IllUlllllllllllIllllllllllllllllllllllllllllllllllllllllll 293 10382 7162 This is to certify that the thesis entitled TISSUE AND CELL CULTURE TECHNIQUES OF ASPARAGUS OFFICINALIS L. AND SELECTION OF CELL LINES TOLERANT TO AN AUTOTOXIC ROOT EXTRACT presented by Karen D. Hassett has been accepted towards fulfillment of the requirements for MS . HORTICULTURE degree 1n ”fig/7% / Major professor Maggi/ 0-7639 OVERDUE FINES: 25¢ per day per it. I RETURNIE LI§RARY MATERMLS: g g: ,{S ..J‘ Ali! Alum}: ”f ' '_ ‘ \_ :.I"'nlufllll . \rllr - ‘ "a :- Place in book return to relieve charge from circulation records TISSUE AND CELL CULTURE TECHNIQUES OF ASPARAGUS OFFICINALIS L. AND SELECTION OF CELL LINES TOLERANT TO AN AUTOTOXIC ROOT EXTRACT BY Karen D. Hassett A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE 1981 .I kw L -(‘A / {’3 ABSTRACT TISSUE AND CELL CULTURE TECHNIQUES OF ASPARAGUS OFFICINALIS L. AND SELECTION OF CELL LINES TOLERANT TO AN AUTOTOXIC ROOT EXTRACT BY Karen D. Hassett Techniques described include: 1) callus and suspension culture of Asparagus officinalis L., 2) suspension cell plating, and 3) regeneration of callus and suspension cultures. Asparagus cell lines tolerant to an autotoxic root extract were selected from cells plated in a modified MS medium containing a dilution series of root extract. Cultures were maintained at 25:20C for 3 — 5 weeks. Of the 303 cell colonies surviving exposure to concentration 1:2 (autotoxinzmedium v/v), 40 were subcultured to medium MS 1:1, where 2.5% cell colonies regenerated shoots and 55% regenerated roots. The remaining 263 putative tolerant cell lines were subcultured to a non-selective medium for one passage before reintroduction to the autotoxin. The tolerant phenotype was expressed in 76.43% of the cell lines reintroduced to the autotoxin. Mutation frequencies of 5.71x10’5, 4.04x10'5 , and 8.13x10-5 were calculated for the three selection experiments, respectively. Possible increased tolerance to the herbicide glyphosate (N-phosphonomethylglycine) is also described. ACKNOWLEDGMENTS . I would like to gratefully acknowledge the help and suggestions of my major professor, Jon F. Fobes, and of the members of my guidance committee; L.W. Mericle, A.R. Putnam, K.C. Sink, and J. Taylor. I extend my thanks to friends and family for their encouragement and to Mark, for his support and unending humor. And thanks to Patty for her assistance in typing this thesis. ii TABLE OF CONTENTS LIST OF TABLES . . . . . . . . . . . . . . . . LIST OF FIGURES . . . . . . . . . . . . . . . CHAPTER I TISSUE AND CELL CULTURE TECHNIQUES OF ASPARAGUS OFFICINALIS L. Introduction . . . . . . . . . . . . . . . . . Procedures . . . . . . . . . . . . . . . . . . Results and Discussion . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . References 0 O O O O O O O O O O O O O O O O 0 CHAPTER II IN VITRO SELECTION OF ASPARAGUS OFFICINALIS L. LINES TOLERANT TO AN AUTOTOXIC ROOT EXTRACT Abstract . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . Materials and Methods . . . . . . . . . . . . Results and Discussion . . . . . . . . . . . . Literature Cited . . . . . . . . . . . . . . . iii CELL Page vi 12 12 l6 17 23 28 35 Page CHAPTER III SELECTION OF ASPARAGUS OFFICINALIS L. CELL LINES IN THE PRESENCE OF THE HERBICIDE GLYPHOSATE Introduction . . . . . . . . . . . . . . . . . . . . . 42 Materials and Methods . . . . . . . . . . . . . . . . . 43 Results and Discussion . . . . . . . . . . . . . . . . 46 Literature Cited . . . . . . . . . . . . . . . . . . . 51 iv Table II III IV VI II III IV LIST OF TABLES CHAPTER I callus initiationmediaOOOOOOOOOOOOOOOOOOOOOOOOO Average settled cell volume during passage 3.... Culture media used for the regeneration of asparagus callus tissue......................... Callus growth and friability rating, 12 weeks after innoculation.............................. Regeneration of roots and/or shoots of Asparagus officinalis L. on 8 various nutrientmedia.0.0.0.0000...00.000.00.000.00.... Regeneration of shoots and roots from suspension cultures transferred to three nutrientmediaOOOOOOOOOOOOIOOOOOOOOOOCOOOOO0.... CHAPTER II Conductivity and pH of the asparagus autotoxin extractOIOOOOOOO0......OOOOOOOOOOO...0.00.0.0... Estimation of cell density, cell viability, and mutation frequency.0.0.0.0...IOOOOOOOOIOOCOO Total number of presumed tolerant cell colonies, number transferred to callus maintenance medium 12d, and number transferred to regeneration medium MSl:l.................................... Reintroduction of presumed tolerant colonies to autotOXinOOOOOOO0.0..0.000000000000000000000. Page 10 12 24 25 26 31 LIST OF FIGURES Figure Page CHAPTER I l Asparagus callus formation on 3 basal media ..... ............... ....... ..... .......... 8 2 Callus regeneration of shoots and roots on 8 nutrient media. ...... . ....... ... .......... ll 3 Regeneration of shoots and roots from suspension cells plated on 3 MS media .......... 13 CHAPTER II 1 Callus response to various concentrations of asparagus autotoxin-containing medium.......... 29 2 Cell colony growth under various concentrations of asparagus autotoxin-containing media........ 30 CHAPTER III 1 Diagram of experimental arrangement where cell colonies surviving an initial exposure to different concentrations of glyphosate were transferred to a divided petri dish containing 10’3M glyphosate medium....... ...... 4S 2 Callus response when plated as described in Figure 1.0......-OOOOOO......OOOOOOOOOOOOO ..... 45 3 Effect of glyphosate-containing medium on the germination and growth of barnyardgrass seedOOOOOOOOOOOOOO0............OOOOOOOOOOIOOOOO 48 4 Effect of various concentrations of glyphosate on callus growth............ ........ ........... 