CELL LEVEL SELECTION FOR SALT TOLERANCE IN SOME TURFGRASS SPECIES AND CONFIRMATION OF WHOLE PLANT SALT TOLERANCE CHARACTERISTICS YU-JEN KUO

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2 CELL LEVEL SELECTION FOR SALT TOLERANCE IN SOME TURFGRASS SPECIES AND CONFIRMATION OF WHOLE PLANT SALT TOLERANCE CHARACTERISTICS BY YU-JEN KUO B.S., National Chung-Hsing University, 1987 THESIS Submitted in partial fulfillment of the requirements for the degree of Master of Science in Horticulture in the Graduate College of the University of Illinois at Urbana-Champaign, 1992 Urbana, Illinois

3 UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN THE GRADUATE COLLEGE AUGUST 10, 1992 WE HEREBY RECOMMEND THAT THE THESIS BY YU-JEN KUO ENTITLED CELL LEVEL SELECTHN FOR SALT TOLERANCE IN SOME TURFGRASS SPECIES AND CONFIRMATION OF WHOLE PLANT SALT TOLERANCE CHARACTERISTICS BE ACCEPTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Director of Thesis Research Head of Department Chairperson t Required for doctor's degree but not for master's

4 UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN GRADUATE COllEGE DEPARTMENTAL FORMAT APPROVAL THIS IS TO CERTIFY THAT THE FORMAT AND QUALITY OF PRESENTATION OF THE THESIS SUBMITTED BY Y_U_-_JE_N_K_U_O AS ONE OF THE REQUIREMENTS FOR me DEGREE OF MASTER OF SCIENCE ARE ACCEPI'ABLE TO THE DEPARTMENT OF HORTICULTURE Fall NturW of DepartIM1Il. DiYi&itm or Unil $'.:.. {7 --t12.. Dille of ApprOVGl

5 Dedication This thesis is dedicated to my dear wife, Li-Chu, for sharing ideas with me and giving me encouragement during my research. Not withstanding, this thesis is also dedicated to my families for their constant support throughout my life, and to my dear father and oldest sister who had passed away before I began my research career. ill

6 Acknowledgements I would like to express my sincerest appreciation and gratitude to Dr. L. A. Spomer and Dr. M. A. L. Smith, my advisors, for guidance and fmancial support in completing this research, which made my program possible. Special thanks is extended to Dr. D. B. Dickinson for serving on my graduate committee. Thanks goes to Dr. A. A. EIObeidy and Mr. Stanley Chen for their technical support of this research. I also wish to acknowledge my sisters for accepting responsibility for our mother in my absence. iv

7 Table of Contents Chapter Page I. Introduction 1 The research objectives of this thesis 4 Chapters included in this thesis 6 References 8 II. ID. IV. The Effects of Explant Type and Culture Conditions on Callus Initiation and Plant Regeneration from Turfgrass 11 Introduction 11 Materials and Methods 12 Results and Discussion 15 References 18 Plant Regeneration of St. Augustinegrass 'Texas Common' from Immature Embryo-derived Callus 38 Abstract 38 Introduction ' 39 Materials and Methods 40 Results and Discussion 42 References 44 Selection of Salt Tolerant Cell Lines of Zoysiagrass and Follow-up Evaluation of Whole Plant Response Under Salt Stress 53 Introduction 53 Materials and Methods 54 Results and Discussion 56 References 58 V. Callus Level and Whole Plant Microculture Selection for More Salt Tolerant Seaside Creeping Bentgrass 68 Abstract 68 Introduction 69 Materials and Methods 70 Results and Discussion 72 References 75 VI. Conclusion References 87 v

