U.t::r

Volume 62, No. 4 ISSN0092-6345 May, 1988 if ISJRA6 62(4);

JUL l l 1988

477

HIBBERD, K. A. Selection for amino acid overproducer mutants in maize: Valine selections...... 479

HORSCH, R. B. and collaborators. Agrobacterium­ mediated gene transfer to plants; engineering tolerance to glyphosate ...... 487

McCOY, T. J. Tissue culture selection for disease resistant plants ...... 503

MEREDITH, C. P., A. J. CONNER, and T. M. SCHETTINI. The use of cell selection to obtain novel plant genotypes resistant to mineral stresses ...... 523

RANCH, J. and G. M. PACE. Science in the art of plant regeneration from cultured cells: An essay and a proposal for a conceptual framework ...... 537

SMITH, R. H. and S. BHASKARAN. Sorghum cell culture: Somaclonal variation/ screening ...... 571

WIDHOLM, J.M. In vitro selection with plant cell and tissue cultures: An overview...... 587 IOWA STATE JOURNAL OF RESEARCH

Published under the auspices of the Vice President for Research, Iowa State University

EDITOR ...... BRUCE W. MENZEL ASSOCIATE EDITOR ...... PAUL N. HINZ ASSOCIATE EDITOR ...... RAND D. CONGER ASSOCIATE EDITOR ...... DWIGHT W. BENSEND ASSISTANT EDITOR-COMPOSITOR ...... CHRISTINE V. McDANIEL

Administrative Board N. L. Jacobson, Chairman J. E. Galejs, I. S. U. Library W. H. Kelly, Collf'gf' of Sciences and Humanities W. R. Madden, Office of Business and Finance B. W. Mf'nzel, Editor W. M. Schmitt, Information Service G. K. Serovy, College of Enginef'ring

Consultants Gerald Klonglan, Consultant for Sociology Faye S. Yates, Promotion Consultant This is the final issue of the Jcm•a State Journal qf RPsearch (ISSN 0092-6435). Publication of the Journal began in 1926. An account of' its history appeared in Vol. 62, No. 2 (Isely, 1987). Throughout its existence, the Jounial primarily published articles from Iowa State University authors and those derived from University­ sponsorf'd symposia and lecture series. Earlier volumPs included abstracts of University doctoral dissertations and lists of faculty publications. Articles in biological and agricultural science were predominant for many years, but in 1972 the Journal was broadened to include the humanities, and its name was changed to the present one. Most back issues of the Journal will be available for a period of one year from the Iowa State University Press, 2121 South State Avenue, Ames, Iowa 50010. Single copies starting with Volume 55 are $8.50 each Prior issues are $5.50 each. Payment is required prior to shipment. The IOWA STATEJOl1RNALOFRESEARCH (ISSN 0092-6435) is published quartffly in August, November, February, and May for $20.00 per year (Canada, $21 00/ other foreign countries, $25.00) by the Iowa State University Press, 2121 South State Avenue, Ames, Iowa 50010. Second-class postage paid at Ames, Iowa f>OOlO. Views C'Xpressed by the authors of contributed papers or of editorials are their own and do not necessarily renect those of Iowa State niversity. POSTMASTER: Send address changes to THE IOWA STATE JOURNAL OF RESEARCH, c/ o Iowa State University Press, 21~1 South State Avenue, Ames, Iowa 50010. Printed by Graphic Publishing Company, 204 N. 2nd Avenue W., Lake' Mills, Iowa 50450. IOWA STATE JOURNAL OF RESEARCH I MAY, 1988 599-602 Vol. 62, No. 4

IOWA STATE JOURNAL OF RESEARCH TABLE OF CONTENTS Volume 62 (August, 1987-May, 1988)

No. 1, August, 1987

From the Editors ...... 1

SCHUSTER, D. H. and L. SCHUSTER. Demographic data support a model for cultural change ...... 3

HECK, P. W. and E. S. TAKLE. Objective forecasts of solar radiation and temperature...... 29

HAMMOND, E. G. Sylvester Graham: America's first food reformer ...... 43

WESSEL, R. I. District heating decisions in Iowa- non-decision decision making ...... 63

GUPTA, S. C. and K. J. FREY. Selection for vegetative growth rate of oats ...... 75

KRAFSUR, E. S. and G. DAVIDSON. Production of semisterile mutants in the Anopheles gambiae s.l. species complex...... 85

YORK, S. L. Fitting human performance data: a compar- ison of three methods of data smoothing ...... 99

DiGRINO, B. N., C. F. YOSHIOKA, L. D. COONEY, and B. C. WILHITE. Purpose and practice among clinical therapeutic recreation settings and community based recreation settings serving special populations...... 109

Note ...... · ..... 121 600 TABLE OF CONTENTS

No. 2, November, 1987

From the Editors ...... 123

ISEL Y, D. Iowa State Journal of Research ( 1926- 1988) ...... 127

BARNUM, S. R. and S. M. GENDEL. Heterotrophic growth of nine strains of filamentous cyanobacteria ...... 14 7

BERAN, J. A. Daughters of the Middle Border, Iowa women in sport and physical activity 1850-1910 ...... 161

DEKKER, J., R. BURMESTER, K. C. CHI, and L. JENSEN. Mutant weeds of Iowa: S-triazine-resistant plastids in Kochia scoparia ...... 183

FLOSI, J. W. and E. R. HART. Endocuticular growth rings as an indicator of age structure in Belostoma flumineum Say (Hemiptera:Belostomatidae) ...... 189

HINZ, P. N. Nearest-neighbor analysis in practice ...... 199

HIRA, T. K. and M, J. MUELLER. The application of a managerial system to money management practices...... 219

JUNGST, S. E. and K.-S. LEE. A ground cover inventory on land-capability classes V-VIII ...... 235

LIN, S.-C. J. and W. D. WOLANSKY. Factors influenc- ing attrition from teacher education programs...... 24 7

MBATA, K. J., E. R. HART, and R. E. LEWIS. Repro­ ductive behavior in Zelus renardii Kolenati 1857 (Hemiptera:Reduviidae) ...... 261

MIZE, C. W. and J. B. PAULISSEN. Yield equations and empirical yield tables for the oak-hickory type in Iowa...... 267 TABLE OF CONTENTS 601

MULDER, P. G. and W. B. SHOWERS. Corn growth response to larval armyworm (Lepidoptera:Noctuidae) defoliation...... 279

OWEN, W. J. Initial analysis of a valid food animal disease database for Iowa ...... 293

TAKEDA , K. , K. J. FREY, and T. B. BAILEY.

Relationships among traits in F 9 -derived lines of oats ...... 313

ZEFF, E. E. Women in the Iowa legislature: A 1986 update ...... 329

ZIEHL, M. A. and W. F. HOLLANDER. Dun, a new plumage-color mutant at the I-locus in the fowl (Gallus gallus)...... 337

**********

No.3, February, 1987

BURKE, K. From Page to Stage: The Use of Shakespeare's Sonnets in Introducing Intimidated Students to His Drama ...... 347

COLEMAN, W. S. E. Anthony Boheme: A Forgotten Tragedian ...... 359

EATON, S. The Rhetoric of (Dis)praise and Hamlet's Mother...... 377

KNIGHT, W. N. Shakespeare Before King James: Betrayal and Revelation...... 387

PRICE, J. E. "Because I Would Followe The Fashion": Rich's Farewell to the Mi litary Profession and Shakespeare's Twelfth Night ...... 397 RELIHAN, C. C. Appropriation of the "Thing of Blood": Absence of Self and The Struggle for Ownership in Coriolanus ...... 407 602 TABLE OF CONTENTS

RUTLEDGE, D. F. The Structural Parallel Between Rituals of Reversal, Jacobean Political Theory, and Measure For Measure ...... 421

SHAFFER, R W. "To Manage Private and Domestic Quarrels": Shakespeare's Othello and the Genre of Elizabethan Domestic Tragedy...... 443

YOUNG, B. W. Haste, Consent, and Age at Marriage: Some Implications of Social History for Romeo and Juliet ...... 459

Afterword ...... 4 75

**********

No. 4, May, 1988

From the Editors ...... 4 77

HIBBERD, K. A. Selection for amino acid overproducer mutants in maize: Valine selections ...... 479

HORSCH, R. B. and collaborators. Agrobacterium­ mediated gene transfer to plants; engineering tolerance to glyphosate ...... 487

MEREDITH, C. P., A. J. CONNER, and T. M. SCHETTINI. The use of cell selection to obtain novel plant genotypes resistant to mineral stresses...... 523

McCOY, T. J . Tissue culture selection for disease resistant plants ...... 503

RANCH, J. and G. M. PACE. Science in the art of plant regeneration from cultured cells: An essay and a proposal for a conceptual framework ...... 537

SMITH, R. H. and S. BHASKARAN. Sorghum cell culture: Somaclonal variation/ screening ...... 571

WIDHOLM, J. M. In vitro selection with plant cell and tissue culture: An overview ...... 587 IOWA STATE JOURNAL OF RESEARCH I MAY, 1988 477-478 Vol. 62, No. 4

FROM THE EDITORS

The Plant Science Lecture Series is held annually at Iowa State University. The lecture series focus each year is on crop improvement, and each series is composed of six to eight seminars related to a theme that is selected to be timely and relevant to genetically improving field, forage, forest, and horticultural crops. Plant breeding had its origin with the rediscovery of Mendel's Laws in 1901. His discoveries, that genes retained their unique identities, even when they are not expressed in phenotypes, and that many of them segregate independently of each other, provided the basis for plant breeders to construct new and ideal genotypes. For the past 80 years, plant breeders have artificially crossed crop genotypes with contrasting alleles, the progenies of which would contain recombinant strains useful to agriculture. Most of the cost and time spent on plant breeding, however, has been devoted to developing strategies for selecting desired recombinants. The progress in improv­ ing crops made by plant breeders, although phenomenal, has been limited because selection was based upon whole plants or progenies. During the past decade there has emerged an array of technologies that will have significant impact on the success of plant breeding. Collectively, these are known as genetic engineering, and they include, for certain traits, the opportunity to practice selection among recombinants at the cellular level in test tubes and petri dishes. Because plant breeding of the future will include certain tech­ niques of genetic engineering in its repertoire of technologies, the Plant Science Lecture Series for 1986-88 has been, and will be, devoted to a series of aspects of what is more broadly called plant biotechnology. The theme for the 1987 Lecture Series, which is published in this issue of the Iowa State Journal of Research, is "Plant biotechnology: In vitro selection upon cells and tissue cultures for traits of importance to agricultural production." In some cases, when certain stresses such as disease toxins or herbicides are applied to cultures of cells in test tubes or petri dishes, only the cells that carry alleles for resistance to the toxins or chemicals can survive. If plants can be regenerated from the surviving cells, the recombinants will give rise to plants that carry the desired alleles. In this series of papers an 478 FROM THE EDITORS up-to-date summary is given of (a) various in vitro selection proce­ dures and techniques, (b) the art and science of plant regeneration from plant cells, and ( c) specific examples of the use of in vitro selection for improving crop plants.

Kenneth J. Frey, Coordinator Plant Science Lecture Series IOWA STATE JOURNAL OF RESEARCH I MAY, 1988 479-486 Vol. 62, No. 4

SELECTION FOR AMINO ACID OVERPRODUCER MUTANTS IN MAIZE: VALINE SELECTIONS

Kenneth A. Hibberd 1

ABSTRACT. Selections were made in tissue culture for alterations in the branched chain amino acids-leucine, valine, and isoleucine. Tissue from a friable maize line was selected for resistance to 6mM valine. Ten presumptive resistant lines were selected from 70 tissue clumps and from these, three lines were found to be significantly more resistant to valine than the parental line. Plants were regenerated from the three selected lines and seeds obtained from two lines and the parental tissue. Seedlings originating from hybrids between valine selected­ regenerated plants and control plants showed segregation for valine resistance in a mature embryo root growth assay. Valine resistance was also expressed in subsequent generations. These results suggest that valine resistance is expressed in maize plants as a heritable dominant character. Isoleucine, valine, leucine, and other free amino acids were increased in some seeds from selected progeny. Index descriptors: Zea mays L., tissue culture, selections, valine.

INTRODUCTION

A variety of selections at the cellular level have been conducted with tissue cultures to improve maize plants in the areas of disease and herbicide tolerance and nutritional quality. The improvements in nutrition have focused on altering the free pool levels of the most limiting amino acids when maize is fed to monogastric animals. These include lysine, tryptophan, threonine, and isoleucine, in order of nutritional limitations. Selection in maize cultures has been made for increases in free amino acids in the aspartate pathway for lysine, threonine, and methionine, and in the aromatic animo acids pathway for tryptophan. Increases in free threonine were obtained in tissue culture selections in the presence oflysine plus threonine (Hibberd et al., 1980; Hibberd and Green, 1982; Miao et al., 1986). Free threonine increases in the selected calli ranged from 8- to 20-fold over that in control tissues. With one lysine plus threonine (LT) selected line, the lysine sensitivity of the enzyme aspartokinase was significantly reduced (Hibberd et al., 1980). Progeny of regenerated plants expressed LT resistance as would be expected for a single dominant nuclear gene. Threonine

1Molecular Genetics Inc., Minnetonka, Minnesota 55343. 480 HIBBERD overproduction was greatest in plants homozygous for the LT resistance trait. Free threonine in these seeds can be high enough to increase total threonine by 50% (Hibberd and Green, 1982). Selections for resistance to the lysine analog aminoethylcysteine have been conducted in several laboratories. However, none of the selected callus lines have increases in free lysine greater than 2 to 4 fold (B. Gengengbach, personal communications, and K. A. Hibberd, unpub­ lished). Selections for increased tryptophan in maize cultures have proven successful. Hibberd et al. ( l 986a, l 986b) and Miao et al. (1986) obtained callus lines with 10- to 50-fold increased tolerance to the tryptophan analog 5-methyltryptophan (5MT). Free tryptophan levels in the calli increased from 2- to over 100-fold. Increased free phenylalanine was also observed in some lines (Miao et al., 1986). Assays in tolerant lines for anthranilate synthase, the branch point enzyme for tryptophan biosynthesis, indicated that there was a reduced sensitivity to feedback inhibition by tryptophan (Hibberd et al., 1986b ). Progeny of regenerated plants expressed resistance to 5MT as expected for a single dominant nuclear trait. Increases in free tryptophan in seed ranged from 2- to greater than 100-fold. The first enzyme unique to the synthesis of branched chain amino acids, acetohydroxyacid synthase is feedback regulated by valine and leucine in higher plants (Millin and Cave, 1972). Growth of seedlings has been shown to be retarded by valine, leucine, or combinations of both amino acids. The growth inhibition by valine or valine plus leucine can be reversed by the addition of isoleucine (Miflin, 1969; Bourgin, 1976). These results have provided an opportunity for selecting in vitro for mutants in the branched chain amino acid pathway. Valine tolerant mutants have been reported in two Nicoti­ ana species (Bourgin, 1978; Vunsh et al., 1982). This report will describe the selection and partial characterization of valine tolerant lines in the monocot Zea mays L.

MATERIALS AND METHODS

Seeds from the maize inbred line Al88 were obtained from the Minnesota Crop Improvement Association, St. Paul, Minnesota. Cul­ tured tissues were initiated from 1 to 2mm immature embryos from self-pollinated ears. The parental culture designated Al was a highly friable and rapidly growing line which would increase 50-fold in fresh weight in 4 weeks. Al was initiated and maintained on MS medium which contains Murashige and Skoog (1962) salts, 50µM 2,4-D, lmM AMINO ACID MUTANTS IN MAIZE 481

AspN, 2.0% sucrose and 0.7% agar. Cultures were maintained at 26°C in the dark Selections were initiated by placing 25 mg clumps of Al tissue on MS medium containing 3mM valine. After one or two 21-day cycles, the surving tissue clumps were transferred to 6mM valine MS plates for an additional three to four cycles. The effects of valine on callus growth were determined by measuring increases in fresh weight after 28 days at 26°C of 100 mg of callus placed on solid MS medium containing various amounts of the amino acid. Plants were regenerated from selected lines by transferring approximately 100 g of tissue to MS medium lacking 2,4-D. After 2 weeks, the tissue on plates lacking 2,4-D were placed on a 14 hr light cycle. Plantlets that developed were then moved to pint Mason jars containing MS medium lacking 2,4-D, allowed to develop to a height of 6 in., and finally transferred to soil. Mature regenerated plants were cross pollinated with inbred maize lines Al88 or N28. For seedling bioassays, seeds were surface sterilized with a 20 min treatment of 2.5% sodium hypochlorite followed by three 5 min rinses with sterile water and then germinated overnight at 26°C in 0.5% Captan. The mature embryos were excised and placed in 25 x 150 mm tubes containing 10 ml MS medium lacking 2,4-D with 0 or 30mM valine and grown at 26°C under 14 hr light for 7 days. Amino acid analyses were conducted on 3 to 4 g of seed from each ear. The seeds were ground in a Wiley Mill to 40 mesh and a portion of the meal was extracted twice for 1 h each with 25% acetonitrile in water. Samples were analyzed as the fluorescent derivative of o-phthaldialdehyde using a Waters HPLC model 721, a Supelco Cl8 column and a Kratos model 950 fluorescence detector (Jones and Gilligan, 1983).

RESULTS

Ten of the original 70 Al clumps survived the selection procedure and showed at least some growth on 6mM valine MS. These survivors were then tested for stable resistance by first transferring to MS medium without valine for one culturing period then onto 0 and 6mM valine MS. Growth after 28 days was determined, and the percent growth on 6mM valine compared to growth on MS for each line. Of the 10 surviving lines, three, Val2, Va45, and Va52, showed significantly better growth responses· than the Al parent. A more complete characterization of two of these lines is shown in Figure 1. At higher 482 HIBBERD

60 """ 0 A1 .....~ 0 Va 12 0 Va52 - 50 ...... ~ ...... J::. CJ) Q) 40 ~ .J::. Q)"' ....~ 30 Q) "'l'O Q) ~ 20 (J c:

1J 0 u. 10

o ...... ______..______.. ______0 3 e 12

Valine concentration (mM)

Figure 1. Growth of control (Al) and select callus lines (Va12, Va52) for 28 days at 26°C on MS medium containing various concentrations of valine. The points on the graph represent the average of 4 samples with standard deviations. AMINO ACID MUTANTS IN MAIZE 483

Table 1. Response of progeny seedlings to valine. 7 Day Root Seedling Valine Seeds Length Response Background Cone. (mM) Assayed (CM± S.D.) Type Al88 X Ala 0 20 7.1 ± 1.4 (20)b 30 20 2.1 ± 1.0 (20) Sensitive

N28 X Va52 0 20 11.8 ± 1.1 (20) 1.3 ± 0.9 (21) Sensitive 30 40 6.3 ± 2.3 (19) Resistant

N28 X Va45 0 20 11.7 ± 1.1 (20) 1.0 ± 0.8 (30) Sensitive 30 40 6.3 ± 2.1 (10) Resistant

a Parental line. b Number of seedlings in responding classification. levels of valine, the selected lines, Val 2 and Va52, grew significantly better than the parental tissue. With l 2mM valine, the selected lines continued to grow while the control tissues stopped after a few doublings and become necrotic. Plants were regenerated from Val2, Va45, Va52, and the parental Al line. Seeds were obtained from Va45, Va52, and the parental line in crosses with inbreds Al88 or N28. The growth of roots from mature excised embryos on 30mM valine was found to be useful in determining the presence of valine resistance in progeny of selected callus lines. Control and progeny seedlings from the parental line were found to be sensitive to this level of valine, being generally inhibited by 80% to 90% of the normal root length development in 7 days. In contrast, progeny seedlings from callus lines Va45 and Va52 showed a varying response to growth on 30mM valine. The growth of some roots was limited and similar in length to control seedlings on 30mM valine while other Va45 and Va52 seedlings developed roots closer in length to what was expected for non-valine treated seedlings. On the basis of root length, it was possible to classify root responses as sensitive or resistant to valine. The results of screening Va45 and Va52 progeny are shown in Table 1. Approximately 50% of the Va52 progeny and 25% of the Va45 progeny 484 HIBBERD

Table 2. Ranges in amino acid levels in seeds from valine selected F2 ears. µg Amino Acid/ gm Seed Control Amino Acid Al88/ N28 (2)a N28/ Va45 ( 13) N28/ Va52 (5) Valine 19-24 21-58 19-59 Isoleucine 10-13 10-66 11-56 Leu cine 11-12 10-39 11-24 Alanine 64-81 68-374 59-213 Aspartic Acid 167-187 179-675 128-508 Glutamic Acid 292-394 213-711 267-412 Serine 35-48 38-114 33-126 a Number of ears sampled.

were found to be more resistant to valine inhibition than control progeny. Va52 seedlings found to be resistant or sensitive in the bioassay were grown to maturity and self pollinated. In the next generation, resistant parents gave rise to seedlings that were signifi­ cantly more resistant to valine than seedlings derived from sensitive parents (data not shown). Seeds from individual F2 Va45 and Va52 ears were analyzed for free amino acids and found to contain from 0 to 6-fold higher levels of free isoleucine and from 0 to 3-fold higher free leucine and valine than was observed in control ears (Table 2). Non-branched chained amino acids were also elevated in some ears. Increases of as much as 2- to 5-fold in glutamic acid, alanine, serine, and aspartic acid were observed. Ears that showed higher levels of isoleucine, valine, and leucine also showed higher levels of the other free amino acids (data not shown). Similarly, Va45 and Va52 ears low in free branched chain amino acid were also generally low in other free amino acids.

