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Somatic embryogenesis and genetic transformation in douglas-fir Jiang, Liwen 1991

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SOMATIC EMBRYOGENESIS AND GENETIC TRANSFORMATION IN DOUGLAS-FIR by LIWEN JIANG B.Sc.(forest Science) South China Agricultural University  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master of Science  in THE FACULTY OF GRADUATE STUDIES FOREST SCIENCES/BIOTECHNOLOGY LABORATORY  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA April 1991 (c) Liwen Jiang, 1991  In  presenting  degree at the  this  thesis  in  University of  partial  fulfilment  of  of  department  this thesis for or  by  his  or  requirements  British Columbia, I agree that the  freely available for reference and study. I further copying  the  representatives.  an advanced  Library shall make it  agree that permission for extensive  scholarly purposes may be her  for  It  is  granted  by the  understood  that  head of copying  my or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department The University of British Columbia Vancouver, Canada  DE-6 (2/88)  Abstract Cell division was obtained from cultured microspores of Douglas-fir on medium supplemented with auxin, cytokinin, and sucrose, but without medium salts. Embryogenic callus was initiated from excised mature and immature zygotic embryos of Douglas-fir on media supplemented with cytokinin and auxin. Precotyledonary embryos produced most of the embryogenic calli in the cultures. Secondary embryogenic callus production, and subsequent subculturing, were required for the establishment of stable embryogenic callus lines for both mature and immature zygotic embryos. Somatic embryos at the precotyledonary stage were obtained in high frequency when Douglas-fir embryogenic callus was transferred onto hormone-free medium supplemented with 1% activated charcoal, while some cotyledonary somatic embryos were obtained from hormone-free medium supplemented with low A B A levels (0-10 «M). The level of A B A in the maturation medium significantly affected the quality of the somatic embryos produced. Cell suspensions were established from embryogenic calli and have been maintained for over one year. Protoplasts were isolated from suspension, cell colonies and calli were regenerated from protoplasts. GUS and C A T genes were successfully introduced into protoplasts of Douglas-fir via electroporation, and their transient expression was obtained 2-4 days after electroporation. The results so far indicate that the production of somatic embryos via embryogenesis in vitro is obtainable, and the application of direct gene transfer via electroporation for genetic engineering of trees in this species is promising.  TABLE OF CONTENTS Abstract  ii  Table of Contents  ,  List of Figures  iii v  List of Tables  viii  ACKNOWLEDGEMENT  ix  CHAPTER 1  .1  G E N E R A L INTRODUCTION  1  CHAPTER 2  7  E V A L U A T I N G MICROSPORE EMBRYOGENESIS WITH MALE-STOBILI IN DOUGLAS-FIR7 2.1 INTRODUCTION 2.2 MATERIALS A N D METHODS 2.2.1 C O N E COLLECTION... 2.2.2 MICROSPORE ISOLATION 2.3 RESULTS A N D DISCUSSION  ...8 10 10 11 16  CHAPTER 3  24  SOMATIC EMBRYOGENESIS IN DOUGLAS-FIR 3.1 INTRODUCTION 3.2 E M B R Y O G E N I C CALLUS F R O M M A T U R E ZYGOTIC EMBRYOS 3.2.1 MATERIALS A N D METHODS 3.2.2 RESULTS A N D DISCUSSION 3.3 E M B R Y O G E N I C CALLUS F R O M I M M A T U R E Z Y G O T I C EMBRYOS 3.3.1 MATERIALS A N D METHODS 3.3.2 RESULTS 3.3.3 DISCUSSION  24 25 29 29 33 43 43 52 68  C H A P T E R 4 PROTOPLAST ISOLATION A N D ELECTROPORATION 4.1 INTRODUCTION 4.1.1 PROTOPLAST ISOLATION 4.1.2 ELECTROPORATION 4.2 MATERIALS A N D METHODS 4.2.1 PLANT M A T E R I A L 4.2.2 PROTOPLAST ISOLATION 4.2.3 C U L T U R E O F PROTOPLASTS 4.2.4 E L E C T R O P O R A T I O N 4.2.5 PLASMIDS  76 77 77 78 81 81 81 83 84 8^4  iii  Table of Contents 4.2.6 PREPARATION OF DNA 4.2.7 GUS ASSAY 4.2.8 CAT ASSAY 4.2.9 CELL SELECTION 4.3 RESULTS 4.3.1 ISOLATION OF PROTOPLASTS 4.3.2 PROTOPLASTS CULTURE 4.3.3 TRANSIENT EXPRESSION OF GUS AND CAT 4.3.4 KILLING CURVE AND SELECTION 4.4 DISCUSSION  ...85 87 ..88 89 i.89 89 .90 98 103 106  SUMMARY  109  Appendix A Media Components  113  Appendix B Abbreviations  113  Bibliography  115  iv  LIST OF FIGURES Fig. 1 Freshly isolated microspores from collection 3, notice that there were two types of microspores, one with exine while the other without, X480 19 Fig. 2 A typical protoplast-like microspore derived from micro-blend isolation, this composed 5% of the isolated population, notice the exine, X560 20 Fig. 3 Cell division was obtained in a very low frequency from cultured microspores of Douglas-fir, X480. 21 Fig. 4 Callus induction from cultured mature zygotic embryos 4-7 days on IC medium with hormone. Callus was first initiated from the radicle region, X40 34 Fig. 5 Embryogenic calli initiation from cultured mature zygotic embryos seven days on IC medium with 2,4-D. Embryogenic calli were either formed from the whole embryo or on cotyledon and hypocotyledon region, X120 35 Fig. 6 Secondary embryogenic calli formation from brown explant or callus of mature zygotic embryos 3-4 subcultures after initiation, X80 36 Fig. 7 Proembryo production on DCR1 + B medium, notice the organized proembryo head and the elongated suspensor. X480 ..39 Fig. 8 Suspension cultures established from mature zygotic embryo-derived embryogenic callus. Notice that there were both round embryogenic cells and long vacillated suspensor cells in the population. X160 40 Fig. 9 Proembryo formation from suspension cultures of Douglas-fir derived from mature zygotic embryos, X480. Fig. 10 Proembryo structure developed from cell suspension of mature zygotic embryos, X240  i41 42  Fig. 11 Embryo development in vivo from preliminary cone collections. A. 12-cell stage proembryo, X160. B. and C. Embryo development from 3 and 7 days after A, respectively, X160. D. Stage I embryo from July 13,1990, X40 48 Fig. 12 Dissection of immature embryo from megagametophyte of Douglas-fir  49  Fig. 13 Classification of embryo development. A. Stage I, X40  51  Fig. 14 Characterization of embryogenic calli initiation from different developmental stages of explants after 7 days in the induction medium (BM2-3 or BM3). A and B, C and D, E and F, G and H, were from Stage I, II, III, and IV zygotic embryos 53 Fig. 15, A and B. Secondary embryogenic callus formation from Stage I and IV swollen, brown zygotic embryos of tree #119 at transfer5 on BM3, X80. C. Single acetocarmine reactive cell from day 2  List of Figures of 2nd E-callus of #119 on BM3, X480. D. Unequal ceU  54  Fig. 16 Somatic embryo development from SEC. Stage I somatic embryo (unorganized) from SEC of genotype #119,7 days on BM3 medium, notice that there are a few proembryos in the suspensor region of the somatic embryo (A,C), X80. Stage I somatic embryo 55 Fig. 17 Somatic embryo development and maturation. A. Stage I somatic embryos were obtained in high frequency when transferred embryogenic calli of #347 line 3 into hormone-free BM medium supplemented with 1% activated charcoal for seven days, X56. B 65 Fig. 18 Somatic embryo development in suspension cultures derived from immature zygotic embryos. A. Elongated binucleate cells from ESM 14 days in BM3 liquid, X120. B. and C. Proembryo production from E S M 20 days in BM3 liquid medium, X120. D - G 66 ;  Fig 19. Flow chart of development of embryogenic calli derived from immature zygotic embryos of Douglas-fir from our study in solid media.  75  Fig. 20 Embryogenic suspension cultures used for protoplasts isolation, X80  91  Fig. 21 Freshly isolated protoplasts from cell suspensions derived from embryogenic calli of Douglasfir, X480 92 Fig. 22 Determination of viability of freshly isolated protoplasts by F D A staining under fluorescence microscope, X320 93 Fig. 23 Protoplast development. A . Protoplast with two nuclear from BM3 medium 4 days after isolation, X480. B. First division 2-4 weeks after isolation, X360  94  Fig. 24 Cell division and cell colonies formation. A. Further cell division from protoplast 4-6 weeks after isolation, X240. B. Production of cell colonies from protoplast 6-8 weeks after isolation in BM3 medium, X240 95 Fig. 25 Unequal cell division from protoplast resulted in the production of proembryo, X240  .96  Fig. 26 Plating efficiency and recovery of calli from protoplasts. A. Small cell colonies recovered from cells of protoplasts 2 months on BM3 solid medium. B. Calli production 3 months on BM3 medium 97 Fig. 27 Protoplasts expressing GUS 3 days after electroporation with pBI 221, assayed with X-gluc substrate, X480  .99  Fig. 28 GUS activity (percentage of transformed "blue" cells over total cells) 5 days after electroporation of Douglas-fir protoplasts with pBI 221 plasmid  100  Fig. 29 Protoplast expressing GUS 3 days after electroporation with pBI 221 under fluorescence microscope with the assay of M U G , X480.... 101 Fig. 30 Stable expression of GUS in cell colonies derived from protoplasts 3 months after  vi  List of Figures electoporation with pBI 221, assayed with X-gluc substrate, X480  102  Fig. 31 Transient expression of C A T activity from protoplasts of Douglas-fir. Protoplasts were electroporated with carrier DNA as control (C), and with or without 60 or 100 ug of pCaMVCAT plasmid. The activity was assayed 2 and 4 days (D2 and D4)after 104 Fig. 32 Killing curve for kanamycin  105  vii  LIST O F T A B L E S  Table 1. Media formulation for microspore cultures  15  Table 2 Embryogenic calli induction from cultured mature zygotic embryos of Douglas-fir from LP medium supplemented with different concentrations of hormones 31 Table 3 Numbers and percentage (%) of different developmental stages of zygotic embryos from the four collections .47 Table 4 Induction media formulation for culture of immature zygotic embryos of Douglas-fir  50  Table 5 Influence of media on embryogenic callus initiation and secondary embryogenic callus production from cultured immature zygotic embryos of Douglas-fir..  .58  Table 6 Influence of developmental stages of explants on embryogenic callus initiation and secondary embryogenic callus production from immature zygotic embryos of Douglas-fir .-. 59 Table 7 Influence of genotype of explants on total and (%) embryogenic callus initiation and total and (%) secondary embryogenic callus production from cultured immature zygotic embryos of Douglas-fir 60 Table 8 Influence of collections on embryogenic callus initiation from immature zygotic embryos of Douglas-fir 61 Table 9 Influence of sucrose and NH4+ on the embryogenic cultures of Douglas-fir, using cell suspension systems  viii  67  ACKNOWLEDGEMENT The education and experiences I have received during my tenure at the University of British Columbia have been extraodinary. Many individuals and organizations have provided invaluable assistance in helping me realize the goal of completing this dissertation, and I would like to thank them all. I express my sincere gratitude to my research supervisor Dr. J. E . Carlson for many helpful comments and suggestions, for answering my many questions, and for patience. I also express my appreciation to Dr. D. Roberts for his useful advice and discussion in my research experiment. I would also like to thank Drs. Y. El-Kassaby, D. Lavender, and J. McPherson for their interest and painstaking task of checking the technical aspects and overall structure of this thesis. In my view, their sharp remarks have considerably improved the quality of this report. I greatly appreciate the assistance given by Victor Luk and Wendy Wong in part of me experiment, and the opening discussion given by W. Lazaroff in the field of somatic embryogenesis. I am grateful to the Forest Research Institute of Guangxi of P.R.C. and the Biotechnology Laboratory of the University of British Columbia for providing me with financial support throughout the course of these studies. My sincerest thanks go to my parents, grandmother, brother, and sister who have had trust in my abilities and have encouraged me to continue my education.  ix  CHAPTER 1 G E N E R A L INTRODUCTION  l  general introduction  The genus Pseudotsuga (Pinaceae) consists of eight species (El-Kassaby et ah 1983). Douglas-fir (Pseudotsuga menziesii_Mirb Franco), a highly-valued timber species on the world market (Hosie, 1979), is of particular importance in western and pacific coastal North America (Mohammed and Patel, 1989). In 1987-88, about 16 million seedlings of Douglas-fir were planted in British Columbia and the number is expected to increase (Mohammed and Patel, 1989). The coastal variety occurs naturally in extensive even-aged stands with trees attaining great heights and girths. Trees 60 m high and 2 m in diameter are common (Sudworth, 1908). Genetic improvement and propagation of Douglas-fir using seeds are slowed by its long life cycle. Asexual reproduction of superior genotypes by traditional horticultural methods has been hampered by poor rooting and plagiotropic growth of cuttings and incompatibility reaction in grafts (Silen, 1978). Development of in vitro propagation methods for Douglas-fir will aid the tree improvement process in several ways. A reliable in vitro propagation system would increase the supply of plantlets from superior seed families for reforestation to ensure the quality of future forests. On the other hand, the application of traditional plant breeding methods to conifers has been successful but limited by the long generation time. Genetic engineering of trees represents a powerful tool for modifying trees and abbreviating the breeding cycle. To exploit this potential, it is essential to develop efficient methods for gene transfer in trees. First, however, an efficient and reliable in vitro technique is essential to permit the clonal propagation of elite genotypes in tree improvement programs and the development of gene transfer techniques in Douglas-fir. There are three approaches to vegetative propagation for achieving more  2  general introduction  rapid tree improvement and increasing productivity: rooted cuttings (macropropagation), organogenesis (micropropagation), and gametic or somatic embryogenesis. Macropropagation is widely used in certain species such as Norway spruce by using juvenile tree materials. Micropropagation involves the treatment of explants with plant growth regulators to induce bud or shoot formation, which may result in large numbers of buds/shoots per culture. Shoot elongation, followed by rooting of shoots, has been difficult to achieve in conifer species (Haccius, 1978). In addition, most successes in micropropagation were obtained only with explants (embryos, cotyledons, hypocotyl pieces, or shoots) from juvenile trees. Commercial application of forest tree tissue culture is presently limited to micropropagation of radiata pine (Aitken-Christie et al. 1987) in conifers. Androgenesis, plantlet regeneration from cultured pollen or microspores, can result from two quite dissimilar pathways of differentiation. In one , pollen develops directly into plantlets after passing through the characteristic stages of embryogenesis (Street and Withers, 1974). In the other, an unorganized callus develops at first from the pollen undergoing division. The callus may be subcultured, and appropriate cultural conditions will then induce organogenesis, or even embryogenesis, through which plantlets are finally obtained (Litchter 1982; Wang et al.1973; Chen 1986). Somatic embryogenesis, a non-sexual developmental process producing a bipolar embryo from somatic tissue (Haccius, 1978) or the production of embryolike structure from somatic tissue under in vitro condition (Becwar et al. 1987), is a recently developed and more promising in vitro technique for mass propagation and genetic engineering programs. Somatic embryogenesis has the advantage over  3  general introduction  organogenesis of true-to-type regeneration and normal seedling development. Somatic embryos can either be formed from a single cell/small groups of cells in somatic tissues (callus or tissue cultures), or be produced from the development of Embryonal-Suspensor Masses (ESMs) (Gupta and Durzan, 1987a) originating from zygotes or callus, thus making highly efficient liquid cell culture techniques available for maintenance and mass production purposes. The traditional plant breeding method of hybridization is an effective approach and transferred genes are generally stably transmitted through the seed. Because of the long generation cycles, hybridization as an approach for gene transfer in forest species seems to have rather limited possibilities (Ahuja, 1988b). Several