8 Chapter n List of Tables Page Table 1. Callus induction from nodal segments of St. Augustinegrass 'Texas Common', 'Floratam', and 'Seville', and bermudagrass 'Tifway II' under a range of 2,4-D concentrations after 4 wk incubation in the dark 23 Table 2. Callus induction from seeds of 'Seaside' creeping bentgrass, common bennudagrass, and zoysiagrass under a range of 2,4-D concentrations after 4 wk incubation in the dark 24 Table 3. Callus induction from immature inflorescences of 'Texas Common' St. Augustinegrass by treatment with different hormones after 4 wk incubation in the dark Chapter V Table 4. Mean root number, root area and shoot area of whole plant microculture plants of creeping bentgrass 'Seaside' derived from seedlings (seed), or plants regenerated from callus on 0, 0.5%, 1.0%, or 1.5% N~S04 stress media. Plants were grown at various concentrations of N~S04 after 4 wk in whole plant microculture screen system 78 vi

9 List of Figures Chapter II Page Fig. 1. Regenerated root (RT) of 'Texas Common' St. Augustinegrass compact callus derived from nodes on plant regeneration medium after culturing as callus for 1 year 26 Fig. 2. Callus induction from segments of unemerged inflorescences of St. Augustinegrass 'Texas Common' and bermudagrass 'Tifway' II at different stages of maturity on 1 mg L- 1 2,4-D MS medium incubated in the dark after 4 wk 28 Fig. 3. Regenerated root (RT) of Sl Augustinegrass 'Texas Common' compact callus derived from immature inflorescences on plant regeneration medium 30 Fig. 4. Callus (CA) was initiated from the axis of embryos of 'Seaside' creeping bentgrass seeds (SE) after 1 wk on' initial medium 32 Fig. 5. Regenerated root (RT) and coleoptile (CL) produced by somatic embryogenesis of zoysiagrass seeds 34 Fig. 6. The percentage of callus induced on agar solidified and filter paper bridge cultures of creeping bentgrass ' Seaside' seeds and St. Augustinegrass 'Texas Common' immature inflorescences after 4 wk incubation in the dark 36 Chapter III Fig. 7. White, friable callus on the surface of yellow, opaque callus. The callus was generated from immature embryos of Sl Augustinegrass 'Texas Common' after transfer to medium with 0.5 mg L- 1 2,4-0 and 0.25 mg L- 1 kinetin for 4 wk 47 Fig. 8. Scanning electron micrograph of somatic embryo from callus induced on Sl Augustinegrass 'Texas Common' immature embryos after transfer to hormone-free medium for 1 wk 49 Fig. 9. Regenerated root and shoot systems on St. Augustinegrass 'Texas Common' plantlet from callus derived from immature embryos, after 3 wk on hormone-free medium 51 vii

10 Chapter IV Fig. 10. The structure of callus derived from seeds of zoysiagrass. (a) yellow and watery callus after 4 wk on initial medium. (b) more compact, non-friable structure after 2 passages of subculture 60 Fig. 11. Somatic embryogenesis of callus derived from seeds of zoysiagrass on 1% N~S04 stress medium for 4 wk 62 Fig. 12. Regenerative capacity of zoysiagrass callus cultured on 1% N~S04 stress medium after 4 wk. (a) living plantlets regenerated on salt stress medium 4 wk. (b) regenerated plantlets dead on salt stress medium after 4 wk 64 Fig. 13. The microcutting-generated zoysiagrass whole plants selected from callus on N~S04 stress medium, then screened in a whole plant microculture system for 4 wk. (a) 0.5% plants from callus selected grown on 0 (left), 0.5% (middle), and 1.0% (right) N~S04 WPMC stress medium. (b) 1.5% plants from callus selected grown on 0 (left) and 1.5% (right) NazS04 WPMC stress medium 66 Chapter V Fig. 14. Callus (CA) was initiated from the axis of embryos of creeping bentgrass 'Seaside' seed incubation in darkness for 1 wk 79 Fig. 15. Regenerated organs from callus derived from seeds of creeping bentgrass 'Seaside' after 8 wk in subculture medium. (a) regenerated leaf (b) regenerated root 81 Fig. 16. The growth of roots and shoots of plants grown in the whole plant microculture system was viewed through gelrite solidified medium 83 viii