DISCUSSION

Tolerance to valine is a heritable trait in maize. Progeny from regenerated Va45 and Va52 plants were more resistant to root growth inhibition by valine than were nonselected controls. Crosses between valine-selected plants and controls as well as self-pollinations of valine-selected plants yielded valine-tolerant progeny. The direction of AMINO ACID MUTANTS IN MAIZE 485 the cross was found not to be important in expression of resistance. These results indicate that valine tolerance is a dominant nuclear trait in maize. The ratio of valine tolerant to sensitive seedlings in Va52 progeny is close to 1:1 (19:21) following crosses with controls, which is consistent with expectations for a single dominant gene. On the other hand, the ratio for resistant to sensitive progeny of Va45 is 10:30. This result may indicate a more complex genetic basis for valine resistance in the Va45 line. Alternatively, the lower than expected ratio may be due to reduced transmission of the Va45 resistance trait through the pollen. Further studies are needed to address this question. Alterations in free amino acid levels were clearly evident in F2 ears. The wide range in amino acid levels is likely due to segregation for this character in this population of ears. The levels of increase of isoleucine in some Va45 and Va52 progeny were significantly higher than in control ears. Overproduction of isoleucine was the desired result of the selection for valine tolerance since isoleucine will relieve valine growth inhibition in plants (Miflin and Cave, 1972). However, the selected progeny also overproduced other free amino acids, which suggests that Va45 and Va52 may have a genetic change affecting general amino acid accumulation. Additional research is needed to clarify the full nature of these valine tolerant lines. The increase in isoleucine in Va45 and Va52 seed is an order of magnitude less than the increases reported for threonine and for tryptophan overproducers (Hibberd and Green, 1982; Hibberd et al., 1986b ). This result may represent inherent limits imposed by the anabolic and catabolic pathway for isoleucine. Another possibility is that these particular valine resistant mutants are not the highest overproducers that can be selected. For example, not all 5MT resistant mutants are equal in levels of tryptophan overproduction (Hibberd et al., 1986b ). Finally the full potential for overproduction by these lines may not have measured. Additional breeding to demon­ strate homozygosity of the valine resistant trait and breeding to remove possible mutations induced by the culture process, which are unrelated to valine tolerance, may greatly affect final free amino acid concentrations in the seed, both in the branched chain and total free amino acids. Valine tolerance has potential uses in areas of metabolism, genetics, and genetic manipulations of plants. In tobacco, valine resistance has been used in fusion experiments (Bourgin et al., 1986). In the future the valine resistance trait will likely be useful as an in vitro marker for genetic engineering of plants. 486 HIBBERD

LITERATURE CITED

Bourgin, J. P. 1976. Valine-induced inhibition of growth of haploid tobacco protoplasts and its reversal by isoleucine. Z. Naturforsch. 31c:337-8. ----. 1978. Valine-resistant plants from in vitro selected tobacco cells. Molec. Gen. Genet. 161:225-230. ----, C. Missonier, and J. Goujaud. 1986. Direct selection of cybrids by streptomycin and valine resistance in tobacco. Theor. Appl. Genet. 72:11-14. Hibberd, K. A., T. Walter, C. E. Green, and B. G. Gengenbach. 1980. Selection and characterization of a feedback-insensitive tissue culture of maize. Planta 148:183-87. ---- and C. E. Green. 1982. Inheritance and expression of lysine plus threonine resistance selected in maize tissue culture. Proc. Natl. Acad. Sci. USA. 79:559-63. ----, M. Barker, and P. C. Anderson. April 15, 1986a. Tryptophan over­ producer mutants of cereal crops. United States Patent Number: 4,581,847. ----, ----, ----, and L. Linder. 1986b. Selections for high tryptophan maize, p. 440. In: D. A. Somers, B. G. Gengenbach, D. D. Biesboer, W. P. Hackett, and C. E. Green (eds.). VI International Congress of Plant Tissue and Cell Culture. Minneapolis, MN. Jones, B. N. and J. P. Gilligan. 1983. o-Phthaldialdehyde precolumn derivatization and reversed-phase high-performance liquid chromatography of polypeptide hydrolysates and physiological fluids. J. Chromatogr. 266:471-482. Miao, S. H., D. R. Duncan, and J. M. Widholm. 1986. Selection of lysine plus threonine and 5-methyltryptophan resistance in maize tissue culture, p. 380. In: D. A. Somers, B. G. Gengenbach, D. D. Biesboer, W. P. Hackett, and C. E. Green (eds.). VI International Congress of Plant Tissue and Cell Culture. Minneapolis, MN. Miflin, B. J. 1969. The inhibitory effects of various amino acids on the growth of barley seedlings. J. Exp. Bot. 20:810-19. ---- and P. R. Cave. 1972. The control of leucine, isoleucine, and valine biosynthesis in a range of higher plants. J. Exp. Bot. 23(75):511-516. Murashige, T. and R. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:473-497. Vunsh, R. , D. Aviv, and E. Galun. 1982. Valine resistant plants derived from mutated haploid and diploid protoplasts of Nicotiana sylvestris and N. tabacum. Theor. Appl. Genet. 64:51-58. IOWA STATE JOURNAL OF RESEARCH I MAY, 1988 487-502 Vol. 62, No. 4

AGROBACTERIUM-MEDIATED GENE TRANSFER TO PLANTS; ENGINEERING TOLERANCE TO GLYPHOSATE

Robert B. Horsch, 1 Robert T. Fraley, Stephen G. Rogers, Harry J. Klee, Joyce Fry, Maud A. W. Hinchee, and Dilip S. Shah

ABSTRACT. We have produced transgenic plants with a vector system that harnesses the natural gene transfer ability of Agrobacterium tumefaciens, the causative agent of crown gall disease. The disease is the result of the transfer and expression of phytohormone biosynthesis genes from the tumor-inducing (Ti) plasmid of A. tumefaciens into the chromosomes of infected plant cells. We have deleted the phytohormone biosynthetic genes that cause crown gall disease from the Ti plasmid, and substituted a vector that contains a selectable marker, neomycin phosphotransferase (NPT II) that confers resistance to kanamycin, and a scoreable marker, nopaline synthase (NOS) that produces a unique and easily scorable metabolite, nopaline. The vector system also permits addition of other genes for co-transfer into transformed plant cells. The vector plasmid is engineered in E. coli and then mated into A. tumefaciens where it is then capable of being transferred into infected plant cells. Transformed petunia, tobacco, and tomato plants have produced by means of a simple leaf disc transformation-regeneration method that couples the gene transfer mechanism of A. tumefaciens with kanamycin selection and de novo shoot generation. Surface-sterilized leaf discs were inoculated with an A. tumefaciens strain containing our vector and cultured for two days. The leaf discs were then transferred to regeneration medium containing kanamycin. Shoot regeneration occurred within 2 to 4 weeks, and transformants were confirmed by their ability to root in medium containing kanamycin. This method should be adaptable to many species that are within the host range of A. tumefaciens and where regeneration of plants from culture is possible. Roundup herbicide (glyphosate) is a potent inhibitor of the enzyme 5-enolpyruvyl-shikimate-3-phosphate (EPSP) synthase in higher plants. A suspen­ sion culture of petunia cells (MP4) was gradually adapted to increasing levels of glyphosate until the culture (MP4-G) was tolerant to 20 mM glyphosate, 20 times more than required to kill the original culture (MP4). MP4-G produced 20 times more EPSP synthase than MP4, which is presumably the basis of its tolerance to glyphosate. The EPSP synthase protein was purified from this culture and a partial amino acid sequence was determined. An oligonucleotide probe was synthesized corresponding to the amino acid sequence, and used to isolate a cDNA clone for EPSP synthase. The MP4-G cell line produced about 20-fold more EPSP synthase mRNA as a result of a 20-fold amplification of the EPSP synthase gene. By constructing a chimeric gene using the cauliflower mosaic virus 35S promoter 1Monsanto Agricultural Company, 700 Chesterfield Village Parkway, St. Louis, MO 63198. 488 HORSCH et al.

to attain high level expression of the EPSP synthase cDNA clone, we were able to produce transformed petunia cells that over expressed EPSP synthase from a single T-DNA insert. These cells and plants regenerated from them were tolerant to glyphosate at concentrations much higher than required to kill all controls. Index descriptors: transgenic plants, transformation, Agrobacterium tume­ faciens, vectors, selectable markers, herbicide resistance, glyphosate resistance, EPSP sythnase.

INTRODUCTION

In the past 4 years, two technologies have been developed for transferring isolated genes into plant cells and regenerating trans­ genic plants from those cells. One method is based on the capacity of plant protoplasts to incorporate and express exogenous DNA when treated with physical or electrical treatments to permeabilize their membranes. The other method is based on the natural gene transfer capacity of Agrobacterium tumefaciens, which can transform cells as well as protoplasts. In theory, the delivery of exogenous DNA to protoplasts is without host limitation (Potrykus et al., 1985), while A. tumefaciens has been observed to transform only dicotyledonous species and a few monocots in the Liliaceae family. In practice to date, a much broader range of transgenic plants have been produced with A. tumefaciens­ mediated transformation since it can transform regenerable tissues with relative ease while protoplasts of most species are still relatively difficult or impossible to regenerate. The difficulties of DNA transfer and selection for transformed colonies during recovery and growth have made low frequency regeneration from protoplasts a major problem. Free-DNA delivery to regenerable protoplasts may be the eventual key to transformation of important crops such as corn, wheat, or rice, but because of the practical ease of A. tumefaciens­ mediated transformation, it is likely to remain the method of choice for routine production of transgenic plants for species within its host-range. Recently, the possibility of using A. tumefaciens for gene transfer to monocot crops has been raised by the finding that maize streak virus (MSV) can be transferred to maize plants by the technique of agro-infection (Grimsley et al., 1987), and the detection of opines in infected maize seedlings (Graves and Goldman, 1986). Application of Agrobacterium-mediated transformation to new species can be difficult to accomplish and to document. This paper discusses the issues of host range and problems with practical GENE TRANSFER TO PLANTS 489 application of the Agrobacterium gene transfer method to crop plants.

AGROBACTERIUM

A. tumefaciens causes crown gall disease by transferring a defined segment of DNA (T-DNA) from its tumor-inducing (Ti) plasmid into the nuclear genome of cells in an infected wound on many dicotyledonous plants (for recent reviews, see Bevan and Chilton, 1982; Depicker et al., 1983; Nester et al., 1984). The T-DNA contains genes that encode enzymes that catalyze the synthesis of the phytohormones IAA (Schroder et al., 1984; Thomashow et al., 1984; Akiyoshi et al., 1984) and IPA (Barry et al., 1984), an auxin and a cytokinin. This overproduction of phytohormones is responsible for the proliferation of wound callus into a gall which can harbor a population of the bacteria. Other T-DNA genes cause synthesis of unique metabolites called opines which Agrobacterium can cata­ bolize, but which cannot be utilized by many other microorganisms or plants. One strain of A. tumefaciens (A208) that we have used produces nopaline synthase, an enzyme that produces nopaline from arginine and alpha-ketoglutarate. The movement of the T-DNA is mediated by genes in another region of the Ti plasmid. These virulence genes (Klee et al., 1983) are not themselves transferred with the T-DNA, but act in trans to cause the transfer (de Framond et al., 1983; Hoekema et al., 1983). The T-DNA is defined by a 25 base sequence, the border sequence, which is present as a direct repeat outside the ends of the T-DNA (Barker et al., 1983). Recent evidence suggests that the border sequence is the site of specific nicking of the DNA by one of the virulence gene products (Stachel et al., 1986). It has been reported that a bacterial origin of transfer can substitute for the border sequence when compatible mobilization functions are also present (Buchanan­ Wollaston et al., 1987). None of the genes or DNA sequences within the T-DNA are required for the transfer process (Garfinkel et al., 1981). These properties of the Agrobacterium DNA transfer system are invaluable for developing a powerful vector system for plant trans­ formation. It is possible to remove all of the original T-DNA which was responsible for the disease and replace it with any DNA, as long as the border sequence is maintained (Zambryski et al., 1983). The DNA is inherently stable once in the plant genome since neither the border nor the virulence genes are transferred. Finally, since the borders 490 HORSCH et al.

define a discrete T-DNA segment, the frequency of co-transfer of the entire segment is very high. This means that your favorite genes are usually transferred along with the selectable marker used to identify the transformed cells or plants.

VECTORS

To harness this mechanism, we designed a shuttle vector (Fraley et al., 1983) that can replicate in E. coli where recombinant manipulations are easily handled, and then transferred in A. tume- faciens in preparation for transfer into plants. There are two basic modes of maintainance of the shuttle vectors in Agrobacterium: either by integration into the Ti plasmid by recombination at a region of DNA homology (Comai et al., 1983; Fraley et al., 1983; Zambryski et al., 1983), or by autonomous replication in trans to the Ti plasmid (Bevan, 1984; An et al., 1984; Klee et al., 1985). The former type of vector is referred to as a cis or integrating vector while the latter is called a trans or binary vector. In most cases they accomplish the same goals of shuttling genes from E. coli to A. tumefaciens in a T-DNA package that can be transferred to plants. The essential components of our vectors are shown in Figure 1. The binary vector pMON530 (a derivative of pMON505; Horsch and Klee, 1986) is shown as an example. They include plasmid functions for replication (pBR322, RK2 Replicon) as well as a bacterial selec­ table marker for spectinomycin and streptomycin resistance (Spc/ StrR) for genetic manipulations with the bacteria. They also include a chimeric selectable marker for plant antibiotic resistance such as neomycin phosphotransferase (NOS-NPTII-NOS) which makes transformed plant cells resistant to kanamycin, and a border sequence (NRB) to identify the end of the new T-DNA. There is also a scoreable marker, the gene for nopaline synthase (NOS), that provides a powerful genetic tool for monitoring the presence of the T-DNA in plants and their progeny. The final feature is the most important of all: a site to add any desired coding sequence between a plant promoter (CaMV 35S promoter) and a poly A addition sequence (NOS3').

SELECTABLE MARKERS

Since only a small proportion of cells are transformed by A. tumefaciens, it is important to have a selectable marker to recognize the transformed cells and suppress the growth of wild-type cells. The GENE TRANSFER TO PLANTS 491 first marker to be used was the neomycin phosphotransferase gene from E. coli. This gene has been used successfully in many systems, including mammalian cells, fungi, and Dictyostellium. In solanacious plants such as petunia and tobacco it provides an excellent, unambig­ uous selectable marker. The features of a good selectable marker are i) high level resistance of the transformed cells to concentrations of the drug that completely inhibit wild-type growth, and ii) efficient selectability of rare transformants from a large excess of inhibited wild-type cells that surround them. Kanamycin-inhibited petunia cells do not appear to interfere with the growth of resistant cells in any way, and may actually facilitate their growth by acting as a nurse tissue. Single resistant clones can be obtained from petunia leaf discs transformed with attenuated mutants of A. tumefaciens (Horsch et al., 1986), demonstrating the efficacy of selection for this marker. A second selectable marker that we have used is the mouse gene for dihydrofolate reductase (DHFR) that encodes an altered protein that does not bind to methotrexate, a potent inhibitor of this essential enzyme. Petunia cells that contain and express this gene are able to grow on concentrations of methotrexate that completely inhibit the control cells (Eichholtz et al., 1987). Selectability also appears to be good for methotrexate resistance, although the efficiency is dependent on the strength of expression of the DHFR gene. A simple technique to improve the selectability of resistant cells is to wait 2 or 3 days after transformation to add selective pressure, thus allowing time to accumulate resistant DHFR enzyme. A third marker we have used is a bacterial gene for hygromycin phosphotransferase which confers resistance to hygromycin (Lloyd et al., 1986). This marker works reasonably well in solancious species as well as Arabidopsis thaliana where it is the marker of choice. The selectability of hygromycin resistance is poor, probably due to the extreme toxicity of the drug. For species such as A. thaliana where kanamycin resistance functions poorly, hygromycin can be a good alternative. However, where kanamycin resistance works, it is clearly a better choice.

LEAF DISC TRANSFORMATION

The simple and powerful interface between A. tumefaciens and plant cells is illustrated by the leaf disc transformation system (Horsch et al., 1985) where gene transfer, selection and regeneration are coupled together in an efficient process. Tobacco is an excellent 492 HORSCH et al.

NAB Nopaline Synthase

pMON530 Spc/StrR 12.0 kb

RK2 Replicon

NOS3 ' CaMV 355 promoter

EcoRI Xhol Clal Kpnl Smal Bglll

Figure 1. Binary vector pMON530.

host for A. tumefaciens, and also responds exceedingly well in culture. While the technique is most easily practiced with tobacco, it has been applied to a number of other species (Table 1). This example will be described for tobacco, using a vector that confers kanamycin resist­ ance, such as pMON200 (Fraley et al., 1985a) or pMON530. Surface sterilized leaf discs or other axenic explants are infected with the appropriate strain of A. tumefaciens carrying the vector of choice, and cocultured on regeneration medium for 2 or 3 days. During this time, the virulence genes in the bacteria are induced, the bacteria bind to the plant cells around the wounded edge of the explant, and the gene transfer process occurs. One empirical observa­ tion we have made is that for species other than tobacco, a nurse GENE TRANSFER TO PLANTS 493

Table 1. Transgenic plants produced with A. tumefaciens-mediated transformation. Species Reference Nicotiana plumbaginifolia Horsch et al., 1984 petunia Horsch et al., 1985 tobacco DeBlock et al., 1984 tomato McCormick et al., 1986 potato Shahin and Simpson, 1986 lettuce Michelmore, R.W. et al., in press poplar Fillatti et al, 1987 Arabidopsis thaliana Lloyd et al., 1986 Medicago varia Deak et al., 1986 flax Basiran et al., 1987 Brassica napus Fry et al., 1987 sunflower Everett, N.P. et al., in press cotton Umbeck et al., 1987

culture of tobacco cells increases the transformation frequency when used during the co-culture period. This may be due to more efficient induction of the virulence genes, or some other factor not yet understood. After the transformation has occurred, the explants are trans­ ferred to regeneration/ selection medium. This contains 500 ug/ ml carbenicillin to kill the bacteria and the appropriate antibiotic to inhibit untransformed plant cells, usually kanamycin. During the next 3 weeks, the transformed cells grow into callus or differentiate into shoots via organogenesis. Between 3 and 6 weeks, the shoots develop enough to remove them from the explant and induce rooting in preparation for transfer to soil. There are a number of technical issues that require careful attention, especially when attempting to apply this technique on species other than petunia or tobacco. First, some of the shoots that grow in the presence of kanamycin do not express and/ or contain the foreign DNA. The reason(s) for these apparent escapes are not clear but may include loss of expression or loss of DNA during plant development, or incomplete selection due to cross-protection of wild­ type cells by transformed cells nearby. Whatever the reason(s), the problem can be dealt with by a second selection for ability to root in 494 HORSCH et al.

the presence of kanamycin (Horsch et al., 1985), and subsequent callus induction assays or opine assays for continued expression of new genes. A second problem arises in the interaction between A. tume­ faciens and explants of some plants. While it is not certain what is happening, it is apparent that some plant species react more strongly to infection by the bacteria. For example, in Lycopersicon escuwtum the titer of bacteria in the inoculum greatly influences the survival and vigor of explants, and overall transformation efficiency (McCor­ mick et al., 1986), while it makes little or no difference in tobacco or petunia (unpublished observations). The choice of Agrobacterium strain can have a significant effect on the efficiency of transformation (Byrne et al., 1987). The factors controlling strain specificity can be difficult to determine (Hood et al., 1987). A third problem that may arise is the efficacy of the selectable marker used with a particular plant species or genotype. For example, the chimeric genes for expressing neomycin phosphotrans­ ferase II in plants work in all species tested, but do not confer kanamycin resistance at a selectable level in all species. In alfalfa, the enzyme can be detected in tumor tissues transformed with pMON200, but the cells do not grow any better in the presence of kanamycin than do wild-type cells, or transformed cells that lack the nptll gene altogether (J. Fry, unpublished data). Position effects, that is, effects of the surrounding DNA or chromatin structure, result in differences in expression of the T-DNA genes in different transformants. The extreme of this is complete loss of expression during differentiation of the plantlet. This occurs in about a quarter to a third of the shoots that grew initially in the presence of 300 ug/ ml kanamycin. One possible reason for failure to select against these escapes is that the kanayrncin is not a good herbicide, and shoots can continue growth in its presence once they are large enough. It is usually necessary to screen several independent transgenic plants to identify the best expression of your favorite gene which sometimes but not always is correlated with the expression of the selectable marker.

INHERITANCE

Progeny of transgenic plants produced using this process always have inherited the T-DNAs found in the parent plant. This means that the L2 cell layer which produces the gametes has always been transformed, and that the Ll and L3 cell layers are not transformed GENE TRANSFER TO PLANTS 495

with a different T-DNA(s). The only remaining possibility is that one or both of these tissues are not transformed at all. While we have not exhaustively searched for examples of this phenomenon, we have never observed it to occur, and would not anticipate any problem after-the-fact since progeny will be clonal anyway. Where this might be troublesome, if it occurs is during the initial selection for trans­ genic shoots, where non-transformed tissues might cause problems with morphogenesis or rooting. Progeny usually inherit the T-DNA according to Mendelian rules for dominant genes. In cases where there is only one T-DNA, selfed progency show a 3:1 ratio for nopaline or kanamycin resistance, while backcrossed progeny show a 1:1 ratio. This is also true for multiple T-DNAs that are present in a tandem array where they are all genetically linked. In a few cases, there are multiple T-DNAs, some of which are unlinked. In these cases, selfed ratios are usually 15:1 (two unlinked loci) or higher (three or more unlinked loci). In nearly all cases, the selected and unselected markers on a single T-DNA are coinherited. We have genetically mapped the chromosomal position of the T-DNA in a number of independent transgenic petunias by using a special hybrid between two multiply-markered genetic testor strains (Wallroth et al., 1986). This Fl hybrid (VR) was produced at the Genetics Department, University of Amsterdam, by crossing strain V23 with R51. VR has at least one genetic marker that is heterozygous on each of its 7 chromosome pairs. In this way, each chromosome is uniquely tagged and can be observed in a single generation by backcrossing to each of the original parents. In a sample of 9 transformants, the T-DNA was found in a different place in each, in four of the chromosomes. This is consistent with a random distribu­ tion, but does not eliminate bias or repeated, dispersed sites for integration. The T-DNA thus becomes a permanent, stable part of the genome of transgenic plants and behaves just as the other endogenous genes. The availability of plants with T-DNA inserts mapped in chromosomal locations brings a valuable new tool to plant genetics. These trans­ formants have an easily scoreable marker which can be used to map new mutants and a selectable chromosome tag that might facilitate breeding or selection of interesting linked genes. 496 HORSCH et al.

HOST-RANGE OF AGROBACTERIUM

A. tumefaciens has been shown to tranform a wide range of dicotyledonous species and a few monocots in the Liliaceae family (De Cleene and De Lay, 1976). Recent reports of transformation of Liliaceae family species (Hooykaas-Van Slogteren et al., 1984 and Hernalsteens et al., 1984) have sometimes been interpreted to mean that A. tumefaciens is virulent on all monocots when, in fact, it means that the host-range boundary is not between monocots and dicots but somewhere else. It really has no bearing on the potential to transform cells of grasses such as corn, wheat, or rice. While there are numerous arguments about this possibility, the data remain scarce. Two lines of evidence have been published that are suggestive of a compatibility between A. tumefaciens and corn. In the study by Grimsley et al. (1987), A. tumefaciens was used to introduce maize streak virus (MSV) into corn plants by the technique that has been coined "agroinfection" (Grimsley et al., 1986). This demonstrated a virulence gene- and border sequence-dependent transfer of MSV DNA from the T-DNA of A. tumefaciens into the corn plants, resulting in a systemic infection. It should thus be a very sensitive assay of gene transfer since a single event could produce the systemic infection. It does not prove integration of DNA into the corn genome or address practical issues related to germline access by cells that may be transformed. The second line of evidence was published by Graves and Goldman (1986), who detected nopaline or octopine in tissues of maize seedlings after infection with nopaline or octopine strains of A. tumefaciens. These studies are suggestive but not conclusive because of the undependable nature of opine production or opine-like sub­ stances in tissues of various plant species, including tissues known not to be transformed (Christou et al., 1986). Thus it remains unclear whether A. tumefaciens is capable of stable gene transfer to crops such as corn. Further analysis and better assay methods should provide a definite answer within the next year. It still remains to be seen if A. tumefaciens can be engineered to transform species such as corn by modification of attachment functions, induction or alterations to the virulence genes, or other modifications of the bacterium or coculture conditions. GENE TRANSFER TO PLANTS 497

ENGINEERING HERBICIDE TOLERANCE

The herbicide glyphosate is a potent inhibitor of an enzyme in the shikimate pathway in higher plants. The herbicide competitively blocks EPSPS (5-enolpyruvylshikimate-3-phosphate syn!hase) and kills plants as well as tissue cultures. We have engineered plants to be tolerant to otherwise lethal doses of glyphosate by a strategy of overexpression of EPSPS in transgenic plants. The method for achieving this goal started in tissue culture to obtain the EPSPS protein, proceeded through molecular biology to obtain and modify the gene, and back into tissue culture to put the gene back into plants. A glyphosate tolerant suspension culture of Petunia hybrida was selected by sequential step-up from 0.1 mM glyphosate to 20 mM glyphosate over a period of several months (Steinrucken et al., 1986). The resistant cell line (MP4G) produces about 20 times more EPSPS than the sensitive parent culture (MP4). The EPSPS protein was purified from the MP4G culture and this pure protein was partially sequenced to obtain an amino acid sequence from its NH2-terminus. A series of oligonucleotide probes was synthesized (in three families) that would cover all possible DNA sequences for the amino acid sequence. These families were then used to probe a northern blot of poly(A)+mRNA from the two cell lines. One of the families of probes hybridized to a specific mRNA from the MP4G culture about 20 times more strongly than to the same size mRNA from the MP4 culture. This strongly suggested that this probe was specific for EPSPS and that the MP4G culture was overproducing the mRNA for this enzyme. The gene was then isolated from a cDNA library prepared from the MP4G culture. The cause of the overproduction of the EPSPS mRNA was an amplification of the gene during the sequential adaptation of the culture to glyphosate (Shah et.al., 1986). Thus the suspension culture had provided an enriched source for purification of the protein, and a means to identify which probe (from a family of redundant probes) was the proper one to use to screen a cDNA library for the EPSPS gene. The culture was also used to prepare a cDNA library which was enriched for the EPSPS sequence. A derivative of the pMON505 vector, pMON530, was used for Agrobacterium-mediated transfer of a chimeric EPSPS gene back into plant cells. It contains the NOS-NPTII-NOS gene for kanamycin resistance and an expression cassette consisting of the cauliflower mosaic virus (CaMV) 35S promoter and a nopaline synthase (NOS) polyadenylation signal. The complete open reading frame of the petunia EPSPS was cloned into pMON530 to create a chimeric EPSPS 498 HORSCH et al. gene capable of high level expression in plant cells (pMON546). The vector was then introduced into A. tumefaciens for transfer to plant cells. Leaf discs transformed with vectors pMON505 and pMON546 both produced large amounts of kanamycin resistant callus. Small pieces of this callus were transferred to medium containing 0.1, 0.25, or 0.5 mM glyphosate. The callus transformed with pMON546 con­ tinued to grow at all concentrations of glyphosate while the callus transformed with pMON505 was inhibited. Glyphosate tolerance could also be demonstrated by direct selection for growth from infected leaf discs on the same concentrations of glyphosate. Four independent transgenic plants containing pMON546 were selected on kanamycin and grown in soil before spraying with a lethal dose of glyphosate. These plants were sprayed with formulated glyphosate (containing a surfactant) at a rate equal to 0.8 pounds per acre, twice the dose needed to kill all control plants. All four transformed plants survived and continued to grow with sight chlorosis at the growing points, from which they later recovered. These experiments demon­ strate that EPSPS is the prime target of glyphosate in plant cells and whole plants, since overexpression of that one gene confers tolerance to the herbicide. These results have been extended to tobacco and tomato using the petunia gene construction in pMON546.

CONCLUSION

Molecular biology and gene transfer technology have opened the door to unprecedented experimental power for understanding and manipulating plants (Broglie et al., 1984; Beachy et al., 1985a, 1985b; Rogers et al., 1986). This power will build on itself as we use it to discover the mechanisms of gene expression, protein structure and function, biochemistry and physiology of plants. This knowledge will rapidly increase our ability to intelligently predict beneficial changes to introduce into crops, as well as the means to do it. Advances in tissue culture regeneration systems and effective interaction with A. tumefaciens will provide a reproducible method for transfer of cloned, engineered genes in an increasingly wide range of crop species.

