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' ‘I l .1 a“, .‘ hug-A m.“ u. . «a 5-014. .<‘~( l A;!f l' “b ,k; ‘ 4. r2233 . - ‘ é . . 4 :4 .v (4'- . .4 r; ‘L z... ‘ . . . v4 4’ . 15:52:35“ 3—“ my - «M81 5 u‘L-‘H 3.2: ‘ 'a-nw 293 009081716 This is to certify that the dissertation entitled Agrobacterium Rhizogenes-Mediated Transformation and Plant Regeneration from Normal and Genetically Transformed Tissues of Robinia Pseudoacacia L, presented by Kyung-Hwan Han has been accepted towards fulfillment of the requirements for Plant Breeding Ph.D. degree in and Genetics ‘ / Major professor Date 9'2'?/ MS U is an Affirmative Action/Equal Opportunity Institution 0-12771 3. LIBRARY Michigan State ‘ University \. PLACE IN RETURN BOX to remove We checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE JUL 2:! 199: 53‘ 4"" 57* $51 u 5-} __iL__ r W zififi I! MSU Is An Affirmative Action/Equal Opportunity Institution emanwt ataxia:.rhizoganesvnnblhrnb TRANSFORMATION AND PLANT REGENERATION TRON NORMAL AND TRANSGENIC TISSUES or Robinia pseudoacacia L. BY Kyung-Hwan Han A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Plant Breeding and Genetics - Forestry 1991 ABSTRACT ctariun.rhizoganes—XBDIATBD TRANSFORMATION AND PLANT REGENERATION IRON NORMAL AND TRANSGENIC TISSUES or Rbbinialpseudbacacia L. BY Kyung-Rwan Han The objectives of this study was to develop the system for introduction of foreign genes into black locust genome. A prerequisite was the need for stable methods for regenera- tion of whole plants in order for genetic manipulation at the cellular and molecular levels to be utilized in a tree improvement program. To devise the regeneration system for callus induction and subsequent shoot regeneration, various tissue sources such as hypocotyl, internodal segments of in vitro shoot cultures, and cambial tissues of mature black locust were used in the experiments described in the first and second chapters of this dissertation. Calli from both juvenile and mature sources were successfully regenerated into whole plants. For the development of gene transfer system, hypocotyl segments were co-cultivated with Agrobacterium rhizogenes strains harboring Ri-plasmid in which kanamycin resistance gene has been inserted. Subsequent hairy rootformation was observed on explants cultured on phytohormone-free medium. Spontaneous shoot regeneration occurred on less than 5% of the hairy roots. Transformed shoots were main- tained and rooted in the presence of 100 ug/ml kanamycin. All transformants showed hairy root phenotype characterized by wrinkled leaf, remarkably abundant root development and the ability of root differentiation on phytohormone-free medium. The presence of T-DNA and NPTII gene sequences in transformed black locust plants was shown by Southern analy- sis. This dissertation is dedicated to my parents, Chang-Seek and Myeo-Yeon Han iv ACKNOWLEDGEMENTS I would like to express my heartful appreciation to my major professor, Dr. Daniel Keathley, for his guidance, support, and confidence in my abilities through the course of this program. I also extend my personal thanks to the members of my committee, Drs. James Hanover, Amy Iezzoni, and Kenneth Sink, for their help,encouragement, and advice. I am also grateful to Dr. Yong-Goo Park for his encour- agement whenever I needed it. My appreciation also extends to Korea Government for the Government Scholarship which allowed me initiate gradu- ate study. A special note of thanks to my close friends and col- leagues, Chishih Chu, Andy David, and John Davis who made helpful discussions and proofreading of manuscript. John Davis kindly provided the bacterial strains and purified PstI 1 kb fragment for the genetic transformation experi- ments. Finally, I express my love to my wife, OkRan, and daughters, SumMee and Sarah, who have been to understanding and supportive during the course of my graduate studies. TABLE OF CONTENTS LISTS OF FIGURES LISTS OF TABLES INTRODUCTION CHAPTER I. Shoot regeneration from callus-derived from explants of juvenile and mature Robinia pseudoacacia L.). Introduction Materials and Methods Results Discussion Literature Cited II. Characteristics of cambium-derived callus cultures and regenerated shoots in mature black locust (Robinia pseudoacacia L.). 35 Introduction Materials and Methods Results Discussion Literature Cited III. Transformation of Robinia pseudoacacia L. via a hypocotyl-Agrobacterium system. 58 Introduction Materials and Methods Results Discussion Literature Cited CONCLUSIONS AND RECOMMENDATIONS vi Page 12 14 16 19 25 31 37 38 40 49 55 60 62 66 74 83 93 CHAPTER II Figure Figure Figure Figure Figure CHAPTER III Figure Figure Figure Figure Figure Figure Figure 1. 2. 3. 6. LIST OF FIGURES Cultures of cambium-derived callus from four mature black locust. Shoot organogenesis from callus of tree #1 on the regeneration medium. Multiplication of the regenerated shoots on Ms + 0.5 uM BAP. Subsequent rooting of the multiplied shoots. Potted black locust which regenerated from cambial tissue of mature tree. Culture of hairy roots derived from hypocotyl segments which cocultivated with Agrobacterium rhizogenes R1601. Cultures of hairy root-derived callus on BCM supplemented with 100 ug/ml kanamycin. Spontaneous shoot regeneration from hairy root cultures on BCM containing 100 ug/ml kanamycin. Spontaneous shoot regeneration from hairy root cultures on BCM containing 100 ug/ml kanamycin. Shoot cultures regenerated from hairy root tissues of black locust. Rooting of shoots regenerated from hairy roots on BSM containing 100 ug/ml kanamycin. Cultures of leaf pieces on callus induction medium in the presence of 100 ug/ml kanamycin. vii Page 54 54 54 54 54 80 80 80 80 80 80 80 Figure Figure Figure Figure Figure Figure Figure Figure .8. Abnormal phenotype in leaf morphology in transformant #771. 9. Abnormal phenotypes in leaf morphology in transformant #772. 10. Abnormal phenotypes in leaf morphology in transformant #773. 11. Potted transformed black locust (#771). 12. Potted transformed black locust (#773). 13. Root systems of both normal and transformed (#771) black locust. 14a. Southern blot analysis of transformed black locust plants. 14b. Southern blot showing hybridization of probe (1 kb PstI fragment from pCIBlO) to DNA from transformed black locust. viii 80 81 81 81 81 81 82 82 CHAPTER I Table Table Table Table Table CHAPTER II Table Table Table Table Table LIST OF TABLES Percent of explants derived from juvenile and mature black locust trees which produced callus after 32 days of culture. The effect of medium phytohormones on the color of calli from black locust explants after 32 days of culture. Observed differences in the proportion of calli grown from black locust explants which regenerated at least one shoot after culture on regeneration medium for six weeks. Shoot proliferation characteristics of cultures derived from M-1 and M-4 explant sources. Root differentiation from the shoots regenerated from J-2, M-1, and M-4 callus cultures on rooting medium. Effect of different phytohormones on callus induction from hypocotyl of random seedlings at 4 weeks of culture. Effect of phytohormone concentration on cambium callus initiation. The effects of different explant sources on cambium callus initiation. Cambium callus growth with different auxins and light intensities in a 4 week interval of cultures. Analysis of variances (GLM-ANOVA) for cambium callus growth with different auxin and light intensities. ix 20 20 21 22 22 39 40 42 44 45 INTRODUCTION Black locust (Robinia pseudoacacia L.), a nitrogen fixing and stress tolerant tree species, is planted global- ly. It has desirable wood characteristics, is fast grow- ing, and many characteristics that facilitate its use as a research organism. The small genome size (Singh and Simi- novitch, 1976), short generation interval (J. Hanover, personal communication), amenability to tissue culture (Chalupa, 1983; Davis and Keathley, 1985, 1987; Barghchi, 1987; Han and Keathley, 1988, 1991a, 1991b; and Merkle and Weicko, 1990) and receptability to Agrobacterium mediated genetic transformation (Davis and Keathley, 1990) make the species suitable as a model organism for genetic engineering of tree species. Utilization. Black locust is an excellent source for the production of good dimension lumber, fenceposts, and poles (Keresztesi, 1988). The heartwood contains high concentrations (6% of dry weight; Smith et al., 1989) of flavonoids such as robinetin and robinin that provide decay resistance (Miller et al., 1987). Black locust fixes atmospheric nitrogen and thrives on poor sites; thus it is widely planted for land reclamation purpose. It provides quick stabilization of disturbed sites, enhances soil devel- opment by furnishing nitrogen and nutrient litter, fosters successional development of high-quality forest stands, and endows improved water quality (Ashby et al., 1985). As an animal feed source, black locust biomass is compara- bly nutritious to alfalfa in terms of crude protein content and is readily digestible since the lignin level is lower than alfalfa (Baertsche et al., 1986). The high heat content, nearly twice that of Populus (Stringer and Carpenter, 1982), rapid juvenile growth, and prolific regrowth after harvest in short rotation intensive culture make the species ideal for biomass production (Miller et al., 1987) and/or multi-purpose plantations (Barrett et al., 1988). Black locust also has good charac- teristics for chemical pulping systems and is currently being used in the paper industry in Michigan (Miller et al., 1987). In addition, black locust has a great potential for beekeepers, as a large amount of quality honey may be gathered (Hayes, 1976; Keresztesi, 1988). Limitation and potential problems. Although black locust is highly desirable in terms of wood quality, indus- trial utilization has been limited in North America by the locust stem borer (Megacyllene robinae) which tunnels into the bole in the larval stage. This ruins stem form, causes vulnerability to windthrow, and provides entry points for fungal pathogens such as Fomes rimosus (Hoffard and Ander- son, 1982). Black locust is so competitive in mixed stands that the trees may overtop or damage other companion trees (Ashby et al., 1985; Chapman, 1935). In addition, the spines are hazardous to people and equipment. Genetic improvement. Both sexual reproduction and vegetative multiplication must be used to achieve the maxi- mum possible gain in a breeding program. Sexual hybridi- zation transfers the genes which are already present in the breeding population, and vegetative propagation enables multiplication of genotypes that are new combination of genes. Typically, black locust breeding programs have been targeted for fast growth, stem straightness, frost resistance, increased nectar yield (Keresztesi, 1983), biomass production (Miller et al., 1987), and spinelessness (Kim, 1974). Such programs have great potential because heritability of these traits is high, allowing efficient selection for their improvement (Kennedy, 1983; Mebrahtu and Hanover, 1989). However, the application of breeding technology has been inefficient largely due to the difficul- ty of conducting controlled crosses (Tesfai Mebrahtu, per- sonal communication). Consequently, the incorporation of biotechnology into black locust improvement programs will have a significant impacts on the attainment of specific breeding goals such as locust borer resistance or a spine- lessness. Tissue culture and genetic engineering. Tissue cul— ture technology provides the means to exploit and capture the maximum genetic gain achieved in a tree improvement program in the shortest possible time (Hasnain and Cheliak, 1986). This is possible through a combined efforts of early selection of desirable characteristics and micropropa- gation of selected genotypes. Micropropagation systems for black locust have been developed using preformed shoot meristems (Barghchi, 1987; Chalupa, 1983, 1987; Davis and Keathley, 1987; Han and Keathley, 1991a,b), and leaf disks of young seedlings (Davis and Keathley, 1985). Other meth- ods for basic research with black locust, include protoplast techniques (Han and Keathley, 1988) and somatic embryogene- sis (Merkle and Weicko, 1990). Although many in vitro techniques have been