Thermal neutron induced changes in Saintpaulia

Environmental and Experimental Botany, VoL 21, pp. 95 to 102

0098-8472181/020095-08 $02.00/0

© Pergamon Press Ltd. 1981. Printed in Great Britain

T H E R M A L N E U T R O N I N D U C E D CHANGES IN SAINTPAULIA* JOHN W. K~.U.Y and n . DANIEL LtNEBERGERt

Department of Horticulture, The Ohio Agricultural Research and Development Center, Wooster, OH 44691 and The Ohio State University, Columbus, OH 43210, U.S.A.

(Received 25 April 1980; accepted in revisedform 16 August 1980) KELLY J. W. and LINEBERGER R. D. Thermal neutron induced changes in Saintpaulia. ENVIRONMENTALAND EXPERIMENTAL BOTANY 21, 95--102, 1981.--Saintpaulia ionantha "Ulery" Wend. cuttings were irradiated with thermal neutrons (250, 1000 and 5000 rad) to determine radiation effects on lethality, root formation, morphology and peroxidase isozymes. Lethality •was high at the high doses. Irradiated cuttings showed increases in the time required for roots to form. Although no morphologically distinct mutants were recovered, separation of peroxidase proteins showed that radiation permanently altered peroxidase isozyme patterns. Radiation-induced mutations increased the number of isozymes which were catalytically active but different in their electrophoretic mobility. Radiation also destroyed the catalytic activity or suppressed synthesis of native isozymes. Thus, the isolation of peroxidase isozymes served as an effective genetic marker for radiation-induced mutants.

INTRODUCTION

MUTATION BREEDING with vegetatively reproduced species offers the advantage of cultivating one or several improved characteristics without altering the remaining genotype. Complications arise when mutations occur in only a portion of the meristematic tissue giving rise to chimeras. ~4'11) Propagation of this meristematic tissue results in multiple genetic sections or layers of tissue growing adjacent to each other. O) NAYLOR and JOHNSON(tT) reported that Saintpaulia leaf cuttings produced shoots which arose from a single epidermal cell. This characteristic is also found in Streptocarpus, Achimenes, and other crops. (4) Propagation of leaf cuttings which form adventitious buds from a single epidermal cell would eliminate chimera formation. SPARROW et al. ~21) used Saintpaulia, and

BROERTJES(4) used Streptocarpus to obtain nonchimerical mutants. The production of improved ornamental horticultural crops by conventional breeding techniques requires long periods of time with infrequent mutations. Ionizing radiation offers a means for increasing the mutation rate and the possibility of producing mutants with desirable characteristics. Identification of mutant cultivars has previously been based on morphological descriptions of the affected plants. Since morphologically similar cultivars may differ in physiological responses, an alternative method of mutation detection is needed. In cases where mutation has altered the structure of an enzyme without altering its catalytic functions, the isozyme can be assayed as a genetic marker. ~I9) Electrophoretic sepa-

*Approved as Journal Article No. 7280 of the Ohio Agricultural Research and Development Center. ]'Graduate Student and Assistant Professor, respectively. 95

