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Storage protein gene expression in zygotic and somatic embryos of interior spruce Flinn, Barry Stanley 1992

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STORAGE PROTEIN GENE EXPRESSION IN ZYGOTIC AND SOMATIC EMBRYOS OF INTERIOR SPRUCE by BARRY STANLEY FLINN  B.Sc., M.Sc.,  Queen’s University, Queen’s University,  1982 1987  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES Department of Botany  We accept this thesis as conforming to the required standard  Signature(s) removed to protect privacy  THE UNIVERSITY OF BRITISH COLUMBIA May 1992  ©  Barry Stanley Flinn  In presenting this thesis in  partial fulfilment of the requirements for an advanced  degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department  or  by  his  or  her  representatives.  It  is  understood  that  copying  or  publication of this thesis for financial gain shall not be allowed without my wiitten permission.  Signature(s) removed to protect privacy  (Signature)  Department of The University of British Columbia Vancouver, Canada  Date  DE-6 (2/88)  Pla..y )  192_-  11  ABSTRACT  Storage proteins from interior spruce complex)  were  identified,  partially  (Picea glauca/engelmanhi  characterized  and  used  as  markers to compare the developmental fidelity between zygotic and somatic  embryos.  The major  storage proteins  expressed  embryo types had molecular weights of approximately 41, 24 and 22 kD.  in both 35,  33,  The 41 kD protein was buffer and low salt-soluble,  whereas the 35—33 kD and 24-22 kD proteins were high salt-soluble and  disulfide  linked.  All  isoelectric variants. characteristics,  of  the  proteins  possessed  Based on solubility and disulfide linkage  as  well  as  cDNA  sequences,  these  proteins were homologous to angiosperm vicilin-type legumin-type Somatic  (35-33 kD,  embryos  several  of  24-22 kD) different  storage  (41 kD)  and  storage proteins. genotypes  matured  accumulated significant levels of storage protein, higher than levels found in zygotic embryos.  on  40  jM ABA  similar to or  Somatic embryos on  10 M ABA displayed initial storage protein accumulation, but the levels did not reach those  found  in  zygotic  embryos  or  somatic  embryos matured on 40 jM ABA. Zygotic embryos and somatic embryos differentiated on 40 ,M or 10  M  ABA  displayed  differential  storage  protein  accumulation,  with the legumin-type proteins apparent before the vicilin-type, although  all  development. of  storage  showed  major  accumulations  during  cotyledon  Zygotic embryos displayed a rapid, transient period protein  accumulation,  with  maximum  storage  protein  levels attained at least 1 month prior to mature seed shed. contrast,  In  somatic embryos differentiated on 40 M ABA displayed a  iii more prolonged, were  still  Somatic  gradual  on the  storage proteins,  increase after 9 weeks  embryos  on  but  these  proteins,  accumulation of  10  jM  were  ABA  of maturation on ABA.  initally  rapidly  which  accumulated  degraded  as  the  storage embryos  germinated precociously. Analysis of storage protein mRNA5 indicated they were present by torpedo stage in zygotic embryos and somatic embryos matured on 40 .LM and 10 I.LM ABA. during  development,  In all cases,  with those  prior to those of vicilin.  of  the transcripts increased  legumin  reaching high  levels  Transcript levels in zygotic embryos  increased during cotyledon development and then declined rapidly to very  low levels  at  least  1 month prior to mature seed shed.  Somatic embryos on 40 ,.LM ABA displayed high transcript levels for a prolonged period,  and these were still present after 9 weeks,  although they had declined to 50% of maximum levels. of on  storage protein transcripts also appeared 10  jM  although  ABA,  they  but were  declined still  during  in somatic embryos  precocious  detectable  after  Low levels  germination,  several  weeks  of  precocious germination. Osmotic medium  stress,  caused  containing  15%  by the  mannitol,  culture induced  storage protein transcript accumulation. by  inclusion  suggesting  of  that  ABA biosynthesis.  the  the  ABA-biosynthetic  increase was  of  due  to  somatic  storage  embryos  protein  on and  This could be inhibited inhibitor, osmotic  fluridone,  stress—induced  iv TABLE OF CONTENTS  Page ABSTRACT  ii  TABLE OF CONTENTS  iv  LIST OF TABLES  vi  LIST OF FIGURES  vii  ACKNOWLEDGEMENTS  x  1.  INTRODUCTION  1  2  LITEP.ATtJIE REVIEW 2.1. Somatic embryogenesis in spruce 2.1.1 General introduction 2.1.2. Somatic embryo induction 2.1.3. Somatic embryo maturation 2.2. Protein reserves during embryogenesis 2.2.1. Regulation of angiosperm storage protein accumulation 2.2.2. Spatial patterns of storage protein accumulation 2.2.3. Storage protein accumulation during conifer embryogenesis 2.2.4. Comparison of storage protein accumulation in zygotic and non—zygotic embryos 2.3. Abscisic acid, osmotic stress and embryo development 2.4. Summary  5 5 5 5 9 13  .  3.  MATERIALS AND METHODS 3.l.Zygoticembryomaterial 3.2.Somaticembryomaterial 3.3. Protein extraction and electrophoresis 3.4. Protein body isolation and analysis 3 . 5 . Microscopy 3.6. Antibody production 3.7. Immunoblotting 3.8. Chlorophyll analysis 3.9. In vivo protein labelling and immunoprecipitation 3.10. 41 kD storage protein cDNA isolation 3.11. Generation of deletion constructs for sequencing 3.12. DNA sequencing 3.13. Genomic DNA extraction, electrophoresis and blotting 3.14. RNA extraction, electrophoresis and blotting  13 17 18 19 21 30 32 32 33 38 40 41 42 42 43 44 46 49 53 55 57  V  Page 3.15.  4  .  5.  6.  cDNA probe production and hybridization to blots  RESULTS 4.1. Identification and characterization of zygotic embryo storage proteins 4.2. storage protein accumulation during zygotic embryo development 4.3. Identification and characterization of somatic embryo storage proteins and comparison with zygotic embryo storage proteins 4.4. storage protein accumulation in somatic embryos and comparison with zygotic embryos 4.5. Identification and characterization of a cDNA encoding the 41 kD storage protein 4.6. RNA gel blot analysis of storage protein gene expression in zygotic embryos and somatic embryos differentiated on 40 M and 10 /iM ABA 4.7. Analysis of storage protein expression in somatic embryos in response to osmotic stress DISCUSSION 5.1. Interior spruce storage proteins 5.2. Zygotic embryo storage protein expression 5.3. Somatic embryo storage proteins 5.4. Developmental expression of storage proteins in somatic embryos on 40 .LM and 10 M ABA 5.5. Osmotic stress and storage protein gene expression 5.6. Concluding statement LITERATURE CITED  59 61 61 71  78 93 101  116 126 136 136 138 143 146 153 157 159  vi LIST OF TABLES  Table  Page  1.  Somatic embryo developmental stages on 40 M ABA  36  2.  The presence (+) or absence (—) of various storage proteins during zygotic embryo development  79  Major storage protein distribution in EK1O zygotic and W29 somatic embryos  94  Developmental changes in storage protein mRNAs in zygotic embryos as determined by scanning densitometry of RNA gel blots  120  3.  4.  5.  6.  7.  Chlorophyll content (mg/g FW) of Stage 4—6 and 9 week ABA somatic embryos of genotypes W29 and W70  ....  121  Developmental changes in storage protein mRNAs in somatic embryos differentiated on 40 jM ABA as determined by scanning densitometry of RNA gel blots  125  Developmental changes in storage protein mRNAs in somatic embryos differentiated on 10 ,AM ABA as determined by scanning densitometry of RNA gel blots  129  vii LIST OF FIGURES  Page  Figure 1.  Coomassie—stained SDS—PAGE of embryo proteins  63  2.  Coomassie-stained SDS-PAGE of zygotic embryo protein body extracts under reduced (A), non— reduced (B) and two-dimensional SDS-PAGE of non—reduced extract under non—reducing conditions followed by electrophoresis under reducing conditions  66  Coomassie-stained SDS-PAGE of protein body samples extracted under different conditions  68  Light micrograph of a longitudinal cotyledon section from a late maturation stage zygotic embryo, stained by the periodic acid—Schiff’s technique and counter—stained with aniline blue b lac]c  70  Silver—stained two—dimensional electrophoretograms of zygotic embryo protein body extract examined using pH 5-8 ampholytes (A) or pH 3-10 ampholytes (B)  73  Silver—stained two—dimensional electrophoretograms of 3 representative zygotic embryo stages collected during the summer of 1988  75  Coomassie-stained SDS-PAGE of developmental stage changes in total protein during EK1O zygotic embryogenesis  77  Abscisic acid-dependent developmental profile of genotype W29  81  9.  Examples of somatic embryo developmental stages  83  10.  Coomassie-stained SDS-PAGE of zygotic embryo, zygotic protein body, 9 week ABA somatic embryo and somatic embryo protein body extracts  86  Coomassie-stained SDS-PAGE of somatic embryo extracts under reduced (A), non—reduced (B) and two—dimensional SDS—PAGE of non—reduced extract under non—reducing conditions followed by electrophoresis under reducing conditions (C)  88  3.  4.  5.  6.  7.  8.  11.  viii Figure  Page Coomassie-stained SDS-PAGE of somatic embryo protein body samples extracted under different conditions  90  Silver—stained two—dimensional electrophoretograms of somatic embryo protein body extract examined using pH 5-8 ampholytes (A) or pH 3-10 ampholytes (B)  92  Coomassie-stained SDS-PAGE of total proteins for two zygotic embryo genotypes and different somatic embryo genotypes derived from them  96  Silver—stained two—dimensional electrophoretograms of total proteins from stage 4-9 (A) and stage 3-2 (B) somatic embryos differentiated on 40 jM ABA  98  Coomassie-stained SDS-PAGE of developmental stage changes in total somatic embryo protein of genotype W29 matured on 40 jM ABA  100  Relative quantification of 41 kD protein immunoblots during zygotic and somatic embryo development  103  Relative quantification of 24 + 22 kD protein immunoblots during zygotic and somatic embryo development  105  19.  Sequence analysis of the spruce 115A.0 cDNA clone  108  20.  Amino acid sequence comparison of 115A.0 with other angiosperm vicilin—type storage protein sequences  110  Pulse:chase labelling of late cotyledonary somatic embryos differentiated on 40 j.LM ABA  113  DNA gel blot analysis of spruce DNA probed with 115A.0 cDNA  115  changes in total proteins and storage protein mRNA during zygotic embryo development  118  Changes in total proteins and storage protein mRNA during somatic embryo development on 40 M ABA  123  Changes in total proteins and storage protein mRNA during somatic embryo development on 10 jM ABA  128  Effects of culture on media containing no growth regulators, 40 M ABA, 15% mannitol or fluridone on somatic embryo development  131  12.  13.  14.  15.  16.  17.  18.  21.  22.  23.  24.  25.  26.  ix Figure 27.  Page changes in total proteins and storage protein mRNA in somatic embryos exposed to no growth regulators, 40 M ABA, 15% mannitol or fluridone  134  x ACKNOWLEDGEMENTS I would like to acknowledge the financial support provided by the  B.C.  Science  Research  Council  Forestry  Canada  Council of and  GREAT  Canada, the  Award as  B.C.  program  well  as  Ministry  of  and  the  National  funds  provided  Forests  through  by the  Forest Resource Development Agreement. Of  the  initially  many  people  I  would  like to thank my parents,  throughout  the  years,  and  I  to  like  would  acknowledge,  for their like  to  love  extend  I  and my  would  support  heartfelt  affection and gratitude to Karen Jackson for her friendship and companionship over the past 1½ years. to  the  members  Research,  of  the  especially  Webster and Dr. discussions  Wayne  the  past  Biotechnology  Lazaroff,  Craig Newton,  over  Susanna Grimes,  Forest  Many thanks are extended  Stuart  Centre  Pritchard,  for their encouragement,  few years.  I  am  at  truly  B.C. Fiona  help and  indebted  to  Stephanie Mclnnis and Jocelyn Steedman for their  help with media preparation during this project. I would like to thank the members of my supervisory committee (Drs. Carl Douglas, Anthony Glass and Ben Sutton)  for their input  and suggestions during this work.  I would like to thank my co  supervisor,  for  glasses extend  of my  Dr.  lain  wine.  E.P.  Last,  appreciation  Taylor, but  to  certainly  my  lab  mate,  being a great friend and supervisor, suggestions LAST!!!  during  the  discussions  course  of  not Dr.  least, Dane  and I’d  numerous like  Roberts,  to for  and for his discussions and this  project.  FINISHED  AT  1 1.  