developed for black locust, no reliable method for the regeneration of whole plantlets from unorganized tissues has been developed. Such a regeneration system must be developed in order for genetic manipulation at the cellular and molecular levels to be utilized. Brown and Sommer (1982) reported regeneration of shoots from callus derived from black locust seedling shoot tips, but culture media and conditions were not de- scribed” ‘The first two chapters of this dissertation describe experimental attempts to create an efficient regen- eration system for black locust. In forest tree species, the use of in vitro techniques has been confined mainly to tissues from juvenile sources (Sanchez and Vieitez, 1991). This limits the application of biotechnology as an alternative to traditional methods because trees are beyond their juvenile phase by the time they express most traits of interest. While a reliable regeneration system is a prerequisite for genetic engineering of plant species, the introduction of foreign genes into a plant genome is a powerful approach for traits that are encoded by a single or few genes. Agrobacterium-mediated transformation has been an efficient way of introducing foreign genes into plant genomes (Klee et al., 1937). A Davis and Keathley (1989) transformed black locust with several strains of Agrobacterium spp. and detected stably incorporated T-DNA sequences in kanamycin resistant calli. However, transgenic black locust plants were not produced. The protocols for the transformation and regeneration of transgenic black locust were explored in the experiments which are described in_this dissertation. Objective. The studies conducted and reported herein were needed for the following reasons: 1) no method was available for shoot regeneration from unorganized tissues of black locust. The objectives addressed in the first chapter were to obtain shoot regeneration from callus tissues, to compare the responses of juvenile and mature explant sources, and to determine if superior response of a genotype to in vitro bud culture persisted in the shoots regenerated from callus; 2) there is a need to circumvent the problems related to the use of mature trees as a starting material in in vitro culture. In chapter 2, the ability to use tis- sues of mature black locust as starting material in a dedifferentiation-regeneration system was examined. Callus induction and subsequent shoot regeneration from the cambial tissues of mature black locust was demonstrated; 3) although the introduction of foreign genes into black locust was successful, no transgenic black locust plants have been reported. Chapter 3 reports the methods for Agrobacterium rhizogenes-mediated transformation and the recovery of transgenic black locust trees. Conclusions and recommendations derived from the find- ings of these studies are presented at the end of this dissertation. LITERATURE CITED Ashby, W.C., Vogel, W.G. and N.F. Rogers. 1985. Black locust in the reclamation equation. Gen. Tech. Rep. NE-105. Broomall, PA: 0.8. Department of Agriculture, Forest Service, Northeastern Forest Experiment Station. pp. 1-12. Baertsche, S.R., Yokoyama, M.T. and J.W. Hanover. 1986. Short rotation, hardwood tree biomass as potential ruminant feed - chemical composition, nylon bag ruminal degradation and ensilement of selected species. J. Anim. Sci. 63: 2028-2043. Barghchi, M. 1987. Mass clonal propagation in vitro of Robinia pseudoacacia L. (black locust) cv. 'Jaszkiseri'. Plant Sci., 53: 183-189. Barrett, R.P., Mebrahtu, T. and J.W. Hanover. 1988. Research on black locust as a multi-purpose tree species for temperate climates. Poster presentation, First National Symposium on New Crops, Indianapolis, IN, Oct. 23-26, 1988. Brown, C.L. and H.E. Sommer. 1982. Vegetative propagation of dicotyledonous trees. In: Bonga, J.M. and D.J. Durzan (eds.), Tissue culture in forestry, Martinus Nijhoff/Dr. W. Junk, The Hague, pp. 109-149. Chalupa, V. 1983. In vitrc propagation of willow (Salix spp.), European mountain-ash (Sorbus aucuparia L.) and black locust (Robinia pseudoacacia L.). Biol. Plant. 25: 305-307. Chalupa, V. 1987. Effects of benzylaminopurine and thidiazuron on in vitro shoot proliferation of Tilia cordata Mill., Sorbus aucuparia L. and Robinia pseudoacacia L. Biol. Plant. 29: 4254429. Chapman, A.G. 1935. The effects of black locust on associated species with special reference to forest trees. Ecological Monographs. 5 (1): 37-60. Davis, J.M. and D.E. Keathley. 1985. Regeneration of shoots from leaf disk explants of black locust (Robinia pseudoacacia L.). Proceedings of the 4th North Central Tree Improvement Conference, 12-14 Aug. 1985, East Lansing, MI. pp. 29-34. Davis, J.M. and D.E. Keathley. 1987. Differential responses to in vitro bud culture in mature Robinia pseudoacacia L. (black locust). Plant Cell Reports 6: 431-434. Davis, J.M. 1989. Responses of different genotypes of Robinia pseudoacacia L. to micropropagation and Agrobacterium-mediated transformation. Ph.D Thesis, Michigan State University, 91 pp. Davis, J.M. and D.E. Keathley. 1990. Detection and analysis of T-DNA in crown gall tumors and kanamycin-resistant callus of Robinia pseudoacacia L. Can. J. For. Res. 19: 1118-1123. Han, K.-H. and D.E. Keathley. 1988. Isolation and culture of protoplasts from callus tissue of black locust (Robinia pseudoacacia L.). Nitrogen Fixing Tree Research Report 6: 68-70. Han, K.-H. and D.E. Keathley. 1991a. Micropropagation of black locust (Robinia pseudoacacia L.): responses to cytokinin and mineral medium. To be submitted to Kor. J. For. Soc.. Han, K.-H. and D.E. Keathley. 1991b. In vitro responses of top and epicormic branches in a mature black locust (Robinia pseudoacacia L.). To be submitted to Tree Physiology. Hasnain, S. and W. Cheliak. 1986. Tissue culture in forestry: economic and genetic potential. The Forestry Chronicle, August, 1986. pp. 219-225. Hayes, B. 1976. The black locust as a nectar source. American Bee Journal, May, 1976. 226, 238 pp. Hoffard, W.H. and R.L. Anderson. 1982. A guide to common insects, diseases, and other problems of black locust. United State Department of Agriculture Forestry Report SA-FR 19, 9 pp. Kennedy, M.J., Jr. 1983. Geographic variation in black locust (Robinia pseudoacacia L.). M.S. Thesis, 10 University of Georgia, 66 pp. Keresztezi, B. 1980. The black locust. Unasylva 32: 23-33. Keresztezi, B. 1983. Breeding and cultivation of black locust, Robinia pseudoacacia, in Hungary. For. Ecol. Man. 6: 217-244. Keresztezi, B. 1988. Black locust: the tree of agriculture. Outlook on Agriculture 17(2): 77-85. Kim, C.S. and S.K. Lee. 1974. Studies on characteristics of selected thornless black locust. Res. Rep. Inst. For. Gen., 11: 1-11 (Korea). Klee, H., Horsch, R. and S. Rogers. 1987. Agrobacterium- mediated plant transformation and its further applica tion to plant biology. Ann. Rev. Plant Physiol. 38: 221-257. Mebrahtu, T. and J.W. Hanover. 1990. Heritability and expected gain estimates for traits of black locust in Michigan. Silvae Genetica 38: 125-130. Merkle, S.A. and A.T. Weicko. 1989. Regeneration of Robinia pseudoacacia L. via somatic embryogenesis. Can. J. For. Res. 19: 285-288. Miller, R.O., Bloese, P.D. and J.W. Hanover. 1987. Black locust: a superior short-rotation intensive culture species for biomass production in the lake states. Paper presented at the Institute of Gas Technology's Eleventh Annual Meeting on Energy from Biomass and 11 Wastes, March 16, Orlando, FL. Sanchez, M.C. and A.M. Vieitez. 1991. In vitro morphogene- tic competence of basal sprouts and crown branches of mature chestnut. Tree Physiology 8: 59-70. Singh, J. and D. Siminovitch. 1976. A reliable method for the estimation of DNA in higher plant tissues. Anal. Biochem. 71: 308-517. Smith, A.L., Campbell, C.L., Diwakar, M.P. and J.W. Hanover. 1989. Extracts from black locust as wood preserva tives: a comparison of the methanol extract with penta- chlorophenol and chromated copper arsenate. Holzforschung 43: 293-296. CHAPTER 1 SHOOT REGENERATION IRON CALLUS-DERIVED IRON EXPLANTS OF JUVENILE AND NATURE Rbbinia pseudbacacia L. 12 ABSTRACT Callus cultures were initiated from explants taken from two seedlings and two mature trees. Explant type had a significant effect on the frequency of primary callus pro- duction. Primary callus colOr was significantly affected by phytohormone level. Regeneration of shoots from green calli occurred on MS medium containing 10 uM BAP alone or in combination with 1 uM NAA. The highest percentage of shoot regeneration was accomplished with primary callus induction on explants placed on MS medium with 1 uM NAA and 10 uM BAP, and shoot regeneration on MS medium containing 10 uM BAP, using seed- lings of tree #2 as source of explants. Differences in growth and rooting between shoots de- rived from callus of the two mature tree sources were appar- ent. Shoots from source M-l produced a significantly higher number of shoots per explant than those of M-4. A significantly higher proportion of shoots regenerated from M-1 callus (57%) produced roots compared to shoots from M-4 calli (25%). These results may reflect genotype specific differences among the explant sources. 13 INTRODUCTION Black locust (Robinia pseudoacacia L.) is a leguminous tree species for which in vitro micropropagation systems have been developed. Chalupa (1983, 1987), Barghchi (1987) and Davis and Keathley (1987) all reported that rooted shoots were produced subsequent to inducing the elongation of preformed shoot meristems. The production of adventi- tious shoots from leaf disks has also been reported (Davis and Keathley, 1985). Such micropropagation procedures preserve the genetic integrity of propagules and thus allow clonal propagation of individual trees (Barghchi, 1987; Han and Keathley, 1991b) for use in basic research and breeding programs. Other tools for basic research of black locust, including protoplast techniques (Han and Keathley, 1988) and somatic embryogenesis (Merkle and Weicko, 1990) have been reported. Along with the development of many in vitro techniques for black locust, Agrobacterium-mediated transformation has been reported for the species (Davis and Keathley, 1989). However, transgenic plants resulting from the genetic manip- ulation at the cellular and molecular level have not been achieved. In order for black locust to be utilized for 14 15 such genetic manipulation, methods for regeneration of whole plantlets from callus tissues must be developed, IBrown and Sommer (1982) reported regeneration of shoots from callus derived from black locust seedling shoot tips, but culture media and conditions were not described. A In forest tree species, regeneration of whole trees from callus cul- tures initiated from mature individuals has been problematic (Bonga, 1982). Many protocols have been explored as a means of circumventing this problem, including preculture hedging of the explant source to rejuvenate the tissue. One way that shoot regeneration frequency has been increased in some herbaceous species is by using genotypes which are superior performers in vitro as explant sources (Bingham et al., 1975; Koornneef et al., 1986). Tree specific responses were observed among five mature black locust trees in vitro (Davis and Keathley, 1987). To further investigate the use of germplasm screening to increase in vitro performance, two different mature trees, both of which appeared to respond well in vitro (Davis and Keathley, 1987), were evaluated for regeneration of shoots from subcultured callus. The first objective of this study was to obtain shoot regeneration from callus tissues. The second objective was to compare the response of these two mature trees to that of black locust seedlings. The third objective was to determine if superior response of 16 a genotype (Tree #1) to in vitro bud culture (Davis and Keathley, 1987) persisted in the shoots regenerated from callus. NATERIAL AND NETEODS Plant materials Juvenile source: Two seedlots (designated J-2 and J-4) were collected from pods borne on mature~trees #2 and #4, respectively as described in Davis and Keathley (1987). . To produce aseptic seedlings, 40 seeds from each seedlot were boiled in water for 20 seconds, immersed in 5.25% NaOCl for 10-15 seconds, placed on sterile filter paper to dry, and to germinate in 