11 CHAPTER I Introduction Turfgrasses are used throughout the world for soil stabilization, absorbing many toxic emissions, reducing dust, enhancing the beauty of a landscape, etc. The need for plants with increased salt tolerance is important since salt accumulation from irrigation water is becoming more serious. At least 25% of currently cultivated land suffers from salt toxicity; also many millions of hectares are not cultivated owing to environmental stresses (Nabors, 1990). Excess soil salinity is a major factor limiting turfgrass growth; therefore, selection of suitable salttolerant (ST) species for available sites can prevent cultivation problems. Selection of ST species using traditional breeding methods is limited since the mutation responsible for conferring ST traits may not be a single gene, so it is difficult to observe the desirable characteristics in the field. It is also difficult to unifonnly apply stress selection pressure in a field population (Nabors, 1990). Tissue culture of turfgrass is a recently expanding research area that may be used for improving the genetic desirability of turfgrass (Krans, 1985; Torello, 1984). One possible use of tissue culture in plant improvement is for the production of ST plants through selection of ST cell lines or callus, as long as ST traits are still expressed in the regenerated plants (McCoy et al., 1987; Smith et al., 1981; Smith et ai., 1989; Torello et al., 1984). The utilities and advantages of in vitro selection have been reported in several other crops. Shepard et ai. (1980) used single leaf cell protoplasts of potato to select a high frequency of variation for several 1

12 horticultural and disease resistance characteristics. However, good correlations between cell level and field stress tolerance are infrequently found. McCoy et ai. evaluated 36 genotypes of Medicago identified for various levels of putative salt tolerance at the cellular level, but failed to fmd a single line with whole plant level tolerance characteristics (McCoy et ai., 1987). The salt-adaptive property of plants always confounds selection of valuable ST mutants by researchers. Hassan et ai. succeeded in selecting salt tolerant cell lines in Lycopersicon peruvianum in vitro by using cell suspension culture. However, the selected cell lines were not stable in the absence of salt, and unfonunately they failed to regenerate plants from the selected cells (Hassan et ai., 1988). The lack of a correlation between whole plant and callus culture expression of ST may be due to the multiple mechanisms for whole plant ST, which are not controlled by a single gene, and may not be discernable at the cellular level. Low frequencies of regeneration and genetic instability are other factors limiting ST line selection after cell culture manipulation. Regeneration of plants from cell culture of several perennial turfgrass species has been reponed (Ahn et ai., 1985, 1987; Anunduaga et ai., 1988; Krans et ai., 1982; Lowe and Conger, 1979; McDonnell and Conger., 1983; Metzinger et ai., 1987; Songstad et ai., 1986; Torello et ai., 1984; Zaghmout et ai., 1988, 1989). In these reports, most researchers are still concentrating on searching for a suitable composition of cultural medium, hormone type and concentration (Krans, 1985), cultural environment (Ahn et ai., 1978; Artunduaga et ai., 1988), explant types (Artunduaga et ai., 1988) and species selection (Artunduaga et ai., 1988; Krans, 1982) for callus induction and plant regeneration. Although some research has aimed at cell level selection for salt and drought tolerance in turfgrass (Torello, 1985), very little progress has been made towards 2

13 new selection. The reasons for failure include problems cited above for ST selection at the cell level: the selected cell lines typically regenerate only at a low frequency, and very quickly lose regenerative capacity after a number of subcultures (Zaghmout and Torello, 1989). Although there are so many constraints on cell level selection, it is still possible to achieve the goal of selecting somaclonal variants in vitro and retrieving somaclonal variants with desirable characteristics. Agronomic improvements were achieved via cell culture selection in potato (Shepard et al., 1980), tomato (Evans and Sharp, 1983), and wheat (Nabors et al., 1983). Heritable somaclonal variation was established using immature embryos of wheat as explants (Larkin et al., 1983). Evans and Sharp (1983) worked on in vitro selection of tomato using leaves as explants. They believed the mutants occurred spontaneously after mutagenic treatment, environmental change and certain chemical and physical treatments, which then increased the frequency of mitotic recombination. The frequency of somaclonal variation in cell culture is determined by the following factors (Pierik, 1987): 1. The method of vegetative propagation. 2. The type of growth regulator used. 3. The type of explant used. 4. The numbers of times calli are subcultured. 5. The chemical nature and concentration of the selective agent. In order to take advantage of potentially useful somaclonal variants, it is impottant to reliably identify these infrequent events in culture, and confmn the presence of valuable whole plant level characteristics. Ideally, this identification and selection would occur at an early stage 3