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Horsch, R. B., R. T. Fraley, S. G. Rogers, P. R. Sanders, A. Lloyd, and N. Hoffmann. 1984. Inheritance of functional foreign genes in plants. Science 223:496-498. ----, J. Fry, N. L. Hoffmann, M. Wallroth, D. Eichholtz, S. G. Rogers, and R. T. Fraley. 1985. A simple and general method for transferring genes into plants. Science 227:1229-1231. ----, H. J. Klee, S. Stachel, S. C. Winans, E. Nester, S. G. Rogers, and R. T. Fraley. 1986. Analysis of Agrobacterium tumefaciens virulence mutants in leaf discs. Proc. Natl. Acad. Sci. 83:2571-2575. ---- and ----. 1986. Rapid assay of foreign gene expression in leaf discs transformed by Agrobacterium tumefaciens: the role of T-DNA borders in the transfer process. Proc. Natl. Acad. Sci. 83:4428-4432. Klee H., F. White, V. Iyer, M. Gordon, and E. Nester. 1983. Mutational analysis of the virulence region of an Agrobacterium tumefaciens Ti plasmid. J. Bacteriol. 153:878-883. Klee, H. J., M. F. Yanofsky, and E. W. Nester. 1985. Vectors for transformation of higher plants. Bio/ Technology 3:637-642. Lloyd, A. M., A. R. Barnason, S. G. Rogers, M. C. Byrne, R. T. Fraley, and R. B. Horsch. 1986. Transformation of Arabidopsis thaliana with Agrobacterium tumefaciens using a gene conferring hygromycin resistance. Science 234:464-466. McCormick, S. , J. Niedermeyer, J. Fry, A. Barnason, R. Horsch, and R. Fraley. 1986. Leaf disc transformation of cultivated tomato (L. esculentum) using Agro­ bacterium tumefaciens. Plant Cell Reports 5:81-84. Nester, E., M. Gordone, R. Amasino, and M. Yanofsky. 1984. Crown gall: A molecular and physiological analysis. Ann. Rev. Plant Physiol. 35:387-413. Potrykus, I., R. D. Shillito, M. W. Saul, and J. Paszkowski. 1985. Direct gene t ransfer-state of the art and future potential. Plant Mol. Biol. Reporter 3:117-128. Rogers, S. G., D. M. Bisaro, R. B. Horsch, R. T. Fraley, N. L. Hoffmann, L. Brand, S. J. Elmer, and A. Lloyd. 1986. Tomato Golden Mosiac virus a component DNA replicates autonomously in transgenic plants. Cell 45:593-600. Schroder, G., S. Waffenschmidt, E. Weiler, and J . Schroder. 1984. The T-region of Ti plasmids codes for an enzyme synthesizing indole-3-acetic acid. Eur. J. Biochem. 138:387-391. Shah, D. M., R. B. Horsch, H. J. Klee, G. M. Kishore, J . A. Winter, N. E. Turner, C. M. Hironaka, P.R. Sanders, C. S. Gasser, S. Aykent, N. R. Siegel, S. G. Rogers, and R. T. Fraley. 1986. Engineering herbicide tolerance in transgenic plants. Science 233:4 78-481. 502 HORSCH et al.

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TISSUE CULTURE SELECTION FOR DISEASE RESISTANT PLANTS

T. J. McCoy1

ABSTRACT. Current plant cell and tissue culture applications to plant pathology include: (1 ) direct selection of resistant plants via selecting cell lines resistant to pathogen-produced toxin(s), (2) screening large populations of regenerated plants for resistant somaclonal variants, (3) evaluating resistance in vitro, ( 4) studies of host-pathogen interactions, (5) transferring disease resistance genes via interspecific hybridization using embryo rescue or somatic hybridization, and (6) transferring genes for disease resistance via genetic engineering techniques following isolation of the genes. Using Fusarium wilt resistance of alfalfa (causal agent, Fusarium oxysporum f. sp. medicaginis) as a case study, results are reported for the selection of Fusarium wilt resistance by selecting cell lines resistant to culture filtrates of the pathogen. Importantly, the regenerated plants were resistant to the pathogen in greenhouse and field evaluations. Inheritance studies indicated that resistance is due to a single dominant gene. In addition to the potential for selecting resistant plants, tissue culture also may offer some advantages in the evaluation of disease resistant genotypes. Comparing the callus response to Fusarium culture filtrate and the whole plant response to the pathogen demonstrated three potential responses: (#1) callus resistant, plant resistant; (#2) callus susceptible, plant resistant; or (#3) callus susceptible, plant susceptible. Inheritance studies indicated that whole plant resistance responses #l and #2 are likely controlled by different genes, and they may represent different resistance mechanisms. Therefore, tissue culture may be a useful tool to the alfalfa plant breeder for evaluation of disease resistance as well as for the selection of new disease resistance gene(s). Index descriptors: alfalfa, Medicago sativa, Fusarium wilt, Fusarium oxy­ sporum f. sp. medicaginis, culture filtrates, disease resistance, host-pathogen interactions.

APPLICATIONS OF TISSUE CULTURE TO PLANT PATHOLOGY

There are several ways in which plant tissue and cell cultures are current ly being utilized in plant pathology research (recent reviews of Wenzel, 1985; Wenzel et al., 1987; and Daub, 1986). One frequently proposed application of tissue culture in plant improvement pro­ grams is the production of disease resistant plants. Disease resistant plants may be recovered from cell culture either by direct selection for resistance to toxin(s) produced by the pathogen (either purified toxin, toxic culture filtrates or toxin analogs) or by regenerating large

1Department of Plant Sciences, The University of Arizona, Tucson, AZ 85721. 504 McCOY numbers of plants and screening for resistant somaclonal variants. Either procedure may rely on spontaneous or induced mutations, although there is no direct evidence that induced mutagenesis improves the recovery of resistant variants. Interest in direct selection of resistant genotypes was generated when tobacco (Nicotiana tabacum L.) plants resistant to wildfire disease caused by Pseudomonas tabaci (Wolf and Foster) Stevens were recovered from cell lines selected for resistance to methionine sulfoximine (Carlson, 1973). Methionine sulfoximine is a methionine analog that produces symptoms on plants similar to the pathogen, and is structurally similar to a toxin produced by the pathogen. Subsequent to the pioneering work on tobacco, the potential of tissue culture selection has been demonstrated by regenerating maize (Zea mays L.) plants resistant to Helminthosporium maydis (Nisikado and Miyake) race T from tissue cultures selected for resistance to H. maydis toxin (Gengenbach et al., 1977; Brettell et al., 1979). Selection of cell lines resistant to culture filtrates of the pathogen (or to purified toxins) followed by plant regeneration from the selected cell lines has also resulted in: oat (Avena sativa L.) plants resistant to Helminthosporium victoriae (Meehan and Murphy) (Rines and Luke, 1985); Brassica napus L. plants resistant to Phoma lingam (Tode ex Fr.) Desm. (Sacristan, 1982); potato (Solanum tuberosum L.) plants resistant to Fusarium oxysporum Sehl. f. sp. tuberosi (Weimer) Snyd. and Hans. (Behnke, 1980a) and resistant to Phytophthora infestans (Mont.) de Bary (Behnke, 1979, 1980b); tobacco plants resistant to Alternaria alternata (Thanutong et al., 1983); and rice (Oryza sativa L.) plants resistant to Helminthosporium oryzae (Ling et al., 1985). In addition, our research on alfalfa (Medicago sativa L.) (Hartman et al., 1984) to be described here has resulted in alfalfa resistant to Fusarium oxysporum Sehl. f. sp. medicaginis (Weimer) Snyd. and Hans. Screening of somaclonal variants has resulted in the identifica­ tion of: regenerated potato plants resistant to Phytophthora infestans (Shepard et al., 1980), and Alternaria solani (Ellis and Martin) Jones and Groat (Matern et al., 1978); sugarcane plants resistant to Fiji disease, Sclerospora sacchari T. Miyake, and Drechslera sacchari (Butl.) Subram. and Jain (Heinz et al., 1977; Larkin and Scowcroft, 1981); maize plants resistant to H. maydis race T (Brettell et al., 1980); and rice plants resistant to H. oryzae (Ling et al., 1985). Considering alfalfa in particular, plants regenerated from unselected protoplasts have been shown to be resistant to Verticillium albo­ atrum Reinke and Berth (Latunde-Dada and Lucas, 1983, 1986) and TISSUE CULTURE SELECTION 505 to a crown rot fungal complex (Johnson et al., 1984). Because resistant plants can be regenerated from selected as well as unse­ lected cell lines, conclusive evidence that resistance can be selected directly in vitro has not been demonstrated. Cell cultures are also being utilized for studies of host-pathogen interactions (Diner et al., 1984; Helgesen et al., 1972, 1976; Huang and van Dyke, 1978; Miller et al., 1984). Several studies using species as diverse as tobacco, white pine and soybean have demonstrated that host-pathogen responses at the whole plant level are the same in tissue culture (Deaton et al, 1982; Diner et al., 1984; Randa et al., 1982; Helgeson et al., 1976; Latunda-Dada and Lucas, 1985, 1986; Maronek and Hendrix, 1978). Miller et al. (1984) used an alfalfa tissue culture system to study host resistance to the alfalfa pathogen Phytophtlwra megasperma f. sp. medicaginis and to the nonhost pathogen P. megasperma f. sp. glycinea. Callus cultures mirrored the whole plant response in regard to ability of the fungus to colonize the callus. Callus cultures from alfalfa plants susceptible to P. mega­ sperma f. sp. medicaginis were quickly colonized by the fungus whereas callus cultures from resistant plants were only minimally colonized. Phytophtlwra megasperma f. sp. glycinea had limited colonization on either alfalfa callus. Miller et al. (1984) found that fungal growth was not absolutely inhibited in any interaction, but they indicated that the callus response was similar to the seedling response. The potential of tissue culture in determining factors that influence expression of alfalfa disease resistance remains to be determined. However, the results of Latunde-Dada and Lucas (1985, 1986) demonstrate the potential of this type of an approach. The differential response of callus lines of alfalfa to Verticillium albo­ atrum is associated with the amount of the phytoalexin medicarpin that accumulates in the infected tissue (Latunde-Dada and Lucas, 1985). In addition, the sensitivity to the fungus, V. albo-atrum, and the amount of medicarpin accumulated in callus lines at low (20°C) and high (28°C) temperature correspond to the expression of the disease jn whole plants (Latunde-Dada and Lucas, 1986). Alfalfa is more susceptible to V. albo-atrum at lower temperatures (20°C) than at higher temperatures (>25°C). Latunde-Dada and Lucas (1986) found that V. albo-atrum grew faster at 20°C than at 28°C on alfalfa callus cultures, and that the phytoalexin medicarpin accumulated earlier and faster at 28°C. In addition, the resistant callus line had a significantly greater medicarpin concentration than the susceptible callus line. 506 McCOY

In situations where the tissue culture response (whether to toxins, culture filtrates, or the pathogen) is precisely correlated with the whole plant response to the pathogen, the in vitro response can be used to evaluate resistance. Hartman et al. ( 1986) reported that a system of screening callus could identify halo blight (Pseudomonas syringae pv. phaseolicola) resistant bean (Phaseolus vulgaris L.) cultivars. The evaluation of resistance was based on the response of callus to media containing toxic culture filtrates of the pathogen. However, negative results concerning in vitro evaluations have also been reported. Vardi et al. (1986) found that callus sensitivity of Citrus cultivars to culture filtrates of the pathogen Phytophthora citrophythora was inversely related to the cultivars whole plant resistance. Calli derived from resistant varieties were highly sus­ ceptible to culture filtrates whereas susceptible varieties produced tolerant calli (Vardi et al., 1986). Interspecific hybridization has frequently been used to transfer genes for disease resistance from wild species into cultivars (Stalker, 1980). In vitro approaches are currently being used to recover interspecific hybrids where conventional sexual crosses are incom­ patible. These approaches include in vitro embryo rescue techniques and somatic hybridization. For example, McCoy and Smith (1986) have recently developed an ovlile-embryo culture method for recover­ ing interspecific hybrids between alfalfa and other Medicago species. A major motivation for producing various interspecific hybrid combina­ tions was the disease resistance present in the wild species (McCoy and Smith, 1986). There are numerous other examples of in vitro embryo rescue of interspecific hybrids (Collins et al., 1984). Frequently, the final goal was transfer of disease resistance genes. Somatic hybridization via protoplast fusion has resulted in the production of interspecific and intergeneric hybrids between sexually incompatible species. Evans et al. (1981 ) observed local lesion-type resistance to tobacco mosaic virus (TMV) in somatic hybrids between tobacco and N. nesophila. The Solanum brevidens genes for potato leaf roll virus resistance were expressed in hexaploid somatic hybrids between potato and S. brevidens (Austin et al. , 1986; Helgeson et al., 1986). However, there are major problems with somatic hybrids for transferring disease resistance genes or other useful traits. When the somatic hybrid is an addition product combining the nuclear comple­ ment of two species, there can be extreme difficulty in backcrossing the gene(s) of interest into elite germplasm. A common initial hurdle is complete male and female sterility of the somatic hybrid. Even if the TISSUE CULTURE SELECTION 507 somatic hybrid is fertile, genetic recombination between the two species genomes may not occur. If genes for resistance can be isolated and cloned, then genetic engineering technology may be useful in transferring the gene of interest. It is likely that disease resistance genes will be cloned in the near future by tagging with transposable elements, similar to the procedures used to clone the bronze locus and the al locus of maize (Fedoroff et al., 1984; O'Reilly et al., 1985). Recently, Agrobacterium tumefaciens mediated gene transfer was used to transfer the coat protein gene of tobacco mosaic virus (TMV) into tobacco plants (Abel et al., 1986). The transgenic plants had delayed disease development when inoculated with TMV. In a similar manner Agrobacterium­ mediated gene transfer was used to incorporate the alfalfa mosaic virus coat protein gene into tobacco plants. The transgenic plants produced alfalfa mosaic virus coat protein, and these plants developed fewer primary infections and had slower systemic infection following inoculation with the virus (Loesch-Fries et al., 1987; Turner et al., 1987).

EXPERIMENTAL RESULTS

Selection for Fusariurn Wilt Resistance We have attempted to produce alfalfa plants resistant to Fusar­ ium oxysporum Schlecht f. sp. medicaginis (Weimer) Snyd. and Hanson by selecting cell lines resistant to toxin(s) produced by the pathogen. At the outset, it must be pointed out that the term toxin is used in a loose sense because we do not know the chemical structure of the component or components that are toxic to alfalfa cells. The results will be divided into three parts. The first will describe the selection of cell lines resistant to toxic Fusarium oxysporum filtrates and the characterization of plants regenerated from the resistant cell lines. The second section will present a preliminary attempt to characterize the toxic component(s) of the filtrate. The third part will describe the use of tissue culture to evaluate resistance to Fusarium wilt, and will include a discussion of the potential separation of mechanisms of resistance to Fusarium wilt.

Selection of Cell Lines Resistant to Fusarium oxysporum Culture Filtrates The selective agent in these experiments was the culture filtrate of Fusarium oxysporum f. sp. medicaginis. The isolate of the fungus used was maintained on soil tubes. An attempt was made to maintain 508 McCOY similar concentration of toxic components in all preparations of culture filtrate. This was done by using similar amounts of incculum from the soil tube to initiate each fungal culture. In all experiments, the fungal inoculum was placed in 250ml of Czapek-Dox broth, and cultures were grown for 12 days at 22°C. After 12 days, filtrate was prepared by sequential filtration through a No. 541 Whatman filter, a Schleicher and Schuell No. 24 glass filter circle, and a 3 micron cellulose acetate filter. Undiluted culture filtrate was used to prepare selective media. To determine if cellular selection for Fusarium wilt resistance is feasible, it was necessary to establish if toxic components were present in the culture filtrate of the pathogen, and if the response to the filtrate correlated with the whole plant response to the pathogen. Initial screening results wit h two known Fusarium wilt resistant alfalfa clones and two known susceptible clones demonstrated that callus cultures of the susceptible clones died on a medium containing 152ml of Fusarium oxysporum culture filtrate per liter. However, callus from the resistant clones were not significantly inhibted on medium containing 152ml of culture filtrate compared to control medium. Based on these results selection experiments were con­ ducted with callus cultures derived from known Fusarium wilt susceptible genotypes. The genotypes used were also known to be capable of plant regeneration following 12 months' growth in culture. Initial experiments used a step-up selection procedure whereby we first selected cultures resistant to medium containing 152ml of culture filtrate per liter. Once resistance to this filtrate concentration was obtained, cell lines were selected for resistance to medium containing 360ml of filtrate per liter. The problem with this procedure is that it was long-term, requiring 15 months growth in vitro. As a result, none of the plants regenerated from these long-term cultures had the normal tetraploid (2n = 4x = 32) complement. All plants were either octoploid or hexaploid (Hartman et al., 1984; and Table 1). Cytogenetic changes in vitro are known to be a common type of somaclonal variation (Larkin and Scowcroft, 1981), and these changes are known to increase with culture age (McCoy et al., 1982). The subsequent experiments were designed to minimize the time in culture. Selections were conducted with 360ml of culture filtrate per liter from the outset. Following the selection of putative resistant lines, cultures were placed on regeneration medium. These shorter­ term cultures produced regenerated plants with the normal tetra­ ploid chromosome number. Table 1. Chromosome number of regenerated plants, callus response to Fusarium osyxporum filtrate and whole plant response to Fusarium oxysporum. No. of Regen- Response to Fusarium oxysporum Original Time in erated plants Chromosome No. Callus Planta Clone Culture (mo.) Evaluated 32 48 64 R s R s Regen-1 15.0 14 0 2 12 12 2 12 2 7.5 15 15 0 0 15 0 15 0 7.5 2b 2 0 0 0 2 0 2

Regen-2 7.5 5 5 0 0 5 0 5 0 7.5 12b 12 0 0 8 4 a Results of field and/ or greenhouse evaluation. R = resistant. S = susceptible. b These plants were regenerated from unselected cultures which had only been grown on control medium.

01 0 '° 510 McCOY

The response to Fusarium ox ysporum filtrate of callus from the regenerated plants was tested to determine if resistance to the culture filtrate was maintained following plant regeneration. Table 1 demonstrates that all but two of the plants regenerated from selected resistant cell lines retained resistance at the callus level following regeneration. It should be pointed out that callus initiated from the original Regen-1 and Regen-2 plants consistently gave a susceptible response to the culture filtrates. Callus from two plants regenerated from control cell lines of Regen-1 was susceptible to the culture filtrate (Table 1). The 12 plants regenerated from control cell lines of Regen-2 were not tested at the callus level. The whole plant response of the regenerated plants to Fusarium ox ysporum was evaluated by standard procedures for evaluating Fusarium wilt resistance (Elgin et al., 1984) except that vegetative cuttings instead of seedlings were used. Vegetative propagules were produced for all plants to be tested by rooting cuttings in a mist bench. The rooted cuttings were placed in sand and allowed to grow for 12 weeks. The cuttings were then washed and immersed in a Fusarium oxysporum inoculum containing 106 spores per ml for 30 minutes. Plants were then transplanted to sterile sand benches for the greenhouse evaluation, or else the inoculated plants were trans­ planted in the field. After 12 weeks the plants were evaluated for susceptibility or resistance. Evaluations are based on a 0 to 5 rating scale where:

0 = no discoloration in the root, 1 = small, dark strands in the stele, 2 =small, dark brown arcs or rings in the stele, 3 =larger, dark brown areas, arcs or rings, 4 =entire outer stele dark brown; plant alive, 5 = plant dead.

With this system, a rating of 0 to 2.0 is considered to be resistant, with 2.0 only moderately resistant. The results of the field and/ or greenhouse tests demonstrated that in most cases a level of resistance capable of field expression was selected (Table 1 ). Two of the plants regenerated from 15-month-old resistant cell lines of Regen-1 were susceptible in t he field tests as well as the callus test. Of interest was the fact that most of the resistant regenerated plants had an average severity index (AS.I.) of 0 to 1.0 indicating a high level of resistance. In the field experiments, the standard resistant check, Moapa 69, and the standard susceptible check, Narragansett, were evaluated by TISSUE CULTURE SELECTION 511 testing 60 plants of each grown from seed. The AS.I. for Moapa 69 was 1.2, and the AS.I. for Narragansett was 3.8. Eight of 12 regen­ erated plants from unselected cell lines of Regen-2 were also resistant to the pathogen in field tests. This result demonstrates that soma­ clonal variation for Fusarium wilt resistance does occur, and is similar to observations of disease resistant somaclones for other diseases of alfalfa (Johnson et al., 1984; Latunde-Dada and Lucas, 1983) and other plant species (Daub, 1986; Wenzel, 1985; Wensel et al., 1987). The critical factor in selecting useful agronomic traits using tissue culture systems is that the selected trait, if expressed in regenerated plants, must be heritable. Our results with Fusarium satisfy this criterion (Table 2). If a single dominant gene for resistance was selected in culture, one would expect a cross to a susceptible plant or to the original unselected clone to segregate one resistant plant to one susceptible plant. This prediction is based on the assumption that the original genotype of Regen-1 or the susceptible clone of Narragansett, NaS-38, is homozygous recessive. Designating the genotype of Regen-1 and NaS-38 as aaaa, then the FR-13, FR-14, FR-19, and FR-20 regenerated plants could be Aaaa. The reason for crossing FR plants to NaS-38 is that the cross to Regen-1 is difficult because of self incompatibility. The results are close to agreeing with the hypothesis of a single dominant gene conferring resistance, except for FR-13 (Table 2). One explanation for FR-13 is that it is in fact duplex, AAaa, and perhaps arose from a somatic recombination event following the original mutation from aaaa to Aaaa. If this were the case, one would expect a 5 resistant:! susceptible segregation, which is what was observed. The results presented in Table 2 clearly show that the selected resistance trait is heritable.

Preliminatry Attempt to Characterize Toxic Component(s) of the Fusarium oxysporum Filtrate Considerable research has been conducted on plant pathogenic toxins and the role of these toxins in plant disease (Durbin, 1980; Scheffer, 1983; Yoder, 1980). One of the toxic components isolated from cultures of species of Fusarium is fusaric acid (Davis, 1969; Gaumann, 1957). Studies on Fusarium wilt of tomato indicated that fusaric acid is responsible for part of the symptoms of the disease. To determine if our selection experiments had resulted in selecting cell lines resistant to fusaric acid, cell viability of the unselected cell line of Regen-1 and the selected filtrate resistant cell line, designated S-A (Hartman et al., 1984) was determined on media containing fusaric 512 McCOY

Table 2. Inheritance of tissue culture selected Fusarium wilt resistance. Number In Vitro Response Chi-square Cross Tested Resistant Susceptible Expect (I:I) N aS-38, selfed 73 0 73 Regen-I, selfed I6 0 I6 Regen-I x N aS-38 58 0 58

FR-I3 x NaS-38 55 45 10 22.27a FR-I4 x NaS-38 39 I3 26 4.33 FR-19 x NaS-38 59 37 22 3.8I FR-20 x NaS-38 3I I4 I7 0.29

NaS-38 x FR-I4 20 11 9 0.20 NaS-38 x FR-20 37 I3 24 3.27 Sum (exclude FR-I3) I86 88 98 0.54

a Chi-square for 5: 1 segregation is 0.09.

acid. Callus cultures of the two cell lines were grown for 7 days on media containing 0, O.I, 1.0, 50, 100, and 1000 ppm fusaric acid. Cell viability was then determined by the fluorescein diacetate staining technique of Widholm (1972). Three callus colonies per treatment were sampled with three slides per colony. A minimum of 400 cells were counted per slide, and percent cell viability was calculated by dividing the number of viable cells by the total number of cells counted. A comparison of the percent cell viability of the unselected cell line and the selected cell line showed no differential response to fusaric acid (Table 3). Both cell lines were severely affected by 100 ppm fusaric acid and a concentration of I 000 ppm fusaric acid was lethal to both cell lines. Therefore, Fusarium filtrate resistance expressed by cell line S-A is not due to resistance to fusaric acid. However, this result does not demonstrate fusaric acid is not produced by the isolate used. The above technique of determining cell viability for Regen-I and S-A cell lines grown on various media was used for additional TISSUE CULTURE SELECTION 513

Table 3. Percent cell viability for the unselected cell line, Regen-I, and the selected Fusarium filtrate resistant cell line S-A, grown on media containing various concentrations of fusaric acid. Fusaric Acid Concentration Mean Percent Cell Viability8 (ppm) Regen-I Selected S-A 0 34.4 ± 2.9 38.9 ± 5.I O.I 38.4 ± 2.3 30.6 ± 4.3 1.0 26.0 ± 3.3 31.3 ± 2.8 50 21.7 ± 4.5 23.5 ± 3.I 100 6.2 ± 3.5 3.8 ± 2.6 1000 0 0

a ± 1 standard deviation.

characterization of the filtrate. Cell viability analysis also demon­ strated that resistance to the filtrate was not due to resistance to fusidic acid or moniliformin, two additional toxic components pro­ duced by Fusarium species (Kern, I972). In addition, cell viability analyses demonstrated that the toxic component(s) in this isolate are highly polar (soluble in water and slightly soluble in methanol and ethanol) and heat-stable (unpublished data).

Evaluation of Fusarium Wilt Resistance

In systems where the tissue culture response (whether to toxins, culture filtrates, or the pathogen itself) is precisely correlated with the whole plant response to the pathogen, the in vitro response can be used to evaluate resistance. Although current procedures for the evaluation of Fusarium wilt resistance have been very effective in developing Fusarium wilt resistant alfalfa varieties, there are several limitations to the procedure. These include: (1) It is long-term, requiring 6 months to complete an evaluation; (2) there is a definite possibility of escapes; (3) the procedure does not allow for a separation of resistance mechanisms, which may be controlled by different genes. Elucidation of different genes controlling Fusarium resistance would greatly aid the deployment of genes for resistance, 514 McCOY as well as making it possible to more accurately define the level of resistance one would expect in a given population. Inheritance studies conducted on artificially inoculated alfalfa plants concluded that Fusarium wilt resistance is controlled by at least two genes (Hijano et al., 1983). According to the model of Hijano et al. (1983), one of the genes is completely dominant and the other gene is additive. Different degrees of resistance are produced by the additive gene in the absence of the dominant gene. If the completely dominant gene is designated A and the additive gene is designated B, then a plant carrying at least one A allele (i.e., Aaaa, AAaa, AAAa, or AAAA) would be resistant; however, in the absence of the A gene (aaaa), for the B gene to confer a high level of resistance, it must be quadruplex (BBBB). Triplex (BBBb) plants are intermediate and duplex (BBbb), simplex (Bbbb), and nulliplex (bbbb) plants are susceptible. Considering just two genes conferring resistance, it is possible to envision the segregation of susceptible genotypes in producing a synthetic variety from initial clones selected for Fusarium wilt resistance. For discussion purposes, consider the simplest scenario where a two-clone synthetic population is to be derived from resistant parent 1, genotype Aaaa bbbb, and resistant parent 2, genotype aaaa BBBB. The two-clone synthetic population derived from parent 1 and 2 will contain susceptible genotypes following segregation of the two genes. It is interesting to note that the number of Fusarium wilt resistant plants in Fusarium wilt resistant, synthetic populations usually does not exceed 75 t o 80% (Hijano et al., 1983). In fact, the Fusarium wilt resistant, check variety Moapa 69, contains approx­ imately 20% susceptible plants. The inability to recover uniformly resistant synthetic populations may be due to the segregation of different genes controlling resistance or to the presence of escapes in the original selections. Tissue cµlture evaluation of Fusarium wilt resistance in alfalfa may allow the development of synthetic populations containing all resistant plants by eliminating escapes and by potentially separating resistance mechanisms. A number of clones from three alfalfa populations, Moapa 69 (the resistant check variety in Fusarium evaluation nurseries), Furez 521 (an experimental population from Hungary bred for Fusarium wilt resistance), and Narragansett (the susceptible check variety in Fusarium evaluation nurseries), were evaluated at the whole plant and callus level. The callus response was visually rated as resistant or susceptible based on growth on media TISSUE CULTURE SELECTION 515 containing 360ml of Fusarium filtrate per liter. Whole plant evaluations were conducted on vegetative propagules grown in the greenhouse as described in the first section. Each clone tested gave one of three responses: (#1) resistant callus, resistant plant; (#2) susceptible callus, resistant plant, and (#3) susceptible callus, susceptible plant. Of the 197 plants tested, 49 gave response #l, 63 produced response #2, and 85 produced response #3. It is important to note that all plants demonstrating callus resistance are also resistant at the whole plant level, and that all susceptible plants at the whole plant level are also susceptible at the cellular level. Response # 1 and #2 that are both resistant at the whole plant level may represent different mechanisms of resistance to Fusarium wilt. Preliminary observations on the callus and whole plant response of progeny of clones representing different whole plant resistance responses appears promising (Table 4). Callus of the progeny was initiated on control medium by excising two-thirds of the cotyledons of aseptically germinated seeds. After four weeks' growth on control medium, callus was transferred to filtrate medium containing 360ml of Fusarium filtrate. Callus was visually rated as resistant or sus­ ceptible after 2 weeks. Progeny which produced poor callus growth were eliminated from the whole plant test. Following removal of the cotyledons for callus initiation, the seedlings were transferred to sand and grown in the greenhouse for approximately 12 weeks. The seedlings were then inoculated with Fusarium oxysporum as described in part 1, transplanted to the field, and rated as resistant or susceptible as described earlier. In this evaluation of whole plants, the seedling itself was used and not vegetative propagules. Although the number of progeny evaluated is limited, the observed segregation ratios indicate resistance response # 1 and response #2 are controlled by different genes, and they may represent different resistance mechanisms (Table 4). Plants RMl-7, -13, and -38 expressed response #l, and plants RM2-6, -22 and -53 expressed response #2. If response # 1 is controlled by a single dominant gene, designated A, and response #2 is controlled by a different dominant gene, designated B, then it is postulated that plants with genotype A __bbbb express response #l, plants with genotype aaaa B __express response #2 and plants with genotype aaaabbbb express response #3. In addition, plants with both dominant genes, A_B__ , express response # 1. If the following hypothetical genotypes are assigned, then the observed segregation ratios Table 4. Callus and whole plant response of progeny produced from crossing plants with resistance response #l or response #2. Number of Progeny progeny with indicated responsea Crossh Tested Response #l Response #2 Response #3

RMl-7 x RMl-13 34_ 27 0 7 RMl-7 x RMl-38 27 25 0 2

RM2-6 x RM2-53 48 0 48 0 RM2-6 x RM2-53 35 0 35 0 RM2 -53 x RM2-22 47 0 43 4

RMl-7 x RM2-6 65 28 37 0 RMl-13 x RM2-6 31 15 16 0 RMl-7 x RM2-53 76 40 25 11 RMl-13 x RM2-53 102 58 21 23 RM2-53 x RMl-13 53 21 18 14 RM2-53 x RMl-7 63 24 13 26 a Response #l =resistant plant: resistant callus. Response #2 = resistant plant: susceptible callus. Response #3 = susceptible plant: susceptible callus. b RMI-plants expressed resistance response #l; and RM2-plants expressed resistance response #2. TISSUE CULTURE SELECTION 517 presented in Table 4 fit the expected segregation ratios based on the above assumptions:

Plant Genotype RMl-7 and RMl-13 Aaaa bbbb RMl-38 AAaa bbbb RM2-6 aaaa BBBb RM2-22 aaaa BBbb RM2-53 aaaa Bbbb

Additional progeny will be evaluated to determine if the hypoth­ esis outlined above is valid. However, the following observations are consistent with different genes controlling response # 1 versus response #2: (a) progeny expressing response #2 were not observed when two plants expressing response # 1 were crossed; (b) progeny expressing response # 1 were not observed when two plants express­ ing response #2 were crossed; and ( c) progeny from crossing response # 1 plants and response #2 plants segregated all three responses. The preliminary data presented here indicate that a callus evaluation can be used to identify Fusarium wilt resistant plants. In addition, use of a callus evaluation may result in developing synthetic populations with all plants expressing Fusarium wilt resistance.

LITERATURE CITED

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McCoy, T. J., R. L. Phillips, and H. W. Rines. 1982. Cytogenic analysis of plants regenerated from oat (Avena sativa) tissue cultures; High frequency of partial chromosome loss. Can. J. Genet. Cytol. 24:37-50. ---- and L. Y. Smith. 1986. lnterspecific hybridization of perennial Medicago species using ovule-embryo culture. Theor. Appl. Genet. 71:772-783. Miller, S. A., L. C. Davidse, and D. P. Maxwell. 1984. Expression of genetic susceptibility, host resistance, and nonhost resistance in alfalfa callus tissue inoculated with Phytophthora megasperma. Phytopathology 74:345-348. O'Reilly, C., N. S. Shepherd, A. Pereira, Z. Schwarz-Sommer, I. Bertram, D. S. Robertson, P. A. Peterson, and H. Saedler. 1985. Molecular cloning of the al locus of Zea mays using the transposable elements En and Mul. EMBO J. 4:877-882. Rines, H. W. and H. H. Luke. 1985. Selection and regeneration of toxin-insensitive plants from tissue cultures of oats (Avena sativa) susceptible to Helmin­ thosporium victoriae. Theor. Appl. Genet. 71:16-21. Sacristan, M. D. 1982. Resistance responses to Phoma lingam of plants regenerated from selected cell and embryogenic cultures of haploid Brassica napus. Theor. Appl. Genet. 61:193-200. Scheffer, R. P. 1983. Toxins as chemical determinants of plant disease, pp. 1-40. In: J.M. Daly and J.M. Deverall (eds.). Toxins and Plant Pathogenesis. Academic Press, Australia. Shepard, J. F., D. Bidney, and E. Shahin. 1980. Potato protoplasts in crop improvement. Science 208:17-24. Stalker, H. T. 1980. Utilization of wild species for crop improvement. Adv. Agron. 33:111-147.

Thanutong, P., I. Furusawa, and M. Yamamoto. 1983. Resistant tobacco plants from protoplast-derived calluses selected for their resistance to Pseudo­ monas and Alternaria toxins. Theor. Appl. Genet. 66:209-215. Turner, N. E., K. M. O'Connell, R. S. Nelson, P. R. Sanders, R. N. Beachy, R. T. Fraley, and D. M. Shah. 1987. Expression of alfalfa mosaic virus coat protein gene confers cross-protection in transgenic tobacco and tomato plants. EMBO J . 6:1181-1188. Vardi, A. , E. Epstein, and A. Breiman. 1986. Is the Phytopthora citrophthora culture filtrate a reliable tool for the in vitro selection of resistant Citrus variants? Theor. Appl. Genet. 72:569-574. TISSUE CULTURE SELECTION 521

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IOWA STATE JOURNAL OF RESEARCH I MAY, 1988 523-535 Vol. 62, No. 4

THE USE OF CELL SELECTION TO OBTAIN NOVEL PLANT GENOTYPES RESISTANT TO MINERAL STRESSES

C. P. Meredith 1, A J. Conner2, and T. M. Schettini3

ABSTRACT. Because plant cells cultured in vitro are genetically heterogeneous and because entire plants can be regenerated from them, appropriately designed selection strategies can be used to isolate novel plant genotypes from cell cultures. Among agriculturally important traits, resistance to specific mineral stresses associated with marginal soils is particularly amenable to this approach. With the use of a selection medium designed to simulate the chemical environment of an aluminum-toxic soil, 246 aluminum-resistant variants were isolated from cell cultures of Nicotiana plumbaginifolia Viv. Sixty-seven of these variants retained their resistance after prolonged culture in the absence of aluminum. Fertile plants could be regenerated from 40 of the 67. The seedling progeny of each of these 40 variants initially all segregated for aluminum resistance in ratios consistent with a single dominant mutation. Subsequent analysis of this material suggests more complex genetic control of this phenomenon. Index descriptors: aluminum toxicity, aluminum resistance, mineral stress, cell selection, Nicotiana plumbaginifolia.

INTRODUCTION

Plant cells cultured in vitro are genetically heterogeneous. While some of this genetic variability may reflect preexisting genetic differ­ ences among the cells of the explant used to initiate the culture, most of the variation arises in culture by mechanisms not yet well­ understood (Karp and Bright, 1985). In some cases, these genetic changes are stable and are expressed and sexually transmitted in plants regenerated from the cultured cells (Evans and Sharp, 1986). Evidence exists for several kinds of genetic change occurring in cultured cells, including gross chromosomal changes (e.g., Orton, 1980), single nucleotide base changes (e.g., Brettell et al., 1986), and gene amplification (e.g., Shah et al., 1986) and deamplification (e.g.,

1Associate Professor, Department of Viticulture and Enology, University of California, Davis, CA 95616. 2Staff Scientist, Crop Research Division, DSIR, Private Bag, Christchurch, New Zealand. 3Postdoctoral Researcher, Department of Viticulture and Enology, University of California, Davis, CA 95616. 524 MERED ITH et al.

Cullis and Cleary, 1986). The mobilization of transposable genetic elements may also generate variability in vitro (Freeling, 1984; Walbot and Cullis, 1985) as well as other as yet undiscovered genetic mechanisms. Fortunately, a complete understanding of these phenomena is not a prerequisite for their exploitation. A novel trait that is stable and heritable may constitute a valuable improvement in a crop species. In vegetatively propagated species, heritability may not even be required. Depending upon the trait, such useful variation may be identified among regenerated plants or among cultured cells. Each of these general approaches has its advantages. A large number of plants can simply be regenerated from cultured cells and then evaluated for variation in traits of interest (e.g., Evans and Sharp, 1983). This approach can be laborious and can require a significant amount of space for the plants, especially if mature plants must be used, as would be the case, for example, if fruit or grain characteristics were of interest. This approach also requires an efficient method for screening a large number of plants, preferably visual. If the plants are grown in the field, environmental variability (such as soil moisture or pathogen distribution) can confound the identification of desirable variants.

CELL SELECTION

On the other hand, some phenotypes can be identified at the cellular level. For these phenotypes, variants can be obtained much more efficiently by cell selection than by screening regenerated plants because large numbers of cultured cells can be maintained in a small space, and, more importantly, selection pressure can be applied to the cell population to enrich the population for the desired phenotype. Plants regenerated from the selected cells can then be further evaluated for expression and transmission of the trait. A major difficulty with this approach is the design of the selection strategy. In order to establish conditions that will permit the preferential survival or growth of the desired cells, one must under­ stand the cellular basis of the phenotype of interest. Selection conditions are most easily developed in the case of resistance phenotypes. The cell population is simply challenged with the stress to which resistance is sought, for example, a chemical stress such as a herbicide or an antibiotic. The range of phenotypes that may be obtained via cell selection is limited to those that have a recognized cellular basis. Many RESISTANT PLANT GENOTYPES 525 agriculturally important traits are not simply cellular; they reside at levels of organization above the cell and are the integrated result of many plant functions (e.g., plant architecture or maturity date). Such characteristics cannot be detected in cultured cells. Other traits may have a cellular basis, but may be developmentally regulated and thus not necessarily expressed normally in cultured cells (e.g., flavor characteristics that result from secondary biosynthetic pathways). Yet another class of agriculturally interesting characteristics com­ prises those that may well have a cellular basis that could be amenable to cell selection, but are presently so poorly understood that we do not yet recognize their cellular manifestation. The membership of this latter group will naturally shrink as our know­ ledge of plant biology expands. A distinct advantage of cell selection is the potential for adding a single attribute to an otherwise well-adapted genotype. While this can readily be accomplished by conventional recurrent backcrossing in many crops, there are others that are not amenable to such breeding programs, by virtue of their long generation time or sterility. Certainly many perennial crops fall into this category. Such backcrossing programs also require an identified and available source of germ­ plasm as a donor of the new trait. Apart from the major agronomic crops, germplasm resources are limited or not well-characterized for many crop species. While genetic transformation technology clearly offers a more directed and specific means to introduce single traits than does cell selection, at the present time the agricultural applica­ tions of this powerful technology are limited to a very few traits for which the molecular basis is well-characterized (e.g., resistance to some herbicides, viruses, and insects) (Comai et al., 1985; Abel et al., 1986; Shah et aL, 1986; Fischhoff et al., 1987; Turner et al., 1987; Vaeck et al., 1987). Until more is known about the molecular basis of plant processes, cell selection will continue to offer a means to isolate certain novel genotypes.

MINERAL STRESS

If the array of agriculturally important plant characteristics is considered in light of their amenability to genetic manipulation by cell selection, one area that stands out is mineral nutrition. Mineral deficiencies and toxicities limit crop productivity worldwide. While certain of these problems can be circumvented by site selection or overcome by fertilization or other soil management practices, such solutions are impractical or unavailable in many parts of the world. 526 MEREDITH et al.

The development of genetically improved crop varieties that are more tolerant of these adverse conditions is in many cases a more desirable strategy (Devine, 1982). Many aspects of plant mineral nutrition are fundamentally cellular and thus amenable to in vitro manipulation. However, very little is known about mineral nutrition at the molecular level and important processes related to the acquisition and utilization of essential elements or the tolerance of toxic ions can not yet be manipulated by genetic transformation. A number of mineral elements play essential roles in plant health, for example as enzyme activators or components of fundamental molecules (Marschner, 1986). Deficiencies of these elements result in a disruption of plant processes. Other mineral elements are signifi­ cant more for their toxic effects when they are present in excess. These elements effect injury by disrupting fundamental cellular mechanisms (e.g., by competing with essential elements for uptake, inactivating enzymes, displacing essential elements from functional sites) (Marschner, 1986). Both the roles played by essential elements and the primary damage effected by toxic elements are primarily cellular. Thus cultured cells can be expected to be sensitive to mineral stresses, both deficiencies and toxicities. Although mineral stresses may have their primary effects at the cellular level, do tolerance mechanisms by which these stresses might be overcome also operate at the cellular level? It is well known, for example, that plants respond to certain mineral stresses via adapta­ tions in root morphology and function (Gerloff and Gabelman, 1983); such adaptations could not exist in cultured cells. However, cellular adaptations to mineral stress are also known. For example, more efficient uptake of a deficient nutrient element can result from altered membrane transport mechanisms, or more efficient utilization can be achieved via higher enzyme affinity for an element (Marschner, 1986). Tolerance to toxicities can be achieved by exclusion at the plas­ malemma or detoxification via binding to an organic molecule (Marschner, 1986). Is it reasonable to expect that cell variants that are tolerant to a particular stress might produce plants that are also tolerant? That is, will tolerance expressed in cultured cells be similarly expressed in intact plants? While genotypic differences in adaptation to mineral stresses are well-known (Gerloff and Gabelman, 1983) and in some cases have even been attributed to single major genes (Devine, 1982), these differences have generally not been studied in cultured cells. There have been a few reports, however, in which in vitro tolerance RESISTANT PLANT GENOTYPES 527 has been compared among genotypes known to differ in tolerance in planta. Differences between Agrostis genotypes in resistance to zinc and copper toxicity were maintained in callus cultures (Wu and Antonovics, 1978). Qureshi et al. (1981) reported similar findings for Anthroxanthum genotypes differing in resistance to zinc and lead, as did Sain and Johnson (1984) for soybean genotypes differing in iron efficiency. Christianson (1979), however, did not observe the expected differences in zinc sensitivity in Phaseolus genotypes. In tobacco, callus cultures of five cultivars differed significantly in manganese resistance, but the differences did not correspond to those observed in seedlings (Petolino and Collins, 1985). Selection strategies that would result in the isolation of cell variants tolerant to particular mineral stresses are simple in concept. The mineral stress to which tolerance is sought is simply simulated by chemically modifying the culture medium. This must be done with great care, however, so as to impose conditions that approach the chemical environment of the soil solution. For example, pH can be of critical importance in that it can significantly influence the solubility and ionic state of the element of interest. Similarly, other interacting elements must be also considered. Simply adding an excess of a toxic element or reducing the concentration of a nutrient may result in a mineral environment quite different than that of the soil in which the mineral stress occurs. Clearly, the likelihood of achieving agricul­ turally significant results will be improved if the mineral stress is simulated as closely as possible. There have been few reports of selection in plant cell cultures for resistance to mineral stresses. In some studies, plants could not be regenerated from the selected cells, and thus neither the expression of the selected character nor its heritability could be investigated. Such has been the case with aluminum-resistant tomato cells (Mere­ dith, 1978), manganese-resistant tobacco cells (Petolino and Collins, 1985), and zinc-resistant cells of Haplopappus gracilis (Gilissen and van Staveren, 1986). In other cases, plant regeneration was perhaps not an objective since no mention of regeneration was made, e.g., mercury-resistant petunia cells (Colijn et al., 1979), cadmium­ resistant Datura (Jackson et al., 1984) and tomato (Bennetzen and Adams, 1984), copper and zinc-resistant Nicotiana plumbaginifolia (Kishinami and Widholm, 1986, 1987). A preliminary report suggested that plants regenerated from aluminum-resistant carrot cells expressed some tolerance (Ojima and Ohira, 1982), but no futher mention of these plants has been made in subsequent reports by these authors (Ojima and Ohira, 1983, 1985; Ojima et al., 1984). 528 MERED ITH et al.

We selected aluminum-resistant variants from cell cultures of Nicotiana plumbaginifolia. The resistance was expressed in whole plants derived from the selected cells and in their sexual progeny and was transmitted in a Mendelian pattern consistent with that expected if a single dominant gene were governing the resistance (Conner and Meredith, 1985b, 1985c). This work represents the first and only successful use of cell selection to date to genetically increase plant tolerance to an agriculturally important mineral stress. We chose to work with Nicotiana plumbaginifolia rather than a crop species because it would allow us to accomplish both in vitro and in planta manipulations in an efficient fashion. Not only can plants be readily regenerated from cultured cells of this species at a high frequency, but it is also quite amenable to genetic analysis because of its diploid nature, small size, short generation time, self­ fertility, and high seed yield. It represented an ideal model species with which to test our hypothesis that genetic adaptation to mineral stress could be manipulated via the identification and isolation of genetic variants at the cellular level.

SELECTION OF ALUMINUM-RESISTANT VARIANTS

Aluminum toxicity is a widespread soil problem associated with many acid soils throughout the world (National Academy of Sciences, 1977; Foy, 1984). It is a particularly serious problem in developing countries-not only because low pH soils are prevalent in these locations but also because cultural practices to improve soil fertility are often prohibitively expensive (Sanchez et al., 1982). Genetic variation in aluminum resistance is well-known (Foy et al., 1978), and its inheritance has been characterized in a number of crop species (Kerridge et al., 1971; Reid, 1971; Lafever and Campbell, 1978; Culvenor et al., 1986). In those crops in which sufficiently resistant germplasm has been identified and which can be readily manipulated genetically, breeding and selection for increased aluminum resistance have been successful (Devine, 1982). We took great care in developing an aluminum-toxic selection medium (Conner and Meredith, 1985a). If aluminum is simply added to standard culture medium, it readily precipitates out of solution, taking with it most of the phosphorus that is essential for cell survival. The mineral stress to which the cells are exposed is thus phosphorus deficiency, rather than the intended aluminum toxicity. To prevent this precipitation, we reduced the phosphorus concentra­ tion in the medium from 1250 µM to 10 µMand replenished it every RESISTANT PLANT GENOTYPES 529 other day. We also lowered the pH of the medium from the standard 5.8 to 4.0. Both of these changes were essential for the expression of aluminum toxicity at agriculturally realistic concentrations. Because agar does not readily gel at the pH 4.0, we used as a cell support filter paper resting on a disk of polyurethane foam saturated with liquid medium (Conner and Meredith, 1984). To further increase aluminum toxicity, we lowered the calcium concentration from 3.0 mM to 0.1 mM and used unchelated iron. Calcium mitigates aluminum toxicity (Munns, 1965; Kinraide and Parker, 1987), and the chelating agent generally used in culture media, EDTA, has a higher affinity for aluminum than for iron (Martell and Smith, 1974). The low phos­ phorus concentration in this modified medium did reduce cell growth by about half, but this was still adequate for our selection exper­ iments. The other modifications did not limit cell growth. Using this modified medium, total inhibition of cell growth was obtained at 600 µM aluminum, considerably lower than the 4 mM required for complete inhibition of carrot cells in an unmodified medium (Ojima and Ohira, 1983). We initially employed a direct selection procedure to isolate aluminum-resistant variants. Cells were plated directly onto modified medium containing 600 µM aluminum. The medium was replaced every other day to maintain phosphorus availability. Aluminum-resistant variants were evident after 5-10 weeks of culture, and after 14 weeks no additional variants had appeared. At this time, the cells were transferred to unmodified medium without aluminum, and a few additional colonies were recovered after an additional 3 weeks. From 100 petri plates, each having been plated with 105 plating units, only 29 colonies were recovered, and in only one of these was the resistance expressed in regenerated plants and transmitted to seedling progeny (Table 1) (Conner and Meredith, 1985c). Much better mutant recovery was obtained with a rescue selec­ tion method. Cells were cultured as suspensions in liquid medium containing 600 µM aluminum for 10 days and then plated onto unmodified medium with no aluminum for the recovery of sur­ vivors. Using this method, we were able to detect variant colonies after 5 or 6 days if feeder cells were employed, or 2 weeks without feeder cells (Conner and Meredith, 1985b). With the rescue selection method, we isolated 217 resistant variants from 115 plates and 39 of these variants ultimately. expressed the resistance in regenerated plants and transmitted it to their progeny (Table 1) (Conner and Meredith, 1985c). 530 MERED ITH et al.

Table 1. Summary of the selection and characterization of aluminum-resistant variants from cell cultures of Nicoti­ ana plumbaginifolia. Approximately 105 colony-forming units were applied to each selection plate (adapted from Conner and Meredith, 1985c). Selection Method Direct Rescue ( 100 plates) ( 115 plates) Total Total variants selected 29 217 246 Reselected from single 9 110 119 cells Resistance retained for 4 63 67 6-9 months in absence of Al Fertile plants regenerated 1 39 40 Seedling progeny segre­ 1 39 40 gate for Al resistance

All variants obtained by both selection methods were grown as callus in the absence of aluminum for 6 to 12 weeks and then reselected from single cell suspensions. These steps eliminated escapes and unstable variants and ensured that the remaining variant lines were genetically homogeneous and not mosaics of resistant and senstive cell types. Less than half of the variants (119/ 246) could be reselected at this point. Callus lines were then maintained for an additional 6 to 9 months in the absence of aluminum and then tested for retention of aluminum resistance. Again, a considerable number of variants ( 48) did not retain their resistance. (Four lines were also lost to contamination or senescence.) Of 67 variants that did retain resistance over this period, plants could be regenerated from 40. All 40 of these variants transmitted aluminum resistance to their sexual progeny (Table 1). In all 40, the ratios of resistant to sensitive seedlings were consistent with those expected for a single dominant mutation (Conner and Meredith, 1985c). We also regenerated plants from 25 of the 48 lines that did not retain their aluminum resistance. As expected, all seedlings of these lines (with one exception) were aluminum-sensitive. We attribute the one case of segregating aluminum RESISTANT PLANT GENOTYPES 531 resistance to a genetic event that occurred after the selection and characterization of the callus lines. When these mutants were originally characterized, we observed discrete classes of resistant and susceptible seedlings. Seedlings were evaluated by sowing them on filter paper over polyurethane foam saturated with a nutrient solution containing 600 µM aluminum (Conner and Meredith, 1985b). Seedlings surviving after 3 weeks were considered resistant. All sensitive (and all wild-type) seedlings died within this period, and resistant seedlings remained green and survived indefinitely in the presence of aluminum. Resistant and suscept ible classes could be easily identified. Since that time, 21 of the 40 resistant lines have been subjected to further study (Schettini and Meredith, in preparation). Aluminum resistance has been tested again in the first generation (R1) seedling progeny of the regenerated plants for all 21 lines. Rz and Rs progeny have been evaluated in five and two of the lines, respectively. In no case have we observed the clear segregation of resistant and sensitive classes of seedlings that we once saw. Instead, a continuous distribu­ tion was observed. All wild-type seedlings were dead at the end of 3 weeks, as before, but so were most of the seedlings from the selected lines. Those that did survive beyond 3 weeks eventually succumbed within another week or two. None survived indefinitely. Lowering the aluminum concentration in search of some critical concentration window did not alter these results, nor did pretreating the seedlings with low levels of aluminum prior to the 600 µM treatment, as might be expected if the resistance mechanism were inducible (Aniol, 1984) and had declined over time in the absence of the inducing factor. Time course studies, in which seedling survival was monitored on a daily basis, were conducted with Rz and Rs seedlings of two lines. These experiments revealed a quantitative difference between wild-type and selected lines. When the percentage of surviving seedlings was plotted over time, the distribution in the selected lines was clearly shifted by 2 to 4 days beyond the wild-type control. The slopes of the survival curves (i.e., the rates at which seedlings die) were similar in both selected lines and controls (Schettini and Meredith, in preparation). Although our original inheritance data were consistent with a single dominant gene for resistance, we are now considering polygenic models that could explain our recent results. We consider it possible that genotype-environment interactions may account for the differ­ ences between our original results and the more recent data. The 532 MEREDITH et al. environment in which the seedlings were originally screened may have favored the expression of a single gene with major effects in our earlier tests so as to result in simple segregation ratios. Although we are not aware of any changes in protocol, our current laboratory conditions may be slightly different such that the polygenic nature of the resistance is now revealed, hence the quantitative expression of resistance that we now observe. Polygenic inheritance patterns have previously been observed in novel genotypes obtained from cell cultures (Karp and Bright, 1985). Alternatively,. the resistance may be con­ ditioned by a single gene that controls a quantitative trait. Further genetic analysis can distinguish between these two hypotheses. We also have considered that the resistance may have resulted from an unstable genetic change, e.g., gene amplification or transposition. However, we consider these latter phenomena less likely since we have observed differences in resistance in the same seed lot over time. Such genetic mechanisms would require cycles of DNA synthesis and cell division that are not known to occur in seed in storage. Clearly, the material we have obtained exhibits and transmits increased resistance to aluminum; however, further work is obviously warranted to elucidate the genetic basis for this phenomenon.

LITERATURE CITED

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Fischhoff, D. A., K. S. Bowdish, F. J. Perlak, P. G. Marrone, S. M. McCormick, J. G. Niedermeyer, D. A. Dean, K. Kusano-Kretzmer, E. J. Mayer, D. E. Rochester, S. G. Rogers, and R. T. Fraley. 1987. Insect tolerant transgenic tomato plants. Bio/ Technology 5:807-813. Foy, C. D., R. L. Chaney, and M. C. White. 1978. The physiology of metal toxicity in plants. Ann. Rev. Plant Physiol. 29:511-566. ----. 1984. Adaptation of plants to mineral stress in problem soils, pp. 20-39. In: D. Evered and G. M. Collins (eds.). Origins and Development of Adaptations. Pitman Press, Bath. Freeling, M. 1984. Plant transposable elements and insertion sequences. Ann. Rev. Plant Physiol. 35:277-298.

Gerloff, G. C. and W. H. Gabelman. 1983. Inorganic plant nutrition, pp. 453-480. In: A. Lauchli and R. L. Bieleski (eds.). Encyclopedia of Plant Physiology. New Series, Volume 15B. Springer-Verlag, New York. Gilissen, L. J . W. and M. J. van Staveren. 1986. Zinc-resistant cell lines of Haplopappus gracilis. J. Plant Physiol. 125:95-103. Jackson, P. J., E. J . Roth, P.R. McClure, and C. M. Naranjo. 1984. Selection, isolation and characterization of cadmium-resistant Datura innoxia suspension cultures. Plant Physiol. 75:914-918. 534 MEREDITH et al.

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IOWA STATE JOURNAL OF RESEARCH I MAY, 1988 537-569 Vol. 62, No. 4

SCIENCE IN THE ART OF PLANT REGENERATION FROM CULTURED CELLS: AN ESSAY AND A PROPOSAL FOR A CONCEPTUAL FRAMEWORK

Jerry Ranch 1 and Gary M. Pace2

ABSTRACT. An overview of an approach to plant cell and tissue culture is presented. An analysis is introduced where emphasis is placed on the factors which most strongly influence the in vitro growth and differentiation of plant cells. These factors include genotype, ontogenic state, and environment. Based on empirical observations and speculation, a model is presented which evaluates in vitro events and attempts to relate them to processes in vivo. This model allows the creation of testable hypotheses for further investigation of in vitro phenomena. Index descriptors: plant cell and tissue culture, morphogenesis, totipotence, regeneration, competence.

WHY DISCUSS APPROACHES TO PLANT CELL AND TISSUE CULTURE?

It has been known for three decades that whole plants can be recovered from cells cultured in vitro (Steward et al., 1958; Reinert, 1959; Vasil and Hildebrandt, 1965a, 1965b). While there has been steady advancement in the theoretical and practical study of this phenomenon, the catalyst behind much of the recent interest in plant cell and tissue culture appears to be derived largely from potential technological applications which exploit the regeneration process (Bliss, 1984; Evans, 1983; Lewis, 1986; Netzer, 1984; Sprague et al., 1980). As a consequence of this driving force, a significant proportion of current plant cell culture literature concentrates on an empirical approach to the production of regenerated plants. It is unusual to see an emphasis on the developmental biology inherent to morphogenic processes in vitro, or the use of this perspective to develop and document in vitro manipulations. We persistently have the impres­ sion that there is a less than adequate appreciation for the parallels and discrepancies between biology in vitro and that familiar to whole plant biologists. In the overwhelming rush to utilize plant cell and

1United AgriSeeds, P. 0 . Box 4011 , Champaign, IL 61820.

2United AgriSeeds, P. 0. Box 4011, Champaign, IL 61820. Current address: Ciba­ Geigy Biotechnology Research, P. 0 . Box 12257, Research Triangle Park, NC 27709. 538 RANCH and PACE tissue culture in commerce, the biology has been superceded by a 'black box' perspective. In our opinion, there is a need to re-establish the relevance of developmental biology to plant cell and tissue culture. In this essay, we hope to demonstrate the value of under­ standing developmental biology as it applies to the characterization and manipulation of morphogenic phenomena in vitro. Numerous reviews concentrate on the manipulations and media formulations used for cultivating various plant tissues (Ammirato, 1983; Evans et al., 1981; Flick et al., 1983). It is not the intention of this essay to discuss such topics. Rather we aspire to put in vitro events and processes into a larger context. When this is accomplished, we hope that the similarity and association between in vivo and in vitro circumstances will be recognized. This exercise will hopefully lead to considerable discussion and new hypotheses. We will consider the current understanding of the growth and differentiation of plant cell and tissue cultures and how we might extrapolate these concepts to test new hypotheses. Since much in cell and tissue culture still remains speculative, we will often add our own thoughts and hypotheses to fill those gaps in our knowledge. Also, we have tended to focus on the major row crops when citing examples as it reflects our particular interests.

REGENERATION AS A BIOLOGICAL PROCESS

Historically, cell and tissue culture had its genesis as a powerful experimental procedure in the study and elucidation of physiology and development (Beasley, 1984; Obendorf et al., 1983; Pareddy and Greyson, 1985; Raghavan and Srivastava, 1982; Thompson et al., 1977; Torrey, 1954; White, 1943). Many crucial considerations were approached by these studies. For example, general questions about the organizational or ontogenic stages at which excised tissues or organs lose developmental autonomy were addressed. Some investi­ gated the nutrition of isolated organs and tissues, and what exogenous influences are required to sustain the tissue. These studies have been very successful as evidenced by the present capability to sustain, in vitro, tissues and organs from many plant species. These important studies provided the groundwork for much subsequent in vitro experimentation. Manipulation of cell cultures, however, inherently lack the usual in vivo inductive and stabilization factors. A general objective of the in vitro strategy is to coerce cell and tissue cultures to behave as if they were part of a larger organized mass, that is, behave as if they PLANT REGENERATION 539 remained in the context of a whole plant. Although the technology associated with manipulation of cell and tissues in vitro has poten­ tially useful capabilities, such as the regeneration of plants from single cells, substantial insights into whole plant biology can also be achieved through the analysis of in vitro growth and developmental behavior. In our view, it is neither necessary nor proper to consider plant regeneration from cultured cells as an artificial process. The capacity for plant regeneration from cell and tissue cultures is based on natural, inherent phenomena. Developmental events reflect what is prescribed in the genome regardless of whether these traits are expressed in vivo or in vitro. While there are in vivo morphogenic processes which we cannot yet duplicate in vitro (e.g., gametogenesis), there probably is no in vitro morphogenic event or process which does not possess an in vivo counterpart. Since plant regeneration from cultured cells represents the totality of developmental biology, it is our challenge to understand and manage these mechanisms in vitro by orchestration of those factors which most strongly impact upon the process.

REGENERATION INTERPRETED, AND CELLS IN VITRO

The term regeneration has been used widely in tissue culture to denote the recovery of a whole organism from cells, tissues, organs, meristems and zygotic embryos, cultivated in vitro. Because of this broad usage, regeneration does not denote a specific developmental process leading to whole plants. The frequent use of this term has prompted us to review its definition. In the classical botanical context, regeneration refers to the replacement of "a lost or otherwise eliminated organ .. _either by the outgrowth of a resting primordium . . . or by a completely adventitious and new formation . _ ." (Strasburger, 1976). This definition was founded on the observation that some cells and tissues in whole plants possess a natural tendency to proliferate and differentiate that is suppressed in vivo. The suppression of this tendency results from their position in the organism, that is, from the correlative effects of neighboring cells. When this suppression is removed, for example by wounding, regenera­ tion may occur. Removal of cells and tissues from the whole plant context and into the in vitro environment permits manipulation of apparent "position," and consequently allows the opportunity to affect directly growth and differentiative processes. 540 RANCH and PACE

Regeneration can be achieved in many ways. For example, in vivo suppression of growth and differentiation can be removed by culture of plant tissues on synthetic medium. Another example is regen­ eration from resting shoot primordia which embraces the in vitro technique of micropropagation. When the release of suppression results in adventitious organogenesis, a redetermination of dif­ ferentiated cells has occurred. This latter process is an exciting aspect of plant cell and tissue culture for it provides the basis for an approach to study and assay for totipotence. These observations serve to reinforce the concept that all growth and differentiation in vitro is a recapitulation of a native, inherent tendency to proliferate. Recovery of plants from in vitro cultures may be most readily achieved and characterized using tissue at a level of organization which retains developmental competence. In these cases, the explanted material is predetermined toward plant organization. Examples of these tissues would be immature embryos or various meristems such as leaf or apical. The notion, however, that single isolated plant cells might also be capable of regeneration into whole plants dates to Haberlandt (1902). The genetic capacity of a nucleated plant cell to organize into a whole plant either directly or through an intervening callus stage, regardless of the morphogenic process, has been termed totipotence. Totipotence is most definitively demon­ strated by regeneration of plants from protoplasts. There has been an explosion of evidence documenting plant regeneration from proto­ plasts from many species and tissue sources (Cocking, 1960; Davey, 1983; Takebe et al., 1971). The concept of a somatic cell cycle easily follows from these observations. The cultured plant cell might then be considered a germ cell and the various culture phases become part of the sporophytic life cycle with morphogenically competent cells being analogous to a zygote. Manipulations in vitro with protoplasts further compares the somatic cell cycle to the sexual cycle by comparing fusion of somatic cells to amphimixis (Kao and Michayluk, 1974). In theory, all cells are totipoent (Krikorian et al., 1986; Steward et al., 1958). In practice, there is substantial variation in the expression of this genetic and biological characteristic. The expression of toti­ potence as a differentiated state is referred to as morphogenic competence. It is the inability of some species, genotypes, or levels of organization to express totipotence which often confounds and limits the capacity for plant regeneration. This variation is probably founded in the differentiated and physiological state of the target cells and is expressed either by the differential receptivity to inductive conditions or by the inability to define precisely the requirements for induction. PLANT REGENERATION 541

Furthermore, cells in vitro are not exposed to the stabilizing and inductive influences of the in vivo environment-the "position" effect. With a reduction in organization, an alteration in the differentiated state of the cells might also be expected. Therefore, the differentiated state which confers morphogenic competence may not be easily retained, or regained, by a cell in a relatively unorganized state. The environment which complements this attribute can be lost. The theory of totipotence predicts, however, that the competent state is not irrevocably lost. Circumstances leading to the induction and maintenance of such a differentiated state may be artifically applied, presumably by complementing the current activity of the cell. In contrast to cells in vitro, morphogenic competence can usually be readily induced from complex primary explants. Although this latter situation may also require an induction, there presumably is a significant, but uncharacterized, contribution from the explanted tissue which retains some memory of the in vivo environment. It becomes enticing, on the basis of recent successes and current concepts of plant developmental biology, to predict that the complexity of a plant can always be maintained at the cellular level. Many technological and commercial applications are pursued on the basis of this prediction. But it is here that technological ambition confronts biological limitations (see Chaleff, 1981). At most, there may be present a cellular component of a whole plant attribute. The contribution of this cellular component to the biology at the whole plant level will likely be modified depending on the level of organiza­ tion and the state of differentiation (Bassiri and Carlson, 1978). Indeed, an important mission of modern plant cell biology is to ascertain in what ways cultured plant cells may be considered similar to a whole plant. Current fundamental and applied interests of cell and tissue culture should reflect both the developmental biology of in vitro events and the need to recover fertile plants from these cultures. Therefore, in the current context, regeneration may be interpreted to include a process composed of morphogenesis and propagation phases. These phases may be further refined into separate but integrated stages (Figure 1 ). Since our approach is reductionistic and aimed at discerning the manner in which plant cells might be considered and treated as complete organisms, the definition of regeneration includes an adventive or de nova morphogenesis phase. In the primary phase the induction of morphogenic processes leading to whole plant organization occurs, whereas the propagation phase mimics standard horticultural practice. Meristem culture without .....,_.------REGENERATION------::m•-11

Donor Plantlet Initiation Maturation Germination Hardening Mature Plant Reproduction Plant Establishment

~ MORPHOGENESIS--•_...1------PROPAGATION ------.~1

Figure 1. A model for the process of induction and recovery of regenerated plants from primary explants via somatic enbryogenesis. It is anticipated that modifications to this scheme will be necessary to describe other regeneration processes which vary in tissue derivation, mode of regeneration, and differences in sexual versus asexual reproduction across species. PLANT REGENERATION 543 adventive multiplication, for example, would not be considered to be regeneration by this definition since it involves the development of an autonomous, preexisting competent structure.

COMPONENTS OF REGENERATION

It is necessary to first consider those factors which most strongly influence rnorphogenic processes from cultured cells and tissues. We have condensed all potential components into three broad elements: the genotype, the ontogenic state, and the environment.

Genetic Constitution Frequently, one of the first steps in examining regeneration from cell and tissue culture is a survey of the genetic diversity for this trait in the species under consideration. This factor has been extensively analyzed, in part, because it can be accurately controlled. Consider­ able amounts of quantitative data can be gleaned from genetic analyses of regeneration competence (e.g., Beckert and Qing, 1984; Maddock et al., 1983; Tornes and Smith, 1985). These and other studies clearly indicate that genotype can exert a significant impact upon the regeneration response. Some of these genetic analyses have demonstrated further that genotype by environment interactions can be significant. Evaluations of regeneration competence in maize, for example, reveal that it is not confined to any particular heterotic family in the dent group (Duncan et al., 1985; Fahey et al., 1986; Hodges et al., 1986). Sweet and flint corn also respond in culture (Lu et al., 1982; Rapela, 1985). Thus it would appear that, at least for the rather extensive cultivars of maize, regeneration competency was not unknow­ ingly selected for, or against, during gerrnplasrn evolution. Interest­ ingly, even in cases of wide genetic divergence, for example, in Nicotiana species, plants can be regenerated under similar conditions (Flick et al., 1983). Compilations of species which undergo somatic ernbryogenesis using remarkably similar conditions are also available (e.g., Ammirato, 1983). This suggests that regeneration is not limited by genetic constitution but by our ability to provide in vitro conditions conducive to the expression of the regeneration pheno­ type. It is possible to specifically breed for regenerable gerrnplasrn. For example, Bingham et al. (1975) and Koornneff et al. (1986) have bred, respectively, alfalfa and tomato with a high and predictable regen­ eration response. In these cases, rnorphogenic competence from a 544 RANCH and PACE specific explant source cultured on a defined series of culture media was used as selection pressure. It is not possible to identify in these studies the loci involved. Unfortunately, it is generally true that more is known about the genetic behavior of most traits, particularly quan­ titative factors, than about the biochemistry and physiology which comprises these same traits. The observation and characterization of regeneration as a genetically quantifiable and heritable trait has, in our opinion, led to an oversimplification of the biology associated with this process. Perhaps the genetic approach leads many to abandon the position that regeneration may be dissected and manipulated by management of the external environment and tissue source. This is manifest in the common notion that breeding for culture response is to be preferred over the "art" of tissue culture manipulations. Introgressing the regeneration trait into unresponsive germplasin would assist, to some extent, the genetic characterization of the regeneration phenotype. Ultimately this strategy might narrow the respective germplasm base and potentially limit the commercial usefulness of the process and the product. In addition, the genetics associated with morphogenesis exist only in the context of the capacity to detect variation in the process. Generally, this variation is assayed under only one or two in vitro conditions. Under the limited conditions of these analyses, it is doubtful that detection of all the genetic determinants which play a role in morphogenesis can be achieved. Consequently, there likely is much more complexity to the regeneration process than genetic studies have indicated. Furthermore, genetic determinants potentially have an influence at all stages of the process up to the recovery of a whole fertile plant. The technique of genetic analysis provides a view of only one dimension of the regeneration process. We learn little about the biology of the regeneration process from simple genetic characterization itself. Since we are advancing the concept that the inability to induce regeneration, quantitatively, from a specific tissue source is due to lack of understanding how to induce morphogenesis, genetic constitu­ tion is viewed as a factor which conditions cell and tissue responses in a certain way to environmental signals. It is not due to a lack of 'regeneration' genes since competence itself is not inherited-only the potential to express morphogenic competence under the test condi­ tions is transmitted. Whether or not that epigenetic sequence which produces competence is expressed depends on many internal and external factors. Ultimately, the goal should be to understand and PLANT REGENERATION 545

manipulate developmental processes without regard to genotype. In the genotype by environment interaction, efforts should be focused on achieving a negligible genotypic contribution. We firmly believe that methods will be developed, either through empirical or inferential studies, which limit the genotype effect in inducing and maintaining the competent state. We must be pragmatic, however, and acknowledge that, as a first step, a survey of the available germplasm represents a viable avenue to initiate studies on morphogenic competence with a new or recalcitrant species. Likely the most productive genetic approach to understanding regeneration competence might be through an empha­ sis on biochemical and developmental genetics. For example, studies of mutants characterized as developmentally or morphologically variant at the whole plant level could be used to investigate, in vitro, specific loci associated with development (Meinke, 1986).

Onto genie State of the Tissue The source of the primary explant and the tissue maintained during subculture are important variables in recovering competent cell and tissue cultures. Plants can be regenerated either directly from primary explants, or from serially propagated cell or tissue cultures. Primary sources include immature embryos, inflorescences, roots and leaves. Thus, even though competent cultures may be generated from different tissue sources, there must be some common biological thread which links these responsive tissues together causing them to behave in a similar fashion. While most any ontogenically juvenile explant would likely be an excellent tissue source from which to establish a competent culture, there may be populations of cells within the explants which vary in the expression of competence (Wenzler and Meins, 1986). Consequently, recalcitrance at one level of organizat ion does not imply recalcitrance at all levels of organization or ontogenic states. The difference in gene expression between different ontogenic states (the internal environment) may result in a differential response to similar inductive conditions. After assaying various cell and tissue types for expression of competence, we should be able to document what commonly possessed characteristics are uniquely associated with competence. A pertinent example of these points is illustrated by the many attempts to establish an in vitro culture of maize that could be serially maintained or from which plants could be recovered. Early efforts focused on using roots and stem sections (Mascarenhas et al., 1965; Mott and Cure, 1978) and various parts of germinating zygotic 546 RANCH and PACE embryos (Green et al., 1974; Gresshoff and Doy, 1973; Harms et al., 1976; Sheridan, 1975) as the explant source. Rhizogenesis was the predominant response, and only in a few cases were whole plants recovered. Use of the immature zygotic embryo as an explant resulted in a dramatic success (Green and Philips, 1975). Although regen­ eration in these cultures was initially found to be through shoot organogenesis (Freeling et al., 1976; Springer et al., 1979), somatic embryogenesis from immature embryo explants was soon docu­ mented (Lu et al., 1982; Vasil et al., 1985). The number of reports of regeneration in maize has since increased with successful regen­ eration of maize from leaf tissue (Chang, 1983; Conger et al., 1987), glumes (Suprassanna et al., 1986), immature inflorescences (Rhodes et al., 1986), and mature embryos (Wang, 1987). In each case, except for glume explants, the mode of regeneration appears to be through somatic embryogenesis. The tissue sources used in early attempts with maize were not in an ontogenic state responsive to the cultural conditions in order to effect regeneration, i.e., they were not competent. This lack of competence may have been due to the influence of surrounding cells or the particular circumstance of differentiation possessed by target cells. Alternatively, there might have been insufficient knowledge of the appropriate conditions to induce competence in the tissue used. Even with a less than model species such as maize, however, when appropriate tissues were used as an explant source competent cultures could be initiated. Just as there are many tissue types in the whole plant, different cell types and levels of organization may be propagated in vitro as cell, tissue, and organ cultures. Many types of morphogenically competent cultures may be initiated and serially propagated (Kri­ korian et al., 1986; Vasil and Vasil, 1980). Vasil (1985) believes that ontogenic state of the tissue is the paramount factor in inducing and maintaining competent cell and tissue cultures. So even though the prior histor:y of cells may be different, and their ongoing develop­ mental programs are different, cells possess the genetic competence to adjust their route of differentiation in response to stimuli. With easily manipulatable species such as tobacco and carrot, virtually any part of the plant may be used to generate a morphogenically competent cell culture. This is compelling evidence that every cell in a plant is genetically identical and is potentially totipotent. However, an imporant caution must be exercised. In complex explants, and even in serially propagated cultures, full documentation as to which cell population possesses morphogenic competence is frequently PLANT REGENERATION 547 lacking. It is possible that only a small fraction of cells react to morphogenesis induction circumstances (Cheng and Raghavan, 1985; Konar et al., 1972b; Nomura and Komamine, 1985). One goal of the cell biologist is to develop a rationale for enriching, or targeting, a cell population for those cells which respond to the inductive conditions. Before this can be accomplished, we need to know how responsive cells are different from unresponsive cells so appropriate selection can be formulated. Although biochemical studies have characterized gene expression during somatic embryogeny (Sung and Okimoto, 1983), there is no compelling evidence which illustrates a cause and effect relationship between specific physiology or biochemistry and morphogenic competence.

Environment In an approach similar to the evaluation of genotype and tissue ontogeny on in vitro behavior, evaluations of media or media components have been used to establish a baseline morphogenic response for a species (de Fossard et al., 1974; Duncan et al., 1985). Media formulations are usually aided by factorial designs, and tech­ niques are available that allow for easy evaluation of large numbers of factors and interactions (e.g., Potrykus et al., 1979). It might be surmised from the proliferation of culture media formulations that the medium manipulation approach holds the most potential for successful induction and maintenance of growth and differentiation (Conger, 1981; Skirvin, 1981). Some trends in the use of specific formulations are apparent, usually depending on the level of organ­ ization. Generally, organ cultures prefer a relatively low ionic strength, low osmolarity medium. Cell and tissue cultures generally favor higher ionic strength media. A majority of documented cases of regeneration via somatic embryogenesis, for example, report success with a Murashige and Skoog (1962) medium with 2,4-dichlorophenoxyacetic acid as an auxin (Evans et al., 1981). In light of these observations, it might be useful to reflect on the role of the environmental component in in vitro and tissue culture. The environment is generally manipulated both to provide appropriate nutrition for the tissue or cells and to regulate mor­ phogenic processes. The chemicals used generally include plant growth regulators, minerals, carbohydrates, and various organic factors such as vitamins, organic acids, protein hydrolyzates, and occasionally, amino acids (Ammirato, 1977; Ammirato and Steward, 1971; Ernst and Oesterheld, 1984; Gamborg and Shyluk, 1981; Murashige and Skoog, 1962; Steward and Krikorian, 1971; Skoog and 548 RANCH and PACE

Miller, 1957; Tazawa and Reinert, 1969; Wochok and Wetherell, 197lb). The physical environment includes factors such as light, osmotic pressure, ionic strength, temperature and physical state of the medium. Physical factors may also influence chemical composi­ tion. Agitation of liquid cultures, for example, has an influence on the concentration of dissolved gasses (Kessel and Carr, 1972). Some studies bear on the nutritional range under which mor­ phogenic processes occur (Dougall and Verma, Wetherell, 1984; Wetherell and Dougall). There are categories of factors, such as activated charcoal and unique carbohydrates, which apparently enhance plant regeneration (Armstrong and Green, 1985; Ben-Hayyim and Newmann, 1983; Fridborg and Eriksson, 1975; Fridborg et al., 1987; Negrutiu and Jacobs, 1978a; Nuti Ronchi et al., 1984). Given all the possible medium components, it is possible to generate a large inventory of factors which have effects on morphogenic processes. Although it may be important to determine which of all these myriad elements is necessary and sufficient for the morphogenic response desired, there does not appear to have been much effort to quan­ titatively investigate the minimal requirements to sustain growth and differentiation in vitro (Murashige and Skoog, 1962; Tazawa and Reinert, 1969). Indeed, most studies focus on initially optimizing media formulations for the most rapid growth. Nutritional and physical requirements for growth and differentiation may not be compatible. It is imperative to be aware under what context a factor was tested. In an effort to mimic the in vivo environment, sequences of different media compositions have been used to effect plant regen­ eration (Evans, et al., 1981; Walker and Sato, 1981). Those situations where plants cannot be regenerated perhaps requires even further r~finements in the number and formulation of media sequences in either the morphogenesis or propagation phases. The nutritional and morphogenic functions of the environment may not be mutally exclusive. For example, Halperin (1966) addressed the different rnorphogenic events which occurred in carrot cell suspension which depended on cell aggregate size and nitrogen composition of the culture medium. In these studies, rhizogenesis was promoted by high nitrate to ammonium ratios, while an embryogenic program was executed under conditions of relatively more ammo­ nium. Walker et al. ( 1979) and Walker and Sato (1981) demonstrated a similar relationship in alfalfa, but additionally described growth regulator requirements for induction of rhizogenesis or embryo­ genesis. This work clearly showed that under embryo induction PLANT REGENERATION 549 conditions the expression of this developmental program required reduced nitrogen. While in either case nitrogen was supplied from inorganic sources, the types of differentiation promoted varied depend­ ing on the quality of the nitrogen source. There are instances where physical factors are critical to success­ ful regeneration from tissue cultures (Murashige, 1974). Physical conditions exert dramatic effects most notably in anther culture. For example, a short period of pretreatment at elevated temperature significantly improved culture response for Brassica spp. (Keller et al., 1982) whereas low temperature pretreatment was found to be beneficial for maize (Genovesi and Collins, 1982; Pace et al., 1987). An evaluation of high and low temperature shocks in the culture response of immature embryo explants of maize indicated a detri­ mental effect (Fahey et al., 1986). Higher levels of sucrose are generally used in anther culture for both osmotic adjustment and suppression of precocious germination (Collins and Genovesi, 1982). The higher sucrose requirement of cultured tissue is supported by direct measurement of anther homo­ genates suggesting that the in vitro behaviour has a physiological counterpart in vivo (Dunwell and Thurling, 1985). Subsequently, the sucrose level is lowered by replacement (Dunwell and Thurling, 1985) or dilution (Marsolais and Kasha, 1985) to produce the different physical environment requirements of induction and survival of androgenic embryos. Increased sucrose concentration (Lu et al., 1983) or addition of mannitol (Fahey et al., 1986) to the medium composition was also beneficial for induction of embryogenic cultures of some maize genotypes. Maintenance of these cultures, however, like anther derived tissue, required transfer to medium formulations with a lower sucrose concentration. Orientation of tissue in culture with respect to the culture medium has also been documented to exert an important influence on regeneration competence. This effect has been most extensively studied with primary expants. For example, cotyledon and leaf tissue explants from Cassava must be cultured adaxial surface away from the culture medium to elicit an embryogenic response (Stamp, 1987). With maize and other Gramineae, the scutellum produces somatic embryos only when it is cultured away from the culture medium (Vasil, 1985). The orientation of barley anthers on semi-solid culture medium exerted a dramatic effect on androgenesis (Shannon et al., 1985). Because of the lack of a predictable and quantitative model for the effect of the chemical and physical environment ·on plant growth 550 RANCH and PACE

and development, the media formulations and growth conditions used in plant cell and tissue culture have been derived from strictly an empirical approach (e.g., de Fossard et al., 1974). It is this limitation that represents probably the greatest obstacle to achieve­ ment in the discipline. Before a model of regeneration which con­ siders nutrition and regulators can be proposed, more knowledge about the biochemistry and physiology of plants is required. We can generate many questions about the relationship of chemicals and differentiation. How does the plant compartmentalize nutrients dur­ ing differentiation and morphogenesis? Do organs and tissues further compartmentalize these nutrients? If there is compartmentalization of nutrients, what role does this activity play in differentiation? Is compartmentalization caused by or a causal agent in differentiation? How do cells communicate with each other? What is the relationship of cell-cell recognition antigens on cell surfaces to morphogenic responses (Raff et al., 1979)? Is physical location related to only chemical effects, or is there a physical cell-cell contact effects in differentiation (Verbeke and Walker; Walker, 1978)? We usually test factors with little regard to dimensionality or the relationship of the factor source and the position of the target tissue. Is the biochemistry and physiology of regeneration polar in nature? Rarely do we see the mode of action of chemicals. We observe only effects. Morphogenic effects of chemicals are first seen days to weeks after exposure. The target or binding sites of growth regulators are uncharacterized. Hopefully, techniques will be established that allow the visualization of morphogenesis at greater levels of chemical resolution. In summary, we can describe three factors in consideration of the manipulation of morphogenic processes relating to plant regeneration from cell and tissue cultures. These factors are genetic constitution, the ontogenic stage of the donor tissue or explant, and the external environment. The potential of developmental events is determined by the genome. The ontogenic stage of the tissue relates to the current action of the genome and is described by the cells biochemical and cytological status. Lastly, the environment refers to the external factors which influence the biochemistry and cytology of the cell. The first two ingredients are properties of cells and predispose primary explants and serially propagated cells and tissues to behave in certain ways. The latter component provides a rationale for the manipulation of in vitro behavior by, for example, alterations in culture medium composition or sequence. PLANT REGENERATION 551

CONCEPTS AND AN OPERATIONAL FRAMEWORK

The three main elements, genotype, ontogenic state, and environ­ ment, are manipulated in vitro to achieve both growth and morpho­ genesis. These factors may not have equivalent impacts on regener­ ation depending on the circumstances of the specific system. They also may have varying impacts depending on the particular phase of regeneration in question. Since we have divided regeneration into morphogenesis and propagation phases, now we can consider how they impact on these distinct but interactive phases. Although we are considering regeneration processes exclusively, these factors also play an important role in the growth of cell and tissue cultures as well as in differentiation in organ cultures.

Morphogenesis Phase It is the interaction of genotype, ontogenic state, and environ­ ment in the morphogenesis phase that has been most extensively studied. The expression of competence and regeneration are initiated in this phase. For the sake of simplicity, it has been vogue to draw parallels between morphogenic patterns and metabolic pathways. In our view, plant development might best be characterized as a network in which these factors operate. Indeed, just as the organism may be described by the interaction of biochemical pathways, developmental patterns are the result of the interplay of differentiation and morphogenic processes. Ostensibly, all developmental phenomena are tied directly to the operation of biochemical pathways. Although there is substantial information concerning the correlation of specific biochemistries with developmental events, it is difficult to resolve the cause and effect relationship of these observations (for example, Brown et al., 1979; Brown and Thorpe, 1980; Ernst et al., 1984; Kochba et al., 1977; Noma et al., 1982; Nomura and Komamine, 1986; Orr and Schoneman, 1986; Rathore and Goldsworthy, 1985; Sung and Okimoto, 1983; Zee et al., 1979). Perhaps, however, we can generalize and speculate that morphogenic processes which are similar in consequence, regardless of genotype or taxa, must be analogous in some regard. In order, then, to encompass all the nuances of anatomy, morphology, physiology, and biochemistry characteristic of regeneration, the rationale in support of an operational approach to regeneration will necessarily be simple and qualitative. Because we cannot develop a priori, a biochemical rationale for attaining or maintaining the compentent state, we must consider regeneration in some other context to approach the formulation of 552 RANCH and PACE a testable model. In our opinion, regeneration should be viewed with a developmental perspective which considers the interaction of onto­ genic state and environmental conditions. After an initial genotype screen, there should be an effort to limit the genotypic effect since it presents a confounding influence to the understanding of the biology of regeneration. We interpret this interaction of ontogenic state and environment in terms of determination and differentiation. While differentiation is the process by which cells become specialized, or become a different cell type, determination is commitment to a specific differentiated or morphogenic state. There likely is a require­ ment for redetermination of some differentiated cell types in order to fulfill a different developmental pathway. In some cases, redeter­ mination may be preceded by dedifferentiation or redifferentiation to some ground or other state. The primary determinations for plant regeneration from cell and tissue cultures are generally regarded to be somatic embryogenesis and shoot organogenesis. Subsequent morphogenic patterns in culture are initiated in these events. It has been argued that shoot organo­ genesis is anatomically and morphologically derived from embryo­ genesis (Wicart et al., 1984). Embryogenesis might therefore be viewed as an archetypal event in plant development, with organogenic patterns being biochemical an<:l morphological modifications of embryo­ genic induction and determination. A corollary to this paradigm concludes that embryogeny is engendered by a seqence of competing organogenic events. The general lack of sharp demarkation between organs suggests this feature of development. This approach is further biologically attractive because embryogeny is ontogenically rooted in a single cell entity (totipotence) and represents the initial develop­ mental events of the sporophytic phase. How, and when, do cells acquire the determination for com­ petence? For somatic embryogenesis, this acquisition of the deter­ mined state has been categorized into direct and indirect depending on the timing of cytokinesis and the determinative event (Evans et al., 1981; Sharp et al., 1980). Possible mechanisms of this determination have been reviewed and discussed by Sharp et al. ( 1980). These classifications of determination are based largely on empirical anatomical and behavioral observations and await corroboration from physiological and biochemical evidence. If determination, like regeneration, is conceived of as a dynamic process rather than an event, it likely is influenced by the historical recollection of the cells and the contribution of the current in vivo cellular environment. The only confident assay for embryogenic determination is the organization PLANT REGENERATION 553 of a somatic embryo. We can characterize the effects of determination, but we may only speculate about the identification of determination itself. In the interaction of ontogenic state and environment, a basis for a continuum of organizational levels which vary in their dependence on the external environment for growth and morphogenesis may be described. The ontogenic state reflects a certain biochemical and physiological status, and each unique differentiated condition is directly related to determination for the competent phenotype. Plants can be regenerat ed and multiplied from propagules in vitro main­ tained in various ontogenic states and levels of organization. This can occur through adventive organogenesis, somatic embryogenesis, or perhaps some other as yet uncharacterized ontogenic hybrid between these regeneration modes. We choose, however, somatic embryo­ genesis as a model for our discussion of regeneration since it is the most well-characterized and offers the most extensive potential for practical and fundamental advance. At one extreme of the organizational spectrum the least organized tissue routinely propagated is the classic cell suspension and callus culture. These cell populations characteristically possess large vacu­ oles, maintain a rapid growth rate, and are apparently metabolically and morphogenically unspecialized (King and Street, 1977; King, 1980; Lindsey and Yeoman, 1985). Although these cell cultures are friable, plant cell cultures do form aggregates of several to hundreds of cells. In some cases these cells are morphogenically competent, but even so, competency is temporally unstable. It is unclear if in these cases there is present a small, almost indiscernible, population of competent cells which confers the appearance of regeneration ability to the popula­ tion as a whole (Nomura and Komamine, 1985). It is likely that the loss of morphogenic competence in typical plant cell culture is related to the manner in which the cultures are maintained. Biochemical and physiological studies historically have been performed with friable, rapidly growing, cell suspensions. This procedure was a powerful selection against more aggregated cell populat ions, possibly more slowly growing, which possessed com­ petence. The competent population, gradually lost by dilution, may explain the temporal loss of competency observed with this type of cell culture. This level of organization then is highly dependent on both the chemical and physical environment provided for growth and differentiation. In order for these cells to regain competence, some induction is required (Maliga et al., 1977; Negrutiu and Jacobs, 1978b; Stavarek et al., 1980; Traynor and Flashman, 1981; Wochok and Wetherell, 197la). These inductions include both acute and chronic 554 RANCH and PACE nutritional or hormone treatments. As might be expected, the conditions for induction of competence likely are not necessarily the same as the requirements for maintenance of the competent condi­ tion. Embryogenic cell cultures, a step upward ontogenically if not organizationally, may be proliferated as a suspension culture or on semi-solid medium formulations. Although these cells are friable, they are frequently characterized macroscopically by a granular appear­ ance. Embryogenic cells exhibit a dense, granular cytoplasm, are smaller than undifferentiated cell cultures, and possess a heter­ opycnotic nucleus with a prominent nucleolus (Halperin, 1966; Halperin and Jensen, 1967; Karlsson and Vasil, 1986; Srinivasan and Vasil, 1986; Thomas et al., 1972; Vasil and Vasil, 1982). They resemble cells in polar meristems. Generally the occurrence of the embryogenic cell type is as a sector randomly segregating upon an even more highly organized embryogenic tissue culture, perhaps derived from an immature embryo explant. Embryogenic cell cultures usually have a precarious existence, and may rapidly lose the capacity for regen­ eration. These cells may be in a metastable condition in which the ontogenic state, and therefore the determination to organize, requires rigorous maintenance of the chemical and physical surroundings. Often, these cultures must be sieved to maintain the competent cell population separate from populations which have differentiated to some other undetermined condition (Halperin, 1966). Indeed, the potential to lose competence in this cell type is reflected by its random generation. Because of this low level of organization and characteristic unstable state of determination, embryogenic cell cul­ tures are referred to as being 'open-ended' since they possess a wide range of morphogenic capacities (Krikorian et al., 1986) depending on environmental circumstances. At the other extreme of organizational complexity, embryogenic tissue cultures are highly heterogeneous and often propagate as tissues possessing visibly developed somatic embryos (Lu et al., 1982; Ranch et al., 1985). If the primary tissue source is properly chosen, these cultures may be produced quite easily. Occasionally special treatments, such as culture of the explant on medium high in osmolality for a short period, are required to induce the morphogenic program (Wetherell, 1984). Because the basis for propagation in these cultures is not at the cellular level, they do not proliferate as friable embryogenic cells and are best propagated on semi-solid medium formulations. In contrast to embryogenic cell cultures, these cultures are not open ended. These tissues inherently possess a terminal PLANT REGENERATION 555

differentiation to produce somatic embryos which often progress to well-advanced stages of embryogeny. In contrast to lower levels of organization, because of this fixed determination, embryogenic tissue cultures are not as fastidious in environmental requirements, and, at least theoretically, may be propagated, using appropriate tissue selection, for long periods without extensive loss of competence. Embryogenic tissue cultures, and perhaps embryogenic cell cul­ tures too, likely reflect a growth and differentiation comparable to in vivo polembryony (Lakshmanan and Ambegaokar, 1984; Vasil and Vasil, 1980). Perhaps like the ontogenic state of primary explants from embryonic sources, embryogenic cell and tissue cultures likely retain certain characteristics of the embryonic state. Haccius (1978) has proposed that embryogenic tissues may be ontogenically related to the suspensor since this organ often exhibits a polyembryonic response in vivo. Cells which confer competence to embryogenic tissues are referred to as proembryonic cell masses or complexes. Under appropriate conditions, somatic embryos are organized, often in a rosette-like fashion, from these meristematic centers. While embryogenic tissue cultures may be readily induced from juvenile primary explants and propagated, embryogenic cell cultures generally occur spontaneously. This is not uncommon, as even undifferentiated callus cultures randomly acquire a wide variety of phenotypes. In many species, there is as yet no way to direct the development of the embryogenic cell type, but once it occurs, it may be serially maintained (Ammirato, 1984; Vasil and Vasil, 1984). As was illustrated with undifferentiated cell cultures, one of the most useful tools in the repertoire of cell culture is the powerful method of tissue selection. For example, when propagating embryogenic tissue cul­ tures, sectors of tissue with a different morphology often appear. Each variant tissue type is subcultured from the parent tissue mass and propagated separately. The ensuing culture is kept pure by fastidious selection. Then, questions about the correlation of morphol­ ogy and level of organization with maintenance of morphogenic competence may be posed. After cells acquire the determination to organize, the induced developmental program requires environmental conditions appro­ priate for its execution. Inducers are no longer required, and a different medium formulation may be used. When the program is executed properly, the resulting morphogenic avenues in vi tro resemble, morphologically, anatomically, and biochemically, correspond­ ing processes which occur in the whole plant (Crouch, 1982; Halperin, 1966; Halperin and Wetherell, 1964; Kamada and Harada, 1981; Konar 556 RANCH and PACE et al., 1972b; Lu and Vasil, 1981; Nomura and Komamine, 1986; Sharp et al., 1980; Tisserat et al., 1979; van Lammeren, 1986; Vasil et al., 1984). This correlation is a direct reflection of the genetic potential in regeneration. At the microscopic level, some researchers nave provided detailed comparisons between zygotic and somatic embry­ ogeny (Haccius, 1978; Haccius and Bhandari, 1975; Street and Withers, 1974; Williams and Maheswaran, 1986). Although somatic embryogeny recapitulates zygotic embryogeny in many ways, there does appear to be some anatomical differences between the two processes, particularly during earlier stages. These differences are of some concern for they appear to contradict the premise that in vitro morphogenic processes are homologous to similar processes in vivo. There is considerable discussion about the unicellular origin of somatic embryos or adven­ tive shoots (Broertjes and Keen, 1980; Broertjes and Van Harten, 1985; Haccius, 1978; Williams and Maheswaran, 1986), and there are observations which describe significant differences in cell division patterns between zygotic and somatic embryogeny (Street and Withers, 1974). These early abnormal growth and developmental patterns in somatic embryogeny may portend abnormalities in sub­ sequent development. For example, somatic embryos in many species often possess an aberrant number of cotyledons. The cause for this deviation has been traced to the number of cells in the morphogenic field which differentiates to form the cotyledons (Ammirato, 1987; Halperin, 1966). Rarely does this particular deviation inhibit plant regeneration. Another type of morphogenic abnormality which arises in cell culture of many species has been termed neomorph (Krikorian and Kann, 1981). Simply put, a neomorph is a structure which has no place in the contextual framework of normal plant development and morphology. Neomorphs also occur at level of the whole organism (Waris, 1959). It is a terminally differentiated structure. What causes this morphological deformation is unknown. Neomorphs may arise by the application of inappropriate inductive signals to the genome, or the presence of conditions not conducive to the proper execution of a program. Neomorphs must be dedifferentiated, and/ or redetermined in order to recover a cell type which is appropriately programmed to develop into a whole plant. The formation of neomorphs and other unnamed morphogenic teratomas is presumed to be epigenetic. However, since we have proposed that growth and development in vitro reflect in vivo processes, we would expect that the essential characteristics of any morphogenic process will be conserved in vitro when a developmental track is accurately rendered. A whole plant PLANT REGENERATION 557 would not be regenerated if in vitro morphogenic processes were not tracking a biologically valid program. Once differences between in vivo and in vitro morphogenic processes are recognized and character­ ized, it might be instructive to interpret this variation as part of the well-known biological plasticity of plants in response to environ­ mental stimulus. We may then generalize that a particular morpho­ genic process may be perturbed to a certain extent and yet yield the same or similar result. In addition, the existence of differences permit analyses of the cause for the variation. Evaluation of these differences in the context of a dynamic process will better our understanding of regeneration biology. We would argue that the methods in cell and tissue culture have not advanced to the level of sophistication which allow the exact duplication of the in vivo environment. It is consequently expected that some anatomical characteristics of regen­ eration are not identical in vivo and in vitro. Under some circum­ stances, some cultured cells may be competent to organize many different type of structures simultaneously (Konar et al., 1972a). These, and other less fatal epigenetic variants, although perhaps inhibiting the organization of a whole plant, yield important informa­ tion about what is necessary for normal morphogenic processes to occur and the range of circumstances under which the process occurs. The occurrence of developmental abnormalities, or different morphogenic processes, may be manipulated by many factors includ­ ing growth regulators (Ammirato, 1977). Since development is funda­ mentally based on the interplay of biochemical pathways (perhaps being dependent on catabolism and anabolism of growth regulators), it might be anticipated that genocopies, mutants which mimic these epigenetic variants, may be generated (Meinke, 1986; Estelle and Somerville, 1987). The genesis of these variants also suggests the interaction of organogenic processes during pattern formation. When morphogenesis induction or expression conditions are inappropriate, the precedence of one organogenic pattern might result in develop­ mentally imbalanced morphogenesis in much the same way as imbalanced growth can occur in batch suspension cultures (King and Street, 1977). Generally, in vitro tissues which reflect the organization of a plant most readily are converted to whole plants. The determination to organize retained in the high organizational level is probably related to multicellularity (Williams and Maheswaran, 1986). Little to no induction is required to drive the developmental pathway toward organization of a whole plant. For example, micropropagation, meri­ stem culture, and zygotic embryo rescue, although not representing 558 RANCH and PACE regeneration under our definition, represent biological systems which have been determined to fulfill a particular developmental program. In many ways they are more autonomous than lower levels of organization such as suspension cultures. But they are also restricted in the breadth of morphogenic avenues they can enter. Cell cultures are more dependent on the environment for nutrition and develop­ mental signals, but potentially are open to programs of the entire developmental library. We agree with Krikorian et al. (1986) that the definitive goal in the manipulative art of plant regeneration would be the establish­ ment of a cell culture which could be maintained indefinitely in a state which conserves the determination to express morphogenic competence. Single cells and protoplasts isolated from such a cell culture would likely be totipotent. This differentiated state might be perpetually embryonic, a state which possesses all the determinative potentials of the complete organism. However, we currently lack the sophistication necessary to approach the manipulation of regenera­ tion in this manner.

Propagation Phase Except in the practice of horticultural propagation, little atten­ tion has been paid to the propagation phase of the regeneration process (Hartmann and Kester, 1983; Murashige, 1974). Multiplication for horticultural goals does not always require a fertile regenerant. Since our intent is the recovery of fertile plants, an understanding of this phase is even more important in our regeneration scenario. Frequently, failure of the propagation phase has been related to lack of capable root development on regenerated shoots or embryos, and lack of fertility. While the former obstacle is generally handled by root­ stimulating treatments, the later difficulty is more complex since it could be due to poor morphogenesis conditions or poor growth conditions for the regenerated whole plant. Often, regenerated plantlets succumb for no apparent reason. Lack of success in recovering whole plants therefore may be due to lack of regard for the regeneration process as a whole or ignoring the importance of the propagation stage. Regeneration is a highly integrated process. Although we have defined stages in regeneration, emphasis should not be placed on the implied discrete nature of the process. Rather, this view of the regeneration process serves to indicate that there is a continuum of development from which we extract symbolic, reductionist stages. The behavior of a cell, tissue, PLANT REGENERATION 559 organ, or whole plant for that matter, is based to a large extent on previous history. The precise cause for failure of the propagation phase, unfortun­ ately, can only be approximated since there can be only an ambiguous determination of the exact cause of the inadequacy. We might predict, however, that when the morphogenesis phase is executed properly, subsequent events in the propagation phase should follow as in vivo. This prediction is founded in the observation that when complexity increases, autonomy from special factors supplied externally will generally increase, and developmental competence is higher. This hypothesis can be readily extracted from organ culture studies. To recover whole plants from somatic embryos, for example, a successful approach might use methods successful for zygotic embryo rescue (Ragahavan, 1983). If a somatic embryo truly recapitulates zygotic embryogeny, then it should behave in all respects like a zygotic embryo. This discussion has ventured to describe how genotype, ontogenic state, and environment relate to in vitro processes in the morpho­ genesis and propagation phases of regeneration. Depending on stage of the regeneration process, these factors vary in their relative importance. In the morphogenesis phase, it is common to observe strong interaction between all the factors. While in the propagation phase, ontogenic state and environment seem to be most relevant.

SUMMARY

Many different processes may be referred to as regeneration. We have adopted an interpretation which suggests a framework for considering regeneration in the context of current uses of cell and tissue culture in 'biotechnology.' Whether the techniques of cell and tissue culture are used to elucidate developmental and morphogenic phenomena, or to produce plants to generate and evaluate soma­ clonal variation, there should be an awareness of the ontogeny of the regenerants. An evaluation to identify the source of cells from which the organization of a whole plant occurs is a valuable asset to effect, evaluate, and understand regeneration from cell and tissue cultures. Previous cell history and the current physiological status of cells, the genotype, and the culture medium, or sequence of culture media, all have an influence on differentiation and morphogenic activity. There should be no surprise that whole plants can be regenerated from cell cultures. Quite the contrary, it should be expected. Since the 560 RANCH and PACE responses of cultured cells reflect those events which occur in vivo, totipotency, as an epigenetic phenomenon, should be expressed in any tissue at any time given the appropriate stimulus-chemical or physical. The potential to express morphogenic competence is inher­ ited as part of the organisms biology. Because we presume that morphogenesis is conditioned by quantitative genetic factors, there likely is a continuum of competence which represents the genotype by environment interaction. Any deficiency in the expression of regen­ eration potential is due to a paucity of understanding how to induce morphogenesis and facilitate further development. Whether or not that epigenetic sequence which determines competence is expressed depends on many internal and external factors. Experimentally, however, through modifications associated with the environment or tissue source, circumstances likely can be defined which liberates the genetic repertory of the reacting cells such that they can be manipulated toward a desired morphogenic avenue. The goal of in vitro manipulations is then to mimic the in vivo environment to evoke the desired developmental response. The 'art of regeneration' describes the myriad manipulations associated with the technical skills of plant cell and tissue culture. These manipulations have acquired a certain mystique and pretense of sophistication. However, adeptness in husbanding cell and tissue cultures in itself does not provide any reasonable rationale for application or understanding. In the study of plant regeneration from cell and tissue cultures, we believe that there exists a scientific basis for understanding, manipulating, and applying in vitro systems. This belief is founded on the disciplines of physiology, biochemistry, genetics, and developmental biology. While gaps in our knowledge may prevent total reliance on this method, it nevertheless provides for the construction of testable hypotheses. In time, the gaps in our knowledge should diminish.

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---- and A. C. Hildebrandt. 1965a. Growth and tissue formation from single isolated tobacco cells in rnicroculture. Science 147:1454-1455. ---- and ----. 1965b. Differentiation of tobacco plants from single isolated cells in rnicroculture. Science 150: 889-890. ---- and Vasil, V. 1980. Clonal propagation, pp. 145-173. In: I. K. Vasil (ed.). Perspectives in plant cell and tissue culture, Academic Press, New York Vasil, V. , C-Y. Lu, and I. K. Vasil. 1985. Histology of somatic ernbryogenesis in cultured immature embryos of maize (Zea mays L.). Protoplasrna 127:1-8. ---- and I. K. Vasil. 1982. Characterization of an ernbryogenic cell suspension culture derived from cultured inflorescences of Pennisetum americanum (pearl millet, grarnineae). Arner. J. Bot. 69: 1441-1449. ---- and ----. 1984. Induction and maintenance of ernbryogenic callus cultures of Grarnineae, pp. 36-42. In: I. K. Vasil (ed .). Cell culture and somatic cell genetics of plants, Vol. 1, Academic Press, New York ----, ----, and C. Lu. 1984. Somatic ernbryogenesis in long-term callus cultures of Zea mays L. (Gramineae). Arner. J . Bot. 71:158-161. Verbeke, J. A. and D. B. Walker. 1986. Morphogenetic factors controlling differentiation and dedifferentiation of epidermal cells in the gynoeciurn of Catharanthus roseus. II. Diffusible rnorphogens. Planta 168:43-49. PLANT REGENERATION 569

Walker, D. B. 1978. Morphogenetic factors controlling differentiation and dedif­ ferentiation of epidermal cells in the gynoecium of Catharanthus roseus. I. The role of pressure and cell confinement. Planta 142:181-186. Walker, K. A. and S. J. Sato. 1981. Morphogenesis in callus tissue of Medicago sativa: the role of ammonium ion in somatic embryogenesis. Plant Cell. Tiss. Org. Cult. 1:109-121. ----, M. L. Wendeln, and E.G. Jaworski. 1979. Organogenesis in callus tissue of Medicago sativa. The temporal separation of induction processes from differentiation processes. Plant Sci. Lett. 16:23-30. Wang, A. S. 1987. Callus induction and plant regeneration from maize immature embryos. Plant Cell Rep. 6:360-362. Waris, H. 1959. Neomorphosis in seed plants induced by amino acids. I. Oenanthe aquatica. Physiol. Plant. 12:753-766. Wenzler, H. and F. Meins, Jr. 1986. Mapping regions of the maize leaf capable of proliferation in culture. Protoplasma 131:103-105. Wetherell, D. F. 1984. Enhanced adventive embryogenesis from plasmolysis of cultured wild carrot cells. Plant Cell Tiss. Org. Cult. 3:221-224. ---- and D. Dougall. 1976. Sources of nitrogen supporting growth and ernbryogenesis in cultured wild carrot tissue. Physiol. Plant. 37:97-103. White, P. R. 1943. A handbook of plant tissue culture, Ronald Press, New York Wicart, G. , A. Mouras, and A. Lutz. 1984. Histological study of organogenesis and embryogenesis in Cyclamen persicum Mill. tissue cultures: Evidence for a single organogenetic pattern. Protoplasma 119:159-167. Williams, E. G. and G. Maheswaran. 1986. Somatic embryogenesis: Factors influencing coordinated behavior of cells as an embryogenic group. Ann. Bot. 57:443-462. Wochok, Z. S. and D. F. Wetherell. 197la. Restoration of declining morphogenetic capacity in long term tissue cultures of Daucus carota by kinetin. Experientia 28: 104-105. ---- and ----. 197lb. Suppression of organized growth in cultured wild carrot tissue by 2-chloroethylphosphonic acid. Plant Cell Physiol. 12:771-774. Zee, S. Y., S. C. Wu , and S. B. Yue. 1979. Morphological and SDS-polyacrylamide gel electrophoretic studies of pro-embryoid formation in the petiole explants of chinese celery. Z. Pflanzenphysiol. 95:397-403.

IOWA STATE JOURNAL OF RESEARCH I MAY, 1988 571-585 Vol. 62, No. 4

SORGHUM CELL CULTURE: SOMACLONAL VARIATION/SCREENING

R. H, Smith and S. Bhaskaran 1

ABSTRACT. Cell culture of sorghum is being evaluated to determine the stability of the variation of somaclones, the reliability of drought tolerance screening, and the potent ial for crop improvement. Eight plant populations (SC2 generation) from individual plants regenerated from cell culture were examined for dif­ ferences in chlorophyll content, plant height, tiller number, days to flower, grain yield, and shoot dry weight. Seven of the somaclones had more tillers than the parent. Three somaclones were shorter than the parent. Six somaclones had more shoot dry weight, and three had higher grain yield. All the somaclones flowered earlier than the parent. Studies of SC2 and SC3 somaclones two years later showed stability in enhanced tillering and height reduction. The traits of earlier flowering, enhanced yield, and increased shoot weight were not maintained. Sorghum cell culture is also being examined as a model system to study t he physiology of osmoregulation and as a system to prescreen cultivars for potential field drought resistance. The data suggest that there is a realistic potential to use plant cell culture to identify cultivars for potential field drought tolerance. Index descriptors: sorghum cell culture, PEG screening, osmoregulation, somaclonal variation, in vitro screening.

INTRODUCTION

Plant cell culture, biotechnology, and molecular genetics have been proposed as a means to change agronomic crop plants in unconventional ways and thereby create superior plants. The potential to achieve unconventional genetic change has been demonstrated in tobacco and petunia plants, but they are much easier to manipulate in cell culture than other dicotyledonous plants and many cereal crop plants. Only within the last 5 to 10 years have research efforts seriously focused on important agronomic species. For this reason it is premature to realistically expect demonstrations of gene transfer into crop plants as dramatic as the transfer of luminescence into tobacco plants (Ow et al., 1986). These efforts in gene transfer probably will result in herbicide tolerance being effectively expressed in agronomic species (Gebhart, 1985; Kempner et al., 1986).

1Department of Soil and Crop Sciences, Texas A & M University, College Station, Texas 77843. Texas Agricultural Experiment Station Manuscript #22371. 572 SMITH and BHASKARAN

Reliable cell culture procedures in many crop plants still remain to be established, and useful agronomic traits must be understood at the metabolic level before genes can be identified and isolated. Cell culture generated variation in specific crop plants shoul\.l be examined. This paper will present some of the results of our studies on somaclonal variation in plants from sorghum cell culture and a study of osmotic stress in vitro and drought stress in vivo.

SOMACLONAL VARIATION

The term somaclonal variation was designated by Larkin and Scowcroft (1981) to describe genetic variability in plants derived from plant cell culture. The reported variation observed from plants derived from cell culture has been very exciting. It is important that the potentially useful variation should be due to heritable genetic alterations. In a recent review by Larkin (1987) evidence for chro­ mosome number changes, chromosome structure rearrangements, gene copy number changes, single gene mutations, altered expression of multigene families, and activity of transposable elements as the basis for observed somaclonal variation is discussed. In cereal crops there are many reports (Larkin and Scowcroft, 1983; Zong-xiu et al., 1983; Oono, 1985; Larkin et al., 1984) of heritable somaclonal variation. In rice (Zong-xiu et al., 1983) the genetic analysis of one dwarf mutant indicated that it was controlled by a single recessive gene. The authors observed a tendency for plant height to decrease and the number of productive tillers to increase in second and third generation somaclones. Oono (1985) reported increased grain number, panicle number, and seed weight in SCs somaclones of rice. Heritable somaclonal variation in wheat (Larkin et aL, 1984) was observed through two seed generations, and the traits appeared to be under simple and quantitative genetic control. Altered expression of a multigene family for gliadins (grain storage proteins in wheat) was discussed (Larkin, 1987) as a mechanism for heritable variation in the electrophoretic pattern of gliadins in wheat soma­ clones. Variation in triticale somaclones was reported by Jordan and Larter ( 1985). Spike length, fertility, and plant height were traits which had the greatest variability.

Somaclonal Variation in Sorghum

The details of this research are described in a paper by Bhaskaran et al. ( 1987} Sorghum bicolor Moench callus cultures which were 4 to 5 SORGHUM CELL CULTURE 573 months old were the source of eight regenerated plants. These eight plants were designated as the SC 1 generation, using the nomenclature of Larkin et al. (1984). To prevent cross pollination the panicles were bagged at exsertion and SD2 generation seeds were collected. Like­ wise, SCs seeds were collected from SC2 generation plants. In order to avoid the initial carry-over effects from cell culture, the first sexually derived progeny (the SC2 generation) was the starting point of this study. Twelve plants from each of the eight somaclone populations were grown to maturity, the panicles bagged at exsertion, and SC3 generation seed collected. The study was conducted over a two year period: fall 1984, ten months later in the summer of 1985, and two years later in the fall of 1986. All studies were conducted in a greenhouse with the populations of plants arranged in a randomized complete block design. The first study, fall 1984, was conducted on twelve plants (SC2 generation) from each of the original eight SC 1 plants and the parent line. These were designated M25 SC2, M26 SC2, M27 SC2 . .. M31 SC2. Agronomic characters which were examined included area of the third leaf, height, tiller number, total shoot weight, seed number, grain yield, days to flowering, and chlorophyll content. Statistical dif­ ferences between populations were tested using LS means, standard error, P difference and Duncan's analysis. The general linear model (GLM) procedure was used for the statistical analysis (Bhaskaran et al., 1987). All clones were similar in chlorophyll content and area of the third leaf. There were differences in some other characters among the somaclone populations and the original parent. All of the somaclones except M32 SC2 had more tillers than the parent. Somaclones which were significantly shorter than the parent were M25 SC2, M28 SC2, and M30 SC2; the rest were similar in plant height to the parent. Shoot dry weight was significantly greater than that of the parent in somaclones except M29 SC2 and M32 SC2 (Fig. 1). All the somaclones flowered 8 to 14 days earlier than the parent. Grain yield in grams per plant was higher in M26 SC2, M28 SC2, and M31 SC2 (Fig. 2); however, single seed weight was 15.6 grams/ 1,000 seeds in the parent and averaged 11.4grams/ 1,000 seeds in the somaclones. These observations in the SC2 generation were similar to those reported in SC2 rice somaclones (Zong-xiu et al., 1983). Some of these traits, if maintained in the SC3 generation, could be important in a breeding improvement program. In the summer of 1985 a study of M31 SC2 and M31 SCs along with the parent was conducted using 574 SMITH and BHASKARAN

10

8 en a: 6 w _J _J ~ 4

2

0 25 26 27 28 29 30 31 32 PARENT

,....., 100 E (.) '-I 80 I- I CJ 60 w I 40 I-z <( 20 _J a.. 0 25 26 27 28 29 30 31 32 PARENT

,....., 50 O> '-I I- 40 I CJ 30 w ~ 20 t- 0 0 10 I en 0 25 26 27 28 29 30 31 32 PARENT

Figure 1. Means of somaclonal populations M25 SC2 through M32 SC2 compared to the original parent line in regard to number of tillers (top ), plant height (middle), and shoot weight· (bottom). Statistical differences among populations, at p<0.05, as determined by Duncan's analysis, are indicated by histogram shading. SORGHUM CELL CULTURE 575

a: 80 w ;: 60 0 _J LL 40 ....0 en >- 20 < 0 0 25 26 27 28 29 30 31 32 PARENT

8

~ C> 1..-J 6 0 _J w >- 4 z a:< 2 (!) 0 25 26 27 28 29 30 31 32 PARENT

Figure 2. Means of somaclonal populations M25 SC2 through M32 SC2 compared to the original parent line in regard to days to flower (top) and grain yield (bottom). Statistical differences among populations, at p<0.05, as determined by Duncan's analysis, are indicated by histogram shading. 576 SMITH and BHASKARAN larger populations (120 plants for each). The results of this study were unexpected. There was no difference among the populations in regard to tiller number, plant height (Fig. 3), shoot weight, or days to flower in the M31 somaclone population. Originally, there were differences between M31 SC2 and the parent in number of tillers , shoot weight (Fig. 1), days to flower, and grain yield (Fig. 2). This study was conducted in the summer when growing condi­ tions for sorghum were optimal (high light, hot temperatures). We felt there might be an environmental interaction involved in the first study since it had been conducted in the fall of 1984 when light intensity was reduced and temperatures were much cooler-a more stressed growing condition for sorghum. A third study was conducted a year later. This experiment involved 28 plants from the parent and each of the somaclones, M31 SC2, M31 SC3, M26 SC2, M26 SC3-7, M26 SC3-9, M28 SC2, M28 SC2-8, M28 SC3-l , and M28 SC3-l 2. The different designations in SC3 clones indicate seed derived from individual plants of the SC2 generation. The data on M31 SC2 and M31 SC3 (Fig. 3) indicate that the original tillering and shoot weight enhancement, and earlier flowering traits were not maintained. The M31 SC3 population did show a reduction in height. In the first study, the M3 l SC2 somaclone was similar to the parent population in height. However, in somaclones M26 SC2 and M28 SC2, the enhanced tillering initially observed (Fig. 1) was subsequently maintained in the SC3 populations (Fig. 4). The original somaclone M26 showed no reduction in height; however, subsequently the SC2 and SC3 populations were shorter (Fig. 5). (Only 12 plants composed each somaclone population in the first study, whereas, 28 plants composed each population in the third study.) Likewise, for somaclone M28, original similarities in the SC2 population were maintained in the SC3 populations except for one plant which was shorter (Fig. 5). Interestingly, differences in days to flower were not maintained in either M26 or M28 populations. From these studies it can be observed that some somaclonal differences were heritable in the SC2 and SC3 generations. These enhanced include tillering and height reduction which can have agronomic benefits. The traits of earlier flowering, enhanced yield, and increased biomass were not maintained.

DROUGHT STRESS AND CELL CULTURE

The use of plant cell culture to examine components of whole plant drought stress has several advantages. Plant cell culture offers b 7 140 ~ Parent rzzzZJ M31 SC2 CSSSS1 M31 SC3 c::.::::J Pa rent a 6 C2'ZZZ2I M31 SC2 ~ M31 SC3

5

(/) a: w ....J w ....J :c I- 1-z <( _J a..

FALL SUMMER FALL FALL SUMMER FALL 1984 1985 1986 1984 1985 1986

Figure 3. Means of tillers (left) and plant height (right) of M31 SC2, M31 SC3 , a nd parent line populations. Within time periods, histograms labeled with the same letter(s) are statistically indistinguishable at p<0.05, according to Duncan's analysis. 8 9 b c::::J Parent b c:=::J Parent l'.ZZZZl M 2 8 SC 2 7 CZZZZJ M26 SC2 8 ~ M28 SC3-2 ~ M26 SC3-7 l'SSSSl M28 SC3-1 ~ M26 SC3-9 ~ M28 SC3-12 7 6 6 Cl) 5 Cl) a: a: w w 5 _J 4 _J _J _J 4 ...... 3 ...... 3 2 2

1 1

FALL FALL FALL FALL 1984 1986 1984 1986

Figure 4. Means of tiller numbers of parent line, M26 SC2, M26 SC3-7, and M26 SC3-9, (left), and parent line, M28 SC2, M28 SC3-2, M28 SC3-l, and M28 SC3-12 (right) populations. Within each growing period, histograms labeled with the same letter(s) are statistically indistinguishable at p< 0.05, according to Duncan's analysis. c=::J Parent c=::J Parent E7ZZ2 M28 SC2 lZ2ZZJ M26 SC2 (;S'SSSI M28 SC3-2 90 ~ M26 SC3-7 90 ES'S'SS1 M28 SC3-1 ~ M26 SC3-9 ~ M28 SC3-12 a a a ab 80 80 a 70 70

60 60 J­ J­ I I (!) 50 (!) 50 w w I 40 I 40 J­ J­ z 30 z 30

FALL FALL FALL FALL 1984 1986 1984 1986 Figure 5. Means for plant height of parent line, M26 SC2, M26 SC3-7, and M26 SC3-9 (left), and parent line M28 SC2, M28 SC3-2, and M28 SC3-l, M28 SC3-12 populations (right). Within each growing period, histograms labeled with the same letter(s) are statistically indistinguishable at p<0.05, according to Duncan's analysis. 580 SMITH and BHASKARAN

Table 1. Ratings of 10 sorghum cultivars to osmotic stress from 15% PEG, and drought tolerance in the field. Tissue Culture Drought Tolerancea Growth in 15% PEG Rating Pre-Flowering Post-Flowering RTx430 BTx623 B-35 RTx7078 RTx7000 1790E RTx7000 RTx7078 RTx430 BTx623b RTx430 RTx432 1790E RTx432 RTx9188 BTx378 1970E RTx7078 B35 B-35 BTx623 RTx432 BTx378 RTx7000 BTx3197 BTx3197 BTx3197 R9188 R9188 BTx378

aCultivars are in order of good tolerance on top to susceptible at bottom of column. bThe cultivars separated by line indicate significant grouping differences using the Scott-Knott Multiple Comparisons procedure (p< 0.05).

the opportunity to study cellular-level responses to osmotic stress. At the whole plant level many mechanisms are involved in overall tolerance. These vary from plant to plant, and include characteristics such as life-cycle timing, deep root systems, leaf orientation, cuticle thickness, stomata! closure mechanisms as well as osmoregulation at the cellular-level (Kramer, 1983). In cell culture, the underlying biochemical mechanisms of osmoregulation can be examined. Cellular­ level mechanisms may be amenable to genetic manipulation. Further­ more, it may also be possible to develop methods of evaluation and/ or screening germplasm for potential drought tolerance. A major question is whether a similarity exists between cellular-level responses and whole-plant responses under field conditions. There are studies which suggest that correlations between whole­ plant and cell-culture responses to salt tolerance can be established. Relative tolerance of grape ( Vitis species) cultivars to salt was the same at the cellular level as for whole plants (Barlass and Skene, 1981). Orton (1980) determined for cultivated barley (Hordeum vulgare L.) and a wild relative that salt tolerance at the cellular level SORGHUM CELL CULTURE 581 is similar to that at the whole-plant level. In tobacco cell culture salt­ tolerant lines were selected, plants regenerated, and salt tolerance was expressed in the whole plant (Nabors et al., 1980). Polyethylene glycol (PEG) has been used as a nonpenetrating osmotic agent that lowers the water potential of media and has been used to simulate drought stress in plants. PEG has been used (Bressan et al., 1981, 1982; Randa et al., 1982) to select Lycopersicon esculentum Mill cell lines which were tolerant to PEG. The tolerant cells grew better than cells never exposed to PEG, but lost resistance in a medium lacking PEG (Bressan et al., 1981).

PEG Screening in Sorghum

Ten cultivars of sorghum were obtained which had been evaluated by the sorghum breeding improvement program of the Texas Agri­ cultural Experiment Station, through field ratings at Lubbock, Big Spring, Amarillo, Chillicothe, and College Station, Texas over a 10-year period for differences in whole plant drought tolerance (Rosenow and Clark, 1981; Rosenow et al., 1983). The ten cultivars were divided into two groups: those with good pre-flowering drought tolerance character­ istics and those with post-flowering drought tolerance. Visual observa­ tions for pre-flowering stress symptoms included leaf rolling, excessive leaf erectness, leaf bleaching, leaf tip and margin burn, delayed flowering, poor head exsertion, head and floret abortion and blasting, and reduced head size. Post-flowering stress symptoms included premature plant (leaf and stem) death or senescence, often accom­ panied by charcoal rotted stalks and stalk lodging and sometimes seed size reduction. Table 1 indicates the relative tolerance of the ten cultivars for pre-flowering and post-flowering drought stress. The ten cultivars were evaluated in vitro (Smith et al., 1985) for their relative tolerance to six levels of PEG (0, 5, 10, 15, 20, and 25% w / v) in medium corresponding to -0.2, -0.35, -0.5, -0.75, -1.15, and -1.62 MPa osmotic stress. Fresh growth weight of the callus was recorded at weekly intervals and initial fresh weights were 250 ± 5 mg. There were differences among the cultivars in response to PEG (Smith et al., 1985). The ranking of the ten cultivars at 15% PEG after 5 weeks growth is shown in Table 1. Cultivars RTx430, RTx7078, RTx7000, and BTx623 were the cultivars which had the best growth at 15% PEG after 5 weeks in culture. These also are the lines which have the best pre-flowering drought tolerance under field conditions (Table 1 ). This observation suggests that at least a component of the 582 SMITH and BHASKARAN

Table 2. Ratings of cultivars screened in vitro by performance on 15% PEG (fresh mean weight at week 5) and stability in the field trials (See Panella, 1986). Cultivar Ranking Cultivar Rank of Performance by Performance on 15% PEG (Fresh Weight t ADN#55 *84V(PDLT161)-l R3224 RTx430 *84V(DLT112)-l *SC719-11E *SC33-14E *SC414-12E *82CV4844-l SC303-14E *84V(PDLT161)-1 84T385 *84Ll0789 *84Ll0789 Bl887 BTx626 80B-2892 IS1598C *SC414-12E *SC33-14E *RTx7078 *84V(DLT112)-1 BTx3197 *82CV4844-l SC599-11E *RTx7078 *SC719-l 1E 84V7696-l 84L6018 *WSMlOO *WSMlOO SC265-14E 84T383 R9188 84L28183 R3224 SC265-14E RTx435 84T385 84T383 BTx626 RTx7000 R9188 84L6018 84V7696-l V615 IS1598C Rio RTx7000 Karper 1597 B35 SC599-11E RTx430 V6146 Rio Bl887 Karper 1597 84L28183 VG15 BTx3197 RTx435 B35 SC303-14E ADN#55 77CS2 77CS2 VG146 SOB-2892

8 Cultivars are ranked from highest to lowest fresh weight on 15% PEG (w/ v).

*Top performing cultivars in both tests. SORGHUM CELL CULTURE 583 early field drought tolerance in sorghum may have a cellular basis. It also suggested that an in vitro screening technique might be useful in selecting germplasm for pre-flowering drought tolerance which could then be field tested. We are currently pursuing both possibilities and the remainder of the paper will present data on the screening possibilities.