18 x 150 mm culture tubes each containing 5 ml MS (Murashige and Skoog, 1962) basal medium with 0.5% (w/v) sucrose and 0.8% Bacto-agar (Difco). The seedlings were grown for 21 days in a growth chamber (50-75 uE/mZ/sec; 18 hr light/6 hr dark). Ten seedlings were chosen at random from each seedlot. Mature source: Shoot cultures from trees #1 and #4 that were initiated during the study reported previously by Davis and Keathley (1987) were maintained for 9 months on MS medium supplemented with 0.32 uM BAP (6-benzylaminopurine) 17 and 0.8% (w/v) Bacto-agar (Difco); referred to herein as shoot multiplication medium (SM). Cultures were initiated with shoot segments 2 cm or more in length. All cultures were incubated in a growth chamber under a 16 / 8 h light- dark photoperiod (50-75 uM/m2/sec). Callus initiation and growth To induce callus, 20 sections, 2-3 mm in length, per treatment were taken from both hypocotyl and internodal segments of the shoot cultures of each clone and placed on callus initiation medium in 15 x 100 mm Petri dishes (10 explants per dish). The initiation medium was MS contain- ing 5 or 10 uM BAP in combination with 1 or 5 uM NAA (naph- thaleneacetic acid), using a 2 x 2 factorial design. After 31 days, the number of explants that produced callus was counted, and green pieces of callus (5-7 mm in diameter) were excised from original explants and trans- ferred to fresh callus medium of the same composition. Green calli were selected for subculture because past exper- iments indicated that morphogenesis did not occur from white callus tissues (Han and Davis, unpublished). After 25 days of further growth, green pieces of callus were removed from the callus mass and transferred to shoot regeneration media. Shoot regeneration and proliferation Shoot regeneration media, 10 ml in each 25 x 150 mm 18 culture tube, was Ms with BAP (10 uM) alone or in combina- tion with NAA (1 uM). The number of calli that produced shoots was counted after 6 weeks of culture. Shoots were excised from the callus tissue and trans- ferred individually to 30 ml SM medium in each 55 x 130 mm culture jar to increase the number and standardize the size of propagules for further study. After 4 weeks, shoots greater than 2 cm in length were transferred to 10 ml of fresh medium in 25 x 150 mm culture tubes. Shoots less than 2 cm were not transferred, eliminating them from the experi- ment. Four weeks later, the number of shoots greater than 1 cm in length that were produced by each 2 cm long explant was counted, and shoots greater than 2 cm in length were again transferred to fresh SM medium. Shoots less than 2 cm in length were eliminated from the experiment. The number of shoots produced by each explant was again counted after 4 weeks of subculture. Shoot cultures were subcultured for another 4 weeks of proliferation. This allowed elongation of the shoots for use in the rooting study. The shoots were cut to a length ranging from 2 to 2.5 cm and were used for the rooting test. Root initiation Shoots regenerated from callus were inserted individu- ally in 10 ml of rooting medium, 0.1 strength MS supplement- ed with 1 uM IBA, in each 25 x 150 mm culture tubes. After 19 4 weeks, the number of shoots with at least one root was counted. Plantlets were potted individually in a peat moss: perlite: vermiculite mix (1:1:1), watered with a fertilizer solution (1 g/l Peter's 20:20:20 in tap water), covered with plastic wrap to prevent desiccation, and placed in a growth chamber under a 16 / 8 h a light-dark regime. After 2 weeks the plastic was removed and the plantlets were fertilized weekly until transfer to a greenhouse. Statistical analysis The callus induction data were analyzed using a conven- tional analysis of variance for a factorial experiment (4 explant sources x 4 culture media). Percentage data were transformed using the arcsin transformation prior to the analysis (Steele and Torrie, 1980). To perform pairwise contrasts between clones, 2 x 2 contingency tables (e.g., "rooted" vs. "non-rooted” for each clone) were constructed, and Chi-square values were calculated using the normal approximation (Steele and Torrie, 1980). RESULTS Callus growth was observed on induction media. Explant source had a significant effect on callus production (Table 1). Only 41% of the explants from tree M-l produced callus; 20 whereas, an average of 97% of the seedling explants produced callus. In contrast, different phytohormone regimes in callus induction media did not significantly affect the number of explants which produced callus (F-test; p>.7). However, phytohormone levels significantly affected callus color (Table 2). Mature explants produced green callus only from segments cultured on callus medium contain- ing 5 uM NAA. Regeneration of shoots from green calli occurred on medium containing 10 uM BAP alone or in combination with 1 'uM NAA (Table 3). Since only green regions of the calli were transferred to the regeneration media, there was un- equal representation of the callus induction treatments on the regeneration media. For example, since virtually all of the callus produced in the treatment with 1 uM NAA and 5 uM BAP was white (Table 2) and not growing vigorously, no calli from this treatment were transferred to regeneration medium. Likewise, callus produced from mature tissue cultured on initiated the medium containing 1 uM NAA and 10 uM BAP was white (Table 2), and hence was not used for regeneration attempt. Consequently, no statistical analysis of these data was attempted due to the lack of data. Shoots were regenerated from callus produced by all four explant sources. Averaged across medium type, shoot regeneration from J-4 explants was poorest (1.1%), and 21 optimum from J-2 explants was best (16.3%). The shoot regeneration (50%) was accomplished with callus initiated from J-2 explants on medium with 1 uM NAA and 10 uM BAP, and shoot regeneration on MS containing 10 uM BAP. Shoots regenerated from J-4 callus failed to grow and develop. The average number of 5 per culture was produced from shoots derived solely from J-2 callus. Differences in growth between shoots derived from callus of the two mature sources were apparent (Table 4). Shoots from M-1 multiplied rapidly after transfer to SM medium, yielding 324 shoots greater than 1 cm in length by 22 Table 1. Percent of explants derived from juvenile and mature black locust trees which produced callus after 32 days of culture on callus induction medium. E ant Explants sallusediilil J-2 94.8 a J-4 98.8 a M-4 73.3 ab M-l 41.0 bc 1)Values followed by the same letter do not differ signifi- cantly (Tukey's test; alpha = .05). Table 2. The effect of phytohormones in MS medium on color of calli from black locust explants after 32 days in cul- ture. Phytohormones Callus gglgrl) Averags (uM) ee 'n MQLQIQ 2212; ) NAA (1) BAP (5) 2.0 1.0 1.5 a NAA (5) BAP (5) 6.0 4.0 5.0 ab NAA (1) BAP (10) 5.5 1.0 3.25 ab NAA (5) BAP (10) 5.5 5.5 5.5 b 1)Callus color was determined subjectively on a scale of 1 (white calli) to 6 (green calli). 2)Values followed by the same letter do not differ signifi- cantly (Tukey's test, alpha = .05). 23 Table 3. Observed differences in the proportion of calli grown from black locust explants which regenerated at least one shoot after culture on regeneration medium for six weeks. Callus Explant Shoot regenerationl) medium source 1 explants 1 total NAA(5) \ J-2 1/16 (.6.3%) BAP(S) / J-4 0/31 M-l 2/16 (12.5%) M-4 0/32 NAA(l) \ J-2 12/32 (37.5%) BAP(10)/ J-4 1/30 ( 3.3%) - Axel 1 NAA(5) \ J-2 0/32 16.3 BAP(10)/ J-4 0/32 1.1 M-l 2/32 ( 6.3%) 8.3 M-4 2/32 ( 6.3%) 3.1 1) Value for the two regeneration media are averaged because the presence of 1 uM NAA in the medium had no effect on ‘ regeneration (pairwise contrast n.s., alpha = 0.05). 24 Table 4. Shoot proliferation characteristics of cultures derived from M-1 and M-4 explant sources. Shoot grpyth Explant Number of shoots interxal eeuree Startlzzeul EBQIZIQBI Mean 1 $1012) 1 2 - - -3) 0 - 4 4 2 - - - 1 23 176 7.7 5.4 4 - 8 4 3 14 4.7 0.6 1 47 324 6.9 3.4 8 - 12 4 4 14 3.5 1.3 1) The number of weeks after excision of shoots from callus. 2) Mean number i SE of shoots produced from a shoot. 3) - , data not collected Table 5. Percent rooting of shoots regenerated from J-2, M- 1, and M-4 callus cultures. Time Explant Rootingl) lueekel seuree 111 Mean 4 J - 2 9/46 (19) 1.6 M - 1 40/72 (56) 1.2 M - 4 3/16 (19) 1.3 5 J - 2 9/46 (19) 1.6 M - 1 40/72 (56) 1.4 M - 4 4/16 (25) 1.3 6 J - 2 11/46 (24) 1.9 M - 1 41/72 (57) 1.6 M - 4 4/16 (25) 1.5 1) The number of explant which produced at least one root is shown in the numerator, divided by the total number of explants. 25 twelve weeks after excision of the shoots from callus. In contrast, shoots from M-4 multiplied slowly; only 14 shoots longer than 1 cm were obtained at the end of the same peri- od. A significantly higher proportion of shoots from M-l callus rooted (57%) compared to both J-2 (24%) and M-4 (25%) calli (Table 5; pairwise difference at alpha - 0.05). Discussion Explant source had a significant effect on callus induction. Almost all of the hypocotyls from the juvenile *tissues produced callus (97%), whereas only 57% of the mature explants produced callus. However, callus induction from M-4 explants did not differ significantly from either of the juvenile explants, but the response of M-l explants did. This indicates the callus induction response was dependent on genotype, not simply on maturity. Similarly, we found a significant genotypic effect on cambial tissue cultures derived from mature black locust (Han and Keathley, 19918) It might be expected that the juvenile explants would exhibit more variability in response on the callus induction media, since the half-sib seed lots were genetically hetero- geneous, in contrast to the shoot cultures that were of clonal origin. This was not observed, and may indicate that 26 hypocotyl explants, due to either anatomical or physiologi- cal differences from stems, form callus more readily. Alternatively, this may reflect a difference between juve- nile and mature tissue. These contentions could be tested if explants of shoot cultures derived from individual seed- lings were compared to mature proliferating shoot cultures. The differential responses of callus induction between juvenile and mature tissues have been observed on cambial tissues isolated from epicormic branches (known as "in juvenile phase"; Bonga, 1982) that produced a higher fre- quency of callus induction than those from crown branches (known as in mature phase) (Han and Keathley, 1991c). Both M-1 and M-4 explants produced sporadic callus on MS medium containing 1 uM NAA, regardless of the BAP level. This was not the case with the juvenile sources, which produced green calli on medium containing 1 uM NAA and 10 uM BAP. This implies that sustained growth of callus from mature black locust tissues may require these higher auxin levels, although it is also possible that this requirement is specific to these two mature genotypes. Regeneration of shoots occurred from callus of both mature explant sources. Although the efficiency of shoot regeneration from the mature explant sources was rather low under these conditions (less than 10%), these experiments demonstrated the capacity of calli derived from mature tis- 27 sues of this species to regenerate in vitro. Previously, shoot regeneration from callus of black locust had been demonstrated only in seedling tissues (Brown and Sommer, 1982). We expected that the calli derived from juvenile ex- plants would regenerate shoots at a higher frequency than that of mature origin. This was the case for calli of the J-2 source. It is interesting, however, to note that M-1 and M-4 calli had a higher shoot regeneration (8.3% and 3.1%, re- spectively) than J-4 calli which had a shoot regeneration percentage of 1.1%. As was indicated by the callus induc- tion data, these results suggest that the juvenility or maturity of an explant source per se is not necessarily a reliable predictor of organogenesis, and should not always be the primary consideration in efforts to develop more efficient regeneration systems for forest tree species. The most efficient shoot regeneration occurred from J-2 calli derived from MS induction medium containing 1 uM NAA and 10 uM BAP. For these calli, the transfer to regener- ation medium was essentially a subculture step, where both callus and shoots proliferated throughout the experiment (even though unorganized callus tissues were selected for transfer at each transfer step). This shoot proliferation was not observed with J-4 calli, calli from hypocotyls of a 28 locally collected, bulked seedlot (Han, unpublished), or calli derived from the mature explants. The difference between the J-2 and J-4 calli could not be attributed to noticeable differences in seedling vigor (personal observa- tion), or average seed weight (data not shown). If this characteristic is common among individuals from the J-2 seedlot, these seedlings may be useful as explant sources for increasing the efficiency of shoot regeneration from protoplast-derived calli of black locust. Shoot multiplication occurred more readily from the M-1 than the M-4 derived calli. This is reflected in both the relative and the absolute increases in shoot number that occurred on SM medium. Eight and 12 weeks after the exci- sion of shoots from callus, the shoots from the M-1 source produced a 7.7- and 6.9-fold increase in shoot number, while shoots from the M-4 source produced a 4.7- and 3.5-fold increase, respectively. Even more pronounced than the relative increases in shoot production were the clonal differences for the total number of shoots produced. By the end of the third inter- val, the M-1 source had produced 23-fold more shoots than M- 4. When dormant bud explants were taken from these trees and compared under similar culture conditions, shoot prolif- eration from M-l was also found to be consistently superior to that of M-4 source (Davis and Keathley, 1987). 