49 vi Readers: The journal format utilized in this thesis meets the requirements stipulated by the Horticulture Department and Michigan State University. Chapter I was prepared in the style of a Michigan State University Agriculture Experiment Station Research Report. Chapter II was prepared in the manuscript style of a journal, and Chapter III is presented in the form of a short communication. vii CHAPTER I: TISSUE AND CELL CULTURE TECHNIQUES OF ASPARAGUS OFFICINALIS L.. INTRODUCTION One of the benefits derived from basic research in the university is the gathering of information useful to other areas of academic research, industry, and to the private sector. Tissue culture techniques now supplement more traditional methods of plant breeding, often cutting time- consuming corners and solving problems unique to certain plant species. Asparagus is one agronomic crop which can benefit from utilization of in vitrg culture techniques. Asparagus is a long-term perennial crop lasting up to and beyond 30 years in the field. It is a dioecious plant, having separate male and female plants, and is therefore an obligately outcrossed species. This outcrossing behavior produces highly variable, heterozygous lines, and severe inbreeding depression results upon self— or sib-pollination to parental plants. Vegetative propagation, usually from crown division, is a slow process yielding few daughter plants of the same genotype per year. Anther culture and in vitrg multiplication of desirable parental genotypes are two culture techniques available for the production of hybrid asparagus plants suitable for use in Michigan. Meristem culture can be used to rid asparagus plants of internal viruses and lateral bud culture is routinely used at Michigan State University as a means of propagating desirable male and female parental plants. The lateral bud technique was defined by Dr. Hsu-Jen Yang at Washington State University and is a simple, effective means of propagation (5). During the course of research to select asparagus cell lines tolerant to an autotoxic root extract of the asparagus plant, certain cell culture techniques have been developed and refined. These include: 1) Asparagus callus culture, 2) Asparagus suspension culture, 3) Plating asparagus cells in a solidified nutrient medium, and 4) Regeneration of asparagus callus and suspension cells. It is our aim to present quick and easily applied techniques which differ somewhat from those previously reported in the literature. PROCEDURES Asparagus Callus Culture. Callus cultures were initiated from young field grown asparagus spears cv. Mary Washington. Spears were sliced 2 — 3 cm thick, surface sterilized for 10 min. in a 10% solution of commercial bleach (5.25% NaOCl), and rinsed 3 times in sterile water. Sterilized spear explants were sliced again, 2 - 3 mm thick, and placed on three different basal media: Murashige and Skoog (MS) (3), Gamborgs (B-5) (2), and Whites (4). Cultures were maintained at 25:20C under 15-20 pEmuzs"l and a 16 hr. photoperiod. Spear discs, prepared as above, were also placed on 12 various MS media (Table 1) for 12 weeks, after which callus was rated visually for growth and texture. Table I: Callus initiation media. Medium Basal .NAAl KIN2 6-BAP3 2.4-1)4 salts (mg/1) (mg/1) (mg/1) (mg/l) MS MS MS MS MS MS MS MS MS 1 0 MS 1 1 MS 12 MS \DCDQOWU'IDWNH Ol—‘O l—‘COOOOOOOOOO OH OOOl—‘Nl—‘Nl—‘l—‘Ol—‘O OOOOOOI—‘Nl—‘l—‘OO OOONl-‘l—‘OOOOOO NAA,4¢naphtha1eneacetic acid KIN, 6-furfury1aminopurine 6-BAP, 6-benzy1adenine purine 2,4-D, 2,4-dichlorophenoxyacetic acid bWNl—J Asparagus Suspension Culture. The three basal media tested for production of friable suspension cultures were the same as for callus cultures above: MS, B—5, and Whites. There were 4 additional MS media tested: 1) 10% v/v cocoanut milk (MS CM), 2) 0.1 mg/l NAA (*-naphtha1eneacetic acid) (MS NAA), 3) 0.1 mg/l KIN (6-furfury1aminopurine) (MS KIN), 4) 0.3 mg/l NAA, 0.1 mg/l KIN, and 0.1 mg/l 2,4-D, (2,4-dichlorophenoxyacetic acid) (12d). Asparagus callus pieces, 1 cm3, were placed into each 125 ml flask containing 40 m1 of sterile medium. The cultures were sealed and maintained at 27:10C in an incubator shaker (100 rpm) and supplied with diffuse light. Suspensions were subcultured every 3 weeks by filtering through a coarse sieve (35 pm), collecting the filtrate, and transferring it to 40 ml of fresh medium. During the third (3 week) passage, cell growth was measured as settled cell volume (Table II). The cultures were gently swirled to evenly distribute the cells and a one m1 aliquot was removed. Aliquots were placed into graduated test tubes and allowed to settle 5 min. before measuring cell volume. Table II: Average settled cell volume during passage 3. Medium Week 1 Week 2 Week 3 MS .026 .05 .054 B-5 .037 .063 .069 Whites .01 .02 .016 MS/CM .024 .059 .08 MS NAA .047 .084 .10* MS KIN .021 .06 .111* 12d .053 .12 .133* Confidence level 90% *Using a pair-wise comparison, media with * are not significantly different from each other. Plating Cells in Solidified Nutrient Medium. Actively growing asparagus suspension cultures were filtered through a coarse sieve (35 pm) and the filtrate centrifuged 5 min. at 100}cg. The pellets were resuspended in a small volume of 12d medium, where cell density was adjusted to 25x104 cells/m1. Average cell counts were determined with a haemocytometer. One ml of cell concentrate was placed in a petri dish and overlaid with 12d medium (autoclaved and held at 400C to prevent solidification). Cells were plated at final densities of 25x104, 15x104, 5x104 and 2x104 cells/m1. Cultures were gently swirled to evenly distribute the cells, allowed to solidify, and sealed. Cultures were maintained at 25:20C in the dark for 3 weeks, until examined. Regeneration of Callus and Suspension Cultures. Asparagus callus pieces, 1 cm3, maintained on callus proliferation medium 12d, were transferred