INTRODUCTION  Forestry is the primary natural resource industry in British Columbia and throughout Canada. the  second  largest  standing  Spruce species timber  (Picea spp.)  volume,  after  provide the second largest log source in B.C. Industries of B.C.  1987).  important  conifers  effort to  ensure  Tissue  culture  prospect of genotypes  is  essential  an  are  widely  of  of  believed  any  national  harvested trees.  to  hold  the  achieving the required mass propagation of  (Cheliak  and  and  (Council of Forest  part  long term replacement  methods  pines,  The propagation of these economically  thus  the  form  Rogers  1990,  Karnosky  1981)  best  superior  which  will  provide the opportunity to maintain and even enhance the quality of  the  forest  embryogenesis rapid  production of  conventional 1990).  provides  of  large  genetic  of  culture  tree  propagation  of  that  breeding  plants  have  programs  embryogenesis  propagation scale  numbers  gains  somatic  of  large  by  somatic  via  production  been  up”  of  fast  economic  because the  by  Rogers  and  the  culture,  “bulk  the  achieved  (Cheliak  improves  tissue and  and  allows  it  selected  It also offers the potential for the genetic engineering  single  production toxin,  of  forest  Use  feasibility  Tissue  (embryo differentiation from somatic cells)  exploitation  stock.  stock.  cells, of  such  for  the  traits  anti—spruce  as  herbicide  budworm  resistance  Bacillus  and  thuringiensis  and their regeneration to plants.  Now that the technologies for somatic embryogenesis have been developed for spruce Hakman  and  von  (Attree et al.  Arnold  1988,  1990ab,  Krogstrup  Becwar et al.  1990,  Lelu  and  1989,  Bornman  2 1990, Roberts et al. 1988, al.  1990ab, Tremblay 1990, von Arnold and Hakman  Webster et al.  1990,  et al.  1990)  and other conifer species  Bourgkard and Favre 1988,  1989,  von Aderkas  Gupta and Durzan 1987, and Bonga  1988),  Durzan and Gupta 1987,  artificial  somatic  embryos  that  resembled their produced  less  seed  zygotic  vigorous  ensure that  emphasis has moved to  (Redenbaugh  produced  et  vigorous  counterparts plants.  Finer  Nørgaard and Krogstrup 1991,  the derived plants are both healthy and vigorous. alfalfa  (Becwar et  al.  1986)  germinants  than did  This  Evaluation of  has  more  the  led  showed  that  closely  embryos  to  the  that  current  wisdom that the potential of somatic embryos to produce vigorous plants  can  be predicted  physiological  and  by determination  morphological  of  similarity  their to  biochemical, zygotic  normal  embryos.  are  The periods  of  critical  for  germination  and  synthesis and deposition of zygotic  embryogenesis,  development  of  robust  conifer embryos, proteins and lipids prominent reserves et  al.  and  1989),  somatic  concerning (see  for  example  development,  (triglycerides)  However,  are the most  Cyr et al.  development  Janick et  al.  in  1991,  there conifer  mature  In  1991,  excellent biochemical markers  embryogenesis.  biochemical  seed  seedlings.  (Bewley and Black 1985,  and are  storage material  of  Flinn  zygotic  few  reports  zygotic  embryos  are  Johnson et  al.  1987)  and  only recently, during the course of this present study have other labs  reported  identification  storage proteins al.  1991,  and  characterization  of  conifer  (Gif ford 1988, Gif ford and Tolley 1989, Green et  Misra and Green 1990, Misra and Green 1991).  Clearly,  3 we need to understand the biochemical processes that occur during conifer  zygotic  embryogenesis  before  we  can  evaluate  conifer  somatic embryos. The involvement of plant growth regulators in the control of storage reserve deposition during angiosperm seed development has been  well  documented  (Bewley  abscisic acid  (ABA)  synthesis  deposition  1985,  Finkelstein  storage 1981,  and  protein  et  in  al.  1985)  1990).  mature,  and  embryos  (Ammirato  1985).  reduces  Endogenous  embryo  1986,  Roberts  Hakman 1988, Webster et al.  and Black  ABA  can  induce  Crouch  and  Sussex  the addition of ABA to  differentiation the  occurrence  1974,  (Bewley  exogenous  Furthermore,  increases  the  studies  and  seeds  (Barratt  somatic  germination,  While most  developing  accumulation  during  precocious  Black  levels increase during the period of reserve  Goffner et al.  cultures  and  number  of  et  of  prevents  embryos  that  morphologically—abnormal  al.  l990a,  von  Arnold  and  1990).  have  been  carried  out with  angiosperms,  little is known about the role of ABA in conifer embryogenesis. Most  of  the  reserve  substance  temporally (Avjioglu Krochko  and/or and  et  information (1989)  and  contained Hakman  angiosperm  and  is  deposition  1989,  1989,  in  Crouch  Stuart  available  Cyr et al. less  have  quantitatively  Knox  al.  studies  from  (1991)  triglyceride  von Arnold  (1988)  et  shown  that  somatic  from  those  1982, al.  found that reserves  embryos in  Shoemaker  No  although spruce  than  et  and von Arnold  1987,  comparable  somatic  and  embryos  al.  Feirer  zygotic  of  differs  zygotic  1988).  conifers,  patterns  et  embryos  embryos  Hakman  al.  and  (1988)  4 showed  that  spruce  somatic  embryos  storage  contained  bodies and protein profiles similar to those of (Hakman  al.  et  developmental  1990).  comparisons  These of  studies  storage  zygotic embryos  did  protein  protein  not  provide  expression  gene  between zygotic and somatic embryos, nor did they report the role of ABA on these reserves. The hypothesis of this present thesis is that somatic embryos matured  in  the  resemble their storage  protein  than somatic  presence  of  high,  adequate  levels  of  ABA  will  zygotic counterparts with respect to patterns of gene  expression  and  overall  embryos matured using low levels  this hypothesis,  embryo  development  of ABA.  the goals of this study will be to:  1)  To test Identify  the major storage proteins of spruce zygotic and somatic embryos; 2)  compare the developmental expression of these proteins at the  protein  and  gene  embryogenesis and;  transcript 3)  level  during  zygotic  and  somatic  Examine the effect of high and low levels  of ABA on the formation of these reserves during spruce somatic embryo development.  5 LITERATURE REVIEW  2.  SOMATIC EMBRYOGENESIS IN SPRUCE  2.1.  General introduction  2.1.1.  advances  The  in  culture  tissue  conifer  past  the  during  6—7  years have led to the production of somatic embryos in species of  Larix (Klimaszewska 1989, Nagmani and Bonga 1985, von Aderkas and Bonga  von  1988,  Aderkas Schuller  1991,  Krogstrup  Krogstrup  1985,  Tremblay  1990,  von  Woodward  1988,  Webb  (Becwar et al.  and  out  et  al.  1991,  Favre  1991,  1989,  Gupta  et al.  1989,  Lamé  Jam  was  the  of  the  interior  and  Pinus  1990),  al.  et  and  Arnold  von  Finer et al.  most  Since  species  complex)  glauca/engelmanhi  1990,  and Sequoia  (Durzan and Gupta 1987)  1988).  Picea  Tautorus et al.  Webster  1989,  Becwar et al.  1990,  1988,  Hakman  Gupta and Durzan 1987,  with  al.  et  Bornman  and  Lelu  1988,  and  Pseudotsuga  and David 1990),  carried  al.  et  Arnold  1990,  and Durzan 1986b,  (Attree  Picea  1989),  1990ab, Roberts et al.  Roberts et al.  (Bourgkard  al.  et  and  (Nørgaard  Abies  1990),  1989, Gupta and Durzan 1986a, Hakman and von  1990a, Becwar et al. Arnold  al.  et  object  of  work  has  spruce  this  been  (Picea  study,  the  following literature review deals only with spruce. 2.1.2.  Somatic embryo induction  Somatic Explant  age  embryo plays  induction a  major  depends  role  in  on the  a  variety  ability  of  to  factors.  respond  to  morphogenic stimuli in culture and somatic embryogenesis has been obtained primarily and  Fowke  which  1991).  specific  from immature embryos There  is  (for review see Attree  commonly a window of  developmental  stages  are  more  competence, amenable  in to  6 induction, with  and  a  increasing  1987,  embryo  Webb et al.  embryos  are  attributed changes  to  capacity  age  1989).  the  that  declining  most  (Hakman  et  responsive.  al.  during  The  1985,  and  Lu  Thorpe  early cotyledonary  loss  competence  of  physiological  embryo  induction  embryogenic  Generally,in Picea,  morphological, occur  for  biochemical  and  (Roberts  maturation  is  et  al.  1989). Culture media commonly used for induction are modifications of the LP medium (von Arnold and Eriksson 1981) or  half-strength  Hakman  1988,  (Hakman  and  Webb et al.  von  1989),  appears to be species dependent embryogenesis Murashige  and  has  Skoog’s  Krogstrup et al. Tremblay  and  formulation  also  1988)  megagametophyte.  on  1986)  and  (Tautorus et al.  obtained  using  (1962)  medium  (Gupta  amounts  and  Arnold  and  1990).  modified and  Somatic forms  Durzan  of  l986a,  and Litvay’s (1985) medium (Tremblay 1990, The  l99lab).  the  Since  angiosperm  Walker and Sato 1981,  von  although optimal media strength  latter  composition  of  total  reduced  and  N,  N,  (David et al.  eiubryogenesis  (Gleddie  Wetherell and Dougall 1976),  ratios  of  medium  chemical  critical for conifer organogenesis al.  1988,  been  Tremblay  based  Arnold  used at either full-  nitrogen  in  the  is  seed  conifer :NH 3 N0 4  1982, et  a  are  Flinn et al.  1983,  the different  different  media  formulations are probably an important factor. Early frequencies However,  studies were  more  with  mature  lower  than  recently,  embryos from  induction  revealed  immature  frequencies  that  embryo similar  induction explants. to  those  from immature embryos have been obtained (Tremblay 1990, Verhagen  7 and Wann 1989,  von Arnold 1987).  The expansion of the window of  competence to include mature embryos has been obtained by various media manipulations. used  (Tautorus  1989,  von  et  Arnold  Half—strength media formulations are often  al.  1990,  1987,  manipulation of 3 NO 4 NH  Tremblay  von  Arnold  levels,  Verhagen  1990,  and  Woodward  Wann  1989).  The  pH and amino acid composition has  substantially enhanced induction from mature embryos  al.  and  (Tautorus et  1990, Verhagen and Wann 1989, von Arnold 1987). Somatic embryogenesis has also been achieved from germinated  seedlings ranging from several days et al.  1990)  Arnold  1991).  cultural  to  3-5 weeks  old  (Lelu and Bornman 1990,  (Attree et al.  Mo  l990a,  Lelu  and von  Induction was achieved by manipulating media and  environment  conditions.  Some  studies  have  suggested  that explant pretreatment with cytokinin enhanced the embryogenic response others  (Lelu  found  and  Bornman  1990,  Lelu  this not to be beneficial  et  al.  1990),  (Attree et  al.  although 1990,  Mo  and von Arnold 1991). While  explant developmental  stage  and media  affect the capacity for embryogenic induction, also  involved.  subsequent Arnold  Cold  induction  1985,  Hakman  from and  of  storage the Fowke  composition may  other factors are  immature  excised  embryos  1987),  although  cones  enhanced  (Hakman  and  Tremblay  von  (1990)  reported that low temperature exposure of mature excised embryos strongly  inhibited  embryogenesis.  storage  significantly  1990).  Furthermore,  decreased  The  duration  embryogenic  of  mature  potential  the vigour of the explant  seed  (Tremblay  (Tremblay  1990),  8 as well  as  its genetic background  Tremblay 1990, Webb et al. Auxin—cytokinin embryogenesis.  used,  other  cytokinins with Krogstrup  et  (BA)  combinations  are  (see  Attree  and  workers  have  found  2,4-D to be  al.  1988).  effective  As  1988,  are important. used  to  While 2,4-dichiorophenoxyacetic acid  —benzyladenine 6 N commonly  1989)  (Hakman and von Arnold  well,  Fowke  initiate  (2,4-D)  1990)  are  combinations  (Gupta  somatic  embryos  most  of  and Durzan  other l986a,  have  induced using other auxins in combination with cytokinins  and  been  (Lu and  Thorpe 1987, Verhagen and Wann 1989, von Arnold and Hakman 1988). Another  important  component  included  the  in  tissue  culture  medium is the carbohydrate source, with sucrose the most commonly used. 3%,  The  optimum sucrose  concentration varies  depending on the basal medium  von Arnold 1987)  influence  found that immature  1%  and  1986,  (von Arnold and Hakman  and may also vary between different seed sources  used for explants also  between  (Webb et al.  the  morphogenic  low sucrose embryos,  1989).  (1%)  while  The sucrose level used can  response.  Webb  et  al.  (1989)  favoured embryogenic induction from levels  higher  (3-4%)  favoured  the  induction of adventitious shoots. The Kvaalen  culture and  environment during  von  Arnold  (1991)  also  induction  is  reported  that  important. gaseous  the  environment of the culture vessel affected induction.  