29 Shoots from M-l were also more likely to root than the M-4 source. This difference between the clones, original- ly reported in the previous study using bud cultures (Davis and Keathley, 1987), was highly significant and was observed on medium containing IBA, NAA, or a combination of these 2 phytohormones. The shoots regenerated from callus reflected the same clonal differences in rooting on MS containing IBA. Rooting of shoots regenerated from callus was less than in shoots derived from bud culture (Han and Keathley, 1991c). This difference in rooting efficiency may be due to physio- logical differences in tissue or to environmental differ- ences such as container size or medium volume. The lower rooting percentage was obtained with shoots produced from callus of juvenile source (J-2) compared to shoots from mature source (M-l). Since juvenile tissues are known to have better rooting ability than mature ones (Sanchez and Vietez, 1991; Hackett, 1987), it is notable that the mature sources produced more roots than juvenile explants. The difference may indicate the genotypic effect of tree #1 rather than physiological differences in tissue. The rooting and shoot proliferation data suggest that the genetic or epigenetic factors responsible for the dif- ferential performance of the two mature sources (Davis and Keathley, 1987) persisted through the somatic cell dediffer- entiation and regeneration processes associated with callus 30 growth. Performance in tissue culture has been related to genotype differences (Reisch and Bingham 1980, Komatsuda and Ohyama 1988). It has been shown previously that explant pretreatments or phytohormones can completely or partially override differential responses due to genotype (Gharyal and Maheshwari 1983, Komatsuda and Ohyama 1988). Our data indicate that different phytohormones in the regeneration of shoots from callus of black locust did not override clonal differences. Although the two mature sources are certainly distinct genotypes, either physiological or genetic factors could be responsible for the differences we observed in the in vitro performance of the two clones. Since the shoot cultures were originally derived from trees of flowering age, crosses could be performed to test such hypotheses. Further studies have allowed the development of more efficient callus growth and shoot regeneration procedures for this genotype (Han and Keathley, 1991a). We hope that sheet cultures of this clone (Han and Keathley, 1991b) can provide a genetically and physiologically uniform explant source for studies aimed at both the regeneration of shoots from protoplasts (Han and Keathley, 1987) and genetic transformation of black locust. LITERATURE CITED Barghchi, M. 1987. Mass clonal propagation in vitro of Robinia pseudoacacia L. (black locust) cv. 'Jaszkis eri'. Plant Sci., 53:183-189. Bingham, E.T., Hurley, L.V., Kaatz, D.M. and J.W. Saunders. 1975. Breeding alfalfa which regenerates from callus tissue in culture. Crop Sci. 15:719-721. Bonga, J.M. 1982. Vegetative propagation in relation to juvenility. In: Tissue culture in forestry, Bonga, J.M. and D.J. Durzan (eds.). Martinus Nijhoff/Dr. W. Junk, The Hague, pp. 109-149. Brown, C.L. and H.E. Sommer. 1982. Vegetative propagation of dicotyledonous trees. In Tissue culture in forestry. J.M. Bonga and D.J. Durzan (ed.), Martinus Nijhoffb/Dr. W. Junk Publishers, Hague, Netherland. pp 109-142. Chalupa, V. 1983. In vitro propagation of willows (Salix spp.), European mountain-ash (Sorbus aucuparia L.) and black locust (Robinia pseudoacacia L.). Biol. Plant. 25:305-307. Chalupa, V. 1987. Effects of benzylaminopurine and thidia- zuron on in vitro shoot proliferation of Tilia cordata Mill., Sorbus aucuparia L. and Robinia pseudoacacia L. Biol. Plant. 29:425-429. 31 32 Davis, J.M. and D.E. Keathley. 1985. Regeneration of shoots from leaf disk explants of black locust, Robinia pseu- doacacia L. Fourth North Central Tree Improvement Conference Proceedings, East Lansing, MI, pp. 29-34. Davis, J.M. and D.E. Keathley. 1987. Differential responses to in vitro bud culture in mature Robinia pseudoacacia L. (black locust). Plant Cell Rep. 6:431-434. Davis, J.M. and D.E. Keathley. 1989. Detection and analy- sis of T-DNA in crown gall tumors and kanamycin-resist ant callus of Robinia pseudoacacia. Can. J. For. Res. 19:1118-1123. Gharyal, P.K. and S.C. Maheshwari. 1983. Genetic and physio- logical influences on differentiation in tissue cul- tures of a legume, Lathyrus sativus. Theor. Appl. Genet. 66:123-126. Hackett, W.P. 1987. Juvenility and maturity. In Cell and Tissue culture in forestry. vol.I. Eds. J.M. Bonga and D.J. Durzan. Matinus Nijhoff Publishers, Dordrecht, pp. 249-271. Han, K.H. and D.E. Keathley. 1988. Isolation and culture of protoplasts from callus tissue of black locust (Robinia pseudoacacia L.). Nitrogen Fixing Tree Research Reports 6:68-70. Han, K.H. and D.E. Keathley. 1991a. Characteristics of cambium callus and callus derived shoots in mature 33 black locust (Robinia pseudoacacia L.). Plant Cell Report. Acepted for publication. Han, K. H. and D.E. Keathley. 1991b. Micropropagation of black locust (Robinia pseudoacacia L.): responses to cytokinin and mineral medium. To be submitted to Kor. J. For. SOC. Han, K.H. and D.E. Keathley. 1991c. In vitro responses of top and epicormic branches in a mature black locust (Robinia pseudoacacia L.). To be submitted to can. J. For. Res. Komatsuda, T. and K. Ohyama. 1988. Genotypes of high compe- tence for somatic embryogenesis and plant regeneration in soybean Glycine max. Theor. Appl. Genet. 75:695- 700. Koornneef, M., Hanhart, C., Jongsma, M., Toma, I., Weide, R., Zabel, P. and J. Hille. 1986. Breeding of a tomato genotype readily accessible to genetic manipulation Plant Sci. 45:201-208. Merkle, S.A. and A.T. Wiecko. 1989. Regeneration of Robinia pseudoacacia via somatic embryogenesis. Can. J. For. Res. 19:285-288. Murashiege, T. and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco cultures. Physiol. Plant. 15:695-700. Reisch, B. and E.T. Bingham. 1980. The genetic control of 34 bud formation from callus cultures of diploid alfalfa. Plant Sci. Lett. 20:71-77. Sanchez, M.C. and A.M. Vieitez. 1991. In vitro morphogenet- ic competence of basal sprouts and crown branches of mature chestnut. Tree Physiology 8:59-70. Steele, R.D.G. and J.H. Torrie. 1980. Principles and proce- dures of statistics. McGraw-Hill Book Company. CHAPTER 2 CHARACTERISTICS OF CANBIUN-DERIVED CALLUS CULTURES AND REGENERATED SHOOTS IN NATURE BLACK LOCUST (Rabinia pseudba- cacia L.) .1) 35 ABSTRACT Primary callus cultures were derived from cambial tissue of mature black locust placed on MS medium containing 5 uM BAP and 10 uM NAA. Genotype had significant effects on callus growth and subsequent shoot regeneration. Diameter growth of callus was not affected by auxins or light inten- sities; however, the color and texture of callus was signif- icantly influenced by genotype, phytohormone, light intensi- ties, and their interactions. Eight month storage of cambial tissue at 4 °C resulted in significant changes in diameter growth but did not affect callus color or medium browning. Sheet regeneration was highly tree specific. 36 INTRODUCTION In vitro micropropagation of black locust (Robinia pseudoacacia L.) has been reported from auxiliary buds (Chalupa 1983; Davis and Keathley 1987; Barghchi 1987) and from leaf disks (Davis and Keathley 1985). Along with these techniques, isolation and culture of protoplasts (Han and Keathley 1988) and regeneration of black locust via somatic embryogenesis (Merkle and Wiecko, 1989) have extended cur- rent in vitro methods for this species.‘ Additionally, Davis and Keathley (1989) were able to transform black locust hypocotyls with several strains of Agrobacterium and to detect stably incorporated T-DNA sequences in calli resistant to kanamycin. Manipulating plants genetically at the cellular and molecular levels for use in plant improvement programs requires the ability to regenerate whole plants from unor- ganized tissues. In black locust, successful shoot regen- eration has been reported from seedling-derived callus and callus derived from shoot cultures of mature trees. Significant tree specific responses were observed in cul- tures from both half-sib seedling and mature origins. We report a protocol for callus initiation and culture from the cambial tissue of mature black locust trees, 37 38 delineates methods for obtaining subsequent shoot regenera- tion from the callus, and discuss persistent tree specific response to in vitro manipulation. NATERIALS AND NETHODS Explant preparation: Branches (0.5 to 1.0 cm in diame- ter) were removed from 4 mature trees (23-26 years-old) in March of 1988. The branches were cut into 3-4 cm pieces and washed with full strength of detergent (Liqui-nox) and placed under running tap water for 1 hr, immersed in 70% ethanol for 1-2 min and soaked in a 50% Clorox solution for 10-15 min followed by 5 washes with sterile distilled water. The branch bark was removed and a cambium layer 0.2-0.5 mm thick and 3 x 4 mm was removed out using a scalpel. Explant culture: Explants of cambial tissue were placed in Petri dishes (15 x 200 mm) each containing 25 ml of MS (Murashige and Skoog, 1962) basal medium supplemented with various phytohormones. Number of explants started and phytohormone levels are given in the Tables for each exper- iment. MS basal medium, 0.8% Sigma-agar, and 2% sucrose were used throughout and adjusted to pH 5.8 autoclaving for 15-20 min at 120° c. Experiments were conducted in a controlled environment chamber at 25° C, with an 18 hr light/6 hr dark lighting 39 regime (50 uE/mZ/sec). Callus was maintained by subcultur- ing at 4 week intervals. Callus growth was scored by measuring diameter, color, browning of medium and texture of the callus. Color, texture, and medium browning were scored in the following manner; color: brown or dead=0, white-1, whitish green=2, greenish white=3, green=4; tex- ture: very friable-1, friable-2, firm-3, very firm-4; medium browning: none=1, light-2, moderate-3, dark-4. Shoot regeneration and rooting: Callus cultures that have been subcultured at least 5 months were used for regen- eration experiments. Five calli per Petri dish were transferred to MS medium containing various concentrations of BAP alone or in combination with NAA (Table 5). The number of shoots regenerated and callus growth were scored at 13 weeks. Shoots (1 cm in length) were excised from the callus tissue and transferred individually to 10 ml of shoot multiplication medium (MS + 0.5 uM BAP) in each 25 x 