96

JOHN W. KELLY and R. DANIEL LINEBERGER

ration of proteins allows the study of altered experiments: potassium metabisulfite, 0.60g; asenzyme properties which may not be pheno- corbic acid, 0.90g; dithiothreitol, 0.10g; Triton typically manifested. Characterization of plant X-100, 4ml of 10% v/v; Tris, 0.90g and water enzymes by polyacrylamide gel electrophoresis to 100ml (pH 6.8). has been used to separate variants of Kentucky Approximately 2 g of leaf tissue were ground bluegrass, (24) soybean, ~la) alfalfa, C15) petunia ~6) with a mortar and pestle in one volume of and roses. (1°) extraction solution ( l m l / g fresh weight) at A genetic marker must be stable for use in room temperature. The extract was centrifuged identification. Changes in peroxidase activity at 4°C for 50 min at 27,000 g. The final cocktail following irradiation are well documented: 6'1s) applied to the gels contained 75% w/v sucrose ENDO~6) determined the variability in isozyme mixed 1:4 with the protein supernatant to give patterns of irradiated maize seedlings from 4 to a final concentration of 15% sucrose. One drop 9 days after germination, gARFIELD eta/. (23) of 0.001% w/v bromophenol blue marker dye assayed Saintpaulia immediately after irradiation was added to each 0.5ml of the sucrose plus to determine the effect of X-rays and ethylene iprotein solution. The amount of protein cocktail on peroxidase patterns. 1applied to the gels was 200#1. A 7.5°/~) separatThe present research was conducted to de- ing gel was made by mixing 5.0 ml of 30o/o w/v termine alterations in morphogenic potential acrylamide (containing 0.8% w/v bisand the stability of radiation induced alterations acrylamide), 8.0ml of 1.0M Tris (pH 8.3) and of Saintpaulia peroxidase isozymes in an effort to 6.6ml distilled water. Polymerization began obtain a genetic marker for use in mutation after the addition of 100#1 of 5% v/v T E M E D detection. and 100#1 of 10% w/v ammonium persulfate. The stacking gel was made by mixing 0.9ml 30% w/v acrylamide (containing 0.8% w/v bisMATERIALS AND M E T H O D S acrylamide), 2.5 ml 0.5 M Tris (pH 6.9), 5.0 ml After a one month acclimation period at 40% w/v sucrose and 1.4ml distilled water. 20°C and 43001x for 18hr each day, 300 leaf Polymerization was initiated using 50 #1 5°/,,o v/v blade and petiole cuttings were taken from T E M E D and 50 #1 10% ammonium persulfate. Saintpaulia stock plants. There were 25 plants The gels were run at 4 m A per tube using a per treatment and three repetitions of each Buchler Electrophoresis power supply. The peroxidase staining solution, containing treatment. The cuttings were thermal neutron 0.50g benzidine dissolved by heating in 4.5 ml irradiated at 500W for 6.8min, 1 kW for 14min of glacial acetic acid was diluted to 100ml with or 10kW for 6.8min to obtain approximately 250, 1000 or 5000 rad doses, respectively, at the distilled water. Just prior to placing the gels in the staining solution, 0.10ml of 30% hydrogen Ohio State University nuclear reactor. After irradiation each of the 300 cuttings peroxide was added to the solution. After the was placed in 12 x 75 mm glass test tubes filled blue peroxidase bands developed (approwith water. The water was replenished twice ximately 20min), the gels were rinsed in distildaily. The glass tube allowed observation of led water, placed in 7°//o v/v glacial acetic acid root formation. Root formation was recorded as for 5min, and rinsed and stored in distilled the time at which a 1 m m root was present. water.~ 10) Each plant assay was replicated three times. Lethality data were based on the number of plants that died prior to forming roots and the Protein bands were identified by R: value and number that formed roots but died before visual evaluation of isozyme density. A range in Rf from 0.002 to 0.014 was observed for inshoots emerged. Electrophoretic analysis of Saintpaulia per- dividual bands. When bands were greater than oxidase was initiated approximately five months 0.5mm in width, upper and lower R: values after transplanting. The following adaptation of were recorded for isozymes. Density was recorded as light, medium, or heavy. Gels were Hare's extraction solution (s) produced the highest quality banding and was used for all placed over a fluorescent light source and

SAINTPAULIA

THERMAL NEUTRON INDUCED CHANGES IN photographed with Kodak Panatomic-X film. As reported by several researchers (5' s, 1o) photographs fail to show all visible bands, therefore, zymograms were drawn using average R/ and density values from three replications (Fig. 1 ). Zymograms depict the density, width and location of bands.

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FIG. 1. Zymograms depict the isozyme banding patterns of the photographed gels. Bands are recorded as light, medium or heavy. The width of the zymogram bands corresponds to the upper and lower R: values of gels which were photographed.

RESULTS

Thermal neutron irradiation markedly affected lethality prior to or after root formation (Fig. 2). Control and 250 rad treatments had ti.u~,r lethals than plants which were irradiated with 5000rad. No lethality occurred after the formation of shoots. In addition to its effect on lethality, neutron irradiation also changed the t i m e required for Saintpaulia root formation (Fig. 3). The delay in rooting of leaf and petiole cuttings was dose dependent at higher radiation levels. The time required for the Saintpaulia cuttings to develop a 1 m m root was significantly different between the 5000 rad treatment and all other treatments. The cultivar used in this study appeared to remain phenotypically stable during the experiment. No morphological alterations were noted in plants from control or radiation treatments. Further examination of the cultivar by polyacrylamide gel electrophoresis of six ran-

domly selected control plants showed no differences in peroxidase isozyme banding patterns (Fig. 4). Eight peroxidase bands can be seen in all gels, indicating that the sampling procedure was consistent. Since the plants were sampled at various times from 9 to 15 months after irradiation it appeared that the isozyme patterns were stable if uniform tissue selection was made. Isozyme patterns of plants irradiated with 250 rad of thermal neutrons demonstrated thai radiation altered electrophoretic mobility and density of Saintpaulia peroxidase isozymes (Fig. 4). High variability can be observed among the plants which were sampled within the treatment. The only bands which appeared consistently in all plants sampled from the treatment were those at R: =0.1 and R: =0.93. Low doses (250rad) of radiation caused an increase in the number of peroxidase isozyme bands. Every plant sampled from the 250 rad treatment had more isozymes than control gels. Ten to twelve bands occurred in each gel sampled from the 250 rad treatment. When compared to a control gel, the only isozyme band which appeared to be common to control and 250rad treatments was at R : = 0 . 9 3 (Fig. 5). This suggests that at least seven of the eight control peroxidase isozymes were altered by low doses of radiation.