approaches to transforming plants now exist, including protoplast fusion (Melchers, 1980), introduction of foreign D N A into protoplasts via electroporation (Gupta et al. 1988), chemically mediated direct DNA uptake into protoplasts (Klein et al. 1988), micro-injection and microprojectile bombardment of D N A into cells or tissues (Kartha et al. 1989), and  Agrobacterium mediated gene transfer (Sederof  al. 1986; Dandeker et al. 1987; Klee et al. 1987). Formulating an efficient gene transfer system for a new species involves several aspects, including: 1) an  in vitro  tissue culture system that is compatible with DNA transfer protocols, 2) a protocol for D N A uptake or transfer into a large number of cells simultaneously, 3) vectors that are suitable to the D N A transfer protocol for carrying the foreign genes into the cells and integrating stably into the nuclear genome, 4) marker genes in the vectors that permit efficient identification and selection of the transformed vs. nontransformed cells, 5) regulatory sequences for the controlled expression of the transferred gene, and 6) a high efficiency of plant regeneration to permit recovery  4  general introduction  of transformed cells (Carlson J.E., 1990; personal communication). Protoplast fusion as an avenue for gene transfer has limited possibilities in conifer species because of their extensive variability and instability in the somatic hybrids. The Agrobacteriwn mediated transformation is limited to those species susceptible to tumor formation. Microprojectile bombardment of DNA into tissues or calli often results in tissue-specific expression of foreign gene. Direct gene transfer is particularly suitable for analyzing gene expression at relatively high levels over brief periods of time, and allows early selection of transformed cells. Totipotent protoplasts are prerequisite for direct gene transfer into protoplasts. Recently, the production of somatic embryos from protoplasts of Douglas-fir and loblolly pine (Gupta et al. 1988), and plantlet regeneration from protoplasts in Picea glauca (Attree et al. 1989a and c) make the regeneration of transformed plantlets from transgenic protoplasts feasible in conifer species. The objectives of this research were: 1) to evaluate a standard angiosperm protocol for microspore embryogenesis with male-stobili in Douglas-fir; 2) to explore the influence of initiation medium, genotype, and developmental stage of immature zygotic embryo explants on the initiation and the stable production of embryogenic callus lines in Douglas-fir; 3) to develop an optimal medium for mature zygotic embryos to produce embryogenic calli and compare their characteristics with those from immature explants; 4) to develop protocol for the differentiation, development and maturation of somatic embryos from embryogenic calli; 5) to test standard protocols for direct gene transfer via electroporation into Douglas-fir protoplasts using 5-glucuronidase (GUS) gene and chloramphenicol acetyl transferase (CAT) gene as marker genes.  5  general introduction  In this thesis, a brief review of previous work on androgenesis, gametic and somatic embryogenesis, and gene transfer via electroporation, focusing on conifer species, is presented, detailed experimental designs and protocols for androgenesis, embryogenesis, and transformation are described. In addition, detailed studies with stable embryogenic callus lines concerning the initiation, proliferation, differentiation, development, and maturation of somatic embryos in both cultured mature and immature zygotic embryo explants are presented.  6  CHAPTER 2 E V A L U A T I N G MICROSPORE EMBRYOGENESIS WITH MALE-STOBILI IN DOUGLAS-FIR  7  chapter 2 microspores culture  2.1 INTRODUCTION Androgenesis, haploid plantlet regeneration from cultured haploid gametic tissues such as anthers or microspores in vitro, has been very successful with some agricultural species. The production of pollen derived plants from anther culture was first reported by Guha and Maheshwari (1964; 1966; 1967) for Datura innoxia, and haploid plants have subsequently been produced in this fashion from 85 genera belonging to 38 families (Srivastava and Johri, 1988). Embryogenic pollens or microspores from different species vary considerably in initial patterns of cell division(Zali and Dickinson, 1990). Four pathways of androgenesis have been observed in agricultural species. Pathway 1 involves embryogenesis via consecutive division of the vegetative cell in Nicotiana tabacum (Sunderland and Wicks 1969; 1971). Pathway 2 occurs in Nicotiana anthers, involving a modification of pollen mitosis, with two equal cells being formed, rather than the asymmetric division normally characteristic of this stage (Nitsch, 1972). Pathway 3 was reported for Datura innoxia (Sunderland and Dunwell, 1974), from which a normal asymmetric mitosis within the microspore occurs, followed by a fusion of the daughter nuclei to form a single, central nucleus which then commences division to form the embryo. Pathway 4 was reported in Hyoscyamus niger (Raghavan 1978), and resembles the pathway 1 until asymmetric division has occurred, when the generative rather than the vegetative nucleus continues to proliferate. Reynolds (1986) reported the direct differentiation of bicellular pollen grains of Solanum carolinense L. into embryoids and plantlets on MS medium supplemented with auxin, and concluded that the type of auxin present in the  8  chapter 2 microspores culture  medium determined the pathways of androgenesis to be either embryogenesis or organogenesis. By using a micro-blender, large numbers of microspores were isolated from whole buds oiBrassica napus (Swanson et al. 1987). Over 700 embryos per bud were obtained and 75% of them developed into normal torpedo-stage structures, and 80% of these structures developed into plants. In conifers, organogenesis and embryogenesis are rare and generally incomplete in callus of haploid tissue origin (Bonga et al. 1987). Androgenesis has been reported only in Picea abies, from which small shoot initials and short roots were obtained from cultured anthers (Simola, 1982); and in Pinus resinosa, from which some callus developed from immature pollen, and small structures, somewhat resembling the early stages of cleavage proembryos, were obtained in microsporophyll cultures of these species (Bonga, 1974). Success in culturing anthers or microspores would enable the production of haploid plants and completely homozygous breeding lines in a shortened time frame compared to conventional plant breeding/selfing. It has been reported that androgenesis is affected by the genotype, the physiological state of the donor plant, developmental stage of microspore, isolation techniques, tissue pretreatment, culture cell density, composition of the culture medium, the culture condition, and the osmolarity of the medium (Swanson et al. 1987; Pescitelli et al. 1990; Huang et al. 1990). In some crops success has been achieved by growing donor plants under a 16-hour high intensity photoperiod (Morrison and Evans 1988; Keller and Stringam 1978), while others require a short photoperiod (Heberle-Bors and Reinert, 1981; Heberle-Bors, 1982). Tissue pretreatment with cold or high temperature for a  9  chapter 2 microspores culture  period of time before culturing appears to be effective for increasing the anther response in wheat (Lazar et al. 1984a and b) and pepper (Morrison and Evans 1988). While most basal media appear effective for supporting development of microspores or anthers into plantlets, supplements to the medium such as elevated sucrose levels (Dunwell and Thurling, 1985) and activated charcoal (Johansson, 1983) have been found to increase the number of plantlets obtained from responding microspores or anthers. Pescitelli et al. (1990) found that the method of blending-isolation of microspore from maize results in less stress on microspores and a 3-fold increase in the yield of embryo-like structures.  2.2 MATERIALS AND METHODS  22.1 CONE COLLECTION Male cones were collected and sent from the CPFP high elevation Douglasfir seed orchard, Saanichton, B.C. by Dr. Y.A. El-Kassaby. One preliminary cone collection was carried out on April 5,1989, designed as P-Cl, in which 10 genotypes were included: 15-C-9,15-C-3, 15-E-7,16-B-9,10-F-2,16-B-12, ll-G-3,16-C-16,150-4, and 16-B-ll. Isolation and culture of microspores from these genotypes were carried out to evaluate their response to the culture media. Six experimental collections were carried out on October 25, December 25,1989; January 22, February 25, March 20, and April 12,1990; respectively. They were designated as CI, C2, C3, C4, C5, and C6, respectively. Only one genotype (16-B-9) which had been determined earlier to be most responsive was involved in each collection from CI to C6. During the collection period, the plantation was under cool over-head  10  chapter 2 microspores culture  water spray to control pollen maturation. The male cones were kept cool after collection and stored at 4°C upon arrival at campus.  2.22 MICROSPORE ISOLATION With cones from P-Cl, CI, and C2 collections, microspore isolation was carried out following the procedures described by Swanson et al. (1987) ioxBrassica spp.. Flower buds selected for a size of approximately 5 mm in length were surface sterilized by soaking in 100% commercial bleach for 10 minutes, followed by three washes in sterile water with 5 minutes each. The sterilized buds were released from the bud coat with a sterile blade, and then transferred into a cool, sterile microblender containing cool (12°C) wash medium (MS salts, 12% mannitol at pH 5.6, 4ml for each 10 buds) with 13% sucrose and blended 2-3 times at high speed for 4 seconds. The slurries were passed through four layers of cheese cloth and collected in a 15-ml centrifuge tube. The blender and filter were rinsed twice with 3ml wash medium and rinses collected in the centrifuge tube. The filtrates were centrifuged at 400 rpm for 10 minutes and the pellet was washed twice with the washing medium at the same speed. After that, the pellet was resuspended in culture medium and transferred to 6-well plates (Corning) with a density of 5X10 cells/ml and 2ml of 4  cells per well. The plates were sealed with parafilm and wrapped with aluminum foil. The cultures were either incubated at 30°C in darkness for 1-3 days and then transferred to 26°C, or incubated at 26°C directly, and maintained on a shaker at 100 rpm. Due to the problem of contamination from fungi spores which attached on the surface of the bud coat, two modified procedures for microspore sterilization  11  chapter 2 microspores culture  and isolation were used in the late collection (i.e. C3, C4, C5, and C6). When the uncoated buds were obtained from the above sterilization procedures, they were soaked in 70% ethanol for 3 minutes, followed by three washes in sterile distilled water; and then the uncoated buds were either transferred into the micro-blender and treated as above, or the microspores released by using a sterile needle with the help of a dissecting microscopy. All media were prepared freshly and used within one month. The media were adjusted to pH 5.7 before autoclaving at 121°C, 104 kPa for 15 minutes, and were stored at 4°C. For all the microspore cultures, liquid media were used; for anther culture solid media gelled with 0.6% agar were used. The agar media were poured in petri plates (100 X 15 mm) with 20ml per plate. The developmental stages of microspores were determined by acetocarmine staining and observing under light microscopy. For liquid cultures the subcultures were carried out every week by sedimenting and replacing half the culture medium with fresh medium, using a sterilized pipet. Subcultures for solid media were carried out every other week. Fluorescence Diacetate (FDA) was used to determine the viability of microspores (modified from Widholm, 1972). Microspores were mixed with F D A working solution (FDA stock solution diluted with culture medium until milk color appeared; F D A stock: lmg/ml in acetone) in a petri dish for 5 minutes, and then slides were made and examined with fluorescence microscopy. As a result, the viable cells immediately began to display a bright-green fluorescence internally. The yield of microspores was determined by counting with a hemocytometer under a light microscope. The number of cells in several of the 1 X 1-mm squares  12  chapter 2 microspores culture  was counted and the total number of cells divided by the number of squares counted and multiplied by 10 , giving the number of cells per ml. 4  Total number of cells . Yield = — X 10 X Vol Number of squares counted 4  The first experiment tested the role of carbohydrate sources in the microspore culture for callus formation or embryogenesis by using the NLN medium (Nitsch and Nitsch, 1967) as the basic medium. Four media were used as culture media: NLN with 0.35M sucrose, NLN with 0.35M maltose, NLN with 0.35M galactose, and NLN basic medium, involving three genotypes from P-Cl and 11 treatments (table 1). The second experiment was to test the influence of low temperature treatment on the microspore response to callus formation or embryogenesis. The cones from P-Cl had been stored at 4 ° C for three months before they were used in this trial. The isolated microspores from the buds were cultured on the same media as in experiment 1, and incubated at 30°C for overnight before being incubated at 26°C. The third experiment was to test different developmental stages of microspores for their callus formation or embryogenesis on a series of standard media from which a 3X3X3 factorial design was used: MS salts (0,1/4X, and 1/2X), 2,4-Dichlorophenoxyacetic acid (2.4-D) (0, lmg/1, and lOmg/1), and 6Benzylaminopurine (BAP) (0, lmg/1, and 10mg/l). All media were supplemented with 0.35M sucrose as carbohydrate source. Materials from collection 1-6 were cultured on these media in 6-well plates. Each treatment had at least 2 wells of  13  chapter 2 microspores culture  cultures as replication in each collection. All the cultures were first incubated at 30°C overnight before being incubated at 26°C.  14  chapter 2 microspores culture  Table 1. Media formulation for microspore cultures  NO.  Genotype  Bud  else  1  15-E-7  8  NLN+G  2  15-E-7  S  NLN+S  3  15-E-7  S  1/4LP+S  4  16-B-9  L  NLN+S  5  15-B-7  L  NLN+M  6  15-E-7  s  NLN  7  16-C-16  s  NLN+S  8  15-E-7  L  NLN+M  9  16-B-9  L  NLN+G  10  15-E-7  L  NLN+G  11  15-E-7  s  NLN+M  1  MediuB  2  S=short bud, 3-4 nun i n l e n g t h ; L=long bud, 4-7mm i n l e n g t h . NLN=Nitsch and N i t s c h (1974) b a s a l medium; G=0.35M g a l a c t o s e S=0.35M sucrose M=0.35M maltose.  15  chapter 2 microspores culture  The last experiment was to culture anthers of genotype 16-C-9 from Collection 3 in solid media MSI and MS2. MSI contained half strength MS salts, 0.4 mg/1 L-glutamine, 100 mg/1 Casein hydrolysate, 100 mg/1 myo-inositol, 34.2 g/1 sucrose, 7 mg/1 BAP, and 7 mg/12,4-D; while MS2 contained the same components as MSI except in hormone levels used, i.e. 0.3 mg/1 BAP, 0.3 mg/1 Kinetin, and 0.7 mg/12,4-D. The media were adjusted to pH 5.7 and gelled with 0.6% agar. After bud surface sterilization, the cone scales were isolated and embedded on the surface of agar media.  2.3 RESULTS AND DISCUSSION Microspores from P-Cl were mostly single-cell stage, with some pollen stage, depending on the size of the flower buds. While in CI and C2, all cells were in the pollen mother cell stage and microspore tetrad stage.  