14 of in vitro cell manipulation, prior to time consuming, expensive field trials. A stable, uniform whole plant microculture (wpmc) system to evaluate turfgrass variants may provide a new dimension to the quest for superior lines, and a valuable pre screen to rigorously screen for ST traits (Pieper and Smith, 1988). The research objectives of this thesis 1. To optimize a complete microculture system for selected turfgrass species (zoysiagrass, bermudagrass, creeping bentgrass and St. Augustinegrass), including continuous callus culture, regeneration, adaptation of stable, uniform whole plant cultures, and acclimation of whole plants to greenhouse and field settings. 2. To obtain stable ST cell lines after in vitro selection, which can be regenerated and subsequently tested at the WPMC level to verify ST. 3. To identify the relative efficiency of cell level and whole plant level screening tests for ST, and establish the effectiveness of a WPMC system as a valid efficient means for pre screening regenerated putative ST lines from cell culture. The overall goal of this research is to develop a system to screen turfgrasses at both the cellular (callus) and whole plant level to verify ST traits, and efficiently provide novel germplasm to breeders. 4

15 The development of the complete microculture system (Objective 1) includes research on: A. Callus induction 1. Explant selection: node, seed, immature embryo, and immature inflorescence. 2. Medium selection. 3. Hormone selection: 2,4-dichlorophenoxy acetic acid (2,4-D), Naphthaleneacetic acid (NAA), and Picolinic acids (Picloram). 4. Microenvironment: dark or light. B. Callus maintenance 1. Medium and hormone selection. 2. Microenvironment: dark or light. C. Plant regeneration 1. Hormone type and level selection. 2. Microenvironment and duration of cultural period. The development of salt tolerance/saline tests at the callus level (Objective 2) includes research on: A. Induction of salt stress in vitro. B. Measurement of the ability of the callus to adjust to high salinity conditions and observation of morphological changes using scanning electron microscopy. C. Regeneration of plants from ST selected calli. 5

16 Testing putative ST lines from cell culture in WPMC (Objective 3), includes research on: A. Screen selected regenerated genotypes in WPMC saline stress. 1. Monitor morphometric and spectral plant responses to stress using video image analysis in shoot and root zone (Spomer and Smith, 1989). 2. Compare putative ST line responses to parent plants. 3. Establish the validity of the 2-phase system. B. Acclimatization and greenhouse testing. Chapters included in this thesis: The chapters listed below address the objectives outlined above: Chapter II describes research on the types and developmental stages of explants, hormone types and concentrations, sucrose concentrations, cultural methods, and the relationship between callus type and plant regeneration of bermudagrass, zoysiagrass, S1. Augustinegrass, and creeping bentgrass. The morphology of callus development was examined by using scanning electron microscopy. Chapter III describes experiments on production of embryogenic callus from S1. Augustinegrass, and procedures for efficient plant regeneration. 6

17 Chapter IV describes callus induction and the morphological change of callus to plandets in zoysiagrass, followed by single step, long term exposure to NazS04 at the cell level, selection of putative salt tolerant plants, and follow-up WPMC screening. Chapter V describes the single step selective method using callus culture in Seaside creeping bentgrass, and the effects of various levels of N~S04 on the growth of seedling plants, non-selected and selected regenerated plants using a WPMC screen system combined with video image analysis. The morphology of callus and plant regeneration was studied using scanning electron microscopy. Chapter IV gives conclusions and evaluates the approaches and results in this thesis. 7