to 8 various media for possible regeneration (Table III). The cultures were sealed, maintained at 25:20C under 15 - 20 }1Em"2’s-l and a 16 hr. photoperiod. and were assessed for root and shoot production after 3 (4 week) passages. Suspension cultures of asparagus, actively growing in suspension medium 12d, were plated in solidified 12d medium (see Plating above). Cell colonies which appeared 3 - 5 weeks after initial plating were subcultured to 3 MS nutrient media for possible regeneration: 1) MS + 0.3 mg/l NAA, 0.1 mg/l KIN, and 0.1 mg/l 2,4-d (12d), 2) MS + 0.1 mg/l NAA and 0.1 mg/l KIN (MS+NAA+KIN), 3) MS basal medium + no hormones (MS). Cultures were maintained under the conditions described above. .mmmum Camcom .mmHIHmauom .maeImeean ucmam .Hoflmmnm can nuBOHm GHQMM Mom Esflwma Ummfl>on m .NmmH .mooxm peep capo whom .mmm Heme .mxm . wonsuaso :oflmcmmmnm mo mucoEonfisqou ucmfluusz .mmma omwxoconmouoH£OH©Iv.~ mcflunmocflamaausmH5MIm caumomwcoamnusmmcl maamo noon cmoQMOm mo .mm mm .q.o .muon5mo .w.z on wine .mHHoU unmam paw Hmawc¢ mo coHum>HDH5U one .mmma .m.m .oufinz .monsuaso osmmflu ooomnou nuaz mammmmoflnm .m can .9 .mmfinmmusza AUOHNHV mEo\mM mv.a um .cflE om ©m>maoousm can .>.m mm on pmumsmcm muo3 waves Haa m.o m.o m.o m.o m.o m.o m.o m.o . . . . . . . .xwv name .HH o.m o.m o.m o.m o.m o.m o.m o.m . . . . . . Awe mmouosm .OH I 0.0 I I I I I I . . x>\>wv xHHe uncoooo .m H.c I I I I I I I . . . . . Aa\mev aIe.m .m H.o I H.o H.o I I I I . . . . . . AH\ma msz .a m.o I H.o I H.o I I I . . . . . . AH\mev eaaz .6 o.~ o.~ o.m o.~ o.~ I o.m o.m . . . . .Aaxmev mcflosao .m m.o m.o m.o m.o m.o o.H H.o m.o . Aaxmev HomImcfixocfluam .e m.o m.o m.o m.o m.o o.H m.o m.o . Aa\msv whom oflcfipooflz .m H.o H.o H.o H.o H.o o.oa H.o H.o . . . . Aa\mav maesmflne .m m2 m2 m2 m2 m2 mIm manage m2 . . . . . . muamm Hmmmm .H UNA so sz zHM aaz mesflcma masheme Hesflcme +mz +¢three nutrient media. No. No. Medium Shoots Roots MS+NAA+KIN 12 18 MS basal medium 7 38 12d 0 6 CONCLUSION The tissue and cell culture techniques presented provide quick and easy methods of manipulating asparagus in vitro. These techniques are research tools useful for the improvement of asparagus as a horticultural crop. REFERENCES 1. Bergmann, L. 1959. A new technique for isolating and cloning single cells of higher plants. Nature 184:648. 2. Gamborg, O.L. gt 2;. 1968. Nutrient requirements of suspension culture of soybean root cells. Exp. Cell. Res. 50:151-158. ‘.‘ M-k3?1p I 13 M51!) M50 l2d Regeneration of suspension cells Figure 3: Regeneration of shoots and roots from suspension cells plated on 3 MS media. l4 Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 15:473-479. White, P.R. 1963. The Cultivation of Animal and Plant Cells. Ronald Press, N.Y. Yang, H-J. 1977. Tissue culture technique developed for asparagus propagation. Hort. Sci. 12:140-141. CHAPTER II: IN VITRO SELECTION OF ASPARAGUS OFFICINALIS L. CELL LINES TOLERANT TO AN AUTOTOXIC ROOT EXTRACT. ABSTRACT Water extracts of coarsely ground Asparagus officinalis L. root tissue exhibit autotoxicity at the cellular level. Callus pieces (1 cm3) exposed to autotoxin concentration 1:2 (autotoxin:nutrient medium v/v) suffer severe necrosis and a visible reduction in growth rate. Asparagus cell lines tolerant to the autotoxic extract have been recovered from culture. Dialyzed water extracts of 10 gm coarsely ground asparagus roots were concentrated under pressure (500C) to final volume of 100 m1 and filter sterilized (.45 p). Asparagus suspension cells were plated in a dilution series of modified MS basal medium containing 0.3 mg/l NAA, 0.1 mg/l KIN, and 0.1 mg/l 2,4-D, and the asparagus autotoxin. Concentrations of 1:2, 1:10, 1:50, 1:250, 1:1250 and 0:1 (autotoxinzmedium v/v) were employed. Cultures were maintained at 25:20C in the dark for 3 - 5 weeks. Of the 303 cell colonies surviving the initial exposure to auto- toxin medium l:2, 40 cell colonies were subcultured to regeneration medium MS 1:1, a modified MS medium containing 0.1 mg/l NAA and 0.1 mg/l KIN. Regeneration of shoots occured on 2.5% of the callus colonies and roots on 55% of the colonies. The remaining 263 putative tolerant cell colonies were subcultured onto a non-selective medium for one passage before reintroduction to the autotoxin. The 16 l7 autotoxin-tolerant phenotype was expressed by 76.43% of the callus colonies reintroduced to the autotoxin. Mutation 5 5 5 frequencies of 5.71 x 10' , 4.04 x 10‘ , and 8.13 x 10' were calculated for the 3 selection experiments,respectively. INTRODUCTION Plant cell cultures are now recognized as an important and exciting tool in plant science research (11, 23, 60). Cell culture techniques, including in yitrg mutant selection, allow for rapid screening of a large cell population in a relatively small space and thus may supplement traditional methods of plant breeding. Carlson and Polacco (11) point out the importance of in vitrg mutant selection as a means of extending genetic variability. Since the late 1960's, the use of cell culture has led to the recovery of a variety of mutants. Some of these mutants include tolerant cell lines in yitrg, whereas others maintain their mutant phenotypes at the regenerated plant level. In particular, a great deal of work has been carried out in the field of herbicide resistance (38). Cell tolerances to the herbicides asulam (20),atrazine (72), amitrole (3, 51), and 2,4-D (38, 42, 72), have been isolated in culture; although none of the above cell lines were regenerated to plants and retested for tolerance in give. However, some cell lines tolerant to various herbicides have been regen— erated to whole plants and inheritance of the resistant trait determined (2, 13, 38, 42). Genetic inheritance was also demonstrated in tobacco plants regenerated from cell lines tolerant to the fungicide Carboxin (45). 