200 mm test tube. The proliferating shoots (2 cm in length) were transferred to rooting medium (1/10 strength MS supplemented with 1 uM IBA and no sucrose). Rooted plantlets were potted in a container using peat moss, pearlite, and vermic- ulite (1:1:1), watered with Peter's 20:20:20 (1 g/l), and covered with plastic wrap. Statistical analysis: The data for callus initiation and growth were analyzed separately for each treatment using 40 a general linear model analysis of variance/covariance (GLM- ANOVA; Hintze 1987). Correlation coefficients among the diameter, color, texture of callus, and browning of medium were calculated as Spearman's rank correlation (Steele and Torrie, 1980). The data for each treatment were separated by Duncan's New Multiple-Range Test (Steele and Torrie, 1980) where the GLM-ANOVA indicated significance. RESULTS Analysis of the data obtained from preliminary experi- ments with hypocotyl indicated that no significant differ- ence (alpha = .05) in callus growth existed between cultures initiated on MS medium containing 2,4-D or NAA (data not included). Both cytokinin type and cytokinin/auxin inter- action had significant effects on diameter growth and callus induction. The inclusion of BAP in the MS medium resulted in significantly greater callus diameter growth, higher callus induction frequency, and greater shoot regeneration (data not included). Based on these results, BAP and NAA were selected for further study. To identify optimal concentrations of the phytohor- mones, four different concentrations each of BAP and NAA were tested (Table 1) using cambial tissues from tree #3, which gave an intermediate response in preliminary experi- ments (data not presented). Analysis of variance showed 41 Table 1. Effect of phytohormone concentration on cambium callus initiation. Phytohormones Explants Average Color Callus NAA BAP (uM) (No.) Diameter (mm) (x) % 0 0 20 no callus dead 0 0 1 20 5.8b 1.1abc 95 0 5 20 6.8bcd 1.2bcd 100 0 25 19 5.6b 0.4a 84 1 0 10 not scored dead 10 1 1 17 ' 9.4e 1.8cde 100 1 5 20 8.9de 2.2ef 100 1 25 19 5.4b 0.8ab 100 10 0 19 6.18bc 1.2bc 73 10 1 15 9.2a 2.0ef 100 10 5 20 12.0f 2.2ef 100 10 25 18 8.2cde 2.1ef 66 50 0 10 12.7f 1.0ab 90 50 1 20 9.1e 1.9def 100 50 5 20 - 8.7de 2.6fg 95 50 25 14 3.0a 3.09 78 1) Values followed by different letters are significantly different by Duncan's NMR test (alpha = 0.05). 42 that BAP, NAA level and their interaction were highly sig- nificant for the diameter of induced callus (F-test; alpha = 0.01). Only the BAP main effect and the interaction term were significant for callus color (F-test; alpha - 0.05 and 0.01, respectively). On the basis of the diameter and callus color data, MS augmented with louM NAA and SuM BAP was chosen as the black locust callus induction medium. Tree specific responses in cambial callus growth were observed for both freshly collected explants and explants from tissues that had been stored for eight months (Table 2). Analysis of variance showed that individual trees differed significantly for callus diameter growth, callus color, and browning of medium (F-test; alpha=0.01). The interaction between genotype and initiation time was highly significant for callus diameter growth and browning (F-test; alpha-0.01). Storage of cambial material did not signifi- cantly affect callus color or medium browning, but did result in significant changes in callus diameter growth (F- test; alpha=0.05). Callus diameter was correlated with degree of green color of callus and media browning (Spearman's rho = -0.43 and -0.69, significant at 1 % level; respectively). Callus color and media browning were not significantly correlated (Spearman's rho = 0.13, sample size of 88). Overall, tree #4 produced the most rapidly growing callus. 43 To test the effect of auxin and light intensities on callus maintenance, two auxins, NAA and 2,4-D, and two light intensities (51 and 8 uE/mzlsec) were applied in a factorial design (Table 3). Different auxins and light intensities did not have significant effects on callus diameter growth. Explant source in combination with auxin was significant (Table 4). The callus color was significantly affected by tree #, tree # by auxin interaction, and light intensity (Table 4). All main effects were significant for callus Table 2. The effect of 8 months storage at 4 0C of branChes collected in March on cambium-derived callus initiation. 'Tree Culture Callus Color Browning No initiationl) Diameter(mm) (x) (x) 1 April (10) 10.8+.6c 2.2+.3b 3.2+.2f2) November (12) 7.6+.6b 3.0+.ZC 3.5+.29 2 April (10) 10.6+.6c 3.4+.3c 1.4+.2a November (11) 15.9+.6e 2.1+.2b 1.3+.2a 3 April (10) 11.7+.6c 3.3+.3c 2.2+.2c November (9) 4.8+.7a 3.0+.2c 3.7+.29 4 April (10) 12.9+.6d 2.5+.3b 2.5+.2d November (16) 14.3+.5e 1.2+.2a 1.3+.2a 1) Number in parenthesis = number of explants. 2) Values with different letters are significantly differ- ent by Duncan's NMR test (alpha = 0.05). 44 texture (Table 4). No significant correlation was found between callus diameter growth and firmness of callus (Spearman's rho = -0.06; sample size of 320) on callus medium. Greenness and diameter growth were not correlated (Spearman's rho = 0.09, sample size of 320), however, the greener callus tended to be firmer in texture (Spearman's rho = 0.24, significant at 1% level). Both the type of auxin and tree from which the explants were taken had sig- nificant effects on callus diameter growth and greenness of the callus following transfer to shoot regeneration medium (Table 6). When NAA was added to the MS medium, callus diameter growth and the greenness of callus rapidly in- creased (Table 6). The only combination that resulted in shoot regenera- tion was when callus from tree #1 was transferred to MS medium that lacked NAA (Table 5). Storage of explant tissue for eight months did not significantly alter shoot regeneration (Pairwise T-test; t = 0.89, df = 413). Shoots were increased on shoot multiplication medium. The number of shoots greater than 1 cm in length averaged 4.75 per shoot culture tube. Twenty-five percent of the shoots rooted (Figure 4) and were subsequently potted and transferred to the greenhouse (Figure 5). 45 Table 3. Increase in cambium callus diameter growth and change in color/texture with different auxins and light intensities during a 4 week interval. Trees Increased in Change Change in No Medium1 Light2 diameter(mm) in color texture 1 MSN Full 13.7de -0.9de 0.0cd3 Dim 10.9ab -1.8ab -0.5b M82 Full 12.1bcd -1.7bc -0.9a Dim 11.3abc -1.8abc -1.0a 2 MSN Full 9.9a -l.93b 0.6f Dim 10.1a -2.1a 0.0Cd M82 Full 10.9ab -1.5bc 0.7f Dim 11.0ab -1.3cd 0.4ef 3 MSN Full 13.9e -0.2f 0.2de Dim 14.2e -1.7bc -0.4b M82 Full 13.4de 0.6ef -0.2bC Dim 14.4e -1.0de -0.3bc 4 MSN Full 12.7cde -0.3f -0.2bc Dim 13.3de -1.0de -0.3bc M82 Full 12.3bcd -0.8de -0.3bc Dim 10.7ab -0.4ef -0.1bcd SE (+) 0.52 0.20 0.12 1) Medium: MSN = M8 + 5 uM BAP + 10 uM NAA; M82 = M8 + 5 uM BAP + 10 uM 2,4-D. 2) Light : Full = 51 uE/m2/sec; Dim = 8 uE/m2/sec. 3) Values followed by different letters are significantly different by Duncan's NMR test at alpha=0.05. 4) Each treatment has 20 explants. 46 Table 4. Analysis of variance (GLM-ANOVA) for cambium- derived callus growth, color, and texture with different auxin and light intensities. Change in Change Change in Source df diameter in color texture Tree 3 162.0** 30.0** 13.0:* Auxin 1 8.128** 1.25** 1.38** Tree x auxin 3 21.03 4.34** 3.04**- Light 1 10.87** 26.5** 4.75* Tree x light 3 21.85 8.04** 0.84** Auxin x light 1 0.253* 12.8* 2.63 Tree x auxin x light 3 14.57 2.06 0.10 Error 304 5.394 0.79 0.28 * Mean square significant at the 0.05 significance proba- bility. - ** Mean square significant at the 0.01 significance proba- bility. 47 Table 5. Shoot regeneration from cambium-derived callus of mature black locust after culture for 13 weeks. Trees Medium Explants % explants Diameter Color No NAA BAP(uM) No produced shoot change change Exp.I Exp.II 1 0 10 12 0.02 8 323 2.7 -3.34 0 25 18 27.8 33.3 7.8 -2.6 0 50 12 0.0 16.7 3.4 -3.4 10 10 18 0.0 0.0 15.4 -0.2 10 25 18 0.0 0.0 15.1 -0.3 10 50 18 0.0 0.0 14.3 -0.2 2 0 10 18 0.0 0.0 2.3 -1.3 0 25 18 0.0 0.0 0.1 -1.4 0 50 18 0.0 0.0 0.4 -1.2 10 10 18 0.0 0.0 14.2 -0.2 10 25 18 0.0 0.0 13.3 -0.2 10 50 12 0.0 0.0 12.5 0.2 3 0 10 18 0.0 0.0 10.0 -3.0 0 25 18 0.0 0.0 3.4 -3.4 0 50 18 0.0 0.0 3.1 -3.6 10 10 18 0.0 0.0 17.0 0.0 10 25 18 0.0 0.0 16.2 -0.3 10 50 18 0.0 0.0 14.7 -0.9 4 0 10 18 0.0 0.0 15.1 -1.1 0 25 18 0.0 0.0 12.6 -1.7 0 50 18 0.0 0.0 11.2 -1.4 10 10 18 0.0 0.0 17.7 -0.4 10 25 18 0.0 0.0 18.2 -0.1 10 50 18 0.0 0.0 17.9 -0.1 Total mean 414 1.2 2.4 10.8 -1.3 1) No. of explants for Experiment 2. 2) Experiment 1: For each treatment, twenty calli from fresh cambial tissues were used for regeneration test. 3) Experiment 2: Calli from cambial tissues stored for eight months were used for regeneration test. 4) Data for diameter and color were taken from Experiment 2. 48 Table 6. Analysis of variance (GLM-ANOVA) for increase in callus diameter growth with different BAP levels on the data from Table 5. Tree x NAA BAP Tree x BAP NAA x BAP Tree x NAA x BAP Error OO‘NO‘NUHU U ‘0 Increase in diameter 1310.8:: 5085.0** 298.59** 114.07* 51.409 17.068* 51.979 23.960 Change in callus color ea 31.33** 245.9 21.45** 0.566 1.898 0.254 0.976 1.165 * Mean square significant at the 0.05 significance proba- bility. ** Mean square significant at the 0.01 significance proba- bility. 49 DISCUSSION The optimum phytohormone levels for the initiation of callus from mature cambial tissue were found to be 5 uM BAP and 10 uM NAA. This combination produced firm primary callus that was competent for shoot regeneration. The fre- quency of callus induction from mature cambial tissues (90- 100 %) was similar to that of juvenile tissues, indicating that unlike organogenesis, cellular growth in vitro is not constrained by explant age. Explant source (tree) is the significant factor in determining callus diameter growth, but light intensity and auxin type become important if we look at the other indicators of metabolic activity, such as the color and texture of the callus. Since the induction of firm, green callus has been a prerequisite for regenera- tion in black locust, these studies showed that culture conditions, as well as explant source, must be considered in identifying an optimal culture condition. Furthermore, significant interactions between the source of explants and environmental factors indicated that not only are both significant, but different trees respond to change in light and auxin levels differently. Differential responses in photosynthetic activity have also been observed among half- sib families of black locust when exposed to different environmental regimes in vivo (Mebrahtu and Hanover, 1990). While the expression of these metabolic and physiological 50 differences in vitro makes it difficult to develop a culture system, it also provides a promising avenue to pursue the mechanisms that underlie them. Callus color and firmness were positively correlated, with the firmer callus greener in color. Diameter growth of callus was not significantly correlated with firmness or greenness of the callus at low cytokinin concentrations. However, in the treatments with high cytokinin levels, the correlation between diameter growth and greenness was highly significant (Spearman's rho = 0.67, significant at 1 % level). The greener color and firmness of callus grown on medium containing high cytokinin concentrations may result from cytokinin-derived plastid development (Partheir, 1979). It has been hypothesized that cell wall loosening triggered by auxin may be due to either the activation of wall loosen- ing factors, such as protons (Cleland, 1987), or increased extracellular cellulase activity (Fry, 1989). Auxin/cytoki- nin balancing effects have been observed in plant tissue culture (Krikorian, et al., 1987), but the mechanism respon- sible for this phenomenon has not been identified. When callus was placed on MS media containing high BAP but lacking NAA rather than callus medium (10 uM NAA and 5 uM BAP), diameter growth and greenness were significantly lowered, confirming the importance of maintaining the auxin/cytokinin balance in in vitro culture systems. Medium 51 browning in tissue culture is generally thought to be a result of the oxidation