18 References Ahn, B. J., F. H. Huang, and J. W. King Plant regeneration through somatic embryogenesis in common bennudagrass tissue culture. Crop Sci. 25: Ahn, B. J., F. H. Huang, and J. W. King Regeneration of bermudagrass cultivars and evidence of somatic embryogenesis. Crop Sci. 27: Artunduaga,1. R., C. M. Taliaferro, and B. B. Johnson Effects of auxin concentration on induction and growth of embryogenic callus from young inflorescence explants of Old World bluestem (Bothriochloa spp.) and bermuda (Cynodon spp.) grasses. Plant Cell Tissue Organ Cult. 12: Evans, D. A., and W. R. Sharp Single gene mutations in tomato plants regenerated from tissue culture. Sci. 221: Hassan, N. S., and D. A. Wilkins In vitro selection for salt tolerant lines in Lycopersicon peruviavum. Plant Cell Rep. 7: Krans, J. V Some applied basics of tissue culture today. Golf Course Management, February: Krans, J. V., and K. C. Torres Callus induction, maintenance and plandet regeneration in creeping bentgrass. Crop Sci. 22: Lowe, K. W., and B. V. Conger Root and shoot formation from callus cultures of tall fescue. Crop Sci. 19: McCoy, T. J Tissue culture evaluation of NaCl tolerance in Medicago species: cellular versus whole plant response. Plant Cell Rep. 6:

19 McDonnell, E. R., and B. V. Conger Callus induction and plantlet formation from mature embryo explants of Kentucky bluegrass. Crop Sci. 24: Metzinger, D. B., C. M. Taliaferro, B. B. Johnson, and E. D. Migchell Jr In vitro regeneration of apomictic bluestem grasses. Plant Cell Tissue Organ Cult. 10: Murashige, T, and F. Skoog A revised medium for rapid growth and bioassays with tobacco tissue culture. Physio1. Plant. 15: Nabors, M. W Environmental stress resistance. p In: Plant Cell Line Selection - Procedures and Applications. Ed. P. J. Dix. VCR Publishers, Inc., NY. Nabors, M. W Increasing the salt and drought tolerance of crop plants. p In: Current Topics in Plant Biochemistry and Physiology. Ed: Randall, D. D. University of Missouri Press. Vol. 2. Pieper, M. A., and M. A. L. Smith A whole plant microculture selection system for Kentucky bluegrass. Crop Sci. 28: Pierik, R. L. M Vegetative propagation. p.218. In Vitro Culture of Higher Plants. Martinus Nijhoff Publishers, Dordrecht, The Netherlands. Shepard, J. F., D. Bidney, and E. Shahin Potato protoplasts in crop improvement. Sci. 208: Smith, M. A. L., L. A. Spomer, and E. S. Skiles Cell osmolarity adjustment in Lycopersicon in response to stress pretreatments. J. Plant Nutrition 12: Smith, M. K., and L. A. McComb Effect of NaCl on the growth of whole plants and their corresponding callus cultures. Austr. 1. Plant Physio1. 8:

20 Spomer, L. A., and M. A. L. Smith Image analysis morphometric measurement for tissue water status and other determinations. Agron : Songstad, D. D., C. H. Chen., and A. A. Boe Plant regeneration in callus cultures derived from young inflorescences of little bluestem. Crop Sci. 26: Torello, W. A Tissue culture research: toward more tolerant turfgrass. Golf Course Management, February:6-16. Torello, W. A., A. G. Symington, and R. Rufner Callus initiation, plant regeneration, and evidence of somatic embryogenesis in red fescue. Crop Sci. 24: Zaghmout, O. M. F., and W. A. Torello Somatic embryogenesis and plant regeneration from suspension cultures of red fescue. Crop Sci. 29: