified Murashige and Skoog med-ium. Placement of embryos on potato dextrose agar for the first two weeks to allow hydration and some growth promoted consistent and rapid callus formation. Figures 1-3 illustrate the growth of the embryo on potato dextrose agar (Figures 1, 2) and show the mesocotyl region excised for initiation of callus (Figure 3). Callus was obtained from all 10 lines in both the N and Terns cytoplasms by this procedure. The callus produced by all lines was yellowish, often grew in compact round clumps (Figure 4), and was friable and easily broken apart by forceps. Frequently, root-like structures complete with root hairs formed (Figure 5), and occasionally fluffy white callus grew (Figure 6). The fluffy white growth indicated rapidly growing callus. Compact yellowish callus occasionally turned brown and died after subculturing. Genetic variability in callus initiation was examined in N and Terns lines. Calli of different lines varied in compact-ness, color, amount of callus formed, proliferation of roots, and the ability to survive subculturing. For example, the cal-lus of Wf9 (both N and Terns) usually grew in compact balls and was yellowish; the callus of CI31A (both N and Terns) was lighter in color, fluffier, and produced more roots. The ability of a line (both N and Terns) to initiate callus was judged on the basis of the proportion of embryo mesocotyl chunks that formed at least 112 cm3 of callus when placed on modified Murashige and Skoog medium. N and Terns calli of each line were classified together because, in general, they were similar in appearance and in growth characteristics. Callus initiation of each line was classified as good ( Oh43, Wf9, CI31A, Bl4; approximately 40 percent of the meso-cotyl chunks formed callus), fair (B37, Mol7, A632, A635, 2Proceedings of the Iowa Academy of Science, Vol. 82 [1975], No. 3, Art. 7https://scholarworks.uni.edu/pias/vol82/iss3/7CALLUS FORMATION 165 A619; 10-30 percent formed callus), or poor ( Cl03; less than 10 percent of the chunks formed callus) . Classifications were based on more than 75 embryos from each line. Cl31A sub-cultured poorly and no calli survived beyond three trans-fers. Bl4, Oh43, and Wf9 calli were still proliferating after seven or more transfers. Green et al. ( 197 4) and Burr and Nelson (1972) found similar variability in the inbred and single-cross com lines they tested for callus initiation. Initiation of our calli was best accomplished at 10 mg of 2,4-D per liter where reasonably fast growth of callus occur-red. At this concentration much of the root formation preva-lent at lower levels of 2,4-D was prevented. Good subcul-turing of initiated calli occurred at 3 mg of 2,4-D per liter. At this concentration there was the best compromise between undue root formation and inhibition of growth. Liquid culturing was attempted without success. Callus chunks turned brown and never grew. No explanation for the lack of growth is available because, using similar methods, Burr an:l. Nelson ( 1972) and Sheridan ( 1972) obtained rapid growth in liquid shake cultures. Calli placed on Murashige and Skoog medium modifie:l for differentiation typically formed prolific roots and several green bud-like stubby protuberances (Figure 7), but no leaves or shoots ever formed. Calli with roots and stubby protuberances were maintained through three transfers on differentiating medium without significant change. Transfer of differentiating calli to sterile vermiculite watered with Hoagland's solution resulted in death of the calli within two weeks. The addition of the toxin extract from H. maydis race T to the agar medium caused a reduction in growth and darken-ing of Bl4 Terns calli. This was determined by visual com-parison of the latter with Terns Bl4 calli on the regular agar medium and N Bl4 calli treated with the same level of toxin extract. The inconsistent growth of calli made interpretation of toxin-treatment difficult and the plotting of a dosage-re-sponse curve impossible. Often as many as half the N and Terns callus chunks died during each subculturing period for no apparent reason, while the remaining calli flourishe:l. Further experimentation with different lines, or perhaps other culturing techniques, might be fruitful in establishing more consistent growth of callus that would be amenable to experimentation with H. maydis race T toxin. SUMMARY Callus cultures wese established from embryo mesocotyl regions of selected lines of Zea mays L. containing cytoplasms with normal (N) and Texas cytoplasmic male-sterile (Terns) factors. Undifferentiated calli and calli with ro:Jts and green protuberances were produced from both the N and Terns lines of Oh43, Wf9, CI31A, Bl4, B37, Mol7, A632, A635, A619, and Cl03. Callus growth varied from good to poor among lines but was relatively consistent within the N anl Terns calli of the same line. Terns Bl4 calli exposed to Hel-minthospor:um maydis race T toxin extract showed less growth and darkening of the calli. Uniform callus growth was difficult to maintain in some of the controls and toxin treat-ments. ACKNOWLEDGMENT The authors wish to thank Dr. C. A. Martinson, Depart-ment of Botany and Plant Pathology, ISU, Dr. W. A. Russell, Department of Agronomy, ISU, and Clyde Black & Son Hy-brid Seed Farms, Ames, Iowa, for providing the seeds, and Miss Carla Holbrook for typing of the manuscript. LITERATURE CITED BURR, B., and 0. NELSON. 1972. Induction and maintenance of maize callus tissue. Maize Genet. Coop. Newsl. 46:202-203. GREEN, C. E., R. L. PHILLIPS, and R. A. KLEESE. 1974. Tissue cultures of maize (Zea mays L.): Initiation, maintenance, and organic growth factors. Crop Sci. 14:54-58. HILDEBRANDT, A. C. 1965a. Growth in vitro of plant single-cell clones of normal and diseased origins. Pp. 411-427 in P. R. White and A. R. Grove (eds.), Proceedings of International Conference on Plant Tissue Culture. McCutchan Publications Corp., Berkeley, Calif. HILDEBRANDT, A. C. 1965b. Tissue culture as an aid in the study of plant disease. Adv. Agric. ~ci. (Madras), pp. 584-597. HOAGLAND, D. R., and D. I. ARNON. 1938. The water culture method for growing plants without soil. Calif. Agric. Ext. Serv. Gire. 347. MuRASHIGE, T., and F. SKOOG. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:473-497. ROGERS, M. A., H. L. GAL, and H. T. HORNER, JR. 1974. Callus formation and differentiation in tissue cultures of normal and cytoplasmic male-sterile sorghum, pepper, sunflower, and to-bacco. In Vitro 9:463-467. SHEIUDAN, W. F. 1972. Induction and growth of callus of corn and wheat. J. Cell Biol. 55:237a, Part 2. TATUM, L. A. 1971. The southern corn leaf blight epidemic. Sci-ence 171:1113-1116. ULLSTRuP, A. J. 1972. The impacts of the southern corn leaf blight epidemics of 1970-1971. Annu. Rev. Phytopathol. 10:37-50. 3Pearson and Horner: Callus Formation and Differentiation in Tissue Cultures of NormalPublished by UNI ScholarWorks, 1975

Callus Formation and Differentiation in Tissue Cultures of Normal and Texas Cytoplasmic Male-Sterile Corn

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Callus cultures were established from 10 lines of Zea mays containing cytoplasms with normal and Texas cytoplasmic malesterile factors. Calli from the different lines varied in their growth characteristics. One callus line was treated with Helminthosporium maydis race T toxin extract.This article is published as Pearson, CB, and HT Horner, Jr.  1976.  Callus formation and differentiation in tissue cultures of normal and Texas cytoplasmic male-sterile corn.  Proceedings of the Iowa Academy of Science 82:163-165. Posted with permission.</p

Pearson, Christine

Horner, Harry

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Callus Formation and Differentiation in Tissue Cultures of Normal and Texas Cytoplasmic Male-Sterile Corn

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