18 In vitro selection of pathogen toxin-tolerant cell lines was begun by Carlson (10), who recovered tobacco mutants resistant to methionine sulfoximide - an analog of Pseudomonas tabaci toxin. Resistant mutants have also been recovered using Helminthosporium toxins (19, 22, 26), Rhizoctonia solani toxin (60), and streptomycin (34). Selection using amino acids or their analogs, such as valine (9), 5-methy1 tryptophan (66), and para-fluorophenylalanine (7), has produced biochemical mutants useful for the study of amino acid biosynthesis. Mutants for improved agronomic characteristics such as increased tolerance to environmental stress and useful morphological or physiological attributes have been found by screening populations of cells in yitgg. Tolerance to increased levels of salt (40, 60, 72), aluminum (37), copper and zinc (70), as well as chilling (18) and flooding tol- erance (60) have opened new possibilities in cultivar development. Also recovered from cultured plant cells were variants for yield capacity (32), morphological features (27), CO2 absorption and chlorophyll content (60). Suggestions have been made for the use of in vitrg mutant selection involving increased accumulation of cyclic hydroxamates in cereals (a pest deterrent), decreased cyanide content in forage grasses, and glucosinolates in Brassica (60). Mutant selection in Kit£g_may offer a unique solution to a horticultural production problem of Asparagus officinalis L.. Asparagus yields per acre have been declining in recent 19 years despite increased production acreage planted (30). In addition to factors such as increasing field age, soil compaction, increasing populations of fusarium, rust and several species of cut worms, recent reports indicate that compounds produced in the root tissues of asparagus may inhibit asparagus plant growth (29, 31, 49, 53); a specialized allelopathic condition referred to as auto- toxicity. Allelopathy refers to the effect, detrimental or bene- ficial, of a donor plant on a receptor plant and is separated from other plant interference in that its effect is exerted through the release of a chemical(s) (47). Autotoxicity is allelopathic self-inhibition. Allelopathic effects, including autotoxicity, are often extended over time, are direct or indirect, and are influenced strongly by environ- mental factors (47). Toxicity of the phytotoxin is dependent on relative rates of accumulation verses the rate of in- activation or decomposition in the environment (50). In this way, the toxicity may depend more on concentration (a factor influenced by stand density) than on specific chemistry. Allelopathic effects in agriculture were first recorded by Democritus in 500 B.C. and then by Theophrastus in 300 B.C. Later, de Candolle (1832) stated that "soil sickness" was the result of root exudates and suggested crop rotation as a means of combating the problem (47). Since that time, plant to plant interractions, detrimental or beneficial, have influenced many agricultural practices such as cover cropping, fertilizer applications, and stubble mulching (61). 20 Autotoxicity is an apparent evolutionary paradox; however, the advantages of self-inhibition must outweigh the disadvantages (64). Some believe that autotoxicity has an evolutionary significance in plant succession (1, 16, 28, 36, 67), although succession cannot explain every instance of autotoxic behavior. Many allelopathic compounds may be evolutionary "relics" or only utilized under specific circumstances. In other cases, autotoxicity may be a competitive edge, keeping plants of the same environmental niche spaced equidistant for best utilization of resources (5, 21, 25, 61, 63). Autotoxic behavior can be seen in seed germination inhibitors that are present in seeds and ovaries (33); they prevent premature germination and enhance survival rates. Regardless of biological function, autotoxic compounds may be produced under adverse environmental conditions such as: water-logging (44), monoculture (5, 21, 63) and increased pathogen population (46). In the intensive culture of perennial crops, autotoxin production may greatly reduce yield (47). Autotoxic behavior has been reported in a variety of plants including successional plant species (1, 5, 16, 28, 36, 67), tropical trees (21, 63), kalanchoe (25), fruit trees (46, 47), and a variety of agronomic crops (14, 15, 58, 59, 62, 68, 69). There are over 10,000 known low molecular weight compounds produced in higher plants and fungi. The majority of these compounds function as protective or defensive mechanisms against predators, competitors and pathogens (57). The major groups of allelopathic toxins include phenolic acids, 21 coumarins, flavenoids, glucosinolates, aromatic compounds, terpenoids, steroids, alkaloids, and organic cyanides. In many instances the chemical compound' is compartmentalized or conjugated in a form that protects the plant from its effects (64). They may be crystalline, polmers, or glycosides (in which case the toxic compound is combined with sugar to render it harmless). The autotoxin(s)* produced by the tissues of asparagus may be composed of a number of phytotoxic compounds. Walnut trees (Juglans spp.) produce allelopathic effects from one compound, juglone, washed from the walnut leaves onto the soil (64). In contrast, nine inhibitory substances have been isolated and identified in red clover (Trifolium pratense) (59). Currently, only one inhibitory compound has been isolated from asparagus tissue. Kitahara et a1. (29) identified asparagusic acid, which is formed in etiolated young asparagus shoots. This compound is reported to have an effect resembling abscisic acid on germinating seedlings of lettuce, rape, rice, radish, carrot and barn— yardgrass. Work by Shafer and Garrison (53) suggested that an autotoxin present in ground root tissue of asparagus had inhibitory effects on the germination of asparagus seed. Water extracts of the root tissue did not, however, influence the germination of asparagus (54). Strong evidence of allelopathic and autotoxic effects observed in asparagus * Hereafter referred to as an autotoxin. 