of phenolic compounds by polypheno- loxidase (Hu and Wang, 1983). In these studies medium browning was positively correlated with the greenness of callus. Since the degree of medium browning varied among genotypes, with the highest degree of browning in tree #1, and since tree #1 has shown consistently better performance, the relationship between the degree of medium browning and shoot differentiation is of interest for further study. The differential in vitro growth response has also been observed in bud cultures of the same trees (Davis and Keath- ley, 1987), shoot regeneration from hypocotyl-derived callus of black locust, and shoots regenerated from callus derived from internodal segments of in vitro shoot cultures of mature black locust (chapter 1). Komatsuda et al. (1989) conducted a complete diallel set of crosses to elucidate the genetic basis of the genotypic differences in callus culture and subsequent shoot regeneration in barley. From their result, significant dominant gene actions with high herita- bility as well as additive gene effects were found in in vitro traits. Considering their findings, our results when combined with previous works with these trees suggest that the superior in vitro response of tree #1 is likely due to true genetic rather than epigenetic effects. Several observation support this contention. First, the differen- 52 tial response observed in mature bud culture (Davis and Keathley, 1987) has been persistent through callus-plant regeneration (Han et al., 1990). Second, the studies re- ported here indicate that the major controlling factor in mature cambium culture is associated with the tree, rather than the culture environment. Finally, Komatsuda et a1. (1989) confirmed that the expression of callus growth and shoot differentiation are governed by different genetic mechanisms, and our results also show a differential re- sponse where tree #4 was best for callus culture; whereas, tree #1 gave optimum shoot regeneration. The use of cambial tissues provide advantages over other tissues from mature trees due to its year-round avail- ability and relatively high chance of avoiding systematic contamination problems caused by soil-born bacteria. Fur- thermore, cambial explants offer the possibility of immedi- ate gene transfer without waiting through an initial cul- ture induction period, as demonstrated in ongoing experi- ments in which cambial tissues of black locust have produced kanamycin resistant calli when co-cultivated with an Agro- bacterium strain which has a kanamycin resistance gene inserted into a disarmed Ti plasmid (Han and Keathley, unpublished). The ability to store cambial tissue facili- tates testing and the development of tissue culture systems by simplifying the logistics of the process. Cambial 53 tissues could also be preserved in tissue banks for conser- vation of tree germplasm by either cryopreservation or cold- room storage methods. 54 Figure 1: Cultures of cambium-derived callus from four mature black locust (Robinia pseudoacacia L.). Figure 2: Shoot organogenesis from callus of tree #1 on the regeneration medium (bar = 3 mm). Figure 3: Multiplication of the regenerated shoots on MS + 0.5 uM BAP. Figure 4: Subsequent rooting of the multiplied shoots. Figure 5: Potted black locust which regenerated from cambial tissue of mature tree (pot diameter = 25 cm). REFERENCES Barghchi, M. 1987. Mass clonal propagation in vitro of Robinia pseudoacacia L. (black locust) cv. 'Jaszkis- eri'. Plant Sci. 53: 183-189. Chalupa, V. 1983. In vitro propagation of willows (Salix spp.), European mountain-ash (Sorbus aucuparia L.) and black locust (Robinia pseudoacacia L.); Biol. Plant. 25(4): 305-307. Cleland, R.E. 1987. Auxin and cell elongation. In: Plant hormones and their role in plant growth and development. P.J. Davies (ed.), Kluwer Academic Publishers. Dordrecht/Boston/London. pp 132-148. Davis, J.M. and D.E. Keathley. 1985. Regeneration of shoots from leaf disk explants of black locust (Robinia pseudoacacia L.). In: Proceedings of the Fourth North Central Tree Improvement Conference, East Lansing, MI, Aug. 12-14, 1985. pp 29-34. Davis, J.M. and D.E. Keathley. 1987. Differential responses to in vitro bud culture in mature Robinia pseudoacacia L. (black locust). Plant Cell Rep. 6: 431-434. Davis, J.M. and D.E. Keathley. 1989. Detection and analysis 55 56 of T-DNA in crown gall tumors and kanamycin-resistant callus of Robinia pseudoacacia. Can. J. For. Res. 19: 1118-1123. Fry, S.C. 1989. Cellulase, hemicelluloses and auxin- stimulated growth: a possible relationship. Physiol. Plant. 75: 532-536. Han, K.H. and D.E. Keathley. 1988. Isolation and culture of protoplasts from callus tissue of black locust (Robinia pseudoacacia L.). Nitrogen Fixing Tree Res. Rep. 6:68- 70. Hintze, J.L. 1987. Number cruncher statistical system. Version 5.0 2/87. Dr. Jerry L. Hintze. Karysville, Utah 84037. Hu, C.Y. and P.J. Wang. 1983. Meristem, shoot tip, and bud cultures. In: Handbook of plant cell culture, vol.1, Techniques for propagation and breeding. Evans, D.A., Sharp, W.R., Ammirato, P.V., and Yamada, Y. (ed.). Macmillan Publishing Co. New York. pp 177-227. Komatsuda, T., Enomoto, S. and K. Nakajima. 1989. Genetics of callus proliferation and shoot differentiation in barly. Jour. Heredity. 80:345-350. Krikorian, A.D., Kelly, K. and D.L. Smith. 1987. Hormones in tissue culture and micropagation. In: Plant hor- mones and their role in plant growth and development. P.J. Davies (ed.), Kluwer Academic Publishers, 57 Dordrecht/Boston/London. pp 593-613. Mebrahtu, T. and J.W. Hanover. 1990. Tree Physiology, in press. Merkle, S.A. and A.T. Wiecko. 1989. Regeneration of Robinia pseudoacacia L. via somatic embryogenesis. Can. J. For. Res. 19: 285-288. Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cul- tures. Physiol. Plant. 15: 473-497. Parthier, B. 1979. The role of phytohormones (cytokinins) in chloroplast development. Biochem. Physiol. Pflanzen 174: 173-214. Steel, R.G.D. and J.H. Torrie. 1980. Principles and proce- dures of statistics. A biometrical approach. McGraw- Hill Book Company. CHAPTER 3 Transformation of Robinia pseudoacacia L. via a hypocotyl- Agrobacteriu- rhisogenes system. 58 ABSTRACT Hypocotyl segments inoculated with Agrobacterium rhizo- genes strains R1500 and R1601 produced hairy roots when cultured on MS medium without phytohormone. Ri-induced hairy roots were faster growing and larger than normal roots. They also had a marked development of lateral roots when cultured on black locust callus culture medium (BCM) supplemented with 100 ug/ml kanamycin. ‘Less than 5% of adventitious roots spontaneously produced shoots. 'The regenerated shoots were multiplied and rooted in the presence of 100 ug/ml kanamycin. Leaf pieces from the shoot cultures were tested for the expression of a kanamycin resistance gene by culturing on BCM with 100 ug/ml kanamy- cin. All leaf pieces taken from transformants induced callus on BCM with 100 ug/ml kanamycin while leaf pieces from normal plants died. The hairy root phenotype which has been characterized as having wrinkled leaves and abun- dant root development was evident after 3 months of shoot development in pot from potted transformants. Southern analysis showed the presence of both TL-DNA and NPTII gene sequences. 59 INTRODUCTION The infection of susceptible plants with virulent strains of Agrobacterium tumefacience or A. rhizogenes re- sults in the formation crown gall tumors or "hairy root" disease (for review, see Bevan and Chilton, 1982; Nester et al., 1984; Zambryski, 1988; Zambryski et al., 1989). These pathogenic responses result from the transfer and expression of the transfer DNA (T-DNA) of the Ti- or Ri-plasmid in the plant genome (Binns and Thomashow, 1988; Klee et al., 1987). Endogenous T-DNA directed synthesis of phytohormones, both auxin (Inze et al., 1984; Schroder, 1984) and cytokinin (Akiyoshi et al., 1984; Barry et al., 1984), forms the basis for phytohormone autotrophic growth of the transformed cells. Intact T-DNA also encodes genes which direct the synthesis of different opines, and these unique amino acid derivatives can be utilized by the bacterium as its sole carbon and nitrogen source (for review, see Tempe et al., 1984). Although the tumor-inducing (Ti) plasmids of A. tume- faciens have been used more frequently as a vector for plant genetic transformation than the root-inducing (Ri) plasmids of A. rhizogenes, in recent years vector systems based on 60 AlIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIlllllllllllllllllllllii 61 the Ri-plasmid have been developed and used for gene trans- fer (for reviews, see Birot et al., 1987; Petersen et al., 1989; Tepfer and Casse-Delbart, 1987). The advantage of Ri- compared to Ti-plasmid mediated transformation is that the presence of the exogenous Ri T-DNA segment does not prevent the regeneration of whole fertile plants from hairy roots. Intact plants of various plant species have been obtained (Ackermann, 1977; Spano and Costino, 1982; Tepfer, 1984). The A. rhizogenes agropine-type strain A4 trans- fers two distinct DNA segments, left (TL) and right (TR), of T-DNA into plant cells (White et al., 1985). The viru- lence of the A. rhizogenes A4 strain was significantly increased by using the vir region of a "supervirulent" plasmid pTiB0542 in trans (Pythoud et al., 1987). Long generation cycles, space requirements for large segregating populations, the large size of trees, and the lack of genetically pure lines are all examples of limita- tions that may be breached with the ability to transform a particular tree species. Even though Agrobacterium can infect a wide spectrum of woody plants, including many angiosperms and some gymnosperms, successful application of an Agrobacterium-mediated transformation system has been limited, partially because the system for the regeneration of whole plants from transformed cells has not been de- veloped (Hassig et al., 1987). Transformation of woody 62 species and subsequent regeneration of transgenic plants has been reported for only a few genera including Populus (Fillatti et al., 1987; Pythoud et al., 1987; McCown et al., 1991), Juglans (McGranahan et al., 1988), Malus (James at al., 1989), and Vitis (Mullins et al., 1990). A number of characteristics make black locust ideal for transformation studies (see 'Introduction' of this disserta- tion). Davis and Keathley (1989) were able to transform black locust with several different strains of Agrobacterium and to obtain kanamycin-resistant calli. However, no transgenic black locust plant was regenerated. The objec- tive of this study was to obtain transgenic black locust plant through a Agrobacterium-mediated transformation. MATERIALS AND METHODS Bacterial strains. Agrobacterium was grown on Luria Bertani (LB) medium supplemented with appropriate antibiot- ics when necessary. The concentrations of antibiotics used were 100 ug kanamycin (Sigma Chemical Company) per ml and 100 ug ampicillin (Sigma Chemical Company) per ml. A. rhizogenes R1500 and R1601 (Pythoud et al., 1987) were kindly provided by J.M. Davis from M.P. Gordon's laboratory. Tissue culture systems and infection procedure. In vitro culture systems for transformation studies have been developed through the experiments described in chapters 1 63 and 2. The small segments (0.5 to 1.0 cm in length) of hypocotyl were soaked for several minutes in an overnight culture of Agrobacterium rhizogenes, and placed on agar (0.6%) solidified Murashige and Skoog (MS) medium (1962) without phytohormones (MSO). After 3 days, the explants were transferred to MSO containing 300 ug/ml cefotaxime (Sigma) and 300 ug/ml vancomycin (Sigma) to kill the bacte- ria. The tissue was subsequently maintained on MSO supple- mented with 500 ug/ml of both antibiotics. Selective culture. Induced hairy roots (> 1 cm in length) were excised and cultured on MS medium containing 10 uM NAA, 5 uM BAP, and 100 ug/ml kanamycin to induce callus and shoot regeneration. Transgenic callus was maintained on the same induction medium. Shoots that regenerated from the hairy roots were inserted in MS medium supplemented with 0.6% agar, 2% sucrose, 1 uM BAP (BSM), and 100 ug/ml kanamy- cin. Rooted putative transgenic black locust plant lets were grown in a peat moss: perlite: vermiculite mix accord- ing to Han et a1. (1990). To test for the expression of kanamycin resistance in the transgenic plants; the following procedures were used: 1) both transformed and normal shoots were transferred to rooting medium (BRM; Han and Keathley, 1991) in the presence of 100 ug/ml kanamycin (Sigma) and survival and rooting ability was evaluated after 4 weeks of culture. 