Microspores from C3 and  C4 were in the 1-cell and 2-cell stages. While those from late collection were at the pollen stages. Ultilizing the micro-blender approach for isolating microspores, yielded two kinds of microspores in suspension (Fig. 1). One had a visible exine thin coat around the cell; the other was slightly larger in size, round in shape, without pollen coat, similar to protoplasts (Fig. 2). The latter occupied about 5% total in the solution. The production of protoplast-like microspores probably was attributable to the micro-blender used in the isolation procedures, because no such products were obtained when isolated by hand as in the control experiment. Compared with other tissues, the pollen grains or microspores are coated with a tough exine containing sporopollenin, one of the most resistant organic substances, and has been  16  chapter 2 microspores culture  considered as the main barrier to pollen protoplast isolation. In addition, it has been reported that the isolation of protoplast from microspore or pollen may be difficult due to the continuous resynthesis of cell wall or exine after digestion (Stanley and Linskens, 1979). So it would be feasible to separate the pollen coat first with the mechanical method, followed by enzyme digestion to obtain protoplasts from pollens or microspores. More than half of the cultures from blender-isolation procedures were contaminated within 4-7 days after isolation. Fungi spores grew and developed in the contaminated cultures. This may be due to the existance of spores inside the bud coat. A better result was obtained from the two modified procedures, using double-sterilization followed by releasing microspores with micro-blender or needles, as mentioned in material and methods. No response of cell division was observed from cultured microspores before and in tetrad stage from all the experiments. In experiment 1, some dense white microspores could be observed in media supplemented with carbohydrates after 4 days in culture. This could be caused by the high osmolarity of the solution, as there were no cells clumps in medium without carbohydrates (NLN only). Cells from these cultures did not develop further as subcultures were carried out. Microspores from C3 were subjected to a series of MS media. At day ten, samples were withdrawn from each treatment, and stained with acetocarmine. 1cell stage, 2-cell stage, and 5-cell stage of microspore were observed from media such as 1/2MS with 10 mg/1 BAP and 10 mg/12,4-D. This suggested that microspore development or cell division can occur in vitro. When two subcultures were carried out on medium supplemented with 10 mg/1 BAP, and 34.2 g/1 sucrose,  17  chapter 2 microspores culture  but without base salt, a few microspore divisions were observed (Fig. 3).  18  chapter 2 microspores  Fig. 1 Freshly isolated microspores from collection 3, notice that there were two types of microspores, one with exine while the other without, X480.  19  chapter 2 microspores culture  Fig. 2 A typical protoplast-like microspore derived from micro-blend isolation, this composed 5% of the isolated population, notice the exine, X560.  20  chapter 2 microspores culture  Fig. 3 Cell division was obtained in a very low frequency from cultured microspores of Douglas-fir, X480.  21  chapter 2 microspores culture  It seems that such divisions occurred adventitiously and were controlled by the culture medium, because they occurred in very low frequency and were only observed in one medium. These cultures did not develop further, no cell colonies were obtained. The viability of microspores as determined by F D A staining was 90% after isolation, but reduced to 60% by the second transfer. A few could survive to 3 months after isolation even if they did not develop or divide further. Calli were initiated from cultured 2 of 10 (20%) anthers of 16-9-B from collection 3 within 14 days in both MSI and MS2 media. Callus could only be induced from buds less than 0.3mm in length and the callus were not embryogenic based on their morphological and histological characteristics, i.e. they were hard, fast growing, and greening when cultured in light. As a matter of fact, callus from haploid tissues are usually slower in growth compared with those from somatic tissues (Ho R.H., 1990, personal communication). So the callus initiated from anthers in this experiment may not be of haploid origin (i.e. from microspores inside the anthers). If so, they were still overgrown by somatic tissues, because their performance in the subcultures were more like those from somatic tissues in this species. An alternative to confirm their haploid or diploid originate is to carry out chromosome counting as haploid tissues have 13 chromosomes while diploid ones have 26 chromosomes in this species. Neither organogenesis nor embryogenesis could be obtained from these cultures. Microspores of Douglas-fir are quite different from those of agricultural species in that multiple cells (5 cells) from mature pollen in Douglas-fir compare to 2 cells (vegetative and generative) from pollen of species such as Brassica (both cells  22  chapter 2 microspores culture  contribute to embryogenesis in Brassica) (Nitsch, 1972). While the mechanism of microspore division and development in vitro is unclear in Douglas-fir, it seems that the one cell stage of microspore can develop into five cells stage by culturing microspores in vitro. During these procedures, single microspore division can perhaps occur to form cell colonies. Further experiments are necessary to capture the optimal developmental stage of microspores for callus production or embryogenesis. Interestingly, cell division was obtained only from medium supplemented with hormone and carbohydrate source, but without medium salts, which may indicate that certain developmental stages of microspore have the balance of microand macro- elements internally, while the application of exogenous hormone would encourage their differentiation and proliferation. Or, a low salt concentration in the medium could favor cell division as has occurred in some embryo cultures in conifer species from which low salt encouraged the induction of embryogenic callus (Gupta and Durzan 1987a; Hakman and Fowke 1985).  23  CHAPTER 3 SOMATIC EMBRYOGENESIS IN DOUGLAS-FIR  24  chapter 3 somatic embryogenesis in Douglas-fir  3.1 INTRODUCTION Since the first success was reported for somatic embryogenesis in coniferous species-Norway spruce (Hakman and Fowke, 1985), the technique has been extended to many other conifer species. Somatic embryogenesis and plantlet regeneration have been reported in Norway spruce (Hakman and von Arnold, 1985), sugar pine (Gupta and Durzan, 1986b), Douglas-fir (Durzan and Gupta, 1987), black spruce and white spruce (Hakman and Fowke, 1987), loblolly pine (Gupta and Durzan, 1987a), eastern white pine (Becwar and Wann 1987), and European larch (Nagmani and Bonga 1985). More recently, somatic embryogenesis was reported in Pinus elliottii (Jain et al. 1989) and Pinus caribaea (Laine and David, 1990). Most of the success in somatic embryogenesis was achieved by culturing immature zygotic embryos, except for two species: Norway spruce and sugar pine, in which mature zygotic embryos were used as explants as well. In general, more success was obtained from culturing immature embryos, concerning the initiation of embryogenic callus, the production, development and maturation of somatic embryos. Cheng and Voqui (1977) first regenerated plantlets in MS medium via organogenesis by culturing embryos in vitro in Douglas-fir. This was also achieved by Aboel-Nil (1987) and Mohammed and Patel (1989). In addition, Gupta and Durzan (1985) reported shoot multiplication from mature trees of Douglas-fir in vitro. In these cases, the rooting of plantlets was unstable and the percentage of rooting was quite different between genotypes: 0-87% rooting in genotypes studied by Mohammed and Patel (1989), while 30-40% of rooting in Aboel-Nil's study  25  chapter 3 somatic embryogenesis in Douglas-fir  (1987), and the best was 80% of rooting obtained from Cheng and Voqui's work (1977). Somatic embryogenesis would bypass the problems of low percentage of elongation and rooting of shoots in micropropagation. Haissig et al. (1987) foresees that within ten to twenty five years organogenesis will be replaced by somatic embryogenesis as it becomes controllable, attainable, and automated for a variety of species. As a matter of fact, organogenesis has alreadly been replaced by somatic embryogenesis in some conifer species such as spruces (Roberts D., 1991; personal communication). Durzan and Gupta (1987) reported polyembryogenesis from suspension cultures in Douglas-fir. Immature embryos were used to induced Embryonal-Suspensor Masses (ESMs) and 27 plantlets were regenerated from the ESMs. This initial result shows promise for a stable regeneration system via somatic embryogenesis in this species. Changes in and optimization of medium component can significantly affect the initiation of embryogenic callus and therefore play a major role in extending the initiation window to more mature tissue (Becwar and Wann, 1989). von Arnold and Hakman (1986) have been able to initiate embryogenic callus (EC) from mature zygotic embryos of Norway spruce cultured in LP medium (von Arnold and Eriksson, 1981) by reducing the sucrose level from 3.4% to 1%. By culturing mature embryos on half-strength B L G medium (Amerson et al. 1985, 1988), Becwar et al. (1987) have been able to induce 25% of the explants to produce embryogenic calli^ while changing to full-strength B L G medium did not result in initiation of embryogenic callus from cultured mature zygotic embryos of Norway spruce. Such results may demonstrate that a low salt concentration in medium would increase the  26  chapter 3 somatic embryogenesis in Douglas-fir  initiation frequence to induce embryogenic callus from cultured mature zygotic embryos in conifer species. The behavior for the initiation of embryogenic callus may be quite different from different materials and species. Less than 0.1% of the mature zygotic embryos produced embryogenic calli in Douglas-fir (Durzan and Gupta, 1987), while 20-40% of the mature zygotic embryos could be induced to produce embryogenic calli in Norway spruce and sugar pine (Becwar and Warm 1989; Gupta and Durzan 1986a and b). Precotyledonary embryos were optimal for the initiation of embryogenic calli in Pinus and embryogenic calli originated from the suspensor region; while embryogenic calli in Picea originated from the hypocotyl and cotyledon region of predominantly post-cotyledonary embryos (Becwar and Wann, 1989). In Douglasfir, embryogenic calli were predominantly initiated from precotyledonary embryos as occurred in Pinus families (Durzan and Gupta, 1987). Several approaches and steps leading to somatic embryogenesis in conifers have been obtained. Somatic embryogenesis (Hakman and Fowke, 1985) in Norway spruce was obtained by inducing calli from explants, and their redifferentiation into asexual ESM, and then differentiation into somatic embryos. Somatic polyembryogenesis (Gupta and Durzan 1987; Durzan and Gupta 1987) in loblolly pine and Douglas-fir involves the production of ESMs directly from the explants which bypasses the callus step, and the proliferation of ESMs and their cleavage and lobing, from which individual somatic embryos could be obtained. Another approach was ESMs induced by contact (Durzan, 1988) which involves the redifferentiation of explant, followed by the induction and production of asexually produced new ESMs. As the cleavage and lobing of ESMs occurred, individual  27  chapter 3 somatic embryogenesis in Douglas-fir  somatic embryos could be obtained. Theoretically, somatic embryogenesis in vitro involves the following procedures: initiation, proliferation, differentiation, development, maturation. germination, and conversion, i.e. the initiation (induction) of embryogenic calli from cultured explants; followed by the proliferation and maintenance of the embryogenic calli either in solid or suspension cultures; embryogenic calli can be differentiated to produce early somatic embryos (or proembryos), they can develop into precotyledonary embryos and then mature into cotyledonary embryos; by germinating the mature embryo, somatic embryos can be converted into seedlings. In embryogenesis, dedifferentiation, induction, and redifferentiation occur naturally in the trees as long as certain conditions (needs for darkness and supply of nutrients) around the zygote are maintained (Durzan, 1988). The induction of embryogenic callus may hot require darkness, but for the development of embryo structure, in general, darkness or dim light is essential. The development of conifer polyembryony involves the free nuclear stage, cleavage, lobing etc (Singh 1978; Dogra 1978). Acetocarmine has been commonly used to detect and confirm different developmental stages in embryogenesis, as proembryos or ESMs are strongly reactive to acetocarmine, which is supposed to be associated with the start of embryo-specific RNA synthesis in proembryo (PE) cells (Durzan, 1988). However, acetocarmine reactivity is not specific for embryogenesis, it is also an indicator for proteinaceous materials in the neocytoplasm and for nucleoproteins in the nucleus. As a result, a double-stain technique has been developed by Durzan and Gupta (1987), from which the proembryo and suspensor can be stained red and blue separately by acetocarmine and Evan's blue,  28  chapter 3 somatic embryogenesis in Douglas-fir  respectively.  3.2 EMBRYOGENIC C A L L U S F R O M M A T U R E Z Y G O T I C EMBRYOS  3.2.1 MATERIALS AND METHODS Open pollinated seeds (collected on August 1988 from 4 trees in campus of U.B.C.) were used in this experiment for sake of convenience. Seeds were stored at 4°C after collection. Seeds were surface sterilized by agitating in 100% commercial bleach for ten minutes, followed by rinsing once with sterile distilled water, and then agitating in 70% ethanol for 30 seconds, and rinsing 3 times with sterile distilled water. The sterilized seeds were then transferred into a 20 X 100 mm petri dish with minimal volume of sterile distilled water (1-2 ml) added, the dish was then sealed with Parafilm and kept at 4°C in the dark for overnight so that embryos could be easily extracted. Embryos were exised the next day with the aid of a dissecting microscope. The first experiment was to optimize medium for the induction of embryogenic callus. Half strength LP medium (von Arnold and Eriksson, 1981), combined with different concentrations of hormones, was tested in this experiment (Table 2). The half strength LP medium consists of half strength of LP salts, 34.2g/l sucrose, and 6g/l agar. Plant growth regulators were prepared as stock solution and stored at 4°C. Media were adjusted to pH 5.7 before autoclaving. A volume of twenty-five ml aliquots were poured into 15 X 100 mm petri dishes and the solidified medium was kept at 4°C and used within 30 days. Ten embryos were plated per  29  chapter 3 somatic embryogenesis in Douglas-fir  dish with 50 embryos per treatment and the dishes sealed with Parafilm and stacked in an incubator set at 26°C in darkness. The experiment was repeated to confirm the best medium for the initiation of embryogenic callus.  30  chapter 3 somatic embryogenesis in Douglas-fir  Table 2 Embryogenic calli induction from cultured mature zygotic embryos of Douglas-fir from LP medium supplemented with different concentrations of hormones.  NO.  Xediua ' ' !  2  3  Embryo inoculated  No. Responsive  1  1/2LP+0.5D  50  0  2  1/2LP+2D  50  0  3  1/2LP+10D  50  0  4  1/2LP+20D  50  0  5  1/2LP  50  0  6  10D+10BA  50  10  7  10D+1BA  50  0  8  100D+100BA  50  0  9  100D+20BA  50  0  10  2D+0.5BA  50  2  11  0.5D+2BA  50  0  A l l media were supplemented with 0.1M s u c r o s e and g e l l e d with 0.61 agar, pH was a d j u s t e d a t 5.8 b e f o r e a u t o c l a v e d . 1  2  D=2,4-dichlorophenoxyacetic BA=6-benzylaminopur ine.  3  The unit for the c o n c e n t r a t i o n of hormone was mg/1.  31  acid;  •  chapter 3 somatic embryogenesis in Douglas-fir  The second experiment was to use the best medium from experiment 1 to induce embryogenic callus for the following proliferation, differentiation and development studies. A total of 300 mature zygotic embryos were cultured on the best medium derived from experiment 1. Once calli were initiated, they were transferred onto either 1/2MS medium (Murashige and Skoog, 1962) supplemented with 5mg/l BAP, 5mg/l Kinetin, 10mg/l 2,4-D, 34.2g/l sucrose, 250mg/l myo-inositol, 300mg/l casein hydrolysate, lOOmg/l L-glutamine (designed as MS3) or 1/2 DCR medium (Gupta and Durzan, 1985) supplemented with 500mg/l casein hydrolysate, 450 mg/1 L-glutamine, 4000 mg/1 myo-inositol, 10 mg/1 BAP, 10 mg/1 2,4-D (designed as DCR1) for 2-3 selective subcultures. The first few subcultures were carried out every seven days on either MS3 or DCR1 media and referred to as the initiation period, while later subcultures were carried out every other week on the same basic medium as above but had lower hormone level (i.e. 0.5 mg/1 BAP, 0.5 mg/1 Kinetin, and 1.0 mg/12,4D; designed as MS3-2 and DCR2, respectively) and referred to as the maintenance period. All the cultures from the initiation and the maintenance period were incubated at 26°C in darkness. Callus derived from each individual embryo was treated as an individual cell line. Cell suspension cultures were established by culturing 0.5 g to 2.5 g of fresh embryogenic or nonembryogenic callus into 20-30 mis of MS3-2 or DCR2 medium in a 125 ml flask and in an environmental shaker at 100 rpm at 26-28°C in darkness. Once established, the subculture was carried out every week by sedimenting and replacing 10-15 ml of medium with fresh medium. For the development of proembryo structures (development period),  32  chapter 3 somatic embryogenesis in Douglas-fir  embryogenic callus or suspension cultures were transferred onto either MS3-2 and DCR2 supplemented with 1/10 of hormone (i.e. 0.05 mg/1 BAP and Kinetin, 0.1 mg/12,4-D; designated as MS3-d and DCR2-d medium) or hormone-free media with a two-week subculture for solid medium or seven-day subculture for suspension cultures.  