22 comes from Putnam et a1, (49). Potted one—year old asparagus crowns were planted in soil amended with ground asparagus root tissue. Maximum shoot and root growth inhibition were 66% and 51%,respective1y. Little is known about the genetic regulation of allelopathic compounds. Genetic differences in the amount of phytotoxin produced can be seen in various plants (43, 47, 50). Putnam and Duke (48) screened 538 cultivated and wild Cucumis accessions for allelopathic activity against 2 weed species. One accession inhibited a weed species 87%, and 25 accessions inhibited a weed species 50% or more. Some studies on the genetic transfer of allelopathic toxins in wheat grass hybrids can be found in Russian literature (50). The suggestion has been made to use Patulin, a toxin produced by microorganisms attaching wheat and rye residues, as a screening chemical on parents and F4 lines of those species (35). Concentrations of toxins can fluctuate quickly, significant changes in concentration occuring in as little as one hour. This rapid flucuation suggests that the turnover and accumulation of toxins may be under genetic regulation (52, 57). The use of asparagus culture techniques as described in Chapter I, and the production of a chemical growth inhibitor which can be extracted from the root tissue of asparagus, led to the selection of autotoxin-tolerant cell lines recovered from culture. To the best of our knowledge, this is the first example of cell lines selected in yitrg which are tolerant to an autotoxic extract of plant tissues. 23 MATER IALS AND METHODS Asparagus Callus Culture. Callus cultures were initiated by removing spears, 2 - 3 cm in diameter, from field grown Asparagus officinalis L. cv. Mary Washington. The spears were sliced 1 cm thick and surface sterilized in a 10% solution of commercial bleach (5.25% NaOCl) for 10 min. and rinsed three times in sterile water. The explants were sliced again (2 — 3 mm thick) and these discs were placed on modified Murashige and Skoog medium (39) containing: -‘-naphthaleneacetic acid (NAA) 0.3 mg/l, 6—furfurylamino- purine (KIN) 0.1 mg/l, 2,4-dichlorophenoxyacetic acid (2,4-D) 0.1 mg/l, and agar 0.8%. This medium, hereafter referred to as 12d, was adjusted to pH 5.7 with 0.1 M HCl and autoclaved at a pressure of 1.46 kg/cm2 (121°C) for 20 min. Cultures were maintained at 25:20C under 15-20 uEm-zs-l cool white fluorescent light on a 16 hr.photoperiod and were subcultured every 4 weeks. Suspension Culture. Suspension cultures were initiated by 3 placing 1cm friable callus tissue into 40 m1 of liquid 12d medium in each 125 m1 flask. Cultures were maintained in an incubator shaker (100 rpm) at 25:20C and were provided diffuse light. Suspensions were subcultured at 21 day intervals by filtering through a coarse sieve (35 um). Single and small aggregate cells in the filtrate were centrifuged at 100 x g for 5 min., and the pellet resuspended in 40 ml of fresh medium. 24 Autotoxin Preparation. Asparagus root material was collected, prepared,and supplied by Dr. A.R. Putnam, Department of Horticulture, Michigan State University. Ten grams of dried, coarsely ground asparagus roots were added to 100 m1 of double distilled water and dialyzed (Mol. wt. cutoff 3500) against 2300 m1 of double distilled water for 24 hours at 1 - 2°C. The dialyzed fraction was reduced under pressure (50°C) to 100 m1 final volume. The pH and conductivity of the autotoxic extract was recorded (Table I), followed by filter sterilization (.45p) and storage at 4°C. Table I. Conductivity and pH of the asparagus autotoxin extract. Extract Conductivity (umhos) pH 1 2400 5.08 2 1620 5.70 3 1800 5.77 Cell Plating and Selection. Suspension cells were plated as described by Bergmann (6) in 12d medium to which sterile autotoxin had been serially added. The concentrations (autotoxin:medium v/v) tested were 1:2, 1:10, 1:50, 1:250, 1:1250 and control 0:1. All media plus autotoxin were held in the liquid state at 40°C until plating. Suspension cultures were filtered through a coarse sieve (35 pm), the filtrate collected, and centrifuged lOOxg for 5 min. The pellet was resuspended and the cell density 25 coumam mHHoU manmfi> .oz Hopoe n hocosvoum moflcoHoo ucmnoaoe .oz Hmuoe coflumusz "mm coumasoamo humongoum coflumusz« mloa x ma.w NNH moa x m.H h.aw eoa x NH om m mloa x vo.v Hoa woa x m.N m.Hm goa x vm om m mica x ah.m om moa x ¢.H m.mo voa x HH om H ewocosvoum moficoHoo maaou mHQMH> wuflaflbma> oumHm\mHHmU moumam cofipomaom cofiumusz ucmnoaoe .oz Hmuoe Hamu w .02 .02 .oz Hmuoa .mocmsvmnw cOADMDDE paw .MDHHHDMH> HHmo .huflmcmp Hamo mo GOHDmEHumm .HH mHDMB 26 ov mmm mom Hmuoe .eIH 2: m3 m 3 mm 2: m 3 me om H Acofiumumcwmonv AmSHHmov pma mmflcoaou unnumaoe cofluomaom Huamz .OZ .Huamz Ezflpme Goflumnocwmmu ou Umuuowmcmnu Hones: Ugo .pmH EdHUoE mocmcoucwme msaamo ou pouummmcmuu Hones: .mmflcoHoo Hamo ucmuoaou UmEsmmHm mo Hones: Houoe .HHH wanes 27 adjusted by dilution with fresh 12d medium (Table II). Cell count averages were obtained using a haemocytometer. The percent viable cells was determined using the fluorescein diacetate method (65) (Table II). A one ml aliquot of cell concentrate was placed in an empty petri dish and was overlaid with approximately 20 m1 of the autotoxin-containing media. The dishes were swirled gently, allowed to solidify and were sealed. Cultures were maintained in the dark at 25:20C for 3-5 weeks, when macroscopic colonies (no less than 2mm in diameter) were counted. Callus colonies, presumed tolerant to selection medium 1:2, were transferred either to solidified 12d medium for subsequent reintroduction to the autotoxin, or placed on modified Murashige and Skoog medium (39) containing NAA 0.1 mg/l, KIN 0.1 mg/l, 0.8% agar, for regeneration (medium hereafter designated MS1:1) (Table III). Callus Response to Autotoxin. Asparagus callus pieces, lcm3, were placed on each petri dish containing solidified 12d medium plus autotoxin concentrations 1:2, 1:10, 1:50 and 0:1 (autotoxin:medium v/v). Cultures were sealed and maintained for one (4 week) passage at 25:20C under 15-20 ;.1Em"zs_'l cool white fluorescent light and a 16 hr. photoperiod. 