2) leaf 64 pieces from both transformed and normal individuals were cultured on callus induction medium (BCM; chapter 2) supple- mented with 100 ug/ml kanamycin (Sigma). Explants were evaluated for survival and callus induction. Plant DNA isolation. Total plant DNA was isolated efficiently as in Draper and Scott (1988). Young leaf material was harvested from the potted plants, frozen in liquid nitrogen and ground to a fine powder using a pre- chilled mortar and pestle. The powder was then thawed in extraction buffer (15 ml/g fresh weight; 2.0M Tris-HCl pH 8.0, 0.5M EDTA pH 8.0, 5.0M NaCl, 10 mM 2-mercaptoethanol), and 1/7.5 volume of 10% SDS was added. After mixing thor- oughly by gentle shaking the suspension was incubated for at least 20 min. in a 65 oC waterbath. 5M potassium ace- tate (1/3 volume of extraction buffer) was then added and mixed by repeated inversions and followed by a 30 min. incubation on ice. The protein/SDS precipitate was removed by centrifugation at 12,000 x g for 30 min (4 °C) in a HB4 rotor. The supernatant was filtered through 4 layers of Miraclothr into a clean tube and DNA was precipitated by adding pre-chilled isopropanol (-20 °C; 2/3 volume of total solution). The precipitated DNA was hooked out using a 1.5 ml Pasteur pipette, transferred to precipitation buffer in a microfuge tube (1 ml/g fresh weight; 76% ethanol, 10mM ammonium acetate), and set for 20 min at room temperature. 65 The DNA was pelleted by centrifugation for 30-60 seconds in an Eppendorf microcentrifuge. The pellet was dissolved in the smallest possible volume of Tris-EDTA buffer (pH 8.0). Southern analysis. DNA was digested with restriction enzyme HindIII under the conditions specified by the manu- facturer (Boehinger Mannheim). The digested DNA was size fractioned by electrophoresis in 0.7% agarose gel with TBE buffer (Sambrook et al., 1987) and then transferred to Zeta- probe blotting membrane by DNA alkaline blotting as speci- fied by the manufacturer (Bio-Rad Laboratory). Two probes were used. The first, a purified PstI fragment, which contains a chimeric Tn5 Kmr gene sequence, from the plasmid vector pCIBlO (Rothstein et al., 1987) was obtained from J.M. Davis. The second was cosmid DNA, isolated from pFW302 (covering TL-DNA) by the alkaline lysis method described by Ausubel et a1.(1987). Cosmid DNA was digested with the restriction enzyme HindIII as specified by the manufacturer (Boehinger Mannheim). Probe DNA was radioactively labeled using a random primed labelling kit obtained from DuPont with [32P1dCTP (3000 Ci/mmol (1 Ci = 37 GBq); New England Nuclear). Hybridization and washing were carried out as specified by Bio-Rad for Zeta-probe membrane. The blots were visualized by exposure to Kodak XAR-S film with an intensifying screen at -700 C for 2 to 10 days. 66 RESULTS Plant transformation. Hypocotyl segments inoculated with Agrobacterium rhizogenes strains R1500 and R1601 (Py- thoud et al., 1987) developed adventitious roots after 4 days culture on MSO medium (Figure 1). No significant effect of seedling age was found for adventitious root formatiOn. The hypocotyl segments inoculated with A. rhizogenes R1601 showed a higher percentage (18%) of hairy root formation than those inoculated with R1500 (10%) (Table 1). The addition of kanamycin (100ug/ml) in M80 after the 3 day cocultivation period significantly lowered the per- centage of explants exhibiting tumorous roots (P < 0.01, Table 2). Significant differences in the number of hypo- cotyl segments producing hairy roots on MSO without kanamy- cin were observed between explants treated with and without IAA (Table 2). However, the addition of 2,4-D did not affect transformation frequency (Table 2). The highest percentage (42%) of transformation was achieved from ex- plants which were inoculated with A. rhizogenes R1601 and cultured on MS medium in the presence of 1 uM IAA (Table 2). Shoot regeneration and selective culture. After transfer to callus induction medium (BCM), containing 100 ug/ml kanamycin, Ri-induced hairy roots exhibited distinc- tive traits. They were fast growing, larger, and showed a 67 Table 1. Effect of seedling age on percentage of explant which produced adventitious roots after 4 weeks of culture following inoculation with Agrobacterium rizogenes strains R1500 and R1601. Seedling age (days) Strains # explants % root 5 R1601 73 24.7 R1500 70 10.0 Control 85 0.0 6 R1601 22 18.2 Control 25 4.0 10 R1601 28 17.9 Control 70 1.4 11 R1601 43 11.6 Control 21 4.8 25 R1601 30 10.0 Control 24 0.0 Total R1601 196 17.9 R1500 70 10.0 Control 225 1.3 68 Table 2. Effect of exogenous auxin and kanamycin in MS medium following co-cultivation with A. rhizogenes on the formation of hairy root. MSO + 1 HM IAA Mso + 1 uM 2.4-0 MSO + 100 ug/ml Km MSO + 1 uM IAA + 100 ug/ml Km R1500 Control R1601 Control R1601 Control R1601 Control R1601 Control 69 marked development of lateral roots (Figure 1). Roots began to form callus in less than a month. While the calli remained white for about two months on callus culture medium (BCM), with kanamycin, they eventually turned green (Figure 2). In the process of forming callus, a small proportion (less than 5%) of adventitious roots gave rise to spontane- ous shoots (Figures 3 and 4). Shoots regenerated from root tumors showed the same growth and proliferation pattern on the shoot culture medium (BSM) containing 100 ug/ml kanamycin as normal shoots derived from in vitro bud culture did on BSM without kanamycin (Figure 5). However, the transformed shoots produced highly proliferating roots on BSM, while normal shoots do not root on BSM (Figure 6). In contrast to the healthy growth of transformed shoots on selection medium, the leaves of normal shoots in the presence of 100 ug/ml kanamycin turned yellow and eventual- ly abscised (Figure 5). The results of the rooting test for putative trans- formed shoots in black locust rooting (BRM) medium (Han and Keathley, 1991) are shown in Table 3. Although all 10 putative independent transformants remained green in the presence of 100 ug/ml kanamycin, only 5 rooted. Based on the rooting performance of transformants #771, 772, and 773, no significant differences in rooting percentage were ob- 70 served for these lines between shoot culture medium (BSM) and rooting medium (BRM) except for #771 (Table 3). Con- trol shoots did not produce roots or survive on the selec- tion medium. Further testing for the expression of kanamycin resist- ance in the transformed plants was conducted by culturing leaf pieces on callus induction medium (BCM) containing 100 ug/ml kanamycin. All of the leaf pieces from the puta- tive transgenic shoots produced callus on the selective BCM (Table 4; Figure 7). The leaf pieces also showed shoot and root morphogenesis on the same medium and each independ- ent transformant showed an individual response (Table 4). Leaf pieces from control plants did not survive the kanamy- cin medium but did produce callus on MSO (Figure 7). Acclimation of transgenic plants to in viva condition: Transformed black locust plantlets were potted (Figure 12) and maintained as described in Han et a1. (1990). Within two weeks after potting, 20 to 30% of the transformed plants died. This represents a higher than normal mortality rate when compared to normal, non-transformed plants. However, until a certain stage of development, about 3 months after potting, the remaining plants were more vigorous, fast growing, and had leaves that were thicker and darker than normal shoots. After 3 months of growth, when the average shoot height reached 60-70 cm, phenotypic alterations in 71 Table 3. Rooting of transformed black locust shoots after 4 weeks of culture in the presence of 100 ug/ml kanamycin. Lines Medium # shoots % root Color 771 BSM +Km 14 100.0 Green BRM +Km 7 42.9 Green 772 BSM +Km 28 7.1 Green BRM +Km 10 20.0 Green 773 BSM +Km 66 63.6 Green BRM +Km 12 66.7 Green 774 BSM +Km 5 0.0 Green 776 BSM +Km 2 100.0 Green 777 BRM +Km 2 50.0 Green 891 BSM +Km 11 0.0 Pale-Green BRM +Km 8 0.0 Green 941 BSM +Km 3 0.0 Green 942 BSM +Km 2 0.0 Green 943 BSM +Km 8 0.0 Green BRM +Km 3 0.0 Green 944 BSM +Km 1 0.0 Green Control BRM +Km 25 0.0 White / Abscised 72 Table 4. Callus induction from leaf pieces of transformed black locust shoot after 4 weeks of culture in MS + 100 ug/ml kanamycin. Lines Explants Callus Root Shoot Color}; No % % % 771 44 100.0 59.1 0.0 G 772 34 100.0 8.8 5.9 G,GW 773 37 100.0 5.4 2.7 G,GW 774 10 100.0 10.0 0.0 G,GW 92008 49 49.0 0.0 0.0 G Control 20 0.0 0.0 0.0 W,Y,D Control -Km 20 100.0 0.0 0.0 G 1) Color: G-green, GW-greenish white, W-white, Y-yellow, D- dark brown (died). 73 leaf morphology were observed (Figures 8 - 11). The abnor- malities in leaf morphology included: wrinkled, furrowed, and variegated leaves, as well as asymmetrical leaflets. Each transformant seemed to have a different aberrant pheno- type in leaf morphology. For instance, wrinkled leaves were found in all transformants, variegated leaves were in #772, and furrowed leaves in #773. The root system of potted transformants was larger than that of non-transformed plants (Figure 13). Southern analysis. DNA was readily isolated from each of three independent transformants, #771, 772, and 773, using the protocol described in above. After digestion with restriction enzyme HindIII, the DNA was size frac- tioned by electrophoresis and transferred to a Zeta-probe membrane following the manufacturer's (Bio-Rad) instruc- tions. The filter bound DNA was then hybridized to 32P- labeled probes (either pFW302 which represent TL-DNA or the PstI fragment which contains the Kmr gene). All three transformants contained fragments which comigrated with fragments 17, 30, and 32 from HindIII digestion of pFW302 (Figure 14a). These results suggest that the TL-DNA se- quences were present in the transformants. The fragment comigrating with HindIII fragment 0.8 was not found in this hybridization pattern. The absence of a fragment comigrat- ing with HindIII fragment 21 was not surprising because 74 R1601 has a transposon insertion in this fragment (Sinkar et al., 1987). Although no fragments that comigrated with TL-DNA fragments were found, some hybridizing bands were present in the control DNA lane. The reason for this hybridization is not currently clear. But, this might suggest that there are homologous sequences between black locust and cosmid (pFW302) DNA. Hybridization patterns using the NPT II gene as a probe are shown in Figure 14b. The DNA of all transformants showed bands which hybridized to the probe. No detectable bands were found in control lane. DISCUSSION Tree species are generally more recalcitrant to genetic transformation and subsequent regeneration of transgenic plants than herbacious species. The successful transfor- mation and regeneration of transgenic plants requires well defined in vitro culture conditions for the particular species. Several woody species transformations have been reported in both gymnosperms (Dandekar et al., 1987; Kar- nosky et al., 1988; Loopstra et al., 1990; Sederoff et al., 1986) and angiosperms (Baribault et al., 1989; Davis and Keathley, 1989; Guellec et al., 1990; Kobayashi and Uchi- miya, 1989; Mackay et al., 1988; Vahala et al., 1989). But 75 until now the regeneration of transformed plant has been reported in only 4 woody species, including poplar (Fillatti et al., 1987; McCown et al., 1991; Pythoud et al., 1987), walnut (McGranahan et al., 1988), apple (James et al., 1989), and grape (Mullins et al., 1990). The results presented in this chapter clearly demon- strate genetic transformation and subsequent regeneration of transformed black locust. Thus, black locust becomes the fifth woody plant species to be regenerated from transformed tissues. Black locust shoots have been regenerated from hairy root tissues in the presence of kanamycin and subsequently cultured on selective medium using kanamycin. Rooting of regenerated shoots on selective medium was a quick assay for the expression of the kanamycin resistance gene in the plant. All of the shoots from transformant #771, 772, and 773 produced roots on BSM supplemented with kanamycin. To further confirm the expression of kanamycin resistance, leaf pieces from individual transformants were cultured on callus induction medium containing kanamycin. All the leaf pieces induced callus while normal leaf pieces died. Abnormal phenotypes in leaf morphology observed among the transformants also confirm transformation because these abnormalalites are closely correlated to the expression of TL-DNA rol genes (Cardarelli et al., 1987; White et al., 76 1985). And finally, the molecular evidence for transformed black locust comes from Southern analysis, which demonstrat- ed the presence of sequences from both the TL-DNA region and the kanamycin resistance gene in the transformants. This research was successful for three main reasons; 1) The basic knowledge of tissue culture procedures, including the dedifferentiation and regeneration of black locust, had been well established (chapters 1 and 2). 