Low 02 in  combination with full strength medium stimulated induction, while on  half-strength medium,  high  also promoted embryo induction. stimulation  of  embryogenic  was  2  better.  High  2 CO  levels  This effect was attributed to a  tissue  growth  or  to  an  inhibitory  9  effect by CO 2  on ethylene synthesis,  since ethylene is known to  inhibit the growth of embryogenic tissue (Kumar et al.  1989).  It has been suggested that light inhibits embryogenic tissue induction. was  Von Arnold  better  (Hakman 1989).  for  induction  Fowke  and  reported that culture in the dark  (1987)  and most workers  1987,  Gupta  and  follow this  Durzan  However, Verhagen and Wann (1987)  l986a,  practice  Webb  et  al.  reported no significant  differences between light and dark treatments on induction. 2.1.3.  Somatic embryo maturation  The consist numerous  embryogenic of  small,  single cells  proembryo—like  cytoplasmic embryos 1988,  cultures  described and cell  structures  with  (Hakman et al.  Webb et al.  1989).  1987,  in  the  above  aggregates, small,  studies  as well  opaque,  as  densely  von Arnold and Woodward  of embryogenic tissues under  Culture  conditions used for induction results in the continued growth and proliferation of these proembryonal structures and the prevention of the  subsequent development. transfer  maturation  to  Further embryo development  conditions  involves  cell  allowing  division  and  requires  maturation. the  Embryo  expansion  of  the  embryonal cells to form globular embryos, which then elongate and develop cotyledon primordia, elongation  (Dunstan  et  al.  followed by cotyledon and hypocotyl 1988,  Hakman  and  von  Arnold  1988).  Developmental progress to maturation or to precocious germination depends  on  the  manipulation  of  media  and  other  culture  spruce  somatic  conditions. In embryos  all past  cases, the  the  continued  proembryo  stage  maturation requires,  of at  the  least,  the  10 reduction or removal of embryo—inducing growth regulator levels. Some  studies  development 1990), and  have  further  Lu and Thorpe 1987,  Tremblay  2,4—D  levels  although embryo quality and yield were low.  the  However,  have  use  of  been  ABA  obtained  alone  Lu and Thorpe  each)  increased  reaching  maturity  Furthermore, agents) above  in  the  (Becwar  Better yield  absence  combination  in  of  2,4—D  with  an  and  auxin.  reported no improvement of somatic of ABA and an auxin (1 J.LM  Low levels  number  buthionine  of  embryos  et  al.  produced  1989,  sulfoximine  (an  and  the  al.  et  Jam  number  inhibitor  1988). reducing  of  tripled somatic embryo maturation in combination with the growth  regulators  concentrations embryo  the  or  (1987)  embryo development by ABA.  (5—16  maturation  M)  (Jam have  (Attree  reduce  regeneration  maturation  and  (von  maturation of  interior spruce  level  there  of  others  Krogstrup  1990).  The  ABA levels  embryos,  most  of  of  ABA  on  lower 1990b).  quality  and  somatic embryos revealed that the  (IBA)  in the range of  which  1988,  and  al.  et  differences  with  germinated  40 in  and  ABA  60  (0.1-10  enhanced  embryo  (Roberts et al.  10-20 jiM resulted in  precociously  M,  maturation  low levels  cotyledon development and morphology  1990a).  promote  al.  et  1988)  (Attree  incorporation of  acid  to  ABA  in excess of this range Hakman  effects  genotype—related  indole-3-butyric  production,  the  by  for most genotypes was between  were  (Webster et al.  and  values  studies  although  levels  Higher  1988).  used  but  Arnold  Comprehensive  optimum ABA  been  1990b,  germination on  al.  et  al.  et  von Arnold and Hakman 1988),  jiM)  for  reduced  (Gupta and Durzan 1986a,  maturation  with  used  (Roberts  fewer et  al.  11 1990a). used  It is evident that a wide range of ABA levels have been  for  number  maturation.  of  et  al.  and  the  Boulay  subcultures  on ABA  (1988)  reported  concentration  that  the  required to  maximize somatic embryo recovery was dependent on the number of previous subcultures under proliferation conditions. In addition to ABA,  ABA analogues have been tested for their (Dunstan et al.  effects on spruce somatic embryo maturation 1991).  Of  those  tested,  abscisyl  alcohol  produced  1988,  results  similar to ABA, while the others were inhibitory. Most  studies  Prolonged Arnold  exposures  and  elongation  have  Hakman  utilized been  1988),  (Boulay  development (1991)  have  et  (Dunstan  et  ABA  reported  inhibit  al.  1988)  al.  exposures to  of  cause  hypocotyl and  1988).  weeks.  swelling and  result  However,  4—5  in  (von  cotyledon poor  Dunstan  root  et  al.  reported that 9 weeks of ABA exposure stimulated epicotyl  development in the resulting plantlets. While ABA influences embryo maturation, carbohydrates,  are also important.  found that 90 mM  (3%)  sucrose  (120-150  levels  Von Arnold and Hakman  that  development  (Lu  6%  mN;  Tremblay l991a). embryo (199la)  sucrose  and  maturation  (1988)  sucrose was optimal for development. 4-5%)  stimulated  development but repressed further maturation. reported  such as  other factors,  Thorpe  was 1987,  optimal  embryo  Other workers have  for  Tremblay  early  High  somatic  1990,  embryo  Tremblay  and  Individual carbohydrates may act differently on in  different  species.  Tremblay  and  Tremblay  reported that 6% fructose was more effective than glucose  12 or sucrose for maturation in red spruce, whereas in black spruce all three carbohydrates were equally effective at the 6% level. Some  of  the  carbohydrate  replaced  by  an  Tremblay  l991a),  osmoticum  supplied during maturation may  (Lu  indicating  and  that  maturation is partly osmotic. levels  of  osmoticum  formation,  and  that  cotyledonary stage. osmoticum  (2-6% ABA  doubled  the  mannitol)  (1991)  for  ABA,  on  reported that low globular  embryo  development  to  the  high levels of  could replace ABA as an inhibitor of  Furthermore, followed  production  and  effect  carbohydrate  promoted  required  Tremblay  1987,  After cotyledon development,  precocious germination. with  the  Roberts  was  (13-20% mannitol)  combination  Thorpe  be  of  by  late  a 1 week mannitol pulse in maturation  on  cotyledonary  ABA  stage  alone, embryos  (Roberts 1991). The  culture  extent of  environment  embryo development.  by combinations of low 02 tM ABA  and high CO 2  influence  embryo  (60 M)  compared to darkness Thus,  This  with  the  stimulated  effect was  ABA levels tested.  a  affects  on medium containing 7.6  1991).  development,  favouring the production of  also  Embryo maturation was  (Kvaalen and von Arnold  evident at the higher  embryo  during maturation  as  Light can also  maturation  greater number  not  in  the  of mature  light embryos  (Tremblay and Tremblay l99lb).  while several factors must be considered during somatic  induction  and maturation,  it  is  possible to  obtain well  developed spruce somatic embryos from a number of species. capability  provides  the  applications  to  species,  spruce  potential as  well  for as  This  biotechnological a  system  to  study  13 factors  that  affect  conifer  embryo  development  without  complications that arise from the effects of embryo excision from the seed, and the presence of the surrounding maternal tissues. PROTEIN RESERVES DURING EMBRYOGENESIS  2.2. 2.2.1.  Regulation of angiosperm storage protein accumulation  Most  mature  seeds  contain  protein,  lipid  and  carbohydrate  reserves which are used by the embryo during germination and post germination processes. and legume reserves,  Most studies have concentrated on cereal  due to their economic importance.  However,  little emphasis has been placed on the study of the major conifer reserve  materials.  reserves,  Since  proteins  are  prominent  conifer  seed  they represent useful biochemical markers to study and  compare development in zygotic and somatic embryogenesis. Storage proteins are synthesized during embryo development and are degraded during germination to supply amino acids, and carbon skeletons to the developing seedling.  nitrogen  These deposits  occur in distinct protein bodies and are confined largely to the embryo  and  surrounding  gymnosperms).  Many  storage  tissue  storage proteins  (megagametophyte  undergo  in  post—translational  modifications during deposition to convert them to their correct size for deposition (MUntz 1989, Storage  protein  expansion phase, and  desiccation  storage  protein  division phase  synthesis  Shotwell and Larkins 1989).  tends  to  be  limited  to  the  cell  following cell division and prior to maturation (Bewley  and. Black  accumulation (Domoney et  al.  has  1985).  been  1980),  Some  noted but  pea  during  it was  not  legumin the  cell  known  if  this occurred in dividing cells or a relatively few non-dividing  14 cells. that  Recent work with pea, using in situ hybridization, storage  protein  mRNAs  only  mitotic activity (Hauxwell et al. Most  seeds  accumulated  cells  in  showed lacking  1990).  contain more than  one class  of  storage protein,  each of which has a distinct temporal accumulation pattern. rapeseed,  the 2S protein, napin,  and the uS protein,  In  cruciferin,  start to accumulate during early embryo development,  with napin  detectable  and  1981,  slightly  Murphy  between  5  et  and  al.  7  earlier 1989).  weeks  Napin  (Crouch  proteins after  accumulate which  ends  when  the  embryo  Sussex rapidly  accumulation  (Crouch and Sussex 1981,  accumulation  begins to decline,  These  post—anthesis,  slows down and levels off 1989).  cruciferin  than  Murphy et al. water  content  while cruciferin accumulation continues until  seed maturity (Crouch and Sussex 1981). In pea, day  accumulation of the 7S protein,  earlier than that of the  first 1987),  few  days,  vicilin  llS protein,  synthesis  vicilin, legumin.  predominates  During the  (Boulter  reaches a maximum by 14/15 days after flowering  then declines.  begins one  et  al.  (DAF)  and  During the period from 18/19 to 20 DAF,  legumin  synthesis and accumulation peaks and remains constant. In soybean,  the 7S 13—conglycinin  —  and cL’—subunit proteins  are detectable 18 to 20 days after anthesis  (DAA),  while the liS  glycinin subunit proteins start to accumulate between 19 DAA. until 1981).  and 21  However, the 13—subunit of 13—conglycinin does not accumulate after  the  onset  of  glycinin  accumulation  (Meinke  et  al.  15 The temporally distinct accumulation patterns described above for  storage  rapeseed,  proteins  napin  maximum at  27  also is  mRNA  occur  for  detectable  DAA and remains  DAA  (Finkeistein  et  al.  by  In  achieves  40 DAA.  its  In contrast,  later at 21 DAA and peaks at  1985).  detectable before legumin mRNA,  levels.  mRNA  DAA,  18  high until  cruciferin mRNA is detected 3 days 40  their  In  vicilin  pea,  peaks at  DAF,  14  mRNA  is  then declines.  Legumin mRNA is detectable about 1 day after vicilin, peaks at 18 DAF,  declines  then  Similarily,  (Boulter  in soybean,  et  al.  1987,  Yang  et  al.  1990).  13-conglycinin mRNA is detectable several  days prior to glycinin mRNA (Walling et al.  1986).  Members of the same protein family may or may not exhibit the same  temporal  mRNA  accumulation  patterns.  In  soybean,  each  glycinin gene is expressed in the same temporal framework during embryo  development  (Nielsen  soybean 13—conglycinin gene for  the o.—  1989).  al.  1989).  In  accumulate prior  In rapeseed,  to  the  contrast,  family shows differences.  and a’—subunits  (Harada et al.  et  The mRNAs  i3—subunit  mRNA  one subfamily of napin  (gNa)  has mRNA levels that peak and decline earlier than those of the other members of the napin family (Blundy et al. The  level of  primarily are  at  storage protein mRNA5 appears to be controlled  the  transcriptional  transcriptionally  relatively  high  activated  transcription  level, early  rates  Storage  in  by  Evans et al.  1984, Harada et al.  protein genes  embryogenesis,  mid-maturation  repressed prior to desiccation and dormancy 1989,  1991).  attain and  are  (Delisle and Crouch  1989, Nielsen et al.  1989,  16 Walling et al.  1986).  roughly parallel those  These changes  in  endogenous mRNA levels. Apart  from  transcriptional  regulation,  post—transcriptional  regulation also plays a role in controlling storage protein mRNA levels  al.  (Delisle and Crouch 1989,  1989,  members  Walling of  the  et  al.  Harada et al.  1986).  glycinin  gene  In  soybean,  family  are  1989, Gy2,  Nielsen et Gy5  and  transcriptionally  activated and repressed at the same developmental stages. DAF,  Gy2  and  Gy5  show  lower than that of  similar  G*.  