28 RESULTS AND D ISCUSS ION The asparagus autotoxic root extract exhibits its effects at the cellular level. Callus, exposed to auto- toxin—containing media concentrations 1:2 and 1:10 (autotoxin:media v/v), displays severe necrosis and a reduction in growth rate (Figure 1). These results may indicate that the asparagus autotoxin is not tissue specific, acting only upon intact, morphologically differentiated tissue such as roots or root hairs. The autotoxin effect may involve a biochemical or physiological function of the cell, such as amino acid biosynthesis, respiration, or membrance permeability. Macroscopic callus colonies, tolerant to the asparagus autotoxin (concentration 1:2), appeared 3 - 5 weeks following initial cell plating (Figure 2). Three selection experiments yielded 303 putative mutant cell lines. According to Meredith and Carlson (38), the first test of presumed cell tolerance recovered from culture, is reintroduction of the toxin after one or more passages on non-selective medium. If the colonies do not survive, the presumed tolerance may be a physiological or biochemical adaptation to the toxin and no genetic change has occured. Enzymes which metabolize or detoxify the toxin may be activated in culture, or toxin-sensitive cells may survive the initial selection and die upon reintroduction to the compound. Tolerance that is maintained after one or more passages on a non-selective medium may be an indication of genetic change but is still not sufficient evidence to conclude that mutation has occurred. 29 autotoxin 1 :50 1:10 Figure 1: Callus response to various concentrations of asparagus autotoxin-containing media. 30 Figure 2: Cell colony growth under various concentrations of asparagus autotoxin-containing media. 31 Of the 303 surviving cell colonies, 263 were placed on a non-selective medium (12d) for one (4 week) passage (Table III). Following the passage on non-selective medium 12d, the callus colonies were reintroduced to the autotoxin (concentration 1:2). Of the 263 callus colonies reintroduced to the autotoxin, 201 (76.43%) maintained the autotoxin- tolerant phenotype (Table IV). Table IV. Reintroduction of presumed tolerant colonies to autotoxin. No. Colonies Reintroduced No. Colonies Selection To Toxin Retaining Tolerance 1 69 63 2 88 77 3 10_6 2; Total 263 201 = 76.43% In vitrg culture techniques induce genetic variability (4, 17, 55, 56), as well as expose the vast inherent variation already present within a cell population. Although the frequency of spontaneous mutation in zitrg is difficult to assess (13), an estimate was calculated for each of the autotoxin selections (Table II). Assuming that each presumed tolerant cell lines resulted from an independent mutation and that the autotoxic extract is not mutagenic, a spontaneous mutation frequency of 5.71 x 10-5, 4.04 x 10-5, 32 and 8.13 x 10-5 was calculated for the three selection experiments, respectively. Mutation frequencies were calculated using estimated population densities. Suspension cultures are usually a mixture of single cells and cell aggregates. The effective removal of aggregates through filtration is hampered by the disparity of cell size and at best yields a mixture of large single cells and equally sized small-celled aggregates. In estimating mutation frequency, it is useful to regard each discrete member of the cell population as a "unit" and assume it is of common cell origin (8). The number of viable cells in a population is considered when determining cell densities. The asparagus cell concentrate used for the autotoxin selection averaged 58.9% viable cells at plating (Table II). And lastly, another problem of estimating mutation frequencies, is that without regeneration of the cell colony and subsequent genetic analysis of the selected trait, the number of tolerant cell lines which are true mutants cannot be known. Regeneration of asparagus plantlets has been reported from callus and suspension cultures (71 and Chapter I). From the original 303 presumed tolerant cell colonies selected from the autotoxin-containing medium, 40 randomly chosen callus were subcultured onto regeneration medium MSl:1. Thus far, one callus (2.5%) has produced a shoot and 22 callus (55%) have rooted. 33 For ultimate utilization, tolerant cell lines which are selected in vitrg must be capable of regeneration to an intact plant. The selected mutation must be expressed in the proper tissue and, for use in a sexual breeding program, be heritable. A high frequency of heritable mutations has been reported in plants regenerated from tissue culture (41). The tolerant trait may be expressed in the regenerated plant or only in tissue cultures derived from that plant. This is because whole plants and in vitrg cell lines are differentially unique and have different gene expression. Although this is still not sufficient evidence to claim mutational change, tolerance expressed in regenerated plants can be effectively used in vegetatively propagated species. It is safe to say that nuclear genetic change has occurred only when the tolerance is maintained in the regenerated plant and is transmitted sexually to the progeny of that plant (38). The tolerance trait may then be used in a sexual breeding program. The basis of current plant breeding is to select recombinants out of a large population of individuals from a broad genetic base (12). On the whole plant level, only major alterations are seen, and these are the end product of a number of biochemical, physiological and develop- mental processes. Selection at the cellular level unmasks these processes. Resistance or susceptibility may depend on cellular permeation, membrance permeability, permeability into a specific organelle, as well as the cell's ability to detoxify the compound enzymatically. These types of 34 resistance or susceptibility can only be utilized if they are expressed in the regenerated plant. Problems arise, using in vitrg selection, when resistance is based solely on morphological characteristics such as cuticle thickness and whole plant processes such as photosynthesis, due to the lack of selection markers at the cellular level. Whereas, in vitrg mutant selection allows for rapid screening of virtually millions of individual genomes in a relatively small space and time, field selection using a chemical compound is often inefficient. Chemical selection pressures in the field can rarely be made to exceed LD90 - LD95 and survivors are usually "escapees" rather than tolerant individuals. Problems of uniformity in application and obtaining the volume of chemical necessary are often limiting factors (24). The genetic inheritance of the tolerant trait must be established before it can be used in a sexual breeding project. Reduction of the autotoxic potential in asparagus may lead to improved yield, longer productive field age, and increased planting densities. Research must also continue in isolating and identifying the chemical compounds responsible for autotoxicity in asparagus, elucidating the mechanisms of resistance in 31339, and examining various cell lines for genetic differences in resistance to the autotoxin. 10. ll. 12. LITERATURE CITED Abdul-Wahab, A. and E.L. Rice. 1967. Plant inhibition by johnson grass and its possible significance in old field succession. Bull. Torrey Bot. Club. 94:486—497. Aviv, D. and E. Galun. 1977. Isolation of tobacco protoplasts in the presence of Isopropyl N-Phenylcar- bamate (IPC) and their culture and regeneration into plants. Z. Pflanzenphysiol. 83:267-273. Barg, R. and N. Umiel. 1977. Development of tobacco seedlings and callus cultures in the presence of Amitrole. Z. Pflanzenphysiol. 83:437-447. Bayliss, M.W. 1973. 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INTRODUCTION Glyphosate (N-phosphonomethylg1ycine) is a broad spectrum herbicide which has a high unit activity, destroys both annual and perennial weed species, and is inactivated by components in the soil (14). Glyphosate is sometimes used prior to asparagus spear emergence and can be used after the harvest season if care is taken not to expose the asparagus plant to the chemical. The effects of glyphosate on the plant begin with a rapid cessation of growth followed by chlorosis, necrosis, proliferation of meristematic growth, shortened internodes, wilt, and death. No known resistance to the herbicide has been reported in the literature although unpublished work may have identified some resistance in alfalfa (9). The use of cell culture as a means of screening for herbicide resistance eliminates the problems associated with cut surface absorption, waxy cuticle layers, the translocation of chemicals through tissues, and uniform application and concentration (7). Herbicide selection using cell lines has led to the recovery of various levels of tolerance or resist- ance. These range from a temporary adaptation to the presence of the chemical to the regeneration of plants capable of genetic transmission of the tolerant trait (16). Tolerance to the herbicides asulam (6), atrazine (18), amitrole (2, 13), and 2,4-D (10, 12, 18), have been isolated in cell culture. 42 43 Cell lines tolerant to the herbicideszlPC, picloram, 2,4-D, 2,4,5-T, and 2,4-DB, have been regenerated to the whole plant and inheritance of the tolerant trait determined (1, 4, 10, 12). The mode of action of the herbicide glyphosate is not known. The theories of glyphosate action include an increase in phenylalanine activity accompanied by an increase in inhibitory phenolics (5), and inhibition of the aromatic amino acid biosynthesis affecting protein synthesis (8). Haderlie 33 31° (7), notes chloroplast ultrastructure is affected and that glyphosate induces chromosomal abberations, while Brecke (3) states that membrane properties involving ion uptake are altered. Using suspension cells of yInIn £323 to study the mechanisms of glyphosate action, Brecke compared the uptake of labeled 10-4M glyphosate and labeled leucine over a 24 hour period. There were only 269 cpm/lO5 cells of glyphosate absorbed into the suspension cells in 24 hours as compared to 14,738 cpm/lO5 cells of leucine absorbed. The conclusion was made that glyphosate uptake by mesophyll cells was minimal. MATERIALS AND METHODS Glyphosate Preparation. Technical grade glyphosate was supplied by Dr. A.R. Putnam, Department of Horticulture, Michigan State University. Aqueous solutions of the herbicide were prepared by dissolving glyphosate in double distilled water, adjusting the pH to 5.7 with 1N HCl, and filter steri- lization (.451fl . Studies on the pH adjustment of glyphosate show no appreciable change in herbicidal activity between pH4 and pH? (3). 