322 RESULTS AND DISCUSSION Calli were only produced from embryos cultured on two media (6 and 10), as far as this experiment to be concerned, with 20% and 4%, respectively (Table 2). These results were confirmed by two other experiments, from which similar results were obtained. It seems that the medium salts, hormone levels and their ratios were important factors for calli induction from cultured mature embryos in Douglas-fir. Embryos cultured on media supplemented with 1/2LP salts (media 1-5) could not produce callus, while calli were initiated from medium supplemented only with optimal hormones and carbohydrate. This again suggested that low concentration of salts in the medium may increase initiation percentage in mature materials for the induction of embryogenic callus (Becwar et al. 1987). Calli started to form after seven days on initiation medium, most initiating from the radicle, and then extended to hypocotyl and cotyledon region (Fig.4), in some cases, calli only formed on hypocotyl or cotyledon region (Fig.5). Most of the calli were white, translucent after initiation; they were composed of elongated cells and acetocarmine-reactive round cell colonies visible under light microscope, indicating embryogenic potential or development from both morphological and histological characteristics.  33  chapter 3 somatic embryogenesis in Douglas-fir  Fig. 4 Callus induction from cultured mature zygotic embryos 4-7 days on IC medium with hormone. Callus was first initiated from the radicle region, X40.  34  chapter 3 somatic embryogenesis in Douglas-fir  Fig. 5 Embryogenic calli initiation from cultured mature zygotic embryos seven days on IC medium with 2,4-D. Embryogenic calli were either formed from the whole embryo or on cotyledon and hypocotyledon region, X120.  35  chapter 3 somatic embryogenesis in Douglas-fir  Fig. 6 Secondary embryogenic calli formation from brown explant or callus of mature zygotic embryos 3-4 subcultures after initiation, X80.  36  chapter 3 somatic embryogenesis in Douglas-fir  Most calli turned brown during the first three subcultures. White calli sometimes formed on the brown calli after several subcultures (Fig. 6), and the stable embryogenic callus lines were established by separating and culturing these new calli on either DCR1 or MS3 medium. No stable embryogenic callus lines were obtained if we failed to separate these new calli from the brown calli and subculture on new medium. This was critical for the establishment of embryogenic calli for Douglas-fir, as occurred from embryogenesis of larch (Cheliak W.M., 1989; personal communication). The embryogenic calli could be maintained for over one year with 14-day subcultures. It seems that DCR medium was better than MS medium for callus subculture in that better growth was observed from cultures on DCR medium. Proembryos could be obtained when embryogenic calli were transferred into either MS3 hormone-free medium or DCR1 medium supplemented with only 1/10 of the regular hormone level for 2 weeks in a 16-hour photoperiod (Fig. 7). They could develop into precotyledonary stage within 4 weeks on the same medium in some cases. No cotyledonary somatic embryos were obtained from cultures derived from mature zygotic embryos. Ten lines of cell suspension were established at the beginning and reduced to four lines after three months because of the browning of the other six cell lines. The four lines have been maintained in MS3 medium for over one year. The suspension was a mixture of single round cells, elongated vacuolar cells, cell colonies, cell clumps, and some polar cell divisions(Fig. 8). The proembryo, an acetocarminereactive round cell connected with a long, vacuolate, Evan's blue-reactive suspensor cell, was continuously produced and observed in these cultures (Fig. 9). It could develop into a few cell stage in the same medium (MS3) (Fig. 10), but they could  37  chapter 3 somatic embryogenesis in Douglas-fir  hardly develop into a stage I somatic embryo even being subcultured on low hormone or hormone-free medium.  38  chapter 3 somatic embryogenesis in Douglas-fir  Fig. 7 Proembryo production on DCR1+B medium, notice the organized proembryo head and the elongated suspensor. X480.  mm  39  chapter 3 somatic embryogenesis in Douglas-fir  Fig. 8 Suspension cultures established from mature zygotic embryo-derived embryogenic callus. Notice that there were both round embryogenic cells and long vaculated suspensor cells in the population. X160.  40  chapter 3 somatic embryogenesis in Douglas-fir  Fig. 9 Proembryo formation from suspension cultures of Douglas-fir derived from mature zygotic embryos,  X480.  41  chapter 3 somatic embryogenesis in Douglas-fir  Fig. 10 Proembryo structure developed from cell suspension of mature zygotic embryos, X240.  42  chapter 3 somatic embryogenesis in Douglas-fir  3.3 EMBRYOGENIC CALLUS FROM IMMATURE ZYGOTIC EMBRYOS.  3.3.1 MATERIALS AND METHODS Four preliminary cone collections were carried out on May 24, June 7, June 20, and July 5,1990, and the zygotic embryo developmental stage determined microscopically (Fig. 11, A-D). Once seed development had reached the proembryo stage (Fig. 11, D), cone collections were carried out every other week throughout the embryo maturation period. Cones from open-pollination of six Douglas-fir trees (numbered 130,623,169, 119,347, and 85) were collected on July 13,23, August 4, and 21,1990, by the British Columbia Forest Service, from the Cowichan Lake Research Station, Mesachie Lake, British Columbia. The cones collected on these dates were designated as CI, C2, C3, and C4, respectively. Cone collections were subsequently reduced from six to four genotypes (623,347,119 and 85) due to the poor seed production in two genotypes. All cones from the four collections had closed scales. The dissection and inoculation of immature embryos were carried out within 3-5 days after cone collection while cones were stored at 4°C. Whole cones were sterilized in 70% ethanol for 30 seconds, followed by several washes in sterile i  1  distilled water for 30 seconds. Explants were then dissected away from the megagametophyte tissue, which remained attached to the seed scale (Fig. 12). Embryo explants were cultured in six-well plates (Corning), with 2ml agar medium and two embryos per well.  Six initiation media were tested (Table 4), with  24 embryos in each treatment from each cone collection. The media used were based on previous studies in culturing both mature and immature zygotic embryos of 43  chapter 3 somatic embryogenesis in Douglas-fir  these species. The media used were modified from MS medium (Murashige and Skoog, 1962), DCR medium (Gupta and Durzan, 1985) and B M medium (Gupta and Durzan, 1987). Induction of embryogenic callus was conducted at 25°C in darkness (plates wrapped with aluminum foil). In the initiation period, the explants were subcultured every 10-12 days onto fresh 6-well plates, and the production of embryogenic callus was recorded at the end of the second transfer. The classification of developmental stages of zygotic and somatic embryos was based on a modified system (Webb et al. 1988,1989) derived from Buchholz and Stiemert's classification (1945). Precotyledonary embryos were classified as stage I or II. Stage III embryos had clearly visible cotyledon primordia that were overtopped by the shoot apical dome, while stage IV embryos had cotyledons which overtopped the shoot apex (Fig. 13). Collection 1 (CI) had stages pre-I, I and stage II embryos with the majority at stages I and II. C2 had mostly stage II and IU embryos with about half the number of stage I embryos as in CI. The majority of zygotic embryos in C3 were at stage IV with a few stage II and III embryos. C4 had only stage IV zygotic embryos (Table 3). Most embryogenic calli turned brown in the third and fourth transfers. While being maintained on the same medium for 1-3 weeks, a piece of white callus often formed on the brown callus. The production of stable embryogenic callus lines was obtained by separating small sections of white, translucent embryogenic calli from the mass of brown calli. This secondary embryogenic callus (SEC) was cultured on fresh medium, most of which could be maintained by 14-day subculture on either BM3 or DCR1 media (Durzan and Gupta, 1987). The calculation and analysis of data from the initiation period were carried  44  chapter 3 somatic embryogenesis in Douglas-fir  out as following: For the distribution of developmental stages in different collection (Table 3), the numbers of embryos cultured were calculated based on each collection, i.e. from all genotypes. The embryogenic calli initiation frequency was only based on individual collections, i.e. counting the initiation from all genotypes and induction media for each collection (Table 8). The influence of media on embryogenic callus (EC) initiation and secondary embryogenic callus (SEC) production (Table 5) was calculated based on embryos from the four collection, all genotypes and stages, comparing among individual induction media for their E C and SEC production capacity, as different genotype and stage of embryos from the same induction medium had simalar induction distribution. The influence of developmental stages of embryos on E C initiation and SEC production (Table 6) was calculated based on embryos from the four collections, all genotypes and induction media, comparing between different developmental stages for their E C and SEC capacity. Similary, the influence of genotype on E C and SEC production was calculated based on embryos of the same genotype from the four collections of all induction media and embryo stages (Table 7). Suspension cultures were established by transferring 1 g of embryogenic callus from solid media to 20 ml of liquid BM3 medium in 125 ml flasks with fluted bases. Cultures were grown at 26°C, shaking at 100 rpm in darkness. Subcultures for maintenance were carried out every week by sedimenting the cells and replacing media with 10-15 ml of fresh BM3 medium. BM3 and DCR1 + B (B = 3% banana powder) media were used as proliferation and maintenance medium for solid cultures, while BM3 medium was used in suspension cultures only. For somatic embryo development, secondary  45  chapter 3 somatic embryogenesis in Douglas-fir  embryogenic callus at the second transfer was subcultured into the same base medium but with or without modified hormone levels (0.5uM 2,4-D, 0.2MM BA, and 0.2w M Kinetin or hormone -free) and with 1% charcoal supplement for 7-14 days in the light with 16-h photoperiod. The third transfer was then transferred onto basal medium plus either luM or lOuM abscisic acid (ABA) . Due to the lack of success in producing cotyledonary somatic embryos from the above experiment, a series of experiments were carried out to test the following factors in the role of somatic embryo development and maturation in Douglas-fir: sucrose concentration (15, 34.2, and 68.4 g/1), arginine (2 and 6 uM), putrescine (500 UM),  and A B A (0, 0.25, 0.5, 1.0,3.5, 5.0, 7.5,10, 20, 30, 40, and 60 uM). All the  cultures were incubated at 20 X 100 mm petri plates at 25°C with 16-h photoperiod. For the studies of A B A regulation on the development and maturation of somatic embryos in Douglas-fir, embryogenic calli with a certain stage of somatic embryos (from proembryos to stage I somatic embryos) were either subjected to preculture on B M hormone-free medium supplemented with 1% activated charcoal followed by exposure to A B A media, or embryogenic calli were transferred from maintenance medium onto A B A media directly for two subcultures.  46  chapter 3 somatic embryogenesis in Douglas-fir  Table 3 Numbers and percentage (%) of different developmental stages of zygotic embryos from the four collections.  Stage Collection  <I  I  II  III  IV  Total  CI  38 (10.73)  189 (53.39)  125 (35.31)  2 (0.56)  0  354  C2  44 (9.26)  89 (18.74)  176 (37.05)  166 (34.95)  O  475  C3  0 (0)  0 (0)  8 (2.23)  8 (2.23)  342 358 (95.53)  C4  0 (0)  0 (0)  0 (0)  0 (0)  297 (100)  These data represent the number of zygotic embryos, from the six genotypes of each collection, used and cultured on the six induction media to induce embryogenic calli in Douglas-fir.  47  297  chapter 3 somatic embryogenesis in Douglas-fir  Fig. 11 Embryo development in vivo from preliminary cone collections. A. 12-cell stage proembryo, XI60. B. and C. Embryo development from 3 and 7 days after A, respectively, X160. D. Stage I embryo from July 13, 1990, X40.  48  chapter 3 somatic embryogenesis in Douglas-fir  Fig. 12 Dissection of immature embryo from megagametophyte of Douglas-fir.  49  chapter 3 somatic embryogenesis in Douglas-fir  Table 4 Induction media formulation for culture of immature zygotic embryos of Douglas-fir.  Medium*  Glutamine Basal medium** ayoinositol (»g/i) (ng/1)  Casein Sucrose hydrolysate (mg/1) (g/D  1/8MS DCR1-1 BM1-3 BM2-3 BM2-3+CM BM3  1/8HS OCR BM BM BM 1/2MS  300.0 0 500.0 500.0 500.0 500.0  a  b  300.0 200.0 1000.0 1000. 1000. 1000,0  100.0 0 450.0 450.0 450. 450.0  34.2 15.0 34.2 34.2 34.2 34.2  A l l media except DCR1-1 and BM1-3 contained 5 uM 2,4-D, 2uM BA and K i n e t i n ; while DCRl-1 and BM1-3 contained 50uM 2,4-D, 20uM BA and Kinetin,and were g e l l e d w i t h 0.61 agar (pH 5.7). Media were autoclaved a t 121°C, 104 kPa f o r 15 min.CM=5% coconut water. 1/8 MS, 1/8 strength macro- and micro-elements o f Murashige and Skoog (1962). DCR, f u l l s t r e n g t h macro- and micro-elements of Gupta and Durzan (1985). BM, h a l f modified MS from Gupta and Durzan (1987). 1/2 MS, h a l f s t r e n g t h macro- and microelements of Murashige and Skoog (1962).  50  chapter 3 somatic embryogenesis in Douglas-fir  Fig. 13 Classification of embryo development. A . Stage I, X40; B l and B2, Stage II, X40; C I and C2, Stage III, X40; and D I and D2, Stage IV, X20.  chapter 3 somatic embryogenesis in Douglas-fir  3.3.2 RESULTS Within 1-2 weeks of initiation, zygotic embryos began to produce a slightly translucent callus from either the embryo proper (or embryo proper, i.e. the upper part of the embryo has high reactive with aceto-carmine, not translucent), from the entire embryo including the suspensor cells, or from just the hypocotyl region, depending upon the developmental stages of the explants in all six media tested (Fig. 14). In more detail, calli were formed from either the embryo proper or the entire embryo including the suspensor for stage I and II zygotic embryos, from hypocotyl and cotyledon region for stage III zygotic embryos, while from hypocotyl region for stage IV embryos. After 10 days in culture (the first subculture), the frequency of embryogenic calli production was recorded based on their macromorphology, i.e. white, mucilaginous, and translucent (Durzan and Gupta, 1987). By the third or fourth transfer, most calli had turned brown. After a further 1-3 weeks in the same medium, new pieces of white, translucent embryogenic calli (defined as Secondary Embryogenic Callus-SEC) were formed on the brown, original swollen explant tissues (Fig. 15, A and B). When these SECs developed, they could be separated and subcultured independent of the brown cells. SEC stained with aceto-carmine at day one after first transfer showed typical embryonic polar cell division and embryonic suspensor masses (ESM) could be observed (Fig. 15, C-F). At the second transfer of SEC onto either BM3 or DCR1 + B media, stage I or II somatic embryos could be observed on the surface of the E-callus. These early somatic embryos varied from either well-organized (Fig. 16, B,D) to unorganized (Fig. 16, A,C). In the latter case, many small proembryos could be observed on the suspensor region of the somatic embryo (Fig. 16, C).  52  chapter 3 somatic embryogenesis in Douglas-fir  Fig. 14 Characterization of embryogenic calli initiation from different developmental stages of explants after 7 days in the induction medium (BM2-3 or BM3). A and B, C and D, E and F, G and H, were from Stage I, II, III, and IV zygotic embryos, respectively; X40.  chapter 3 somatic embryogenesis in Douglas-fir  Fig. 15, A and B. Secondary embryogenic callus formation from Stage I and IV swollen, brown zygotic embryos of tree #119 at transfer5 on BM3, X80. C. Single acetocarmine reactive cell from day 2 of 2nd E callus of #119 on BM3, X480. D. Unequal cell division, X480. E. Formation of embryonal and suspensor initials resulting from unequal cell division, X360. F. ESMs from Secondary Embryogenic Callus (SEC) day 3 on BM3 medium, X120.  54  chapter 3 somatic embryogenesis in Douglas  Fig. 16 Somatic embryo development from SEC. Stage I somatic embryo (unorganized) from SEC of genotype #119, 7 days on B M 3 medium, notice that there are a few proembryos in the suspensor region of the somatic embryo (A,C), X80. Stage I somatic embryo (wellorganized) from SEC of genotype # 3 4 7 , 7 days on B M 3 medium (B,D), X80.  55  chapter 3 somatic embryogenesis in Douglas-fir  Marked differences were observed in efficiency of embryogenic callus initiation and production of stable embryogenic callus lines between culture media, developmental stages, and genotypes tested (Table 5-7). Based on an analysis from the perspective of the induction media, the best production of stable embryogenic callus lines was obtained from media BM3 + C M and BM2-3, with the frequency of somatic embryos formed at 15% and 4.4%, respectively (Table 5). Relative to the developmental stages of explanted embryos, stage II and III zygotic embryos produced the best results both in initiation and production of stable embryogenic callus lines, with 93% and 90% in initiation, and 4% and 3% in stable line production, respectively (Table 6). Because the studies concerning induction media and stages were conducted separately, we can not directly compare 15% SEC production on BM3 + C M medium with 4% SEC production from Stage II embryos. Relative to the genotypes tested, a high rate in initiation did not necessarily mean efficient production of stable embryogenic callus lines. While collections from trees #119 and #347 yielded 4% and 2% secondary embryogenic callus, respectively, stable embryogenic callus production was not obtained from either #130 or #169, although all 4 genotypes initiated embryogenic callus similar well (Table 7). So far, 26 lines of secondary embryogenic calli of Douglas-fir have been established , from which at least four homogeneous calli have been maintained in each line. Although these lines are all embryogenic based on their morphological and histological characteristics, differences among them persist. Some lines produce well-organized embryos, while some others produce only elongated, unorganized proembryos. Also, embryogenic callus lines maintained on DCR1 medium were  56  chapter 3 somatic embryogenesis in Douglas-fir  more translucent and grew slightly faster than those on BM3 medium, but produced fewer well-organized somatic embryos or ESMs from DCR1 medium, indicating an antagonism between proliferation and organization (see also Laine and David, 1990).  57  chapter 3 somatic embryogenesis in Douglas-ftr  Table 5 Influence of media on embryogenic callus initiation and secondary embryogenic callus production from cultured immature zygotic embryos of Douglas-fir.  Medium  No.embryos inoculated  Total and (%) EC i n i t i a t i o n  Total and (%) seconary EC  1/8MS  238  173 (73)  1  (0.4)  DCR1-1  339  125 (37)  2  (0.6)  BM2-3  315  200 (63)  14 (4.4)  BM2-3+CM  108  9 (8)  0  BM1-3  140  72 (51)  0  60  46 (77)  9  BM3+CM  These data represent the cumulative analysis of each induction media from different genotypes and developmental stages of zygotic embryos at the time of second transfer after initiation.  58  (15)  chapter 3 somatic embryogenesis in Douglas-fir Table 6 Influence of developmental stages of explants on embryogenic callus initiation and secondary embryogenic callus production from immature zygotic embryos of Douglas-fir.  Develop. stage  No.embryos Inoculated  T o t a l and (%) EC i n i t i a t i o n  <I  80  34  (42)  0  I  238  153 (64)  3  II  305  285 (93)  13 (*)  III  174  157 (90)  5  (3)  IV  602  51  5  (0.8)  (8)  These data represent the cumulative analysis of individual developmental stages of zygotic embryos from different induction media as showed in table 4 and different genotypes at the time of second transfer after initiation.  59  T o t a l and (%) seconary EC  (1)  chapter 3 s o m a t i c embryogenesis in Douglas-fir  Table 7 Influence of genotype of explants on total and (%) embryogenic callus initiation and total and (%) secondary embryogenic callus production from cultured immature zygotic embryos of Douglas-fir.  Genotype  No.embryos inoculated  T o t a l and (%) EC i n i t i a t i o n  T o t a l and (%) secondary EC  #85  280  175 (62)  3  1119  314  134 (42)  12 (4)  #130  78  62 (79)  0  #169  49  30 (61)  0  #347  336  150 (45)  8  (2.4)  #623  342  129 (38)  3  (0.9)  These data represent the cumulative analysis of different geneotypes from different induction media and developmental stages of zygotic emrbyos at the time of second transfer after initiation.  60  (1)  chapter 3 somatic embryogenesis In Douglas-Or  Table 8 Influence of collections on embryogenic callus initiation from immature zygotic embryos of Douglasfir.  Collection  No.embryos inoculated  T o t a l and (%) EC i n i t i a t i o n  CI  354  260  (73.45)  C2  475  388  (81.68)  C3  358  46 (12.85)  C4  297  16 (5.39)  These data represent the cumulative analysis of the four collections from different induction media, genotypes , and developmental stages at the time of second transfer after initiation.  61  chapter 3 somatic embryogenesis in Douglas-fir  An analysis of results based on the cone collection source, shows that the 1st and 2nd collections gave the best results for embryogenic callus initiation, at the second transfer, with 73% and 81%, respectively. Collections 3 and 4, however, yielded only 12.8% and 5.4 % embryogenic callus initiation by the second transfer, respectively (Table 8). This result indicates that there is a genetic component between embryogenic callus initiation and developmental stages of zygotic embryos. Cultures of stable embryogenic calli on either DCR or B M based hormonefree or low hormone medium supplemented with 1% activated charcoal for 7-14 days in 16h photoperiod light produced pigment accumulation on the surface of the embryogenic callus, accompanied with a high frequency of visible proembryo production. This could perhaps be a signal of further embryo development as has been reported in spruce embryogenesis (Roberts D., 1990; personal communication). Light red anthocyanins appeared in some cells, some proembryos, and a slight greening of the embryo proper both occurred. Large late stage proembryos and stage I and II somatic embryos with cotyledon initiation could be obtained with a high frequency in some lines (such as line 2 of #347, line 4 of #119) by this treatment. Late stage I and stage II of somatic embryos were obtained in high frequency from 5-6 lines when embryogenic calli from 5-6 subcultures of secondary embryogenic calli were cultured on BM hormone-free medium supplemented with 1% activated charcoal for seven days (Fig. 17, A), or they could be obtained directly from proliferation medium (DCR1 + B) but in low frequency (Fig. 17, B). Embryogenic calli with late stage I and/or stage II somatic embryos from pretreatment on charcoal medium were then transferred onto media supplemented  62  chapter 3 somatic embryogenesis in Douglas-fir  with different concentrations of A B A for two subcultures. The development and maturation of somatic embryos were significantly regulated by the A B A level on the medium. By the end of the second subculture on A B A media (i.e. 28 days on A B A media), thin greening cotyledonary embryos with translucent suspensor were obtained from BM hormone-free medium without A B A (Fig. 17, C), while "shooty embryos" were formed from BM medium supplemented with 0.25 uM A B A (Fig. 17, D). Cotyledonary somatic embryos similar to zygotic embryos (except for slight greening) were produced from BM medium supplemented with 0.5 and 1 uM A B A (Fig. 17, E). In addition, polyembryos with yellowish cotyledons form from B M medium supplemented with 3.5 and 7.5 uM A B A (Fig. 17, F), media supplemented with A B A higher than 10 uM A B A resulted in the destruction of somatic embryos. Embryogenic calli on ABA media could still grow and some browning and hardening of callus could be observed from cultures on A B A higher than 20 uM. On the other hand, no development and maturation of somatic embryos could be obtained from ABA media after transfer of callus with somatic embryos younger than stage I embryos. Similar results were obtained from three other maturation studies using the same cell lines. In suspension, the culture cell lines were maintained in liquid BM3 medium with weekly subculturing. Late stage proembryos with well-organized suspensors were obtained by transferring the cultures into DCR1 medium for the final two subcultures (Fig. 18, A-G). Cotyledon stage embryos have not yet been observed in our suspension lines. Using embryogenic cell suspension as a system and half-strength MS as base medium to study the influence of sucrose and nitrogen ( N H 4 ) concentration on +  63  chapter 3 somatic embryogenesis in Douglas-fir  the production of proembryos, at the time of the 4th subculture, we found that the optimal concentration was 34.2 g/1 or 68.4 g/1 for sucrose and half strength or 2 times MS for nitrogen content, respectively (Table 9).  64  chapter 3 somatic embryogenesis in Douglas-fir  Fig. 17 Somatic embryo development and maturation. A, Stage I somatic embryos were obtained in high frequency when transferred embryogenic calli of #347 line 3 into hormone-free B M medium supplemented with 1% activated charcoal for seven days, X56. B. Late stage I somatic embryos formed in low frequency from proliferation medium (DCR1 + B), X120. C. thin greening cotyledonary embryos with translucent suspensor formed from B M hormone-free medium without ABA, X80. D. Shooty somatic embryos formed from B M hormone-free medium supplemented with 0.25 uM A B A for two subcultures of line 24, X120. E . Yellow-greening cotyledon stage of somatic embryos formed on BM hormone-free medium supplemented with 0.5 uM A B A for two subcultures, X240. F. Polyembryos with yellowish cotyledons fromed from BM medium with 3.5-7.5 uM ABA, X120.  i 1  •  a  '  • J  ' 65  I.  chapter 3 somatic embryogenesis in Douglas-fir  Fig. 18 Somatic embryo development in suspension cultures derived from immature zygotic embryos. A. Elongated binucleate cells from ESM 14 days in BM3 liquid, X120. B. and C. Proembryo production from ESM 20 days in BM3 liquid medium, X120. D-G. Somatic embryo development in BM3 liqiud medium after 4 subcultures for D and E; 6 subcultures for F and G, respectively, X80. H . Showing Stage I somatic embryos after 6 subcultures as G, X20.  66  chapter 3 somatic embryogenesis in Douglas-fir  Table 9 Influence of sucrose and NH4 on the embryogenic cultures of Douglas-fir, using cell suspension systems. +  1  2  4 5 Proemb. Organize I Brown 3  6  Media No.  Sucrose NH4+ 9/L)  8  34.2  0.5X  No  No  Yes  None  7  68.4  0.5X  No  No  Yes  Low  3  68.4  l.OX  No  No  Yes  Low  4  34.2  2. OX  Few  No  Partial  Low  1  34.2  l.OX  Few  No  Partial  Low  5  12.0  2. OX  Yes  Partial  No  Low  2  12.0  l.OX  Yes  Partial  No  Low  6  68.4  2.OX  Yes  Yes  No  Low  BM3  34.2  0.5X  Yes  Yes  No  Low  1. half strength MS was used as base medium; 2. the amount of nitrogen in MS medium; 3. proembryo formation; 4. the organization of proembryos; 5. the state of cell suspension based on color change; 6. hormone level, i.e. low level was 1 mg/12.4-D, 0.5 mg/1 BAP and 0.5 mg/1 Kinetin.  67  Horn.  chapter 3 somatic embryogenesis in Douglas-fir  3.3.3 DISCUSSION Two different pathways for embryogenesis were observed from solid vs. suspension cultures. In suspension, the early stages of embryony involve detection of an early transient binuclear stage as described by Durzan and Gupta (1987) (Fig. 18, A), followed by the formation of proembryos with suspensors (Fig. 18, B) and the further development of both the proembryos (Fig. 18, C,D) and suspensors (Fig. 18, E,F). An individual stage I somatic embryo is then formed on each suspensor (Fig. 18, G). Two somatic embryos sharing a common suspensor were also occasionally observed (Fig. 18, G), as also reported for Picea glauca (Hakman and Fowke, 1987) and slash pine (Jain etal. 1989). However, on solid medium, the single primordial embryo cell (Fig. 15, C) divided by polar or unequal cell division (Fig. 15, D) to produce two unequal daughter cells (Fig. 15, E). From such polar cell division, ESMs were obtained (Fig. 15, F). Stage II embryos yielded the best results both in E-callus initiation and stable production of embryogenic lines. Success was also obtained, but with lower efficiency, with stage III and stage I explants. This observation of greatest competence for somatic embryogenesis with stage II zygotic embryos agrees with Durzan and Gupta's earlier work (1987) in Douglas-fir and Pinus taeda, and Laine and David's work (1990) in Pinus caribaea, from which precotyledonary embryos were the best source of explants for embryogenic calli or ESMs induction. However, our established stable embryogenic callus lines of Douglas-fir derived from different developmental stages of zygotic embryos have the same performance on both proliferation and maintenance medium and maintain equal embryogenic capacity. In addition, there were some differences among embryogenic callus  68  chapter 3 somatic embryogenesis in Douglas-fir  derived from different genotypes based on their morphological and histological characteristics. Our data from the induction study indicated that the genotype, developmental stage of explants, and the induction medium are all critical factors for both the initiation of embryogenic callus and the production of stable embryogenic callus lines. These observations agree with various published results in conifer embryogenesis (Jain et al. 1989; Becwar and Warm 1989; Durzan and Gupta 1987). The fact that a high initiation rate of embryogenic calli was obtained from genotypes of #130 (78%) and #169 (62%) but that no stable embryogenic callus lines were produced from either genotype indicates that there is a genetic component in both the establishment of embryogenic callus lines and for stable embryogenic capacity (also see Becwar and Warm, 1989). Only 10% of the embryogenic calli from stages II and III survived to become stable embryogenic lines. Also, the best two media (BM3 and BM2-3) gave 60% less long term secondary embryogenic calli than primary embryogenic calli. This distinction between initiation of embryogenic callus and stable culture production indicates that improvement in the efficiency of stable embryogenic callus production is both essential and obtainable. A possible approach is to use the optimal induction media (BM3 + CM and BM2-3) with the optimal explants (stage II zygotic embryos) in initiating embryogenic calli in this species. The addition of coconut water in the initiation medium did not benefit embryogenic calli induction, but once the secondary embryogenic calli were initiated, coconut water was one of the factors that did encourage the growth and maintenance of stable embryogenic callus lines, and the addition of 3% banana powder in the maintenance medium also encourages  69  chapter 3 somatic embryogenesis in Douglas-fir  the growth of embryogenic callus. In addition, we found that the growth of secondary embryogenic callus from BM2-3 medium could be much improved by culture on either DCR1 or BM3 media in later transfers. Interestingly, the ratio between the numbers of vacuolar, narrow and long suspensor cells and the acetocarmine-reactive embryo cells or ESMs increased as subcultures were carried out on the proliferation media, and it seems that a certain period for several subcultures on proliferation medium is essential after SECs initiation before differentiation, development and maturation of somatic embryos occur. The fact that fast growth and more translucent embryogenic calli could be obtained from DCR1 medium but that less well-organized proembryos were produced, again, suggest an antagonism between proliferation and organization (also see Laine and David, 1990). Comparing these results with our previous attempts to culture mature zygotic embryos, which had been stored at 4°C for over one year, we found that less than 20% of mature zygotic embryos were induced to produce embryogenic calli in our experiments, and only a few of these could be maintained stably in subsequent subcultures. The initiation of secondary embryogenic calli from either the hypocotyl or radicle region of the brown explants was essential for the stable production of embryogenic callus lines from mature zygotic embryos just as we report here for immature zygotic embryos. In agreement with Becwar and Warm (1989), we could increase efficiency of embryogenic callus initiation from cultured mature zygotic embryos by reducing the concentration of basic medium salt to 0 strength (i.e. without medium salt). In general, embryogenic calli from mature embryos were different from those derived from immature embryos. In other words, embryogenic  70  chapter 3 somatic embryogenesis in Douglas-fir  callus from immature zygotic embryos was better than those from mature ones which yielded lower frequencies, fewer ESMs or proembryos, a longer period was required for their production and susbequent establishment, and the development and maturation of somatic embryos were slower and more difficult. To obtain a good regeneration system via somatic embryogenesis in vitro, immature zygotic embryos are the best option to initiate and establish the cultures. It required 28 days (2 subcultures) on B M hormone-free medium supplemented with 1% charcoal for the proembryos to develop into stage II somatic embryos or, in some cases, embryos with visible cotyledon primordia. No maturation of somatic embryos (i.e. cotyledonary embryos) was obtained from charcoal medium alone. The timing, duration, and concentration of abscisic acid (ABA) applied in the maturation medium was critical in Douglas-fir for a normal pathway for embryogenesis to be obtained. Preliminary data indicated that A B A requirement differs drastically from that for white spruce. It seems that the optimal levels of A B A for the maturation in Douglas-fir in our studies were between 0.5 to 10 u M depending on the cell lines. A B A level higher than 10 uM resulted in the destruction of embryo structures, while the optional A B A level for the maturation of somatic embryos in spruce was 40-60 uM (Becwar and Wann 1989; Dunstan et al. 1988; Hakman and Fowke 1987; Roberts et al. 1990). In agreement with Roberts et al. (1990), A B A regulated the maturation of somatic embryos in Douglas-fir. Shooty embryos were produced from medium supplemented with 10-20 uM A B A in interior spruce, while 0.25 uM ABA in the maturation medium resulted in shooty embryos in Douglas-fir from our studies. The significant difference in the requirement of A B A level for the maturation of somatic embryos in vitro between  71  chapter 3 somatic embryogenesis in Douglas-fir  Douglas-fir and other coniferous species such as spruce could be due to the high endogenous A B A level in Douglas-fir cells. This hypothesis should be studied with immunological techniques, using antibodies for A B A to determine the endogenous A B A level from calli with different developmental stages of somatic embryos. In addition, the result that no somatic embryo development and maturation was obtained from A B A treatments with embryos either earlier than late stage I or later than stage III indicates that somatic embryos must reach a certain developmental stage before A B A will affect their normal development and maturation; and this could be controlled by the genetic components during the embryo development in this species. Our results from this study show that stage II zygotic embryos were the best stage of explants for embryogenic calli initiation and stable production, while stage II somatic embryos were the most sensitive stage to be affected by A B A treatment for their normal maturation, from which we could conclude that stage II embryos are the most sensitive stage to be affected by exogenous factors in this species. Again, this could be controlled by the genetic components in the developing embryos. In summary of the results from our studies in somatic embryogenesis from immature zygotic embryos, Fig. 19 shows the procedures of somatic embryogenesis in vitro from solid media in our studies in Douglas-fir. In comparing our results of somatic embryogenesis in Douglas-fir with other reports in conifers, particular white spruce, concerning initiation of embryogenic calli and maturation of somatic embryos, we can summarize the following: 1. induction media, genotype, and developmental stages are critical factors for embryogenic calli initiation. Precotyledon embryos were the best stage of explants  72  chapter 3 somatic embryogenesis in Douglas-fir  for E C induction as reported in Durzan and Gupta's study (1987). However, because no data were presented in their paper, we can not compare our result with theirs. In contrast, post-cotyledonary embryos were the best explants for E C induction for spruce (Hakman and Fowke 1987; Webb et al. 1989), and higher initiation rate of embryogenic callus was obtained from spruce than Douglas-fir. These difference may be controlled by genetic components. 2. Secondary embryogenic calli were critical for the establishment of stable callus lines in our studies. Durzan and Gupta (1987) mentioned that both embryogenic and nonembryogenic calli were initiated, while in our cases, SEC was obtained from brown calli or explants 4-6 weeks after initiation. 3. In agreement with Durzan and Gupta (1987), SECs were embryonal suspensor masses rather than undifferentiated calli, which would result in low frequency of somacolonal variation in the procedure for somatic embryogenesis in Douglas-fir (Gupta P.K., 1990; personal communication). 4. Calli on A B A containing media could still grow even though no maturation of somatic embryos developed on these media. This phenomena was also reported in other studies with Pines (Laine and David 1990). This was most probably due to the existence of hormones carried over from the pervious media after transfer. An improved approach was to transfer calli into charcoal media for one week before being transferred into A B A media, because activated charcoal can absorb the cell wastes and hormones. By using this approach, better embryo development was obtained from our studies. 5. Concerning the quality of somatic embryos produced, yellow-greening cotyledon somatic embryos were obtained from B M media with 0.5-1.0 uM A B A in our studies, while the greening of cotyledons during the maturation of embryos is not observed from zygotic embryos in vivo. This  73  chapter 3 somatic embryogenesis in Douglas-fir  greening could be due to the low A B A concentration in the media which resulted in precocious germination. A B A is supposed to prevent precocious germination and stimulate the accumulation of storage protein in the embryos. By increasing the A B A level to 30-40 uM, Roberts et al. (1990) obtained opaque cotyledonary embryos in spruce which closely resemble to their zygotic counterparts, while shooty embryos predominated in lines at low level of A B A (1-10 uM). 10-20 uM ABA promoted the formation of bipolar embryos that germinated precociously in interior spruce. They concluded that storage protein accumulation in the embryos was dependent on the concentration of ABA. This relation between embryo development and A B A level was also observed from our studies in Douglas-fir, i.e. shooty embryos formed on 0.25 uM A B A and cotyledonary embryos formed on 0.51.0 wM ABA. In addition, the embryos produced from our studies in Douglas-fir germinated precociously, and only the cotyledons and hypocotyls elongated while no roots developed.  74  chapter 3 somatic embryogenesis in Douglas-fir  Fig 19. Flow chart of development of embryogenic calli derived from immature zygotic embryos of Douglas-fir from our study in solid media.  immature zygotic embryos 1- 2 weeks  induction media 2- 4 weeks  t  embryogenic callus secondary embryogenic callus  t  t  maintenance on BM3  differention on DCR1+B 4 weeks (poly- ond proembryos  hormone-free medium + charcoal 1 week (lote SI or 91 embryos)  ABA (0.5-10 uM) media 4 weeks  cotyledonary somatic embryos  75  CHAPTER 4 PROTOPLAST ISOLATION A N D ELECTROPORATION  76  chapter 4 protoplast isolation and electroporation  4.1 INTRODUCTION  4.1.1 PROTOPLAST ISOLATION The use of protoplasts in the tree improvement programs via techniques such as somatic hybridization and gene transfer has great potential (Ahuja 1988a; 1988b). However, protoplasts have been regarded as recalcitrant to culture until Vasil and Vasil (1980) successfully recovered adventitious embryos from a previously nonregenerable monocot  (Pennisetum purpupreum) using an emb  derived from immature embryos as the protoplast source. Embryogenic calli are good sources for protoplast isolation in conifer species because of their regeneration potential. Development of techniques for the isolation, culture, and regeneration of forest trees from protoplasts is in many cases a prerequisite to development of efficient transformation protocols. In order to facilitate the use of protoplasts in forest biotechnology, efficient systems for regeneration of trees from protoplasts must be developed. Protoplast isolation, followed by the recovery of cell wall, cell division, cell colonies production, and regeneration of somatic embryos have been obtained in  Picea glauca (Bekkaoui et al. 1987; Attree et al. 1987), Pinus taeda ( Durza, 1987b), Pseudotsuga menziesii (Gupta et al. 1988), Abies Kohlenbach, 1989), and Picea mariana Mill. (Tautorus et al. 1990). After the development of cell colonies, the cell suspension cultures from protoplasts entered proembryogenic differentiation and plantlet regeneration in al. 1989a, b, and c), and  Picea glauca (Attree  Pinus caribaea (Laine and David, 1990).  Protoplasts have also been isolated from non-embryogenic sources in conifer  77  chapter 4 protoplast isolation and electroporation  species, such as cotyledons of Pseudotsuga menziesii (Cheng, 1975) and Pinus pinaster (David, 1987). Calli were the most advanced stage observed from these protoplasts, no organogenesis was reported (David, 1987). Enzymes are commonly used for protoplast isolation from conifer species. The time required for enzymatic release of protoplasts differs with the species utilized, the nature of the starting material, and the enzymes employed. A high yield of protoplasts (4.5 X lO** protoplasts/g fresh cells) was obtained from embryogenic suspension cells by using 1% enzymes for 3-4 hours in white spruce (Bekkaoui et al. 1987). The time for the recovery of somatic embryos from protoplasts in vitro is quite dependent on the species and the culture medium. The first division was observed 24 hours after protoplast isolation in Picea glauca (Attree et al. 1987) in LP medium and Picea mariana Mill. (Tautorus et al. 1990) in modified MS medium , and it took 4 and 8 days to obtain cell clusters and recognizable somatic embryos, respectively, the total time to plantlet recovery, following protoplast isolation, was 15 weeks in Picea glauca. In Pinus caribaea (Laine and David, 1990), the time for the first division, cell clusters production, and the plantlet regeneration was 2 days, one month, and 5 months, respectively. While in Pseudotsuga menziesii (Gupta et al. 1988), the first cell division occurred 4-6 weeks after protoplast isolation in DCR medium.  4.12 ELECTROPORATION Electroporation is the application of one or more short electrical pulses to a membrane system causing holes or pores to form in the outermost membrane  78  chapter 4 protoplast isolation and electroporation  (Potrykus et al. 1985). In the past few years, electroporation of DNA or R N A into cells has became a very popular method for direct gene transfer and transfection of cells. The high efficiency of this technique was first demonstrated by Neumann et al. (1982) and Potter et al. (1984) with animal cells. Fromm et al. (1985,1986) showed that this technique could be used to transfer genes into plant protoplasts. Since these early reports, direct gene transfer into protoplasts via electroporation has been successful in many agricultural species, such as potato (Masson et al. 1989), sugar beet (Lindsey and Jones, 1989), and Nicotiana tabacum (Shillito et al. 1985). A chimeric gene, encoding resistance to kanamycin was introduced into protoplasts of Nicotiana tabacum and transformed plantlets were obtained (Shillito et al. 1985). Transient expression of a luciferase gene has been obtained in tobacco cells and plants (Ow et al. 1986). In conifer species, a luciferase gene was introduced into protoplasts of Douglas-fir and loblolly pine via electroporation, and transgenic protoplasts developed into somatic proembryo structures (Gupta et al. 1988). Transient chloramphenicol acetyl transferase (CAT) gene expression was obtained in white spruce protoplasts via electroporation (Bekkaoui et al. 1990) and polyethylene glycol (PEG)-mediated DNA uptake (Wilson et al. 1989). In plant transformation experiments, B-glucuronidase (GUS) gene from bacteria, luciferase gene from fire flies, and chloramphenicol acetyltransferase (CAT) gene encoded with or without selectable genes have commonly been used as marker genes to standardize transformation protocols, to obtain high transformation efficiency, and apply instant selection for transformed cells. The GUS gene of E. coli has been developed as a gene-fusion marker for higher plants by Jefferson (1988) and Jefferson et al. (1987). It has been widely used as a reporter gene in the  79  chapter 4 protoplast isolation and electroporation  study of foreign gene expression (Benfey and Chua, 1989) and in crop plant genetic engineering (Wenzler et al. 1989). C A T gene was developed for mammalian cells by Gorman et al. (1982). Because rapid, sensitive assays for both GUS and C A T activities are available, the monitoring of 5-glucuronidase (GUS) and Chloramphenicol acetyltransferase (CAT) activities provides a relatively quick and easy verification of the uptake and transient expression of D N A by protoplasts. It has been reported that the transformation efficiency of a foreign gene into plant protoplasts via electroporation and its transient expression in the protoplast is affected by the concentration of foreign DNA used, the amplitude and duration of applied electric pulse, the presence of Polyethylene glycol (PEG) in the electroporation buffer and other medium components used, the type of promoter in the vector, and cell line (genotype) used (Bekkaoui et al. 1990; Tyagi et al. 1989; Nea and Bates 1987; Tautorus et al. 1989; Lindsey and Jones 1989; Gupta et al. 1988). In white spruce protoplasts (Bekkaoui et al. 1990), approximately equal magnitude of transient C A T gene expression was obtained from the C A T gene fused to either the cauliflower mosaic virus (CaMV) 35S promoter or the nopaline synthase (NOS) promoter; while eightfold higher C A T enzyme activity was obtained from the C A T gene fused to a tandem repeat CaMV 35S promoter, as compared with the previous two promoters. Higher levels of transient C A T expression were obtained in protoplasts of black spruce and jack pine by increasing the plasmid DNA concentrations from 20 to 150 ug/ml (Tautorus et al. 1989). The addition of 3% Polyethylene glycol (PEG) in the electroporation buffer promotes the uptake of the luciferase gene into the protoplasts of Douglas-fir and loblolly pine (Gupta et al. 1988).  80  chapter 4 protoplast isolation and electroporation  42 MATERIALS AND METHODS  4.2.1 PLANT MATERIAL Cell suspensions initiated from embryogenic calli were maintained on MS3 or BM3 medium for over six months, as described in chapter 3, and used as source for protoplast isolation. The suspension contained a heterogeneous population of cells including free cells, clusters of isodiametric cells, cell clumps, and proembryos/proembryo bridges (Fig. 20).  4.2.2 PROTOPLAST ISOLATION Protocols for protoplast isolation were modified from Gupta et al. (1988) and Bekkaoui et al. (1987). Two enzyme solutions were used. Enzyme solution I included: 2%(w/v) cellulysin, 2%(v/v) pectinase, l%(m/v) macerase, 0.5%(w/v) potassium dextran sulphate, 12%(w/v) mannitol, half strength DCR salts at pH 5.6; enzyme solution II consisted of 0.5% (w/v) Cellulose R-10, 0.25% each of Macerozyme and Pectinase, 5mM CaCl2-2H20 and 0.5 M mannitol. Enzyme solutions were prepared freshly and filter-sterilized using disposable Nalgene filterware before use. The first procedure involved harvesting 3-5 day-old suspension cultures in a 15-ml centrifuge tube by centrifuging at 400g for 5 minutes. To each 2ml packed cells, 10 mis of enzyme solution I added to resuspend the cells and transferred into a 25 X 100 mm petri dish, the dish was sealed with Parafilm and incubated at 26°C in darkness on a shaker set at 100 rpm for 3-12 hours (the time for enzyme digestion was flexible, the cells were monitored with an inverted microscope every hour so  81  chapter 4 protoplast isolation and electroporation  that a complete digestion of cell wall could be obtained). The digestion solution was filtered through a 50 mesh screen with 4 layers of cheese cloth, collected in a 15-ml centrifuge tube and crude protoplasts pelleted at 400 rpm for 5 minutes. The pellet was washed twice with washing medium (DCR salts, 12% mannitol, 0.5% potassium dextran sulphate at pH 5.6, autoclaved) and the purified protoplasts were resuspensed in culture medium. The second procedure was carried out as following: Cells from 6-day-old subcultures were collected on sterilized Miracloth and transferred to a 25 X 100 mm petri dish containing enzyme solution II (10 ml for 2 g cells). Cells were incubated in enzyme solution for 3-12 hours on a shaker (100 rpm) at 26-28°C in the dark. The resulting suspension was then passed through a 65 mesh screen to remove debris and undigested cells and was centrifuged at 100 g for 5 min. The pellet was then gently resuspended in 5 ml of 20% (w/v) sucrose on top of which 1 ml of 0.5M mannitol was layered. Following centrifugation at 100 g for 5 minutes, viable protoplasts formed a loose band at or above the interface between sucrose and mannitol and were collected and washed twice with a 0.5M mannitol solution before being finally resuspended in culture medium. Viability of the protoplasts was tested with fluorescence diacetate (FDA) (modified from Widholm, 1972). A drop of protoplasts was mixed with a drop of F D A working solution (diluted F D A stock at 1 mg/ml in acetone, stored at -20°C, with culture medium until milk color appeared) for 5 min. in a petri dish, and then examined under a fluorescence microscope. Viable cells immediately began to display a bright-green fluorescence within each cell. The presence of cell walls was confirmed by calcofluor white staining  82  chapter 4 protoplast isolation and electroporation  (Galbraith, 1981). A drop of 0.1% (w/v) calcofluor white (stains cellulose) was mixed with protoplasts on a slide, and examined under a fluorescence microscope. As a result, cell walls fluoresced, while no fluorescence was observed from protoplasts that were free of cell wall.  4.2.3 C U L T U R E OF PROTOPLASTS Purified protoplasts were cultured as suspension (1-2.5 ml) at a density of 2 X 10 per ml in either 6- or 12-well plates. Plates were sealed with parafilm and 5  incubated in a shaker set at 100 rpm at 26°C in the dark. Two culture media were tested. Medium 1 (designed as DCR-P) contained: DCR salts, 3% sucrose, 2% glucose, 6% myo-inositol, 1% sorbitol, 450 mg/1 glutamine, 500 mg/1 casein hydrolysate, 5 mM 2-(N-morpholino) ethanesulfonic acid (MES) buffer (pH 5.7), 5uM 2,4-D., 2uM kinetin and BAP. Medium 2 (designed as MS-P) consisted of 0.5 MS salts, 12% mannitol, 1% myo-inositol, 0.1M sucrose, 450 mg/1 glutamine, 500 mg/1 casein hydrolysate, 5uM 2,4-D., 2wM Kinetin and BAP. The percentage of cells with division potential (the presence of two nuclei) was estimated after 4 days in culture according to the formula: number of cells with two nuclei Percentage = X 100 initial plating density At least 200 protoplasts per treatment were scored for the estimation of division potential frequency and 2 replicates were prepared for each treatment. All the experiments were repeated at least twice.  83  chapter 4 protoplast isolation and electroporation  42.4 ELECTROPORATION An electroporation protocol modified from Gupta et al. (1988) was tested. Freshly isolated protoplasts as described above were resuspended in 0.8 ml of electroporation buffer (lOmM Hepes, 140 mM CaCl2, 6% (w/v) myo-inositol, and 3% (w/v) PEG) at a density of 4X10^ protoplasts per ml. This was mixed with or without 50 «g of sonicated calf thymus DNA as carrier DNA, and with or without either 60 ug or 100 ug of plasmid DNA (PBI221, PBI121, or pCaMV.CAT). The mixture was transferred into a pre-cooled disposable electroporation chamber (BRL) and set on ice for ten minutes. Electroporation was then applied at 330 uF and a series of voltages, either 100, 200, 300, and 300V or 200, 300, 300, and 400V. After ten minutes at room temperature, the electroporated protoplasts were diluted with 1-2 ml of culture medium (i.e. MS3 or DCR1) and cultured in 6-well plates (Corning) with a density of 5 X 10^ per ml. The plates were sealed with Parafilm (American National Can) and incubated in a shaker set at 50 rpm at 26°C in the dark.  4.2.5 PLASMIDS Three plasmids were used: 1) pBI 221, the plasmid incorporating the CaMV 35S promoter and the 5-Glucuronadase (GUS) gene from E . coli; 2) pBI 121, a binary vector carrying an 800 bp fragment containing the CaMV 35S promoter controlling the Kanamycin resistance-NTPII gene together with the GUS gene of pBI 221; and 3) pCaMV.CAT, a PUC8 derived vector that includes a C A T (Chloramphenicol acelty transferase) coding region under the control of the CaMV 35S promoter and a NOS polyadenylation region (Jefferson et al. 1987; Gorman et  84  chapter 4 protoplast isolation and electroporation  al. 1982). In between the promoter and C A T coding region of pCaMV.CAT, a linker with multiple coding sites has been introduced. The pUC8 plasmid DNA was obtained commercially. All plasmids were filter sterilized before being added to electroporation buffer.  4.2.6 PREPARATION OF DNA Plasmid preparations were conducted according to the procedures from Young and Davis (1983): Inoculated 10 ml of LB medium containing the appropriate antibiotic with a single bacterial colony and incubated at 37°C overnight with vigorous shaking. The following morning, inoculated 25 ml of LB medium in a 100-ml flask containing the appropriate antibiotic with 0.1 ml of the overnight culture. Incubated at 37°C with vigorous shaking until the culture reached late log phase (OD600=0.6), and then inoculated 25 ml of the late log culture into 500 ml of LB medium prewarmed to 37°C with the appropriate antibiotic in a 2-liter flask. Incubated for exactly 2.5 hours at 37°C with vigorous shaking and 2.5 ml of a solution of chloramphenicol (34 mg/ml in ethanol) added. The final concentration of chloramphenicol in the culture was 170 ug/ml. And lastly, incubated at 37°C with vigorous shaking for a further 12-16 hours. The bacterial cells were harvested by centrifugation at 4000 g for 10 minutes at 4°C. The supernatant was discarded and the pellet was washed in 100 ml of icecold STE (0.1 M NaCl, 10 mM Tris-Cl[pH 7.8], and 1 mM EDTA). The bacteria were lysed by alkali (Birnboim and Doly, 1979): the bacterial pellet from a 500-ml culture was resuspended in 10 ml of solution I (50 mM glucose, 25 mM Tris-Cl[pH 8.0], 10 mM EDTA) containing 5 mg/ml lysozyme; and then  85  chapter 4 protoplast isolation and electroporation  transferred to a Beckman SW27 polyallomer tube and left at room temperature for 5 minutes. A total of 20 ml of freshly made solution II (0.2 N NaOH, 1% SDS) were added. The tube was covered with parafilm and mixed by gentle inversion and then left on ice for 10 minutes. A volume of 15 ml of an ice-cold solution of 5 M potassium acetate (pH 4.8) was added from 60 ml of 5 M potassium acetate, plus 11.5 ml of glacial acetic acid and 28.5 ml of H20, so that the resulting solution was 3 M with respect to potassium and 5 M with respect to acetate. The tube was covered with parafilm and mixed by inversion and then left on ice for 10 minutes. The lysate was centrifuged on a Beckman SW27 rotor at 20,000 rpm for 40 minutes at 4°C. The cell D N A and bacterial debris formed a tight pellet on the bottom of the tube. Equal quantities of the supernatant were transferred into each of two 30-ml Corex tubes, and 0.6 volumes of isopropanol were added to each tube, mixed well and left at room temperature for 15 minutes. The DNA was recovered by centrifugation in a Sorvall rotor at 12,000g for 30 minutes at room temperature. The supernatant was discarded and the pellet was washed with 70% ethanol at room temperature. As much ethanol was discarded as possible, then the nucleic acid pellet was dried briefly in a vacuum desiccator. The pellet was dissolved in a total volume of 8 ml of TE(pH8.0). The plasmid D N A was purified by centrifugation to equilibrium in cesium chloride-ethidium bromide density gradients: the volume of the DNA solution was measured and for every milliliter exactly 1 g of solid cesium chloride was added and mixed gently until all of the salt dissolved. A volume of 0.8 ml of a solution of ethidium bromide (10 mg/ml in H 2 O ) was added for every 10 ml of cesium chloride solution and mixed well. The final density of the solution was at 1.55 g/ml, and the  86  chapter 4 protoplast isolation and electroporation  concentration of ethidium bromide at about 600 ug/ml. The cesium chloride solution (together with the protein aggregates) was transferred to a tube suitable for centrifugation in a Beckman Type-50 and the remainder of the tube was rilled with light paraffin oil and centrifuged at 45,000 rpm for 36 hours at 20°C. The cap from the tube was removed and the lower band of D N A was collected into a glass tube through a #21 hypodermic needle inserted into the side of the centrifuge tube.  4.2.7 GUS ASSAY Transient expression of the GUS gene in electroporated protoplasts was assayed by two methods. 1) A method modified from Jefferson et al. (1987): Dissolved 5 mg X-gluc (5-bromo-4-chloro-3 indolyl glucuronide) into 5 ml of assay buffer (30.5 mM Na2HPC>4, 19.5 mM NaH2P04 diluted to 1000 ml with distilled water) to give a final concentration of 1 mg/ml X-gluc, which was filter sterilized. Beginning at day one after electroporation, 500 ul of electroporated cells were taken out every day up to day 5 from each treatment, and mixed with 208 ul of X-gluc stock solution to give a final concentration of 300 ug/ml for the assay, and then transferred into a 24-well plate (Corning), sealed with Parafilm and incubated at 26°C in the dark. Observations were carried out every day using an inverted microscope and photographs were taken using Kodak Ektachrome 160 color slide film. The transformation efficiency was estimated by counting blue cells over total cells on the observation field, in comparison with no D N A controls, at least 10 focal fields of cells were counted for each treatment of the day. 2) A non-destructive assay for GUS described by Gould and Smith (1989): Added 50 ul M U G (4-Methylumbelliferyl-B-D-Glucuronide) assay solution (7 mg  87  chapter 4 protoplast isolation and electroporation  M U G and 10 ml of assay buffer consisted of 30.5 ml M Na2HPC»4, and 19.5 ml M NaH.2PC»4, diluted to 1000 ml with distilled water; filter sterilized the amount of M U G assay solution to be used just before assay) were added to 500 ul aliquots of electroporated cells in 1.5-ml micro-centrifuge tubes. The tubes were incubated at 37°C overnight. 250 ul of stop buffer (0.3 M Na2CC»3) was added to ionize the methyl-umbelliferone product and the cells were immediately evaluated with U V light or under a fluorescence microscope, and the comparison between the experimentals and the controls was made.  4.2.8 C A T ASSAY C A T assays were carried out using a protocol modified from Gorman et al. (1982): 48 and 72 hours after electroporation, the electroporated cells were collected into 1.5-ml micro-centrifuge tubes (2 ml cells, 2 tubes for each treatment), the cells were pelleted and washed twice with 0.25 M Tris-hydrochloride (pH 7.8) at 600 rpm for 15 min at 4°C. The pellet was resuspended with 50 ul of 0.25 Trishydrochloride (pH 7.5) and the cells were ground mechanically using a pestle. After the homogenates were spun for 15 min in a microcentrifuge at 4°C, the supernatants were removed and assayed for enzyme activity. The assay mixture contained (in a final volume of 180 ul) 100 ul of 0.25 M Tris-hydrochloride (pH 7.5), 20 ul of cell extract, 1 uCi of [ C]chloramphenicol, and 20 ul of 4 mM acetyl 14  coenzyme A. All of the regents except coenzyme A were preincubated together for 5 minutes at 37°C. After equalization was reached at this temperature, the reaction was started by adding coenzyme A. The reaction period was 30 min at 37°C. The reaction was stopped with 1ml of cold ethyl acetate. The organic layer (upper layer)  88  chapter 4 protoplast isolation and electroporation  was dried under vacuum and taken up in 15 ul of ethyl acetate, spotted on silica gel thin-layer plates which had been incubated at 65°C for 60 min, and run with chloroform-methanol (95:5, ascending) which was prepared one night before the assay. Separated spots of chloramphenicol and its acetylated forms were visualized by autoradiography on X - O M A T Kodak film for 1-3 days.  4.2.9 C E L L SELECTION Before the selection for transgenic cells was carried out, a killing curve for kanamycin was established to determine the lethal concentration of kanamycin to Douglas-fir cells. A series of concentrations of kanamycin (0,10,20,50,80, and 100 ug/ml) were screened with two repeats of 1 ml cells with a density of lO** per ml. The percentage of survival was based on viability and cell division determined. After the lethal concentration of kanamycin to Douglas-fir cells was determined from the killing curve, the pBI 121 electroporated protoplasts were cultured on medium supplemented kanamycin as determined for selection of resistance cells, i.e. transgenic cells.  4.3 RESULTS  4.3.1 ISOLATION O F PROTOPLASTS The embryogenic suspension culture consisted of cell aggregates composed mainly of isodiametric cells and elongate suspensor-like cells (Fig. 20). Some proembryo bridges or individual proembryos were identified. The protoplasts were isolated from six-day-old cell suspension cultures (Fig.  89  chapter 4 protoplast isolation and electroporation  21). The suspension culture yielded 0.5-1.5 X 10^ protoplasts per gram fresh weight of suspension as determined by haemocytometer counts. The period of incubation required for the complete removal of the cell walls, confirmed with Calcofluor white staining, was 3-5 hours for enzyme solution I and II. The viability of the protoplasts tested with F D A staining varied from 85-90% after fresh isolation (Fig. 22).  4.3.2 PROTOPLASTS C U L T U R E Within two days many protoplasts exhibited changes in shape indicative of a functioning cell wall and the recovery of cell wall. Many protoplasts with two nuclei were observed at day four (Fig. 23, A), up to 11% of protoplasts in the cultures, and these were treated as cells with division potential. The first division was observed at 2 weeks after isolation (Fig. 23, B). When diluted with half volume of fresh culture medium, both division resulting in isodimetric cell colonies (4-10 cell stage) (Fig. 24, A and B) and unequal division or elongation of one of the daughter cells following first division resulted in elongate highly vacuolar suspensor-like cells (Fig. 25). These cell colonies and suspensor-like cells could not develop further and mostly turned brown while being maintained as suspension cultures. Proliferating cell clusters or callus could be obtained by plating cell suspension onto agar medium (MS3) or embedding into agarose (0.5%) MS3 medium in 20 X 50 petri plates for 24 weeks (Fig. 26, A and B). Due to the existance of cell colonies at the time of plating and the browning of most of the cells, it was difficult to determine the plating efficiency. No proembryo structures have been obtained from these protoplast-derived cell clusters yet.  90  chapter 4 protoplast isolation and electroporation  Fig. 20 Embryogenic suspension cultures used for protoplasts isolation, X80.  91  chapter 4 protoplast isolation and electroporation  Fig. 21 Freshly isolated protoplasts from cell suspensions derived from embryogenic calli of Douglasfir, X480.  c  92  chapter 4 protoplast isolation and electroporation  Fig. 22 Determination of viability of freshly isolated protoplasts by F D A staining under fluorescence microscope, X320.  93  chapter 4 protoplast isolation and electroporation  Fig. 23 Protoplast development. A. Protoplast with two nuclear from BM3 medium 4 days after isolation, X480. B. First division 2-4 weeks after isolation, X360.  94  chapter 4 protoplast isolation and electroporation  Fig. 24 Cell division and cell colonies formation. A. Further cell division from protoplast 4-6 weeks after isolation, X240. B. Production of cell colonies from protoplast 6-8 weeks after isolation in BM3 medium, X240.  