transcription  However,  is  more  unstable  conglycinin,  the  (Nielsen  et  By 35  which  are  and Gy5 mRNA  suggesting that the G* mRNA  al.  1989).  B—subunit  With  transcriptionally activated at the same developmental stage,  show  although the c’/o.’-subunit  mRNA levels are higher than 13-subunit mRNA  These  results  subunit  mRNA  cruciferin However, levels  suggest that is  less  transcription  at this  stable. rates  (Harada et al.  developmental  In are  on  rapeseed the  stage,  both  decline  1989). the B-  napin by  38  and DAA.  cruciferin mRNA levels remain elevated while napin mRNA drop,  suggesting  that  cruciferin  mRNA  during the later stages of embryo maturation 1989).  which  13-  also  similar transcription rates by 25 DAF,  genes,  soybean are  cL/0.’_  and  rates,  steady state Gy2  levels are higher than those of G*,  G*  is  more  stable  (Delisle and Crouch  All of these above studies suggest that developmentally  specific changes  in mRI[A stability may affect steady state mRNA  levels. While  storage protein accumulation can be regulated by mRNA  availability,  it  can also be regulated at the  level  of protein  17 synthesis. amounts than  In developing oat  of  seeds  (Chesnut et al.  avenin storage protein mRNA are  globulin  storage  equal  protein mRNA during most  or greater  to  of  the  1989),  development,  although globulin is the predominant storage protein in the seed. The  high  proportion  transcriptional translational, message  of  regulation,  which  some  is  polysomes  into  Post—translational  protein  suggests  of  type  believed  postbe  to  because there is a greater proportion of globulin  incorporated  1985).  globulin  accumulation  (Fabijanski  regulation  (Shuttuck-Eidens  also  Altosaar  regulates  Beachy  and  and  storage  1985).  These  workers used pulse:chase—labelling to detect the synthesis of 13— conglycinin 13—subunit protein well before its accumulation during embryo over  development. by  proteolysis  maturation.  As  during  early  proceeded,  proteins  and  are  cotyledon  of  stability  differentially  qualitatively  different cells within the  in  the  of  various  both  expressed, tissues,  and  in In  same tissue during development.  the relative proportions of 7S and llS proteins differ  between embryonic axes and cotyledons. storage protein,  oc. —subunit  and  no  conglycinin subunit  al.  stages the  turned  rapidly  allowing its accumulation.  storage  quantitatively  liS  was  Spatial patterns of storage protein accumulation  Seed  soybean,  protein  this the  maturation  protein increased, 2.2.2.  However,  1981).  In  Axes contain very little  as well as reduced levels of  13—subunit. that  maize  is  not  In  addition,  found  endosperm,  13—conglycinin  axes  contain  in cotyledons  storage  protein  a  13—  (Meinke  et  composition  varies with increasing distance from the aleurone layer.  Cells  18 adjacent to the aleurone accumulate 13— and —zeins, but little or no °‘—zein,  whereas cells further away from the aleurone contain  more oc-zein of  (Lending and Larkins 1989).  Arabidopsis  patterns  also  (Guerche et al.  gene family, embryo,  thaliana  at2S2,  while  The 2S storage proteins differential  show  1990).  Of  the  4  members  expression of  the  at2S  at2S3 and at2S4 are expressed throughout the  at2Sl  is  strongly expressed only  in the  axis.  A  recent study using in situ hybridization pointed out the changing cellular patterns of embryo development  storage protein expression during rapeseed  (Fernandez et al.  1991).  Napin and cruciferin  mRNAs accumulated initially in the cortex of the axis during late heart  stage,  then  torpedo stage,  in  during  maturation  outer  face  of  cotyledons  the  during  followed by a “wave—like” spread to the inner face  of the cotyledons. meristem  the  No expression occurred in the root or shoot  the  early  drying,  both  stages mRNAs  of  embryogenesis,  were  detected  in  but  during  the  shoot  meristem. The  results  accumulation  of  of  the  storage  above  proteins  studies during  development is a highly regulated event, is  controlled  at  a  variety  of  indicate  that  the  and  seed  embryogenesis  in which gene expression  levels  both  temporally  and  spatially. 2.2.3.  storage protein accumulation during conifer embryogenesis  It is well known that conifer seed embryos contain prominent protein bodies Durzan  1974),  proteins  have  (Flinn et but only  al.  qualitative been  1989,  Green  et  descriptions  reported  over  al. of  the  1991,  conifer past  few  Mia  and  storage years,  19 (Gif ford 1988,  Gif ford and Tolley 1989,  course  been  carried  (1987) red  of  this  present  during  out  followed changes  and white  pine  work.  in buffer-soluble protein  embryos.  However,  are  buffer—insoluble  solubilization  al.  1991),  (Gifford 1988,  this  study  accumulation patterns. changes during  in  embryo  loblolly  accumulation  pine  was  biochemistry,  length,  not  dry  seed  seed  require  weight  did  SDS  or  Green et  major (1991)  protein followed  acid  content  storage  protein  fatty but  for  urea  1989,  the  and  development,  addressed  molecular  and since most conifer  Janick et al.  Recently,  al.  et  deal  examine  not  have  in developing  study  Gifford and Tolley  did  during  not  and  this  Misra  studies  Johnson  embryogenesis.  specifically with storage proteins, proteins  1990)  biochemical  Few  conifer  1991,  Stabel et al.  and Green 1990, Misra and Green 1991, the  Green et al.  specifically.  the  Therefore,  biology and developmental  regulation  of  conifer storage proteins requires further study. 2.2.4.  Comparison of storage protein accumulation in zygotic and  non—zygotic embryos  The  studies  described  zygotic embryogenesis. storage  protein  gene  microspore—derived) embryo  storage  quantitatively Shoemaker et al. synthesis, parallelled  expression  embryos  previous  sections  and  patterns  non—zygotic  in have  expression  the  (1987)  the  dealt  with  Only a few studies have concentrated on  protein from  in  shown  differs  observed  that  (somatic  non—zygotic  temporally in  and  zygotic  and/or embryos.  noted that the pattern of storage protein  processing and accumulation in cotton somatic embryos that  reported  in  zygotic  embryos,  although  somatic  20 embryos  accumulated  smaller  their  amounts.  proteins  somatic embryos,  observations  Similar  microspore—derived rapeseed  embryos  Stuart et al.  (1988)  while Krochko et al.  stages  were  in  and  reported  1982).  (Crouch  storage proteins accumulated to only zygotic embryos,  earlier  at  In  for  alfalfa  found that both 7S and uS of the  10%  level  found  in  found that alfalfa  (1989)  somatic embryos contained altered proportions of the 7S and llS storage proteins. none of the above studies included ABA during their  However,  differentiation embryo  maturation  section 2.3 could  protocols.  storage  and  the  these embryos.  altered  is  storage  known  gene  to  influence  expression  protein  treated  embryos  mRNA  stage zygotic embryos,  induction however,  to  protein  expression  found  with that  displayed  ABA  observed  in  in  Rapeseed similar  equivalent  storage protein accumulation was  The developmental timing of  storage protein gene  expression was also similar to that of zygotic embryos al.  (see  the absence of exogenous ABA  Recent evidence may support this view.  microspore-derived  not examined.  ABA  protein  of Literature Review),  explain  storage  Since  (Wilen et  1990). few  The embryos  showed  contained  investigations that  distinct  of  those  protein  carried  reported  that  proteins  to  (1991)  found  Norway  those  spruce  found  that  Norway  bodies  Hakman and von Arnold 1988).  in white  out  conifer and  spruce  (Von Arnold  In addition, somatic zygotic  with  embryos,  spruce  white  and  contained and  somatic  spruce  Hakman  Hakman et al.  embryos  somatic  Joy  1988, (1990)  similar  IV et  embryos  al. that  21 differentiated on  low ABA levels contained  less  lipid and total  protein, but more starch than their zygotic counterparts. The work to date with conifer somatic embryos has not included developmental gene  comparisons  expression.  These  of  storage  studies  are  protein  required  accumulation  if  are  we  to  and make  comparisons between zygotic and somatic embryos and increase our understanding of conifer embryonic gene expression. 2.3.  ABSCISIC ACID,  The  plant  OSMOTIC STRESS AND EMBRYO DEVELOPMENT  growth  regulator  ABA  is  believed  to  modulate  numerous aspects of plant growth and development (for reviews see Creelman  1989,  Zeevaart  in  such diverse  implicated and  bud  dormancy,  inhibition. limited  to  and  stress  Creelman  processes  as  adaptation,  1988).  It  has  been  stomatal  closure,  seed  gravitropism  For the purpose of this review, detailed  studies  of  ABA  and  growth  discussion will be  effects  on  seed/embryo  development. Abscisic seed  development,  followed  by  desiccation al. a  acid has  1987,  growth  pool 1991).  showing decline  (Ackerson  Pence 1991). inhibitor.  precociously medium,  a  when  conditions  (Ackerson  during  1984a,  medium  to  or  increase  two  late  during  peaks  of  embryo  Finkelstein  et  angiosperm  accumulation,  development  al.  1985,  and  Galau et  It is commonly considered to function as Immature  which  1984b,  found one  cultured  Furthermore,  ABA-containing  been  in  zygotic vitro  allow depletion  Finkelstein et the  on  continued  prevents  this  al.  embryos  will  germinate  growth  regulator—free  of  endogenous  the  1985,  culture  of  Rivin these  germination  ABA  and Grudt embryos  (Eisenberg  on and  22 Finkelstein et al.  Mascarenhas 1985, Application  of  germination 1988,  the  of  Roberts  endogenous  exogenous somatic  al.  et  ABA  in  1985, Rivin and Grudt 1991).  also  ABA  embryos  (Amrnirato  1990a).  Further  the control  the  prevents  Boulay  1974,  support  precocious  the  for  role  embryo germination comes  of  (vp)  of  from  such as  study of ABA-deficient and ABA-insensitive mutants,  the maize viviparous  al.  et  which are characterized by an  mutants,  uninterrupted progression from embryogenesis to germination (Kriz et al.  1990, Rivin and Grudt 1991).  While these studies suggest that ABA is inhibitory during the later embryo  stages  of  development,  maturation.  Crouch  it and  also  Sussex  promoted embryo growth in rapeseed. cultured  in  the  presence  of  between ABA levels and growth,  has  ABA  a  (1981)  found  Similarily, showed  role  promotory  a  that  in ABA  soybean embryos  close  correlation  with a stimulation of growth and  dry weight accumulation during early phases of embryogenesis, and growth  suppression  l984b). embryo  A  further  by  mid—stage  promotory  differentiation  embryo  effect  systems,  has  where  development been ABA  noted  enhances  number and morphological normality of the embryos Kamada and Harada 1981, Roberts et al.  (Ackerson in  somatic  both  the  (Ammirato 1974,  l990a).  Abscisic acid has also been implicated in the development of desiccation tolerance during embryogenesis. Abundant  Embryogenesis  (Lea) proteins accumulate during the mid to later stages  of embryo development,  al.  Late  1987).  just prior to maturation drying  (Galau et  These proteins, because of their structure and timing  of accumulation,  are believed to serve a protective, role during  23 desiccation  et al.  (Dure  Lea mRNA5 mirror changes do  not  these  (Galau proteins  1986). with  ABA  al.  found  show  and  induced  excised  enhanced  crosses ABA  embryos  partial  for  development  the  treated complete  or  mutants. for  (abi3),  of  Using  Roberts (1989)  desiccation recombinants  ABA deficiency  these  of  al.  et  Koornneef et al.  1990).  mutations  sensitivity  to  (Galau  1991, Kim and Janick 1991,  thaliana  containing  appearance  ABA  somatic  of the  while others  levels,  exogenous  tolerance  some  of  precocious  zygotic or  required  Arabidopsis  in  the  by  Senaratna et al.  that ABA was  reduced  1987),  be  expression  in endogenous ABA  (Bochicchio et al.  l990b,  tolerance from  can  Furthermore,  desiccation et  al.  et  The  1989).  workers  (aba)  and that  found  desiccation intolerance in seeds only occurred when both maternal and  embryonic  homozygous  recessive  sensitivity an  genotypes  recessive  for  (aba/abi3)  individual for  were  double  both  ABA  recessive;  deficiency  seeds  ie.  and  reduced  ABA  that arose from the self-fertilization of  heterozygous reduced ABA  for  ABA  sensitivity  deficiency  (aba/ABA,  and  homozygous  abi3/abi3)  were  still capable of desiccation and were viable. In addition to the above effects on seed/embryo development, ABA  has  also  compelling  been  implicated  evidence  Arabidopsis mutants. ABA  sensitivity  Koornneef et al.  for  this  Mutants  display 1984).  in  again  comes  deficient  reduced  induction.  dormancy  dormancy  Furthermore,  from  the  The  study  in ABA or with (Karssen  et  most of  reduced  al.  1983,  the use of double mutants  has shown that dormancy induction in the developing seed requires embryonic ABA.  Maternal ABA or exogenously applied ABA did not  24 induce  dormancy  in  seeds  mutation (Koornneef et al. Different  stages  characterized by has  been  at  development expressed affected Galau  al.  development  are  at  al.  1989).  et  most  those  al.  and  development  are  gene  expression.  It  diverse  Several have  1991, et  highly  the  of  aba  particular  any  Hatzopoulis  The  for  the  20,000  development  (Bartels  1988).  growth  differential  of  embryo  1986,  homozygous  approximately  (Goldberg et  by ABA  seed  level  mRNA  during  et  Quatrano  patterns  the  were  1989).  of  suggested that  expressed  that  stage  proteins been  1990,  expressed  et  that are  al.  1989).  The potential  role  of ABA  to  al.  be  1991,  during  and seed  whose mRNAs  represent at least 50% of total mRNA at mid-maturation et  seed  Williamson  genes  storage proteins,  are  of  found  Bochicchio  al.  genes  in the  can  (Goldberg  regulation of  expression of these genes has been studied intensively. Evidence  for  the  situ  in  regulation  of  storage  protein  accumulation by ABA in zygotic embryos is based primarily on the fact  that  endogenous  development reserves  and  are  ABA  correlated  (Bewley and Black 1985,  work with  ABA induces  Bopp  with  increase the  during  synthesis  Finkelstein et al.  of  1988),  and  soybean  1985)  and wheat  broad  bean  Sussex  1981),  (Ackerson  (Barratt  mustard  1984a,  (Williamson et al.  cotyledons  storage Most  cultured  This type of experiment has shown that  storage protein accumulation in  (Crouch  embryo  1985).  zygotic embryos has utilized excised embryos  on ABA-containing media.  rapeseed  levels  (Croissant—Sych  Eisenberg  1985),  1986)  immature embryos  and  and  of and  Mascarenhas  as well as in cultured endosperm  of  Solanum  25 (Smith  species  and  Desborough  characteristic is not true for all plants, to  associated  be  either  in  vivo  or  with  in  storage  vitro  protein  (Dure  However,  1987).  this  as ABA does not appear expression 1981,  and Galau  in  cotton,  Galau  et  al.  1987) In embryos where ABA does affect storage protein accumulation, response  the  to  ABA  varies  with  developmental  embryos are more responsive to ABA, so  (Bray and Beachy 1985, Mascarenhas  and  1985,  stage.  while older embryos are less  Croissant-Sych and Bopp 1988,  Finkelstein  Younger  et  al.  1985),  decline in ABA sensitivity during maturation.  Eisenberg a  suggesting  In addition, while  ABA is required for us storage protein and mRNA accumulation in early  and  decline  mid—maturation  cultured  in uS protein mRNA  embryos,  soybean  it  (Eisenberg and Mascarenhas  a  causes 1985)  at  younger stages. In embryos that do show storage protein response to ABA, all  storage proteins are equally affected.  In  not  cultured soybean  cotyledons, the accumulation of the 13-subunit protein and mRNA of oL/o.’-subunits are  13-conglycinin is stimulated by ABA, while the not  (Bray  and  Beachy  accumulation  by  (Davies  Bedford  and  1985).  cultured  pea  1982),  Also, embryos whereas  legumin is  not  pea  storage  stimulated  vicilin  enhanced by exogenous ABA application to seeds  protein by  synthesis  ABA is  (Schroeder 1984).  The enhancement of storage protein accumulation by ABA appears to be due to increased storage protein mRNAs 1985, These  Goffner et al. increases  are  1990, Kriz et al.  1990,  dose—responsive,  with  (Finkeistein et al. Taylor et al.  higher  ABA  1990). causing  26 increased  levels  mRNA  (Finkeistein  et  al.  1985).  Different  storage protein mRNA5 are increased to different degrees by ABA, as  seen  in  cultured  levels  mRNA  were  27  rapeseed  DAA  increased  2—fold  increased  4-fold  addition,  Eisenberg and Mascarenhas  of  different  by  1  ABA  IM  members  of  embryos,  and  napin  (Delisle  the  and  (1985)  soybean  where mRNA  cruciferin levels  were  1989).  In  Crouch  noted that the mRNA5 glycinin  gene  family  stimulates  storage  accumulated to different degrees in response to ABA. The mechanism by which ABA maintains  or  protein synthesis and accumulation is complex. ABA  appears  1990,  primarily  transcriptional  The response to  (Mansfield  and  Williamson and Quatrano 1988), with the higher mRNA levels  parallelled 1989).  by  higher  However,  transcription  regulation  and  (1988)  Quatrano  storage  rates  post—transcriptional,  translational  protein)  have  noted  also  that  mRNA  been  suggested.  ABA-mediated  by  ABA  translational ABA,  Em  occurred. protein  Finkelstein  the  regulation  synthesis This  and  at  et  is  al.  implicated,  continued but  suggests  allow  translational  its  that  ABA  accumulation  (1985)  found  that  (an  However,  level.  help  to  (Williamson excised  post—  albumin  Em protein  suggesting no Some  in the  appreciable  may  and  stabilization,  because,  no  Crouch  Williamson  protein  Em  synthesis rates closely parallelled Em mRNA levels, control  and  translational  wheat  showed  (Delisle  suggesting post—transcriptional regulation.  of  Raikhel  postabsence  accumulation  stabilize et  al.  rapeseed  the  1985). embryos  cultured on 1 M ABA accumulated cruciferin at the same rate as in  seed  embryos,  although  the  mRNA  levels  were  lower  in  the  27 cultured  embryos.  These  workers  enhanced cruciferin translation, mRNA  but  more  cruciferin  suggested  that  ABA  may  have  since there was less cruciferin  protein  accumulated  embryo  per  at  increased ABA concentrations. The previously described studies used exogenous ABA to study effects ABA  storage  on  effects  mutants.  on  protein  storage  Groot et al.  accumulation.  proteins  (1991)  have  Further been  insight  gained  into  from  ABA  noted that tomato mutant sitiens,  which has strongly reduced ABA levels,  did not differ from wild  type in seed fresh weight, dry weight or storage protein content. While this suggested no role for ABA in seed development, were  indications  that  endogenous ABA to (1989)  had  Arabidopsis  this  mutant  was  “leaky”,  allow normal development.  examined double  this  mutants  poposal using  with  enough  Koornneef et al.  earlier  recombinants  aba with ABA-insensitive abil or abi3.  there  by  constructing  of  ABA-deficient  They found that aba, abi3  double mutants did not display storage protein accumulation and that  aberrant  seed  development  occurred  when  the  reduced  ABA  level of the aba mutant was combined with the reduced sensitivity of the abi3 mutant. Arabidopsis levels  wild  of  Finkelstein and Somerville (1990)  type  storage  significantly  less  and  abil  and  protein, storage  but  protein  abi2 abi3 and  mutants mutants  storage  found that  had  similar  contained  protein  mRNA.  These results indicated that the abi3 locus is important for seed responses  to  important  for  development.  ABA  and  storage  that  ABA  protein  levels  gene  and  sensitivity  expression  during  are  embryo  28 The water  normal  process  content.  internal  It  osmotic  maturation.  of  development  seed  has  been  environment  suggested  of the  includes  role  a  differentiation systems,  Roberts 1991). subculture  medium  (Litz  1986,  development past this point may require  of  lower  osmolarity  (Litz  1986).  In  later stages of embryo development can be enhanced and (Nadel  et  al.  inhibited by osmotic stress Xu et al.  1989)  precocious  and  to  zygotic  germination  (Finkelstein and Crouch 1986,  stimulate  storage  protein  osmotic stress has been  accumulation  in  (Finkelstein and Crouch 1986, Goffner et al.  1990)  and  Roberts  1990).  Apart from morphological development,  al.  in embryo  osmotic stress during early development  However,  to  accelerated  shown  the  In somatic embryo  can enhance the number of globular embryos produced  1991,  in  of  In support of this, numerous studies have shown that  osmotic stress influences embryo development.  addition,  loss  changes  that  seed play  a  microspore-derived  embryos  (Wilen  excised Xu et  1990,  et  al.  1990).  Since osmotic stress can cause elevated ABA levels in vegetative tissues  (Skriver  and  Mundy  1990),  it  has  suggested  been  that  osmotic effects on embryo development are mediated via increased ABA  levels.  Some  studies  support  this.  Wilen  et  al.  (1990)  noted that rapeseed microspore—derived embryos cultured on 12.5% sorbitol  showed  induction  of  storage  protein  mRNAs,  slower rates than after exogenous ABA application. of  endogenous ABA changes  increase  in  suggesting  ABA that  that  during sorbitol  preceded  storage  osmotically-induced  at  Measurements  treatment revealed an  protein ABA  albeit  mRNA  induction,  stimulated  storage  29 protein  gene  expression.  Similarily,  cultured on high osmoticum legumin storage proteins fluridone  broad  bean  cotyledons  (18% sucrose)  accumulated vicilin and  (Barratt 1986).  The combined effect of  (an inhibitor of endogenous ABA biosynthesis)  with the  high osmoticum treatment inhibited the storage protein increase. Exogenous ABA counteracted this inhibition. ABA-deficient vp5 mutants after exposure to ABA, alone did not  Additionally,  accumulated globulin  storage  maize  proteins  whereas those cultured on high osmoticum  (Rivin and Grudt 1991).  This was attributed to the  fact that these embryos could not synthesize  ABA in response to  osmotic stress. of  All  these  studies  suggest  that  osmotic  stress-induced  alterations in ABA levels are responsible for the changes in gene expression, In  and that ABA is directly involved  contrast,  occur  in  (1986)  other  workers  response  noted  accumulated transient  to  that  storage  increase  have  osmotic  that  stress.  rapeseed protein  found  changes  cultured  storage  in ABA content was  ABA  Finkelstein  embryos  and  in this response.  protein  observed.  and on  do  not  Crouch  sorbitol  mRNA, In  but  no  sunflower,  llS storage protein and mRNA increased during culture on sorbitol (Goffner  et  al.  pronounced  for  stimulation  of  1990).  The  culture  on  ABA  levels  increase sorbitol  or  ABA  response to the osmotic stress. pea plus  pods  cultured  fluridone  for  14  contained  translatable mRNA,  days no  in  mRNA  than  on  biosynthesis  Barratt et al. on medium  differences  level  was  ABA,  was (1989)  more  and  detected  no in  found that  containing ABA or ABA in  legumin  or  vicilin  suggesting that ABA did not play a role in pea  30  storage protein gene expression. that  alfalfa  Finally, Xu et al.  embryos  cultured  did  germinate,  embryonic  and  osmoticum  synthesized  not  storage proteins).  embryo  on  ABA  but  or  osmoticum  only  remained  cultured  embryos  developmental  noted  (1990)  on  (including  proteins  This suggested that the osmotic prevention of  germination and maintenance of developmental gene expression was not mediated through ABA. Since ABA and osmoticum do inhibit water uptake and Crouch  1986,  Schopfer  and Plachy  1984),  involvement of ABA in the above responses the  major  effector  causing  changes  (Finkeistein  it may be that the  is  indirect,  and that  developmental  in  gene  expression is the water status of the embryo, which is altered by osmotic stress or by ABA. It is evident that the role of ABA in the regulation of gene expression identify  is  still  putative  unclear.  ABA  transduction pathways. regions help  to  of  receptors  the  and  work  is  elucidate  required  to  signal  the  The identification of upstream regulatory  ABA—responsive  clarify  Further  role  and of  osmotically—induced ABA  and  osmotic  genes  stress  should in  gene  regulation. 2.4.  SUMMARY  Major advances in tissue culture have made the attainment of conifer somatic embryos and plants a reality. the  quality  of  physiological  in  embryos  comparisons  embryo—derived proteins  these  material  angiosperm  between  is  and  zygotic  required.  zygotic  plants,  and  In order to gauge biochemical  embryo—  Comparisons somatic  and  and  somatic  of  storage  embryos  revealed  31 several differences between the two systems.  These differences  may be attributed to the absence of ABA during differentiation in vitro.  Work to define storage protein accumulation patterns and  the effects molecular addressed.  of ABA on storage proteins at both the protein and  level  during  conifer  embryogenesis  has  still  to  be  32 3.  3.1.  MATERIALS AND METHODS  ZYGOTIC EMBRYO MATERIAL  Interior  spruce  from the  interior of  British Columbia  mixture of two closely related species Picea glauca and Picea engelrnanhi Parry were  collected  during  (Owens and Molder  1988  and  1989  at  a  (Moench) Voss Seed cones  1984).  the  is  British  Columbia  Ministry of Forests Kalamalka Research Station, Vernon, B.C.  from  source tree EK1O, which was open pollinated and possessed a Picea engelmanii during EK11.  maternal  1991,  seed  background.  cones  were  Due  to  collected  poor from  cone  EK1O  the  yield  adjacent  tree  Cones were received the day after collection and stored at  4°C until embryo excision, which took place within a few days of receipt.  Embryos  developmental by  Buchholz  stage and  required.  based  were  classified  according  on morphological  Stiemert  Embryos  (proembryos)  were  with  (1945)  and  suspensors  obtained  in  very  criteria  stored  and  to  small  established  at  small  their  -80°C  until  embryonal  heads  numbers.  Stage  2  embryos were torpedo—shaped,  possessed a visible apical dome but  lacked cotyledon primordia.  Stage 3 embryos possessed cotyledons  that had not developed above the apical dome. during  1989  were  abundant  enough  classification into Stage 3A but not very large) clefts  between  Collections embryos  to  grouped  together.  allow  further  allow  sub—  (cotyledons visible as small stubs,  and Stage 3B (cotyledons larger with distinct  them,  during  to  Embryos collected  but  1988  this  not  and  overgrowing  1991  separation,  Stage  3-4  did so  embryos  not  all had  the  shoot  contain  Stage  3  apex).  sufficient  embryos  cotyledons  that  were had  33 overgrown the shoot apex, apical  dome.  Stage  enclosed  the  1989  1991,  and  embryos. Stage  embryos  cotyledons  Collections  dome.  with the  had  that  were made  completely  weekly during  last dated collection being mature seed  Collections made during 1988 were not as frequent,  obtained stages  apical  embryos  4  4  but had not yet completely covered the  at  of  3  (excluding mature desiccated embryos) collection  embryo  age  dates,  (stage  4-1:  collected July 27; Stage 4-3:  which  were only  represented  collected July  increasing Stage  13;  were surface sterilized in 10% (v/v)  distilled Imbibed  Tween 80 water  for  and  (v/v)  15 mm,  imbibed  Chicago,  washed three times with sterile  overnight  at  4°C  in  the  Corp.,  Chicago,  with a  light intensity of  IL)  dark.  (Seedburo  inside a Magenta GA7 vessel  IL)  seeds  commercial bleach containing  seeds were placed on a water—saturated Kimpak  Equipment Co.,  4—2:  collected August 24).  To study the effects of germination on embryo proteins,  0.1%  and  (Magenta  and germinated at 27°C in a 16-h photoperiod 30 2 ILE/m / sec.  seeds were examined at  three—day intervals and classified according to extent of radical emergence.  Germinants  of  similar  development were  removed  and  stored at -80°C. 3.2.  SOMATIC EMBRYO MATERIAL  All culture media used were autoclaved,  all growth regulators  were added prior to sterilization and all media were with  0.6%  otherwise  Noble  agar  stated.  trans-Abscisic  acid  The  (Difco ABA  (Sigma  Laboratories, used  in  Chemical  Detroit,  experiments Co.,  St.  was  Louis,  solidified MI)  unless  (+,—)—cis, MO).  All  cultural manipulations were carried out under sterile conditions  34 and all cultures were grown at 27°C in either the dark or light (25—40 2 bLE/m / sec). Somatic embryo cultures were available which had been induced from individual zygotic embryos of trees EK1O and PG118  al.  1989),  and identified as distinct lines  from EK1O;  W70  maintained  in the dark on Litvay’s medium  and W46  from PGll8).  (W29,  (Webb et  W74,  Embryogenic  W76,  cultures were  (Litvay et  al.  using half—strength macronutrients containing 1% sucrose medium)  and 10 tM 2,4-D and 5 M BA.  W77  1985)  (1% ½LM  Cultures were subcultured  to fresh media every 2 weeks. Somatic embryo differentiation was carried out in the light, and  initially  used  the  protocol  Roberts  of  al.  et  (1990a).  Embryogenic tissues containing proembryos were subcultured to VE medium  (von  Arnold  (3.4% VE medium), week.  Eriksson  1981),  containing  no growth regulators and 1%  3.4%  sucrose  charcoal,  for one  The ABA-dependent developmental profile was obtained from  embryogenic placed acid  and  tissues  onto  (IBA)  3.4%  (approximately VE medium  and various levels  150  mg  containing (0,  20,  pieces)  1  jM  40 or 60 M)  of ABA for 5  with subculture to fresh medium every 2 weeks.  this  maturation,  morphological  appearance  were  (Roberts  according  al.  as  1990a)  (remained opaque with well—developed cotyledons) germinating  abnormal  to  mature  or precociously  (exhibited extensive greening and elongation of the  cotyledon—hypocotyl axis). an  Following  classified et  were  indole-3-butyric  weeks,  embryos  that  elongation  of  Aberrant structures that appeared as cotyledons  classifed as shooty structures.  from  a  basal  callus  were  The number of visible structures  35 were  counted  after  differentiation was developmental levels,  weeks.  All  performed  using  5  study  to  compare  subsequent 40  M  effects  somatic  ABA,  of  embryo  except and  high  for  the  low  ABA  in which case 10 M ABA was also used.  Somatic  embryos  on  40  ,M ABA were  classified  according  to  developmental stage (Table 1), using a modification of the scheme described somatic  by von Arnold  embryos  were  and Hakman  designated  (1988).  Stage  cotyledons had overgrown the shoot apex  3  Early cotyledonary embryos.  Once  (Stage 3—4),  the  the somatic  embryos resembled Stage 4 zygotic embryos, and were classified as Stage 4 somatic embryos.  Somatic embryos reached maximum length  after approximately 6 weeks on ABA and did not lengthen much more during  the  time  classified as (Stage 4—9) protein  of  these  7-week  (Stage  embryos.  experiments, 4-7),  and  8-week  were  (Stage  thereafter  4-8)  or  9—week  In order to compare the pattern of storage  accumulation  during  the  later  stages  of  maturation,  zygotic and somatic embryos were identified on the basis of time since onset of cotyledon development. Somatic embryos on 10 M ABA developed in a similar manner to those shoot  on  40  ABA,  apex  (Stage  but  once  4-2),  the  the  cotyledons  embryos  had  showed  overgrown the  the  extensive  chlorophyll accumulation and hypocotyl/cotyledon elongation that was  characteristic  could  not  be  of  precocious  classified  using  the  applied to non-germinating embryos referred  to  comparisons,  as  germinants  embryos  (Germ  cultured  germination.  on  These  length/morphology  on  40  criteria  jM ABA and hence were  1—6). both  embryos  For 10  the  and  40  developmental jM  ABA  were  36  TABLE 1.  Somatic embryo developmental stages on 40 jM ABA  Stage  Weeks after onset of cotyledon development  Pe Glob Round F 1 at Early Cotyledon  Late Cotyledon  3-1  0  3—2  0.2  3—3  1.0  3—4 4—1 4—2 4—3 4—4 4—5 4—6 4—7  1.4 1.7 2.1 2.4 2.9 3.3 4.1 4.6  4—8  5.6  4-9  6.6  Description  Proembryos on maintenance calli Embryos with globular heads Torpedo embryos with round heads Torpedo embryos with flat heads Small cotyledon primordia barely visible Primordia enlarged with clefts visible between them, but not overgrown the shoot apex Cotyledons enlarged and slightly overgrown the shoot apex, but embryo less than 1 mm long Embryos 1.0-1.25 mm long Embryos 1.3-1.5 mm long Embryos 1.55—1.75 mm long Embryos 1.8-2.0 nun long Embryos 2.05-2.25 mm long Embryos 2.3-2.5 mm long Embryos 2.55-2.75 imu long Embryos 2.55—2.75 mm long and on ABA for 7 weeks Embryos 2.55—2.75 mm long and on ABA for 8 weeks Embryos 2.55—2.75 nun long and on ABA for 9 weeks  37 collected for analysis after the same exposure time to ABA.  Germ  1  were  (2-2.25  nun  in  length)  precociously  germinating  collected at the same time as Stage 4-3 mm  in  the  length)  same  advanced  time  as  precocious  Stage  4—8  embryos,  germinants  embryos.  The  embryos  (8+  and Germ 6  were  collected  intermediate  at  stages  formed a developmental continuum of increasing size. Embryos  differentiated  developmental protocol.  comparison Embryogenic  suspension culture 2,4-D  and  BA  filtered  through  2  Jolla,  CA)  growth  regulators.  (20%  in  3  The  (Millipore  the  a  of  were  rpm.  resulting  1%  The  1%  Corp.,  from the  for  1  week  in  ½LM medium containing suspensions  were  (Calbiochem  Corp.,  tissue  mass  was  ½LM  1%  MA),  with  absorbant backing to remove excess medium. separated  ABA  differentiation  then La  a  The  resuspended The  medium.  was plated onto black  Bedford,  jM  ½LN medium containing no  regulator-free  (750 jl)  M/lO  grown  Miracloth  times with  40  modified  in liquid  120  layers  growth  resuspended tissue discs  tissues  at  and washed  w/v)  used  (10% w/v)  rotating  during  0.8  j.un filter  filter  paper  filter disc was  absorbant pad and placed onto  solid  VE  3.4%  medium containing 1 M IBA and 40 M ABA as described previously. For  the  osmotic  stress  experiments,  embryogenic  cultures  obtained via the suspension culture method were differentiated on solid  3.4%  VE  medium  containing  1  ,M  IBA  and  40  ,M  ABA.  Embryogenic tissues containing Stage 3—4 embryos were removed and placed onto solid 3.4% VE medium containing no growth regulators, 40 M ABA, Fluridone  fluridone, (kindly  15% mannitol or combinations of the above.  supplied  by  Dr.  Franklin  Fong,  Texas  A  &  N  38  University)  was made up as a 2.5 mg/mi stock solution in acetone  and added as required. the  prior  media  to  All of the above compounds were added to  autoclaving.  Cultures  were  grown  for  a  further 2 weeks and then embryos were removed and stored at —8 0°C until required. 3.3.  Four cultures were used per treatment.  PROTEIN EXTRACTION AND ELECTROPHORESIS  All protein and immunoblot analyses were performed using line W29  except where  indicated.  For analysis of proteins by sodium  dodecylsulfate-polyacrylamide  electrophoresis  (SDS-PAGE),  embryonic tissues were homogenized in Eppendorf tubes with 30-40 j.l 4x SDS sample buffer  [562.5 l 0.5 N Tris—HC1 pH 6.8,  2—mercaptoethanol,  Ll  225  bromophenol blue] centrifuged stored  at  (10  glycerol,  per mg tissue. mm;  -80°C  14,000  until  was  dot-blotted,  in  needed.  incubated  in  SDS,  0.5  mg  Samples were boiled for 7 mm,  rpm)  a  bench  Protein  modified method of Ghosh et al.  (w/v)  9%  225 j.Ll  (1988), 1  ml  was  top  microfuge  determined  in which 2  1%  (w/v)  measured spectrophotometrically at 595 nm.  SDS  by  and the  jl of sample overnight  and  Bovine serum albumin  (BSA) was used as a standard. For two—dimensional electrophoresis, buffer  were  water,  frozen  dialyzed at  resuspended  in  [9.6  buffer  ml  Inc.,  of  Montreal,  13  —80°C 1  and  modified  contained  QU),  200 1 Triton X—lOO, (DTT),  overnight  175  at  extracts in 4x SDS sample  4°C  against  lyophilized. 2D-NH 100  mg NaC1,  jl  (Mayer  et  al.  L  deionized  samples 1987)  3-10  mg Na EDTA, 2  8.8 ing ascorbic acid,  100 g leupeptin,  The  Pharmalyte  3.7  4  were buffer  (Pharmacia  3.8  mg  EGTA,  154 mg dithiothreitol  100 go-macroglobulin,  2%  (w/v)  SDS and  39 deionized  water]  application,  containing  9.5  M  0.5 p1 Pharmalyte (5-8 or 3-10)  For one-dimensional SDS-PAGE analysis, separating system were  gel  and  into  consisted  a  5%  stacking  1970).  (Laemmli  run  10  a  4.86  For  cm  x  urea,  g  Triton X-100  and  500  p1  aimnonium  20%  solution.  was added.  gel,  with  the  Laenunli  analysis,  nun  The  2  tube ml  T,  (31%  gel.  deionized 4.3%  Pharmalytes  persulfate  gel  2.03  C),  ml  buffer samples  solution  water,  (Pharmalyte  and  sample  gels consisted of a 12%  Pharmalyte 5—8 + 50 p1 Pharinalyte 3-10), p1  before  two—dimensional  2.88  acrylamide-bis-acrylamide  2  Just  urea.  1.18 10%  3-10  or  ml  (v/v) 450  p1  and was polymerized with TEMED  3p1  ml  per  of  gel  The sample was applied at the cathode end and covered  with  overlay  (500  p1  buffer  Pharmalyte  Pharmalyte 3—10),  [2  ml  3-10  (v/v)  10% or  450  Triton  p1  X-lOO,  Pharmalyte  7.5 ml deionized water].  Pharmalytes  5—8  +  50  p1  The cathode solution  was 0.1 N NaOH and the anode solution was 0.06% phosphoric acid. Gels were run at  400 V constant voltage to a total  After electrophoresis, gels extruded, applied (w/v)  to  a  tubes were placed on  ice  gel.  The  tube  gel  agarose in 12.5 mlvi Tris-HC1 pH 6.8.  was  with  the  for 20 mm  and  Coomassie  R-250  or  silver  overlaid with  1%  After electrophoresis,  gels were fixed with aqueous 40% methanol + stained  7100 Vh.  for 20 mm,  incubated in 4x SDS sample buffer stacking  of  10% acetic acid and  reagent  (Wray  Ektachrome  160  (Kodak,  of  protein  et  al.  1981) The Toronto, bands  was  gels ON)  were colour  carried  photographed slide  out  by  film  using and  scanning  quantification  densitometry  of  each  colour  40 slide  560  at  Instruments  nm  using  Inc.,  individual  total  percentage  of  DU—64  a  Mississauga, protein  the  ON).  profiles  major  Spectrophotometer  storage  Results were  from  used  proteins  to  (Beckman  scans  of  calculate  relative  3  the  total  to  protein. 3.4.  PROTEIN BODY ISOLATION AND ANALYSIS  Protein bodies were isolated from whole embryos based on the technique mature  of  Chrispeels  zygotic  et  embryos  or  al. 50  Approximately  (1982). stage  4—9  somatic  embryos  placed on ice in a petri dish containing 2 ml Medium A Tris-HCL pH 7.8,  1 mM EDTA),  12 %  (w/w)  80-100 were  (100 mM  sucrose and 2 mM MgC1 . 2  Embryos were macerated and the resulting slurry filtered through one layer of 53 ,.t.m nylon mesh. ml of the same medium. Medium  containing  A  The petri dish was rinsed with 1  This 3 ml filtrate was layered over 8 ml 16%  (w/w)  sucrose  centrifuge tube and centrifuged (40 mm:  and  2  mM  2,800 rpm)  2 MgC1  resuspended  in  Mississauga,  80-100  4x  ON).  SDS  a  at 4°C, using  a SW41 swinging bucket rotor in an L8-M Ultracentrifuge Instruments Inc.,  in  (Beckman  The protein body pellet was  sample  buffer,  treated as described above for SDS—PAGE.  and  the  sample  Extractions under non—  reducing conditions were performed in 4x SDS sample buffer minus 2—mercaptoethanol. reducing  The  conditions  by  following SDS-PAGE and  They  were  then  non—reduced cutting  samples were  slices  from  incubating them in  placed  on  top  of  electrophoresed under standard conditions.  non—reducing  4x SDS  the  analyzed under gels  sample buffer.  stacking  gel  and  41 Buffer—soluble  and  buffer—insoluble proteins  were  separated  from the protein body pellet by extraction with 35 l of sodium  phosphate  14,000  rpm).  extracted  and  buffer The  (pH  7.5)  supernatant  centrifuged,  and  and was  the  centrifugation removed,  pooled to give the buffer-soluble fraction.  mm;  (10  pellet  the  supernatant  0.05 M  re—  fractions  were  This was mixed with  an equal volume of 4X SDS sample buffer and treated as described previously for SDS-PAGE.  The insoluble pellet was extracted with  100 1 of a 1:1 mixture of sodium phosphate buffer:4x SDS sample buffer  and  treated  for  SDS-PAGE  as  described  previously.  The  solubility characteristics of the storage proteins were examined using  Osborne’s  extracted  (1924)  twice  with  phosphate  buffer,  containing  1 M NaC1.  criteria. 25  jl  buffer  of  The either  containing  protein  body  deionized 0.2  M  pellet  water,  NaC1  The extracts were mixed with  or 50  was  sodium buffer  ,l  of 4x  SDS sample buffer and treated for SDS-PAGE as described above. 3.5.  MICROSCOPY  Embryos from late August seed collections were excised, overnight in 25 mM potassium phosphate buffer 2.5% (w/v)  (v/v)  tetroxide.  acetone  series,  Specimens  60°C,  resin.  sections  acid—Schiff’s  were  transferred  propylene oxide:Spurr’s resin Spurr’s  containing  glutaraldehyde and post-fixed for 2 hr in buffered 1%  osmium  graduated  (pH 6.8)  fixed  (1:1)  to  dehydrated propylene  (Spurr 1969)  (PAS)  reagent  were cut, (Jensen  oxide,  a  then  and embedded in  After polymerization of the resin (1 p.m thick)  through  for  24  hr  at  stained using the periodic 1962)  for  polysaccharides,  42 and  counter—stained  with  1968)  for proteins.  3.6.  ANTIBODY PRODUCTION  aniline  blue—black  (Jensen  and  Fisher  The 41 kD or 24 + 22 kD bands were excised from SDS-PAGE gels of  protein  body  extracts,  dried,  ground  to  a  fine  powder  in  liquid nitrogen and added to a 1:1 mixture of phosphate-buffered saline:Freund’s complete adjuvant.  A total of 100—200 j.g protein  was injected into New Zealand white rabbits.  Booster injections  of  phosphate—buffered  100—200  jg  protein  saline:Freund’s later.  in  incomplete  a  mixture  adjuvant  of  (1:1)  Rabbits were bled 1 week later,  were  given  weeks  4  the blood allowed to clot  overnight at 4°C then the supernatant was removed and centrifuged (15 mm;  4,000 rpm).  The  straw—coloured supernatant  was removed and used for subsequent immunoblots. serum,  which  molecular  displayed  weight  embryo  slight protein,  method of Smith and Fisher 3.7.  nitrocellulose (Bio-Rad, stock  separated  overnight  Mississauga,  buffer  [glycine  by  at  30  ON)  high  a  affinity-purified  100  Tris-buffered  g/L,  saline  each with TBS + 0.5% TBS  containing  1%  V using  Tris  pH  7.4  (CM)  CM  were  the  transfer base  (1:2:7).  (v/v)  (w/v)  SDS-PAGE  and  Carnation powdered skim milk  in  with  by  the  (1984).  SDS]:methanol:deionized water  mm  was  The 41 kD anti  IMMUNOBLOTTING Proteins  with  cross-reactivity  anti—serum  80  onto  apparatus  consisting  g/L pH  8.7,  2  of g/L  The filters were treated  (TBS)  Tween 80, a  Transblot  buffer  for 1 hr,  and  blotted  containing  3%  (w/v)  washed 3 times for 5  then incubated overnight  1:400  dilution  of  primary  43 antibody with  solution. +  TBS  containing goat  Tween, a  developed  3  incubated  in  1:3000  anti-rabbit  then rinsed  Filters were rinsed  3  dilution  IgG  times  and  of  (Bio-Rad, for  following  photographed  for  alkaline  for +  TBS  5 mm  1%  each  (w/v)  CM  phosphatase—conjugated Filters  ON).  were  each with TBS + Tween and colour  Bio-Rad  scanned by  hr  Mississauga,  5 mm  the  1  times  protocol.  The  densitometry of  blots  photographic  were slides  as described previously. 3.8.  CHLOROPHYLL ANALYSIS  Chlorophyll protocol  of  analysis  Hiscox  and  of  somatic  Israelstam  embryos  (1979).  was  based  Somatic  placed  sulfoxide for  10  screw-capped  (DMSO)/mg  mm.  solvent,  into  and  centrifuged  fresh weight,  Samples  were  incubated  at  (14,000  rpm)  65°C  for  a  measured  at  645  and  663  containing  that  shaken  droplets condensed on the side, was  vials  to  for few  had  20  been  45  mm.  seconds  to  l  heated  submerge  the  embryos  lines W29 and W70 were collected at Stages 4-6 and 4-9. were  on  Embryos dimethyl at  65°C  the  embryos  The  vials  recover  of  in  were  solvent  and the absorbence of each sample nm  against  a  DMSO  blank.  Total  chlorophyll was calculated using the equation: Total  [Chlorophyll)  in mg/g  =  (20.2 0645 + 8.02 ) 663 D  x V  (ml)  1000 x W (g) Where D  =  absorbence at the wavelength stated  V  =  total volume of the chlorophyll solution  W  =  weight of the fresh tissue extracted  Analyses experiments.  were  carried  out  twice  with  samples  from  separate  44 3.9.  IN VIVO PROTEIN LABELLING AND INMUNOPRECIPITATION Stage  4—7  somatic  sterile  Eppendorf  mixture  of  70%  Mississauga,  embryos  tube  of  containing  L-methionine,  ON;  loll  line 30  15%  Ci/mmole)  pCi  L-cysteine; in  300  to label for 4 hr at room temperature, embryos  were  rinsed with medium  methionine  and  unlabelled  methionine.  cultured  500  in  p1  Embryo  (immediately after the rinse),  described  previously  and  the  of  were 16  10  The  mm.  disc was  placed SDS  and  into  and  Aquasol  allowed a  in hot  (Du  to  Pont  radioactivity Scintillation  The  dry.  scintillation vial  allowed  stand Corp.,  was  for  2  SDS  Briefly,  pM  after  hr of  0  chase.  sample buffer acid  (TCA)  five p1 of each  allowed to dry,  (Beckman  (w/v)  10%  (w/v)  sample  containing hr.  Mississauga,  determined  Counter  24  10  and  TCA containing 1 mg/mi methionine for  boiled  to  and  trichioroacetic  washed by vacuum suction with cold 10% ethanol,  mM unlabelled  1  collected  4x  sample was spotted onto a filter paper disc, (w/v)  VE  3.4%  containing  medium  8,  precipitable radioactivity determined.  incubated in cold 10%  (a  Embryos were allowed  Proteins were extracted from embryos using as  a  Biomedicals,  liquid  containing  4,  5-Labei 35  ICN  of  p1  into  the solution was removed,  samples 2,  placed  Tran  of  medium containing 1 pM IBA and 40 pM ABA.  the  were  W29  using  TCA  spots  were  1 ml  was  of  mm,  added  Inc.,  out,  cut  (w/v)  0.1%  this,  LS  an  Instruments  10  TCA followed by 95%  Following ON)  for  1801  10  ml  and  of the  Liquid  Mississauga,  ON). Total of  labelled protein profiles were obtained by application  samples  containing  equal  TCA-precipitable  radioactivity  to  45 each  gel,  followed  Following  fixation,  deionized  (Enlightning; (v/v)  to  water Du  SDS—PAGE  by gels  were  remove  Pont  described  rinsed  excess  Corp.,  glycerol for 1 hr,  as  twice  (30  fixative,  Mississauga,  previously. each)  mm  placed  in  fluor  containing  ON)  dried at 60°C for 2 hr,  in  10%  and allowed to  expose at -80°C using Kodak X-Omat AR film. For  iinmunoprecipitation,  radioactivity sample, of BBS  of  inN  250  dilution)  of  incubated  for  4°C.  The  each  (w/v)  BSA,  methionine  hr  1  whole  For each individual  60 jl of 10%  and  BBS.  with mixing solution  11.5  Montreal, of  TCA-precipitable  160 mM NaCl pH 7.9].  5  QU), This  of  mg)  at  was  Protein  final  to  a  kept 20  A-Sepharose 3  1:275  overnight  il CL4B  1  for  14,000 rpm).  at  volume  bed  (Pharmacia  times with  shaken gently  temperature and centrifuged (2 mm;  a  give  and  which had been washed mixture was  Triton X-l00,  The reaction mixture was  37°C,  added  To this were  (v/v)  (to  jl  41 kD protein antiserum.  (approximately Inc.,  of  11-15 l of labelled protein extract was added to 1200 ,l [10 mM sodium borate,  l  amounts  were used for each reaction.  added 36 j.Ll of 16.5% 60  equal  hr  100  l  at room  The supernatant  was discarded and the Protein A-Sepharose washed 3 times with BBS containing 10% by  a  single  buffer  was  (v/v)  wash added  Triton X-lOO and 250 mM methionine,  with to  the  for 7 mm,  centrifuged  containing  the  each  0.3x  BBS.  Fifteen  jl  Protein A—Sepharose,  (5 mm;  14,000 rpm)  immunoprecipitated  immunoprecipitated sample was  protein,  of  4x  the  followed  SDS  sample  sample  boiled  and the removed.  supernatant, Ten  j.Ll  of  analyzed by SDS-PAGE and the  gel processed for fluorography as described above.  46  3.10.  41 kD STORAGE PROTEIN CDNA ISOLATION  A  cDNA  library  constructed  using  poly  cotyledonary somatic embryos cultured on 40 from Dr. into  Craig Newton at B.C.  the  I  Sma  site  competent SURE cells The  cDNA  library  containing  of  Research.  pUC  18  and  g/L  partially  approximately  bacto-yeast  ampicillin  7500  (Bio-Rad, plates  extract,  plasmids  screened  colony  covered  on  by  g/ml  the  using  forming  NaC1]  ,l  (IPTG).  in  were a  of  100  into  La Jolla,  CA).  plates,  2  units.  each  Colonies  [8 g/L bacto-tryptone, containing  100  tg/ml  overnight  lysed  for  TLC  tank  and  They in  30  growth  mm  in  equilibrated  at  .B-D— 37°C,  chloroform  30  mm  with  Filters were placed in a solution of 50 mM Tris-HC1  lysozyme  agitation.  Isopropyl  inN  After  150 inN NaC1 and 5 mM MgC1 , 2  incubated  transformed  and solidified with 15 g/L agar.  50  filters  suspension  chloroform. pH 7.5,  obtained  and placed colony side up on YT + amp  ON)  with  Thiogalactopyranoside  vapour  g/L  late  filters of the plates were made using nitrocellulose  Mississauga,  colonies  5  (YT + amp medium)  Replica  M ABA was  (Stratagene Cloning Systems,  was  from  RNA  Inserts had been cloned  were grown overnight at 37°C on YT medium 5  (A)  3%  (w/v)  were  TBS-Tween  CM,  rinsed  and 3  containing  purified 41 kD antibody.  containing 1 g/ml DNase,  Washing,  incubated  times a  1:400  with  overnight TBS-Tween,  dilution  of  40  with then  affinity  secondary antibody incubation  and colour development of filter immunoblots was as described for SDS-PAGE immunoblots. An filter,  antibody-positive  colony  was  identified  on  the  replica  the corresponding colony sampled and inoculated into 2 ml  47 of  +  YT  dilution  for  overnight  series was made  hundred jAl grown  broth  amp  procedure  a  replica  repeated colonies  as  this,  and  the  previously.  isolated,  a One  colonies/mi was plated,  filter made  described  were  Following  and plated onto YT + amp plates.  of a dilution containing io 2  overnight,  positive  growth.  