Field vs. In Vitro Screen

Panella ( 1986) compared drought tolerance under field condi­ tions and in vitro osmotic tolerance to PEG using 34 sorghum cultivars. Four different field locations were studied, two of which involved pre-flowering drought. Table 2 is a summary of some of his findings. Panella (1986) used the model of Finlay and Wilkinson to describe stability as performance of a cultivar over a series of environments. The top 17 cultivars in the field ranking are compared to the top 17 cultivars in the in vitro screening; the cultivars indicated by an asterisk fall in both groups (Table 2). If 34 cultivars are screened in vitro and the top 50% selected for further field testing, it appears that there is a 53% chance that these cultivars would rank in the top half of a field evaluation. This is an indicator that the in vitro screening may have merit as a predictive evaluation of field performance. We are currently study­ ing the cellular physiology of callus and seedling tissue under osmotic and drought stress to understand what, if any, cellular mechanisms may be common to both systems. If a specific mechanism can be established, in vitro screening could become a useful predictive tool for cultivar performance in the field. In summary, research is continuing on addressing the question of how cell culture approaches can be integrated and have a positive impact on traditional sorghum improvement plant breeding pro­ grams. There are some aspects of somaclonal variation that could be utilized for enhanced biomass production. Additionally, there appears to be the very interesting observation that the initial somaclones all flowered significantly earlier. Yet, the same seed batches stored for a period of time totally lose this ability. Many researchers have observed earlier flowering in plants from cell culture. Why does this disappear over time of seed storage? This is an observation that merits further study; if it is a real phenomenon, is it possible to ultimately regulate flowering from seed? Certainly this would be of agronomic impor­ tance. 584 SMITH and BHASKARAN

Our studies on in vitro screer..ing for potential drought tolerance at this point are interesting. It seems that there is an aspect of cellular reaction to osmotic stress that is reflected in the plant's overall performance in the field under drought stress. Studies at the molecular level on both seedling and callus tissue are examining similarities at the cellular level to whole plant response. Comparisons of whole plant reactions to stressful environments to cellular level responses in tissue culture are difficult and it is impossible at this time to make direct comparisons. The above studies, however, do indicate some similarities in response which may have a common metabolic pathway at the cellular level. Should a common ground be determined between isolated cellular response and the more complex whole plant reaction, then a better position will be established to regulate and ultimately modify whole plant response.

LITERATURE CITED

Barlass, M. and K. G. M. Skene. 1981. Relative NaCl tolerance of grapevine cultivars and hybrids in vitro. Z. Pflanzenphysiol. Bd. 102:147-156. Bhaskaran, S., R.H. Smith, S. Paliwal, and K. F. Schertz. 1987. Somaclonal variation from Sorghum bicolor (L.) Mo ench cell culture. Plant Cell Tissue & Organ Culture 9:189-196. Bressan, R. A., P. M. Hasegawa, and A. K. Handa. 1981. Resistance of cultured higher plant cells to polyethylene glycol-induced water stress. Plant Sci. Lett. 21:23-30. ----, A. K. Handa, S. Handa, et al. 1982. Growth and water relations of cultured tomato cells after adjustment to low external water potentials. Plant Physiol. 70:1303-1309. Gebhart, F. 1985. Calgene obtains first patent for genetically engineered crop gene. Gen. Eng. News 5:3. Handa, A. K., R. A. Bressan, S. Handa, et al. 1982. Characteristics of cultured tomato cells after prolonged exposure to medium containing polyethylene glycol. Plant Physiol. 69:514-521. Jordan, M. C. and E. N. Larter. 1985. Somaclonal variation in tritical (x Triticosecale Wittmack) cv. Carman. Can. J. Genet. & Cytol. 27:151-157. Kempner, D. H., G. W. Schnell, and B. Wolnak. 1986. Biotech's impact on agriculture will be evolutionary, not revolutionary. Gen. Eng. News 6:18-19. Kramer, P. J. 1983. Water stress research-progress and problems, p. 129. In: D. D. Randall (ed.). Current Topics in Plant Biochemistry and Physiology. Univ. of Missouri Press, Columbia. Larkin, P. J. 1987. Somaclonal variation: history, method, and meaning. Iowa State J. of Res. 61:393-434. SORGHUM CELL CULTURE 585

----, S. A. Ryan, R. I. S. Brettell, and W. R. Scowcroft. 1984. Heritable sornaclonal variation in wheat. Theor. Appl. Genet. 67:443-455. ---- and W. R. Scowcroft. 1981. Sornaclonal variation-a novel source of variability from cell cultures for plant improvement. Theor. Appl. Genet. 60:197-214. ---- and ----. 1983. Sornaclonal variation and crop improvement, pp. 289-309. In: T. Kosuge, C. P. Meredith, and A. Hollaender (eds.). Genetic Engineering of Plants: An Agricultural Perspective. Plenum Press, New York. Nabors, M. W. , S. E. Gibbs, C. S. Bernstein, et al. 1980. NaCl-tolerant tobacco plants from cultured cells. Z. Pflanzenphysiol. Bd. 97:13-17. Oono, K. 1985. Putative homozygous mutations in regenerated plants of rice. Mol. Gen. Genet. 198:377-384. Orton, T. J. 1980. Comparisons of salt tolerance between Hordeum vulgare and H. • jubatum in whole plants and callus cultures. Z. Pflanzenphysiol. 98:106-118. Ow, D. W., K. V. Wood, M. DeLuca, J. R. DeWet, D.R. Helsink, and S. H. Howell. 1986. Transient and stable expression of the firefly luciferase gene in plant cells and transgenic plants. Science 234:856-859. Panella, L. 1986. A comparison of field and in vitro screening methods for drought resistance in Sorghum bicolor (L.) Moench. M. S. Thesis, Texas A & M University, College Station, Texas. Rosenow, D. T. and L. E. Clark 1981. Drought tolerance in sorghum. Arn. Seed Trade Assoc. Proc. 36:18-30. ----, J. E. Quisenberry, C. W. Wendt, et al. 1983. Drought-tolerant sorghum and cotton gerrnplasrn. Agric. Water Manage. 7:207-222. Smith, R. H., S. Bhaskaran, and F. R. Miller. 1985. Screening for drought tolerance in sorghum using cell culture. In Vitro Cell. & Dev. Bio. 21:541-545. Zong-xiu, S., Z. Cheng-zhang, Z. Kang-le, Q. Xiu-fang, and F. Ya-ping. 1983. Sornaclonal genetics of rice, Oryza sativa L. Theor. Appl. Genet. 67:73.

IOWA STATE JOURNAL OF RESEARCH I MAY, 1988 587-597 Vol. 62, No. 4

IN VITRO SELECTION WITH PLANT CELL AND TISSUE CULTURES: AN OVERVIEW1

Jack M. Widholm2

ABSTRACT. In vitro techniques can be used to select a wide variety of variants or mutants using plant cells, callus or protoplasts. These would include strains capable of growing under the culture conditions employed, like photoautotrophic, or different hormonal or mineral regimes. Strains capable of producing valuable secondary compounds can also be selected for by several methods. Several auxotroph selection methods are also available, and resistance selection systems are usually easily devised. The proper application of in vitro selection techniques can produce materials of value for basic research, for compound production, as well as for crop improvement. Index descriptors: auxotrophic mutants, crop improvement, in vitro selection, resistance mutants.

INTRODUCTION

The selection of mutants using cultured plant cells is in principle similar to that done with microorganisms, but in practice is much more difficult. The reasons for the difficulties include the usual clumpy nature of plant cell cultures; single cells or protoplasts usually cannot be easily grown to form clones, cell growth is slow and the cells are usually not monoploid. Despite these problems a large number of successful selection experiments have been carried out to produce mutants of value for producing compounds, for biochemical and molecular biology studies, for markers in genetic experiments and for improving crop plants. Part of the reason for the success is that cell systems readily allow the screening of millions of cells for the desired trait. Whether the selected phenotype is under genetic or epigenetic control can most easily be determined by regenerating plants and by following the phenotype in progeny. Genetically controlled phenotypes would be inherited by progeny and would generally be more stable at the cell level in comparison to epigenetically controlled traits. A large 1Supported by funds from the Illinois Agricultural Experiment Station, National Science Foundation and USDA Competitive Research Grants Program. 2Professor, University of Illinois, Department of Agronomy, Turner Hall, 1102 South Goodwin, Urbana, IL 61801. 588 WIDHOLM number of in vitro selected traits have been shown to be expressed in regenerated plants and to be passed on to progeny. In this review I will discuss only a small sample of the published information on in vitro selection since space does not allow a complete review.

SELECTION FOR GROWfH

Growth in culture could be described as an unnatural situation for plant cells and one which would select for those growing most rapidly. This is evident in many suspension cultures where often for a month or so, the cultures are very lumpy and grow slowly, but in time they become finer and growth accelerates. This can be possibly explained by a selection process. I also think that there is a selection process for growth under the specific media conditions used, like salt formulations and hormone types and concentrations. Evidence for this comes from observations that media used in different laboratories do not support good growth of cell strains from other labs. This would indicate that either a selection for cells adapted to the specific media occurred or that cells can actually adapt in mass to different media. Another example of selecting or adapting cells for growth under specific conditions is the case of photoautotrophic cultures, i.e., cultures which can grow photosynthetically solely with C02 ~nd light. In our experience developing photoautotrophic suspension cultures of soybean (Horn et al., 1983, S. Rogers and J. M. Widholm, unpub­ lished), cotton, Nicotiana and Amaranthus (L. C. Blair and J. M. Widholm, unpublished), visual selection for green callus is usually necessary. In subsequent incubation in liquid medium with the sucrose concentration decreased, the more intensely green cells are selected for by their ability to survive and after many months usually form a relatively fine, highly chlorophyllous suspension with a doubl­ ing time of approximately 4 days.

SELECTION FOR VALUABLE COMPOUND PRODUCTION

An area which is still in its infancy is the selection of cell strains which accumulate valuable compounds like drugs which are com­ mercially obtained, in many cases, from plant materials. Even though scientists have been attempting for about 30 years to obtain plant tissue cultures which produce commercially important compounds at levels making their production economically feasible, the amount of success has been minimal. I feel that this lack of success is in part due PLANT CELL AND TISSUE CULTURES 589 to the general approach used, which is to culture tissues and then try to induce synthesis of the compound rather than looking for naturally occurring or induced mutant cells which are producing the com­ pound at high levels. One way to select such overproducing strains is to do visual selection if the compound in question is colored. This was done to initiate the only system which is now in commercial production, shikonin, which is a red pigment (Tabata and Fujita, 1985). As discussed in a recent review (Widholm, 1987), there are a number of other ways to identify overproducers or attempt to manipulate the biosynthetic pathways to create overproducers. If the compound is not colored then sensitive, rapid chemical assays, spot tests, micro­ spectrophotomet ric or flow cytometric analysis can be used. As described later, pathways may be manipulated to select over­ producers using analogs or by using toxic compounds which can be detoxified by a certain biosynthetic enzyme which would thus select for increased levels of that biosynthetic enzyme. While extensive efforts at selecting for cells which accumulate compounds as described above have not been carried out as yet, some studies like those of Zenk (1978) have shown that such strains can be unstable in their compound-accumulating ability. One can assume that the selected variants in these cases are not mutants but are epigenetic variants in an unstable developmental state. Since plant regeneration is not needed, the use of mutagens to induce the desired mutations might be called for in such experiments.

AUXOTROPH SELECTION

Auxotrophs can be defined as mutants which cannot synthesize a compound needed for growth, usually caused by a deficiency in a biosynthetic enzyme. Auxotrophs can only survive if the deficient compound is added to the culture medium. Since auxotrophy would normally be recessive, monoploid cultures must be used in the selection. Anther culture techniques are usually used to obtain the monoploid starting material. The most successful method for auxotroph selection has utilized the "specific directed suicide" procedure in which chlorate, acting as a nitrate analog, is converted by nitrate reductase to the more toxic compound chlorite. Thus cells with nitrate reductase activity produce chlorite and commit suicide, leaving cells which lack nitrate reductase alive. These nitrate reductase deficient cells can be rescued by adding a reduced form of nitrogen like amino acids. This system was first 590 WIDHOLM used by Muller and Grafe (1978) with tobacco cultures. Other directed systems include allyl alcohol to select for alcohol dehydro­ genase deficiency (Schwartz and Osterman, 1976) and indole analogs to select for tryptophan synthase deficiency (Widholm, 1981). The allyl alcohol system has been used successfully with pollen but not with cultured cells. We have recently, however, used allyl alcohol with Nicotiana plumbaginifolia suspension cultures to select lines with less than half the normal alcohol dehydrogenase activity (I. Kishinami and J. M. Widholm, unpublished). The allyl alcohol tolerant cells lack two of three activity bands on starch gels indicating the loss of activity of a gene forming one subunit of the dimeric enzyme. The cells with decreased alcohol dehydrogenase activity also showed increased sensitivity to anaerobic conditions as do Zea mays mutants which lack alcohol dehydrogenase (Schwartz, 1969). There are also "general suicide" methods which would include 5-bromodeoxyuridine (BudR) which is toxic due to incorporation into DNA and arsenate which is a phosphate analog. These compounds kill growing nonauxotrophic cells on minimal medium while the nongrow­ ing auxotrophs must be rescued by supplementing the medium with the required compounds. Carlson ( 1970) used BudR to select six leaky tobacco auxotrophs which showed a partial requirement for hypoxanthine, biotin, para-aminobenzoic acid, arginine, lysine or proline while Polacco ( 1979) used arsenate to select a soybean cell line which required added proteins or amino acids for growth. The widest variety of auxotrophic mutants have been obtained using "brute force" methods whereby cell clones are individually tested for their inability to grow on minimal medium. As an example, Gebhardt et al. (1981) tested 28,872 clones obtained from muta­ genized Hyoscyamus muticus protoplasts for their ability to grow on minimal medium at a higher than normal temperature of 32°C. Clones which did not grow were rescued on complete medium at 26°C and upon further testing, two required histidine, one tryptophan, three nicotinamide, two undefined amino acids, while two lacked nitrate reductase and two were temperature sensitive, i.e., they could grow at 26°C but not at 32°C.

RESISTANCE SELECTION

Selection for resistance should be the easiest kind of selection to accomplish and from the number of reports in the literature this would appear to be true. As described above, resistance selection can yield auxotrophic mutants using specific inhibitors, but the more PLANT CELL AND TISSUE CULTURES 591

Table 1. Agriculturally valuable traits which may be selectable with cultured plant cells. Trait Selection Agent Resistance to

Herbicide Herbicide Disease Pathogen toxin Salt Salt Metals Metal High or low pH High or low pH Flooding Anaerobic Heat Heat Cold Cold Drought High osmotic Insects Select for high toxic chemical Low nutrient Low nutrient

Increased levels

Free amino acids Amino acid analog

general approach can utilize almost any compound or condition which inhibits cell growth. Table 1 contains a list of agriculturally valuable traits which might be selected for in culture, and I will describe some examples. One necessary requirement of such selection schemes is that the trait selected for at the cell level be expressed in the regenerated plant if plant improvement is desired. Certainly not all traits desired at the whole plant level are expressed at the cell level such as yield, seed color, etc. Many agronomic traits are also under multigenic control so simple selection seems unlikely. We are also very deficient in our knowledge of the biochemical processes which control most traits of interest so rational selection approaches are not always evident. Selection for tolerance to herbicides is, however, an example which is straightforward since many herbicides which kill plants can also kill cell and tissue cultures. Sometimes, however, herbicides which act by inhibiting photosynthesis do not kill cultured cells easily 592 WIDHOLM since the cells are usually grown on sucrose and so are not photosynthetic. Resistance to a common photosynthetic herbicide type, the triazines, was selected for using green cultures of Nicotiana plumbaginifolia (Cseplo et al., 1985). The regenerated plants were two to three orders of magnitude more resistant than the wild type to triazine herbicides, and the resistance was maternally inherited. Non-green Nicotiana tabacum callus was selected resistant to the sulfonylurea herbicides, chlorsulfuron and sulfometuron methyl, and plants were regenerated which also expressed resistance (Chaleff and Ray, 1984). The resistance was inherited as dominant or semi­ dominant nuclear genes with two different loci being identified in six different selected mutants. The resistance was due to an altered form of the target enzyme, acetolactate synthase, which was less sensitive to inhibition by the two sulfonylurea herbicides (Chaleff and Mauvais, 1984). Acetolactate synthase is the first enzyme in the biosynthetic pathway of the branched chain amino acids, isoleucine, leucine and valine. Selection for resistance to another herbicide, glyphosate (N­ phosphonomethylglycine) has shown that gene amplification can be selected for with plant cell cultures. We selected carrot suspension cultures by a gradual procedure to be more than 50-times more tolerant than the wild type (Nafziger et al., 1984). The resistance was due to a 12-fold increase in the glyphosate target enzyme, 5-enolpyruvylshikimic acid-3-phosphate synthase (EPSPS), which we have recently found by Southern blotting to be caused by an amplification in the number of EPSPS genes (Ye et al., 1987). A glyphosate resistant Petunia culture with 15- to 20-times the normal EPSPS activity, had increased EPSPS messenger RNA and a 20-fold amplification of the EPSPS gene (Shah et al., 1986). The only other case of selected gene amplification in higher plants is the alfalfa suspension cultures selected for resistance to the herbicide, L-phosphinothricin, which inhibits glutamine synthetase (Donn et al., 1984). The glutamine synthetase activity was elevated 3-to 7-fold, the messenger RNA was elevated 8-fold, and one gluta­ mine synthetase gene was amplified 4- to 11-fold. Successful selection for disease resistance can be possible when the disease symptoms are caused by a toxin produced by the pathogen. The first case of such a selection was reported by Gengenbach and Green ( 1975) where maize callus with T-cytoplasm was treated with partially purified toxin from Helminthosporium maydis race T, the fungus which causes southern corn leaf blight. Callus with T-cytoplasm was much more sensitive to the toxin than PLANT CELL AND TISSUE CULTURES 593 was callus with N-cytoplasrn. All plants regenerated after 5 or more cycles of recurrent selection were resistant to the toxin and to the pathogen, and this resistance was inherited maternally (Gengenbach et al., 1977). Unfortunately, in no case was the link between cyto­ plasmic male sterility and disease susceptibility broken. The change to disease resistance was associated with a change in mitochondrial DNA from the T-type to some new form different from the N-type (Gengenbach et al., 1981). We have been able to devise a selection scheme with soybean callus for resistance to brown stern rot which is caused by Phialophora gregata (Gray et al., 1986). Culture filtrates inhibit the growth of callus from susceptible genotypes but not resistant ones, while culture filtrates from nonpathogenic P. gregata isolates have no effect on callus from either susceptible or resistant genotypes. Selection has been in progress with organogenic soybean callus which is capable of plant regeneration (Barwale et aL , 1986) to attempt to select resistance in sensitive genotypes and to increase the resistance of resistant genotypes. So far nothing with proven resistance has been regenerated. This laboratory has been involved in the selection for amino acid analog resistance for a number of years. This selection work can be important for plant improvement since we usually recover resistant lines which overproduce the corresponding normal free amino acid. Such increases are a potential means of increasing the concentrations of certain essential amino acids such as lysine, tryptophan or methionine in which plants are often deficient, or the content of proline which is associated with stress responses. Increased levels of free amino acids can also lead to the increase in certain secondary compounds like phenolics which could impart insect and disease resistance. The selection for amino acid analog resistance is able to produce mutants which overproduce the corresponding normal free amino acid since these high levels can generally compete with the analog for its site of toxic action, such as incorporation into protein. Our earliest work showed that tobacco suspension cultures could be selected as resistant to the tryptophan analog, 5-rnethyltryptophan (5MT), that this resistance usually was associated with a large increase in free tryptophan (trp) and that this increase in free trp was caused by an altered form of the trp biosynthetic control enzyme, anthranilate synthase, which was less sensitive to feedback inhibition by the endproduct, trp (Widholrn, 1972). When plants were regen­ erated from another 5MT-resistant tobacco suspension culture, the plants did not contain the altered form of anthranilate synthase, but 594 WIDHOLM cultures initiated from leaves did (Brotherton et al., 1986). Thus it appears that in tobacco in vitro selection produces variants with increased levels of a feedback insensitive anthranilate synthase, but this form is not expressed in regenerated plants. However, in the cases of 5MT selection with Datura innoxia (Ranch et al., 1983), rice (Wakasa and Widholm, 1987) and corn (Hibberd et al., 1986; Miao et al., 1986), the trp overproduction is expressed in the regenerated plants, the roots show resistance to 5MT, the trait is inherited by progeny and significant increases in tryptophan are found in the seed. We have also selected tobacco suspension cultures resistant to the methionine analog, ethionine (Gonzales et al., 1984). These lines accumulate over 100-fold the normal levels of free methionine due to an increase in activity of the lysine sensitive aspartokinase. Plants could not be regenerated from these cultures. Carrot suspension cultures which accumulate from 15- to 30- times the normal concentration of free proline have also been selected using the proline analog, hydroxyproline (Widholm, 1976). The mechanism for the proline accumulation has not been elucidated since the proline biosynthetic enzymes have not been well studied. In preliminary studies the carrot strain with elevated free proline levels was not more tolerant to salt, mannitol or polyethylene glycol stress or to controlled freezing. Plants were not regenerated due to the age of the culture, and so far no plants have been produced with significantly increased free proline levels to determine if this is beneficial for stress tolerance. Cell lines of several species including carrot and tobacco have been selected as resistant to the phenylalanine analog, P-fluoro­ phenylalanine (Palmer and Widholm, 1975). While different resistance mechanisms have been noted, many of the resistant lines contain elevated levels of phenylalanine ammonium lyase (Berlin and Widholm, 1977), which probably confers resistance by detoxifying the P-fluorophenylalanine to P-fluorocinnamic acid. By having increased levels of phenylalanine ammonia lyase, the first committed enzyme in phenolic biosynthesis, the cells accumulate at least 6-fold the normal phenolic levels. Since certain phenolics have been associated in certain instances with insect and disease resistance, we need to regenerate plants from selected cultures to determine if the high phenolic trait is expressed and if it confers resistance to insects or disease. Besides the instances discussed above, there also have been reports of selection for resistance to other compounds like metals, salt, PLANT CELL AND TISSUE CUL TURES 595 high osmotic concentrations, antibiotics, nucleotide analogs, plant hormones and other inhibitors. In many studies mutagenic treat­ ments were used and often increases in selection frequencies were documented, so mutagens certainly have at least an auxiliary role in in vitro selection work Mutagens can increase the frequency of the desired trait but also would increase the frequency of undesirable traits, so one should balance one against the other. This short overview of in vitro selection studies with cultured cells has shown that successful selection for ability to grow under various culture conditions, for compound production, for auxotrophy and for several kinds of resistances are possible. One item which was not discussed was the "artistic" nature of plant tissue culture, i.e., the fact that cell systems of some species and even genotypes are more difficult to manipulate than others and that some individuals or laboratories can do some things while others cannot. However, I feel that with the great interest now being expressed in in vitro methods that even the crop species systems will become more reproducible and amenable to manipulation so that all possible selections can be accomplished. Then the value of these alterations can be determined.

LITERATURE CITED

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Berlin, J and J. M. Widholm. 1977. Correlation between phenylalanine ammonia lyase activity and phenolic biosynthesis in P-fluorophenylalanine-sensitive and -resistant tobacco and carrot tissue cultures. Plant Physiol. 59:550-553.

Brotherton, J. E. , R. M. Hauptmann and J. M. Widholm. 1986. Anthranilate synthase forms in plants and cultured cells of Nicotiana tabacum L. Planta 168:214-221. Carlson, P. S. 1970. Induction and isolation of auxotrophic mutants in somatic cell cultures of Nicotiana tabacum. Science 168:487-489. Chaleff, R. S. and C. J. Mauvais. 1984. Acetolactate synthase is the site of action of two sulfonylurea herbicides in higher plants. Science 224:1443-1445. ---- and T. B. Ray. 1984. Herbicide-resistant mutants from tobacco cell cultures. Science 223: 1148-1152. Cseplo, A. , P. Medgyesy, E. Hideg, S. Demeter, L. Marton and P. Maliga. 1985. Triazine-resistant Nicotiana mutants from photomixotrophic cell cultures. Mol. Gen. Genet. 200:508-510. 596 WIDHOLM

Donn, G., E. Tischer, J. A. Smith and H. M. Goodman. 1984. Herbicide-resistant alfalfa cells: an example of gene amplification in plants. J. Mol. Appl. Genet. 2:621-635.

Gebhardt, C., V. Schnebli and P. J. King. 1981. Isolation of biochemical mutants using haploid rnesophyll protoplasts of Hyoscyamus muticus II. Auxotrophic and temperature-sensitive clones. Planta 153:81-89.

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