44 Asparagus Culture and Plating. Asparagus suspension cells were established by placing 1cm3 callus pieces into modified MS medium (11) containing: 0.3 mg/l NAA (.L-naphthaleneacetic acid), 0.1 mg/l KIN (6-furfury1aminopurine), and 0.1 mg/l 2,4-D (2,4-dichlorophenoxyacetic acid)(hereafter referred to as 12d). Cultures were maintained at 27:10C in an incubator shaker (100 rpm) and provided with diffuse light. The suspension cells were prepared for plating by filtering cultures through a coarse sieve (35}mn , centrifuging the filtrate at 100xg for 5 min. and resuspending the pellet in a small volume of medium 12d. Plating density was 29 x 104 cells/m1 as determined using a haemocytometer, while 50.25% of the cells were viable as determined by the fluore- scien diacetate method (17). Cells were plated in 12d medium containing the following concentrations of glyphosate 3 4 5 (10’ M, 10' M, 10’ M, 10'6M, and 12d + no glyphosate). All media were autoclaved and held at 40°C until plating to prevent solidification. Cultures were swirled gently to evenly distribute the cells, allowed to solidify, and sealed. Cultures were maintained in the dark at 25:20C for 4 weeks until observation. 3 Reintroduction of Callus to 10- M Glyphosate. Callus colonies 3M, lo'Gn and 12a + no surviving initial exposures to 10 glyphosate were subcultured to subdivided petri dishes con- 3 taining 12d medium + 10' M glyphosate (See Figures land 2). Cultures were maintained at 25:20C in the dark for 6 - 8 weeks. 45 Callus Callus transferred transferred ‘ f 12d d' from 10 M rom me 1um glyphosate (no glyphosate) 10'3M glyphosate medium Callus transferred from 10'6M glyphosate Figure 1: Diagram of experimental arrangement where cell colonies surviving an initial exposure to different concentrations of glyphosate were transferred to a divided petri dish containing 10'3M glyphosate medium. 1mm 12¢ (MW) rm w'3u Wu .mw‘unfi-‘ul Figure 2: Callus response when plated as described in Figure l. 46 Seed Assay. Seeds of barnyardgrass (Echinochloa crusgali) were surface sterilized in a 10% solution of commercial bleach (5.25% NaOCl) for 10 min. and rinsed 3 times in sterile water. Five hundred seeds were placed on 2 media, 1) 12d + no glyphosate, 2) 12d + 10-3M glyphosate. Cultures were sealed and maintained at 25:20C under 15 - 20}umn-zs—l cool white fluorescent light and a 16 hr. photoperiod for 5 days. Callus Response to Glyphosate. Callus pieces, 1 cm3, were placed on 12d medium containing the following concentrations 3 4 5 6 of glyphosate: 10‘ n, 10' M, 10' M, 10' M and 12d + no glyphosate. Cultures were maintained at 25:20C under 15 - 20 pEm-zs-1 and a 16 hr. photoperiod for 4 - 6 weeks. RESULTS AND D I SCUSS ION Four weeks after the initial plating on glyphosate- containing medium, the cell colonies had grown to visible size and could be observed. Cell colonies on the 10-3M glyphosate had ceased to grow at some point and remained 4 5 6M and the smaller than cell colonies on 10- M, 10- M, 10- control. Observations made 4 weeks later revealed that the cell colonies on 10-3M glyphosate had resumed growth and were eventually indistinguishable from the other glyphosate concentrations. Questions arose as to whether the glyphosate remained active in culture when combined with the nutrient medium, and the barnyardgrass seed assay was devised. The seed assay revealed that the glyphosate remained active in culture 47 (Figure 3). Seed germination on both media was between 93% - 95% but seedling growth on the glyphosate medium was severly inhibited. Growth of callus exposed to the dilution series of 12d medium + glyphosate, was inhibited at concentrations 10-3M and 10'4 M glyphosate (Figure 4). No apparent reduction in callus growth could be seen on lower concentrations of glyphosate as compared to the control. Callus colonies, removed from the plating experiment 3 above and placed on 12d medium + 10' M glyphosate in a subdivided petri dish produced an interesting response. Cell colonies, having survived initial exposure to 10-3M glyphosate increased in size when subcultured to 10-3M glyphosate, whereas colonies transferred from 10-6M glyphosate and no glyphosate, did not (Figure 2). The increase in growth may be attributed to an increase in glyphosate tolerance. The increased tolerance to glyphosate, however, may be a tissue culture artifact, a biochemical or physiological adaptation to the presence of glyphosate In yInng, and may be lost once the selection pressure is removed (16). Absolute levels of resistance in organized tissue and unorganized cell cultures differ considerably, and the presumed tolerance may have no expression in regen- erated tissue. Further research must be done to determine if the presumed tolerance is an artifact only relevant to the In vitro situation. Thomas 2E nI. (16) suggest that if the suspected tolerant cell line has a higher yield (i.e. growth 48 ‘9 9 gr” 12d 12do1o'3M Glyphosate Figure 3: Effect of glyphosate—containing medium on the germination and growth of barn- yardgrass seed. 49 Glyphosate ' ' C‘r ' r 1' .. Figure 4: Effect of various concentrations of glyphosate on callus growth. 50 rate) than the original cell line, in the absence of the selection agent, the tolerance is actually an adaptation to the culture environment or presence of the selection pressure. Such is the case with several 5-methy1tryptophan resistant cell lines isolated from culture. They were shown to be auxin autotrophs, hormonally independent lines, which appeared more resistant to the amino acid analog than the parent culture cells (15, 16). In addition to the possible benefits of a glyphosate resistant plant in a breeding program, glyphosate resistance isolated In XEEEQ could be an important research tool in the study of herbicide metabolism, mode of action, and in the mechanisms of resistance and susceptibility in plants. 10. 11. L ITERATURE C I TED Aviv, D. and E. Galun. 1977. Isolation of tobacco protoplasts in the presence of Isopropyl-N-phenylcar- bamate and their culture and regeneration into plants. Z. 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