98

JOHN W. KELLY and R. DANIEL LINEBERGER o

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Fro. 3. Cumulative percentage of survivors which rooted from each dosage of radiation (HSD=4.8).

Variability in isozyme banding patterns was present in plants which were irradiated with 1000 rads of thermal neutrons (Fig. 4). Isozymes at R I = 0 . 3 9 , R I = 0 . 4 5 and R I = 0 . 5 0 appeared in all gels of the plants sampled. Comparison to protein isozyme patterns of control gels show

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that the same three bands (Rf=0.39, 0.45 and 0.50) which were observed in the 1000rad treatment were also present in controls (Fig. 5). The bands which correlated to controls have the higher electrophoretic mobilities. More variability in banding is seen in the bands which Control

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Saintpaulia plants exposed to 250, 1000 and 5000 rads of thermal neutrons when compared to control plant isozymes.

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T H E R M A L N E U T R O N I N D U C E D C H A N G E S IN SAI.\,TPAUL1A

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Fl(~. 4. Isozyme banding patterns of control, 250 rad, 1000 rad and 5000 tad treatments of Sainlpaulia.

99

THERMAL NEUTRON INDUCED CHANGES IN SAINTPAULIA have lower electrophoretic mobilities. Seven to nine bands were present in the 1000 rad treatment compared to eight bands in controls, therefore one or more isozymes have lost catalytic activity when subjected to radiation. Plants which were irradiated with 5000rad produced three isozymes with similar RI and density values (Fig. 4). Isozymes present at Rf values of 0.1, 0.39 and 0.45 were found in all gels of the plants which were sampled. When the 5000rad treatment was compared to controls the only consistent bands occur at Rf =0.39 and Ry=0.45 (Figs. 5). The number of bands produced due to 5000rad irradiation ranged from six to ten, which indicates the variability present in radiation effects on isozymes.

DISCUSSION

It is well known that lethality increases with higher radiation doses. LOVE(12) observed the effects of fast neutrons on survival of eight poinsettia cultivars. He found that all cultivars survived 600rad, five cultivars survived 800 rad and complete lethality occurred at 1000rad. BROERTJESTM reported 50% lethality when Saintpaulia leaf and petiole cuttings were irradiated with X-rays at an exposure of 5000rad. WARFIELI) et al. ~z3) found 5500rad of X-rays to be lethal for 50% of the Saintpaulia cuttings irradiated. Only 20% lethality was observed in this study with 5000rad of thermal neutrons. Differences due to cultivar radiation sensitivity may account for the low lethality reported here. WARFIELD et al. ~23) and BROERTJES~3) used X-rays instead of thermal neutrons, therefore the form of radiation may be a factor in the lethality differences. The preand post-irradiation environments are known to affect radiation sensitivity, but neither BROERTJES(3) n o r WARFIELD and co-workerst23) discuss the environmental conditions imposed in their experiments, so no comparisons can be made.

The

radiation induced alteration in Saintpaulia rooting pattern is consistent with the previous work of SPARROW and co-workers. ~21) Using leaf and petiole cuttings they noted no effect with 2000rad of X-rays while 3000rad

lO1

delayed rooting. They do not show data supporting their results, therefore no quantitative comparisons can be made between the studies. Although no other developmental or morphological alterations were observed, distinct differences were noted in peroxidase isozymes. Peroxidase isozyme patterns can change during growth and development, °'1'25) but selection and sampling of tissue of approximately the same physiological state of maturity should alleviate this problem. ~13'z3) By the selection of tissue of a similar physiological state, we experienced no problems in repeating the banding patterns for each experiment. Determination of isozyme banding patterns is a more direct measurement of mutation than morphological evaluation. ~s) If changes in isozyme patterns can determine disease susceptibility,~8, 22) genetic variability~7,14, 2m and growth potential, ~x'2'251 they may serve as an important genetic marker for mass irradiated populations.

SUMMARY

Thermal neutron radiation increased lethality and time required for rooting in Saintpaulia. These increases were dose dependent with higher levels of thermal neutrons having a greater effect. No other developmental or morphological changes due to radiation were observed. Irradiation did alter the isozyme banding patterns of Saintpaulia peroxidases. These alterations were independent of dose since low levels of radiation (250rad) increased the number of isozymes while higher levels caused changes in the number of isozymes which were unpredictable. The isozyme changes were stable during the nine to fifteen month sampling period, indicating the possibility of their use as a genetic marker.

REFERENCES

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