2) Use of the proper vector system. We also tried a disarmed Ti-plasmid (pGV3850HPT::pKU2, Baker et al., 1987) which was not suc- cessful in getting transgenic black locust. This is not too surprising since vector specific effects are also found among Ti-plasmids (Guri and Sink, 1987). 3) Use of proper plant materials in terms of species, genotype, and juvenili- ty. We have found that black locust can be easily manipu- lated in vitro (Han and Keathley, 1988, 1989; Han et al., 1990; see also previous chapters). We also used explants from juvenile materials. When IAA was added to the medium on which explants co-cultivated with A. rhizogenes were incubated, the per- centage of explants that produced hairy roots was signifi- cantly increased. This result is interesting in relation to the recent findings that have determined that hairy root formation is not the result of the activity of the TR-DNA borne auxin gene or due to a substantial imbalance of en- 77 dogenous phytohormones, but it is a result of an increased sensitivity to auxin conferred by the TL-DNA (Cardarelli et al., 1987; Shen et al., 1988; Spano et al., 1988). Since a high concentration of kanamycin was used (100 ug/ml; most other reports used 5-50 ug/ml), in vitro selec- tion was very efficient, all normal plants were killed and transformants were still able to undergo growth and organo- genesis. These results suggest that there is enough NPTII activity in the transformed tissues to allow growth and organogenesis. The level of kanamycin being used is important because if it is too low, the selective culture might end up with untransformed escapes. Consequently, if it is too high, then the selective culture might kill trans- formants which have a low level of NPTII enzyme activity. The concentration of kanamycin used in the present studies is suggested for future transformation studies involving black locust. Typical phenotypes of hairy root derived plants have been described as having dark wrinkled leaves, an extremely abundant and plagiotropic root system, reduced apical domi- nance, reduced internode length and leaf size, and the ability of leaf explants to differentiate roots in a phyto- hormone-free medium (Cardarelli et al., 1987; Hamamoto et al., 1990; Spano et al., 1987; Tayler et al., 1985; Tepfer, 1983, 1984). Reduced apical dominance, internode length, 78 and leaf size were not found in black locust transformants. In all three transformants the hairy root phenotype was not expressed at the early shoot growth stage, but rather became evident after 3 months growth. 1 Interestingly, in addition to the wrinkled leaf mor- phology, asymmetrical and variegated leaves were found in black locust transformants. These phenotypes were not reported earlier as a hairy root phenotype, but Kim et al. (1973) found the same abnormalities in their black locust breeding efforts which utilized mutations generated by X-ray and thermal neutrons. Therefore, these abnormalities have possibly originated from the genomic disturbance due to the insertion of foreign DNA, rather than from the expression of T-DNA genes in the transformants. This hypothesis might explain why each transformant has a different abnormal phenotype in addition to wrinkled leaves which is closely correlated to the presence of the TL-DNA rolB locus (Cardar- elli et al., 1987; White et al., 1985). Southern analy- sis, utilizing pFW302 (covering TL-DNA) as the probe, showed the presence of TL-DNA in all three transformants. The absence of a fragment comigrating with the HindIII 0.8 fragment of pFW302 in the hybridization pattern may suggest incomplete incorporation of T-DNA. But it remains to be studied further. In summary, the transformation and regeneration system 79 reported here is repeatable and effective, allowing routine introduction of genes into the black locust genome. Successful use of this system may provide the development of locust borer resistant and/or spineless black locust, which substantially saving time and effort, when compared to typical tree improvement program. Figure 1. Culture of hairy roots derived from hypocotyl segments which cocultivated with Agro bacterium rhizogenes R1601. Figure 2. Cultures of hairy root-derived callus on BCM supplemented with 100 ug/ml kanamycin. Figure 3. Spontaneous shoot regeneration from hairy root cultures on BCM containing 100 ug/ml kanamycin. Figure 4. Spontaneous shoot regeneration from hairy root cultures on BCM containing 100 ug/ml kanamycin. Figure 5. Shoot cultures regenerated from hairy root tissues of black locust. Figure 6. Rooting of shoots regenerated from hairy roots on BSM containing 100 ug/ml kanamycin. Figure 7. Cultures of leaf pieces on callus induction medium in the presence of 100 ug/ml kanamycin. Figure 8. Abnormal phenotype in leaf morphology in transformant #771. 8O Figure 9. Abnormal phenotypes in leaf morphology in transformant #772. Figure 10. Abnormal phenotypes in leaf morphology in transformant #773. Figure 11. Potted transformed black locust (#771). Figure 12. Potted transformed~black locust (#773). Figure 13. Root systems of both normal and transformed (#771) black locust. 81 82 Figure 14a: Southern blot analysis of transformed black locust plants. Total black locust DNA was digested with HindIII, size fractioned on agarose gel by electropho- resis and transferred to Zeta-Probg membrane (Bio-Rad). Southern blots were hybridized with 2P-labeled pFW302 DNA (covering T -DNA). Lanes: 1) DNA (10 ng) from pFW302; 2) transforman #771 (0.7 ug); 3) transformant #772 (2 ug); 4) transformant #773 (6 ug); 5) DNA (3 ug) from untransformed black locust. 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Edited by 91 D.P.S. Verna and T. Hahn. Springer-Verlag, Wein and New York. Tepfer, D.A. 1984. Transformation of several species of higher plants by Agrabacterium rhizagenes: sexual transmission of the transformed genotype and phenotype. Cell 37: 959-967. Tepfer, M.E. and F. Casse-Delbart. 1987. Agrabacterium rhiZagenes as vector for transforming higher plants. Microbial. Sci. 4: 24-28. Vahala, T., Stabel, T., and T. Erikssan. 1989. Genetic transformation of willows (Salix spp.) by Agrabacterium tumefaciensL Plant Cell Reports 8: 55-58. White, F.F., Ghidassi, G., Gordon, M.P., and E.W. Nester. 1982. Tumor induction by Agrabacterium rhizagenes involves the transfer of plasmid DNA to the plant genome. Proc. Natl. Acad. Sci. U.S.A. 79: 3193-3197. White, F.F., Taylor, B.H., Huffman, C.A., Gordon, M.P., and E.W. Nester. 1985. Molecular and genetic analysis of the transferred DNA region of the root-inducing plasmid of Agrobacterium rhizagenes. J. Bacteriol. 164: 33-34. Zambryski, P. 1988. Basic processes underlying Agrobacteri- um-mediated DNA transfer to plant cells. Annu. Rev. Genet. 22: 1-30. Zambryski, P., Tempe and J. Schell. 1989. Transfer and function of T-DNA genes from Agrabacterium Ti and Ri 92 plasmids in plants. Cell 56: 193-201. CONCLUSION AND RECOMMENDATIONS. Application of plant genetic engineering technology in a black locust improvement program holds great promise for accomplishing specific breeding goals such as insect re- sistance or spinelessness. The results of the experiments described in this dissertation clearly show the feasibility of the application of such technology into ongoing tree improvement programs. The results of the experiments described in the first chapter illustrate that the regeneration of black locust can be achieved from callus tissues derived from either hypoco- tyl or internodal segments of in vitra shoot cultures. This plant regeneration system was simple and repeatable. Whale plant regeneration from unorganized tissues is there- fore no longer a barrier for genetic manipulation of black locust at the cell or molecular level. This system can be utilized in many ways in tree breed- ing efforts such as in vitra selection, somaclanal varia- tion, somatic hybridization, somatic embryogenesis, and genetic transformation. Since the successful application of these techniques relies on the regenerability of manipu- lated cells and tissues, practical application of this system should be considered at the early stages of a 93 94 breeding program if biotechnology is to be employed. The results of the experiments described in Chapter 2 indicate that cambial tissue of mature black locust can be used to generate callus and from this callus shoot regenera- tion is possible. It was important to be able to regener- ate shoots from mature trees, because in vitra techniques have been confined to seedling materials (Bonga, 1982) and this limitation has been an unsolved problem in the applica- tion of biotechnology as an alternative to traditional methods. Now, it is clear that direct use of mature trees as starting materials in in vitra culture, and manipulation of tree species is feasible by using cambial tissues. Furthermore, cambial tissues are available regardless of season so that the culture can be initiated year-around. The results described in Chapter 2 also demonstrate that the cambial tissues can be stored without substantial lose of regenerability. The ability to store cambial tissues has two practical applications: 1) in the short term, it facilitates testing and the development of tissue culture systems by simplifying the logistics of the process, and 2) cambial tissues could be preserved in tissue banks for conservation of tree germplasm by either cryopreserva- tion or cold-roam storage methods. The results described in Chapter 3 report the regenera- tion of transformed black locust. It is important to 95 report this, because there are currently only 4 woody spe- cies (apple, grape, poplar, and walnut) capable of regener- ating transformed plants. Hypocotyl segments inoculated with Agrabacterium rhizo- genes produced hairy roots on phytohormone-free medium. Transformed plants were obtained by spontaneous shoot regen- eration from hairy root tissues cultured on callus induction medium containing kanamycin. Consequently, shoot regener- ation from transformed callus has not been attempted. Regenerability of transformed callus remains to be tested. All transformants clearly showed ralB phenotype such as wrinkled leaf, remarkably abundant root development, and rooted on phytohormone-free medium. It is of interest to note that only the explant inoculated with Ri-plasmid vec- tors produced transgenic trees. Therefore, the use of an Ri-plasmid based vector system may be recommended for future transformation work with black locust. With this Ri-plasmid mediated gene transfer system, genetic manipulation of certain traits, such as locust borer resistance or spinelessness, is possible. However, since this system utilizes seedling materials, it currently has limited use in a breeding program. The development of a .protocol for the transformation of mature cambial tissues utilizing this transformation system will provide the means to overcome the limitation of using seedling materials. REFERENCES Bonga, J.M. 1982. Vegetative propagation in relation to juvenility. In: Tissue culture in forestry, Bonga, J.M. and D.J. Durzan (eds.). Martinus Nijhoff/Dr. W. Junk, The Hague, pp. 387-412. 96 nICHIceN STATE UNIV. LIBRARIES llWWIW[Ill"IUIIIHWIIWH“WNWHIWI 31293009081716