chapter 4 protoplast isolation and electroporation  Fig. 25 Unequal cell division from protoplast resulted in the production of proembryo, X240.  chapter 4 protoplast isolation and electroporation  Fig. 26 Plating efficiency and recovery of calli from protoplasts. A. Small cell colonies recovered from cells of protoplasts 2 months on BM3 solid medium. B. Calli production 3 months on BM3 medium.  97  chapter 4 protoplast isolation and electroporation  4.3.3 TRANSIENT EXPRESSION OF GUS A N D C A T The viability of the protoplasts was reduced from 85-90% to 70% after electroporation. GUS expression (tested with X-gluc substrate resulting in visible blue cells) was first detected at day 2 after electroporation with plasmid pBI 221 (Fig. 27), and the GUS activity as determined by counting the blue cells over the total cells plated, increased as the days passed. The highest GUS activity was observed at day four after electroporation, with 4% of the cells turning blue, and then dropped to 1% at day 5 (Fig. 28). No blue cells were observed from control protoplasts electroporated without plasmid but with carrier DNA. No blue cells could be detected from any of the experiments when the concentration of X-gluc substrate was lower than 100 ul per ml. Slight differences in GUS activity existed between the two concentrations of pBI 221 plasmid used in the electroporation. However, a much lower percentage of blue cells (the highest was less than 0.5%) were obtained from protoplasts electroporated with plasmid pBI 121. In addition, cell colonies derived from electroporated protoplasts 2-3 months after electroporation with plasmid pBI 221 still exhibited GUS activity when new X-gluc substrate was added in the suspension cultures in some cases, which suggests the stable transformation of GUS gene in the cells(Fig. 30). GUS was also detected at day 2 after electroporation under fluorescence microscope with the M U G assay (Fig. 29). It was difficult to distinguish transgenic protoplasts from nontransgenic ones under the U V light using the M U G assay, perhaps due to the small size of the protoplasts. Under fluorescence microscope, some dull green cells were observed, while no such cells could be detected from the controls. No quantitative calculation was carried out from the M U G assay.  98  chapter 4 protoplast isolation and electroporation  Fig. 27 Protoplasts expressing GUS 3 days after electroporation with pBI 221, assayed with X-gluc substrate, X480.  99  chapter 4 protoplast isolation and electroporation  Fie. 28 GUS activity (percentage of transformed "blue" cells over total cells) 5 days after electroporation of Douglas-fir protoplasts with pBI 221 plasmid.  0U9 activity {% blu* ctlO  1  2  3  4  Tim* (day)  100  5  chapter 4 protoplast isolation and electroporation  Fig. 29 Protoplast expressing GUS 3 days after electroporation with pBI 221 under fluorescence microscope with the assay of M U G , X480.  101  chapter 4 protoplast isolation and electroporati  Fig. 30 Stable expression of GUS in cell colonies derived from protoplasts 3 months after electoporation with pBI 221, assayed with X-gluc substrate, X480.  102  chapter 4 protoplast isolation and electroporation  No acetylated chloramphenicol products were detected from protoplasts 48 hours after electroporation for either controls or the two experiments, while two weak bands were detected by T L C (thin layer chromatography) assay from cell extracts derived from protoplasts 96 hours after electroporation from the two experiments(Fig. 31). Both protoplast electroporations with 60 and 100 ug/ml of pCaMV.CAT plasmid exhibited two bands which represented 1-AcCAP and 3AcCAP products, respectively. There was no direct correlation between the amount of DNA introduced and the expression of C A T activity from the plasmid pCaMV.CAT as far as these experiments are concerned. Almost no acetylated chloramphenicol products were detected in the Douglas-fir protoplasts electroporated with carrier DNA alone, which was used as control in this experiment.  4.3.4 KILLING C U R V E AND SELECTION The lethal concentration of kanamycin for the cells of Douglas-fir was determined to be 80 ug/1 (Fig. 32). When protoplasts electroporated with plasmid pBI 121 were cultured on MS3 liquid medium supplemented with 80 ug/1 of kanamycin, few cell colonies could be obtained, instead, most cells turned brown and died. No cell colonies developed from plating cultures, following electroporation.  103  chapter 4 protoplast isolation and electroporation  Fig. 31 Transient expression of C A T activity from protoplasts of Douglas-fir. Protoplasts were electroporated with earner D N A as control (C), and with or without 60 or 100 ug of pCaMVCAT plasmid. The activity was assayed 2 and 4 days (D2 and D4)after electroporation. The T L C plates were exposed to X-ray film for 3 days. CAP=Chloramphenicol; l-AcCAp = l acetate chloramphenicol; 3-AcCAP=3-acetate chloramphenicol.  104  chapter 4 protoplast isolation and electroporation  Fig. 32 Killing curve for kanamycin.  e«ii viability («) 1001  0  20  40  60  80  kanamycin* (ug/ml)  105  K>0  120  chapter 4 protoplast isolation and electroporation  4.4  DISCUSSION  The first cell division occurred at two weeks after isolation of Douglas-fir protoplasts in this experiment, and it took 2-3 months to obtain cell colonies composed of 4-10 cells. This slow development was previously described for Douglas-fir (Gupta et al. 1988), and Pinus taeda (Gupta and Durzan, 1987b), from which the recovery of somatic embryos took place only after 12-24 weeks and 8-10 weeks, respectively. In contrast, protoplasts from white spruce, regenerated new cell walls, exhibited high division frequency, formed embryos by 23 days and within 35 days had produced numerous colonies containing many somatic embryos (Attree et al. 1987). The browning of protoplast cultures may be due to the liquid medium without shaking which may inhibit gas exchange. The plated cultures and the agarose bead cultures (Shillito et al. 1985) resulted in the production of cell colonies. This method also benefited the protoplast cultures in Douglas-fir (Gupta et al. 1988) and white spruce (Attree et al. 1987). This is most likely due to enhanced gaseous exchange and more even distribution of nutrients and waste substances (Attree et al. 1987). It took a longer time to digest cell walls from suspension cells than by digesting callus directly. This may due to the stable response of cells to the osmolarity of the liquid or to stress induced cell wall thickening. As high as 4% of the protoplasts expressed GUS at day four after electroporation with plasmid PBI 221. This was achieved by applying 60 ug per ml of PBI 221, four electric pulses of 300 voltages each, and 3% P E G in the electroporation buffer. Though the transformation efficiency in this experiment  106  chapter 4 protoplast isolation and electroporation  could not be compared with some published results due to the different assay approach, the efficiency obtained here was still considered to be reasonable. The curve of GUS activity after electroporation suggests that the pattern of GUS staining may reflect cell division activity in these cells (see also Jefferson et al. 1987). A reasonable transformation efficiency had been obtained by applying the similar electroporation conditions in white spruce (Bekkaoui et al. 1990), black spruce and jack pine (Tautorus et al. 1989), and Douglas-fir and loblolly pine (Gupta et al. 1988). A lower transformation efficiency from plasmid pBI 121 may due to its large size as compared to pBI 221, because both selectable marker genes were driven by the same promoter. By addition of methanol at 20% volume to a GUS reaction mixture, Kosugi et al. (1990) could enhance the activity originating from the introduced GUS gene, enabling more reliable assay for GUS expression and suppressing the endogenous GUS activity. This method should be tried in future experiments in Douglas-fir. Two weak bands representing 3-[* C] acetyl chloramphenicol (3-Ac CAP) 4  and 1-[* C] acetyl chloramphenicol (1-Ac CAP) products, respectively, were 4  detected by T L C (thin layer chromatography) assay from sample extracts 4 days after electroporation of Douglas-fir protoplasts with vector pCaMV.CAT. No products were detected from extracts 2 days after electroporation nor in controls. The low level of the products 3-Ac CAP and 1-Ac CAP may either result from the low transformation efficiency as indicated from GUS assay (4%) which limited these products to the few transformed cells, or from the long period needed for the occurrence of the first cell division from protoplast in Douglas-fir, which perhaps slowed and postponed the production of 1-Ac CAP and 3-Ac CAP within the  107  chapter 4 protoplast isolation and electroporation transgenic cells. Another possibility could be the method of grinding cells for extracts rather than sonicating cells as is done in many other experiments. In addition, the slow development of Douglas-fir protoplasts may also delay the production offirstdetectable Ac CAP products beyond 2 days after electroporation. The Ac CAP products could be higher after day 5, etc., and further experiments are necessary to confirm that. In contrast, transient C A T expression was obtained by T L C assay from protoplast extracts 24 hours after electroporation in both black spruce and jack pine (Tautorus et al. 1989). The time required for thefirstdivision of protoplasts was 2 and 3-4 days in these species, respectively. Some cell colonies derived from protoplasts electroporated with plasmid pBI 221 still exhibited GUS expression as detected by X-gluc substrate, indicating the possibility of stable transformation of GUS gene into protoplasts of Douglas-fir. This could make the regeneration of transformed plantlets feasible in this species. In conclusion, the results presented here indicated that both GUS and C A T genes were successfully introduced into protoplasts of Douglas-fir, and transient expression of each was obtained and confirmed by different assay approaches. This supports the view that electroporation is usable with coniferous species. Further investigation is needed to determine the optimal conditions for plasmid concentration, promoters, voltages applied for electroporation and other factors for enhanced GUS and C A T expression in Douglas-fir protoplasts after electroporation. Future experiments should aim at obtaining stable transformation and plantlet regeneration from transformed protoplasts.  108  SUMMARY  109  summary Cell division and cell colonies were obtained from cultured microspores of Douglas-fir (Pseudotsuga menziesii Mirb Franco) on medium supplemented with sucrose, cytokinin and auxin, but without medium salts. Embryogenic calli were initiated from both cultured mature and immature zygotic embryos of Douglas-fir on media supplemented with cytokinin and auxin. Embryogenic calli originated first from the radicle after 7 days and then extended to the hypocotyl region from cultured mature zygotic embryos; while embryogenic callus induction was observed after 4-7 days from either the embryo proper (embryo head), the entire embryo including suspensors, or just the hypocotyl depending on the developmental stage of the explants from cultured immature zygotic embryos. Secondary embryogenic calli production, and their subsequent subculturing, were typically required for the establishment of stable embryogenic callus lines for both cultured mature and immature zygotic embryos. The culture media determined the degree of organization of somatic embryos produced. Different capacity of somatic embryo production existed between embryogenic calli originated from mature explants and immature zygotic embryos. Somatic embryos at the precotyledon stage have been obtained in high frequency when Douglas-fir embryogenic calli were transferred onto low hormone or hormone-free B M media supplemented with 1% activated charcoal. Precotyledonary embryos were the most advanced stage obtained from this treatment. Cotyledon stage somatic embryos were obtained by transferring late proembryo or stage II somatic embryos onto hormone-free B M medium supplemented with low concentrations of abscisic acid. The timing of A B A exposure and the handling of the cultures markedly influenced the development of somatic embryos into cotyledonary embryos. The A B A level in the maturation  110  summary  media significantly affects the maturation of somatic embryos in this species. The optimal A B A levels were between 0.5 uM to 10 uM in this study. About 30 cotyledonary somatic embryos have been obtained. Trials for somatic embryo germination and conversion to seedlings are in progress. Cell suspension cultures of Douglas-fir were established by transferring 1 g of embryogenic calli from solid media to 20 ml of liquid modified MS medium (BM3 + CM) and have been maintained for over one year on this medium. Protoplasts were obtained from the suspension cultures by enzymic digestion using cellulase and pectinase. Cell colonies were regenerated from protoplasts of cell ; suspension cultures at a frequency of 0.1%. Protoplast regeneration proceeded with recovery of cell wall within 2 days, cells with two nuclei at 4 days, and cells with first division at 2 weeks. 11 % of the protoplasts had the potential for cell division and a viability of 90% as determined by Fluorescence Diacetate (FDA) staining. Thefl-Glucuronidase(GUS) gene and the chloramphenicol acetyl transferase (CAT) gene were successfully introduced into protoplasts of Douglasi-fir by electroporation with 3% polyethylene glycol (PEG) buffer. The viability of protoplasts was reduced from 90% to 70% by electroporation. Transient expression of the GUS gene in protoplasts was detected using X-Glucuronide substrate within 36-48 hours after electroporation, while the expression of C A T gene was detected using a T L C assay. As high as 4% of the electroporated protoplasts showed GUS expression. No GUS activity was observed in the electroporation controls. The detection of 3- and 1-acetyl chloramphenicol products was obtained from extracts 4 days after electroporation with plasmid pCaMV.CAT. Stable transformation of the GUS gene was observed in protoplast-derived cell colonies 2-3 months after  ill  summary  electroporation with plasmid pBI 221. Further research should be focus on the following directions: 1) improve the regeneration efficiency via somatic embryogenesis (a feasible alternative could result from cell suspension derived from embryogenic calli); 2) after electroporation and selection of protoplasts (cells), try to recover transformed calli and transformed plantlets, and confirm the stable transformation using molecular biology techniques such as southern blot and polymerase chain reaction (PCR); 3) studies of A B A regulation on somatic embryogenesis in this species using molecular biology techniques.  112  Appendix A Media Components COMPONENTS  KNOS  NLN  MS  OCR  (m|/L)  (m/L)  BM  («|/L)  LP  («|A)  125.0  SJ.O  »7O0  170.0  340.0  I2S.0  23310  1900.0  34O0  1940.0  2750  1650.0  4oao  1200.0  NH4NOJ M|S04 7H20  I2S.0  185.0  370  37O0  37O0  H3B03  tao  3.1  62  62  06J  Za EDTA  4.05  EDTA  19.0  J7J  )7J  Nt2EDTA  37J  1165  FeS04 7H20  m  13J  27J  27J  14.0  0.415  013  0J)  075  KJ Ni2Mo042H20  0.25  0125  0L2S  0.25  0025  CuS04 5H20  oars  0.0125  0025  025  0.025  C o 0 2 6H20  0.025  0012  0025  0.025  00025  O02S  NrCB  $56.0  C»(N03)2 4H20 ZnS04 7H20  10.0  4JI3  1616  16 04  45O0  L-Gluiamine L A ) mine  0.05  L-CyjitniixHa  0.02  L-Apmiot  aoi  L-Ltucioc  0.0!  L-Ph«nyU)aninc  O01  UTvrounc  0.01  Glycine  20  100  20  20  20  D-SuaoM  3420.0  D-CluroK  110  L-AnbinoK  1$0  Nicotinic l a d  so  ii  Oi  Oi  Pyndount HC1  0J  2i  Oi  Oi  10  Mcso-mcniiol  1000  10000  1000  2000  100  ThiimintHG  Oi  10.0  0.1  10  50  Bioiin  00$  Folic arid  Oi  MnS04 H20  25.0  11 153  22306  22J  2.2  220.0  4400  85 0  180.0  CaCU  113  2.0  Appendix B Abbreviations 2,4-D  2,4-dichlorophenoxyacetic acid  ABA  abscisic acid  BAP  6-benzylaminopurine  Carrier D N A  sonicated calf thymus DNA  CAT  chloramphenicol acetyl transferase  EC  embryogenic callus  ESMs  embryonal suspensor masses  FDA  fluorescence diacetate  GUS  5-glucuronidase  HEPEs  N-(2-hydroxyethyl)piperazine-N'-(2 ethanesulfonic acid)  MES  2-(N-cyclohexy lamino) ethanesulfonic acid  MUG  4-Methylumbellifery 1-B-D-Glucuronide  NAA  naphthyl acetic acid  PEG  polyethlene glycol  SEC  secondary embryogenic callus  TRIS  tris (hydroxymethyl) aminomethane  X-gluc  5-bromo-4-chloro-3 indolyl glucuronide  114  Bibliography Aboel-Nil, M.M. 1987. 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