grown  immunoscreening  Several  overnight  antibody—  in  +  YT  amp  broth and their plasmid DNA isolated by the alkaline lysis method described by Sambrook et al.  (1989).  Briefly,  1.5 ml of bacterial culture was centrifuged the  medium  Solution  I  Following NaOH,  removed [50  mM  this,  1%  and  glucose,  200  (w/v)  the  il  SDS]  of were  pellet 25  mM  acid,  Tris-HC1,  freshly added  transferred to  mm;  of  14,000 rpm),  mixed,  8].  [0.2  mixed  N by  ice cold  the tube kept  14,000 rpm)  (1:1),  pH  of  11.5 ml glacial acetic  fresh tube,  phenol:chloroform  II  and then 150 jl of  supernatant volume  EDTA  contents  and centrifuged (5 mm;  equal  mM  jLl  100  the  and  were added,  a  with  Solution  on ice for 5 mm was  10  14,000 rpm),  prepared  [60 ml 5 M potassium acetate,  28.5 ml deionized water]  (30 sec;  resuspended  inverting the tube rapidly 5 times, Solution III  for each sample,  at 4°C.  extracted with  mixed,  centrifuged  The an (5  and the aqueous phase removed and mixed with 2  vol of 95% ethanol.  The DNA was allowed to precipitate for 2 mm  at room temperature,  the sample centrifuged  at  4°C,  the  vacuum dried HC1,  pellet  washed  with  95%  and resuspended with  50  (15 mm;  ethanol, ,.L1  TE  then  buffer  14,000 rpm) 70%  ethanol,  [10 inN Tris  1 inN EDTA pH 8].  The plasmid cDNA inserts were excised using restriction enzyme digests.  All restriction enzymes were used with the appropriate  48 buffers  and  conditions  described  by  the  plasmid DNA samples were treated with 5%  suppliers,  (v/v)  and  all  of 10 mg/mi RNase  during digestion to destroy contaminating RNAs.  Two p1 aliquots  of the plasmid DNA samples isolated above were digested with Eco RI—Pst I and analyzed by agarose gel electrophoresis. each  digest mix was  bromophenol blue,  added to  0.25%  (Pharmacia)  in water],  in  [50x  lx  TAE  containing buffer light  0.5  TAE  consisting revealed  fragment  (1.7  kb).  dye mix  xylene cyanol FF,  and separated on a 1.2% 2  ix  N  Tris-acetate,  TAE.  they One  designated as 115A.0. Pst  Ficoll  p1  ethidium bromide,  of  that  (w/v)  =  pg/mi  1  with the  contained  colony was  (w/v)  0.05  Examination  all  15%  chosen  [0.25%  Ficoil  agarose gel EDTA  pH  under  sized  further  insert  band  was  use  volume  visualized  under  UV  light,  cut  out  and  perchlorate,  and  3  1 mM EDTA,  vol  The gel  (Bio-Rad, Mississauga, ON).  the gel fragment was placed into an Eppendorf tube, estimated  and  This plasmid DNA was digested with Eco RI  purified using the Prep-A-Gene method Briefly,  UV  insert  and the insert fragment separated on an agarose gel.  I  8],  electrophoresis  same  for  (w/v)  (w/v)  samples  of  the  N  Ten p1 of  binding  buffer  50 mM Tris-HC1 pH  7.8]  [60 was  the  sodium  mM added.  The  sample was heated at 37—55°C for several minutes to dissolve the agarose, added.  after  which  p1  of  Prep-A-Gene  binding  matrix  was  The sample was mixed by vortexing, then shaken for 10 mm  at room temperature. rpm),  10  The sample was centrifuged  (30 sec;  14,000  the supernatant removed and the pellet rinsed 3 times with  1 ml of a 1:1 mixture of 95% ethanol:wash buffer mM EDTA,  20 mM Tris-HC1 pH 7.4],  [400 mM NaCl,  2  followed by suspension with TE  49 buffer. above,  This  was mixed  by vortexing,  centrifuged  and the supernatant TE transferred to a  as  described  fresh tube.  One  tl of the TE fraction was diluted to 100 jl with fresh TE buffer and  the  DNA  quantified  spectrophotometrically  gel-purified DNA was used for 115A.0 probe.  at  260  nm.  The  Other gel purified  probes  (XI5H and lBS rRNA) were obtained from Dr.  3.11.  GENERATION OF DELETION CONSTRUCTS FOR SEQUENCING  Craig Newton.  Two hundred ml of YT + amp broth was inoculated with bacteria containing the 115A.0 insert. 37°C,  nucleic  procedure. buffer  acids  and  the  (4.2 g)  plasmid  isolated  using  plasmid  DNA  isolated  (CsC1-EtBr)  temperature  in  Mississauga,  ON).  with 8 ml of  gradient  using  chloride  centrifugation.  Briefly,  and  centrifuged  J2—2l  a  (5  Centrifuge  mm;  filled with light CsC1 solution,  at  hr;  needle  the  bottom  40,000  of  rpm)  and  extracted  isobutanol.  transferred to a clean tube, of 95% ethanol were added, at  room Inc.,  this  in  an  tube.  and the plasmid The  tube  was  sealed and centrifuged L8—M  Ultracentrifuge.  the lower band was removed from the tube using a  water—saturated  mm  at  Instruments  (63 g/lOO ml),  balanced,  Following this,  rpm)  A Ti70..l centrifuge tube was partially filled  light CsCl solution  (18  6,000  (Beckman  placed  30  lysis  cesium  solution  21—gauge  alkaline  and 400 l of 10 mg/ml EtBr were added to the 2.4 ml  solution,  overnight  the  The resulting pellet was resuspended in 2.4 ml of TE  ethidium bromide CsC1  were  Following overnight incubation at  -80 C 0 .  The  4  times The  3  with  lower,  an  equal  aqueous  volume phase  of was  vol of deionized water and 2 vol  and the DNA allowed to precipitate for  sample  was  centrifuged  (15  mm;  14,000  50 rpm),  the  pellet  washed  with  ethanol,  dried  under  vacuum  and  resuspended with TE buffer. The resulting DNA preparation was used for deletion construct formation.  In order to  sequence both  was required in both orientations. by Eco  RI-Sal  fragment  gel  fragment,  I  double  strands,  and  blunt-ended.  500 ng of 115A.0 DNA in 3  pH 7.9],  2  [125  .LM  This  phase  was  was  extracted  mixed  precipitated  with  with 2  with  0.5  vol  of  ethanol,  dried,  blunt  7.5  95%  dCTP,  ainmonium  M  ethanol  resuspended  end  the  30 mM  100 inN Tris—acetate dGTP,  phenol:chloroform,  vol  resulting  dTTP]  and incubated for 15 mm  at  obtained following centrifugation (15 mm; with  the  [1.5 inN DTT,  each of dATP,  and 1 unit of Klenow DNA polymerase, 37°C.  insert  of TE buffer was added to  200 InN potassium acetate,  j.tl dNTP mix  and To  a solution containing 6 ul of reaction buffer magnesium acetate,  cDNA  The 115A.0 insert was excised  restriction digestion  purified  the  with  acetate The  14,000 rpm)  TE  buffer  aqueous  the  -80°C.  at  and  pellet  was washed  and  the  DNA  content was determined. Ten jg of pEMBL 8 Sma  I,  and  intestinal  the  resulting  alkaline  recircularization. sample  was  vector  linearized  phosphatase  Briefly,  extracted  (without  with  insert) plasmid  (CIP)  to  was digested with treated prevent  with  calf  plasmid  following restriction digestion, the phenol:chloroform,  the  aqueous  phase  mixed with ammonium acetate and precipitated with 95% ethanol as described previously. j1 of  10 mM Tris-HC1  CIP buffer  The resulting pellet was resuspended in 90 (pH 8.3).  [10 inN ZnC1 , 2  To this was added  10 mM MgC1 , 2  10 jil of  lOx  100 inN Tris-HC1 pH 8.3]  and  51 1  ul  (22  mm,  units)  CIP.  The  sample was  followed by the addition of  C for 45 mm. 0 55  of CIP and  l  The  for  37°C  15  incubation at  after which the sample was  (pH 8),  phenol:chloroform,  the  aqueous  ammonium acetate and precipitated with 95% above.  at  CIP was inactivated by heating at 65°C for 1 hr  in the presence of 5 mM EDTA extracted with  1  incubated  pellet  was  resuspended with  phase  mixed  with  ethanol as described  TE  buffer  and  the  DNA  content determined. The vector and insert were ligated using a 3:1 ratio of insert:vector.  (300 ng:100 ng)  The insert + vector were contained in a  total of 3 tl of TE buffer, to which was added 1 l of lOx ligase buffer 10  mM  [500 mM Tris—HC1 pH 7.6, DTT  and  1  l  of  T4  DNA  100 mM MgC1 , 2 ligase  10 mM ATP],  5 l of  units).  Ligation was  allowed to proceed overnight at room temperature,  then the 10 il  ligation  mixture  (Stratagene incubated  was  Cloning  for  30  added  to  Systems,  mm  on  ice,  (5  of  100  l  La  Jolla,  heated  at  competent CA),  42°C  then placed on ice  for 2 mm.  g/L bacto-tryptone,  5 g/L bacto-yeast extract,  g/L glucose]  was added,  mixed  cells  gently,  minutes  and  SOC broth  [20  0.5 g/L NaC1,  3.6  for  2  Two hundred ,l of  the sample warmed to  SURE  37°C for 5 mm  and  shaken at 37°C for 1 hr.  Cells were plated onto YT + amp medium  and  37°C.  grown  removed,  overnight  the  plasmid  digested with Barn HI,  at DNA  Ten  isolated  by  individual alkaline  colonies lysis,  and separated on an agarose gel.  the  were DNA  Samples  were compared to Barn HI-digested plasmid containing II5A.0 in the forward orientation.  A colony containing altered fragment sizes  52 expected  from  the  reverse  was  orientation  identified  and  were  for  designated as RII5A.0. Plasmids  containing  115A.0  RII5A.0  and  used  unidirectional deletion construct formation following the Promega system  Erase-A-Base (5-10 g)  (Promega  Madison,  Corp.,  was digested with Sal  I  WI).  Plasmid  and a  overnight,  DNA  small amount  was analyzed by agarose gel electrophoresis to assay for complete digestion. Pst  I  the plasmid DNA was digested with  After completion,  for  6  and  hr,  phenol:chloroform  extracted  (1:1).  The  with  aqueous  an  equal  phase  was  ammonium acetate and precipitated with 95%  ethanol  previously.  30  buffer  The pellet was resuspended  [lOx Exo III buffer  2.5  ,1  carried out  aliquots  were  mixed as  jl of  4.6,  2.5  nuclease)  M  ix Exo  10  at  35°C.  collected  After a  every  30  25  sec  lag period,  sec  for  up  to  mM  , 4 ZnSO  [0.3 M Tris base,  50%  determined  by  analysis  electrophoresis. mix  of  2  1  of  mm.  [172 jl  units  Si  then 1 jl  was added and the  The extent of digestion was each  sample  by  agarose  gel  Samples were heated to 37°C and 1 l of Klenow  [30 l ix Klenow buffer  (20 mM Tris-HC1 pH 8,  5 units Kienow DNA polymerase] for 3 mm,  60  glycerol),  0.05 M EDTA]  samples heated at 70°C for 10 mm.  5  (0.3 M potassium acetate pH  and incubated at room temperature for 30 mm,  Si stop buffer  III  and then 250 units of Exo III added.  27 1 7.4x Si buffer  NaC1,  with  described  These were placed into tubes containing 7.5 l of Si mix deionized water,  of  6.6 mM MgC1 ], 2  660 mM Tris-HC1 pH 8,  =  heated at 35°C for 10 mm, Digestion was  in  volume  was added,  the  100 mM MgC1 ), 2  samples  followed by the addition of 1 j1 dNTP mix  incubated  [125 ,M each  53  of  100  10 mM ATP),  , 2 100 mM MgC1  7.6,  was  l)  5  ligase (500  buffer  used  then  to  [890  jl  Tris-HC1  pH  mix  mM  5 units T4 DNA ligase]  10 .tl DTT,  100  transform  Samples  mm.  Ligation mix  ligation allowed to proceed overnight.  and  added,  of  ,l  40  ligase  lOx  ,l  for  incubation  temperature,  room  water,  deionized  (10  to  moved  were  and  dTTP]  dGTP,  dCTP,  dATP,  l  of  cells.  competent  Ten  These were plated onto YT + amp medium and grown overnight. individual  colonies  from each deletion time point were used for The resulting DNA was  plasmid DNA isolation by alkaline lysis. digested with Eco RI-Hind  double  analysed  by  gel  agarose  III  excise  to  the  insert,  containing  Colonies  electrophoresis.  and  The deletions obtained  deletions were identified in this manner.  spanned the entire length of the 115A.0 and RII5A.0 inserts. DNA SEQUENCING  3.12.  DNA  sequences  were  obtained using  sequenced,  each colony representing a deletion time point to be plasmid DNA was culture using  isolated from a 2-4 ml YT + amp broth overnight The DNA was resuspended in 100 jll  alkaline lysis.  with 1 l RNase  of TE buffer and then treated  hr at 37°C to digest contaminating RNA. 2.5  M NaCl/20%  PEG  8000  was  room temperature for 15 mm, 4°C and the supernatant was  resuspended  in  100  ammonium  acetate  added,  the  ul  and  buffer,  for 1  60 l of  contents mixed,  left at  (10 mm;  centrifuged  TE  (10 mg/nil)  Following this,  (containing the RNA)  volume of phenol:chloroform, with  For  dideoxy method.  the  14,000 rpm)  The pellet  removed.  extracted  at  with  an  equal  the aqueous phase removed and mixed precipitated  with  95*  ethanol  as  54 The pellet was  described previously.  in  resuspended  buffer  TE  and the DNA quantified.  TE  was  buffer  to  added 10  temperature for  4  1  1  of  M  at  incubated  NaOH,  and precipitated with  mm,  in 16 ,.tl  (2-4 g)  A portion of the template DNA isolated above  vol  0.5  of  7.5 M  The pellet  ammonium acetate and 2 vol of 95% ethanol at -80°C.  dried  obtained following centrifugation was washed with ethanol,  1 jl  and resuspended in a solution of 5 jl deionized water,  100  mM  [500 mM Tris-HC1  sample buffer  sequencing  and  ] 2 MgC1  1  1  (5’ GTAAAACGACGGCCAGT ). 31 briefly  centrifuged  1  100  il  of  Klenow  mM  ON;  Mississauga,  This collect the  to  2  ng/tl  was  mixed  forward  primer  vortexing,  by  incubated  and  sample  mM NaC1,  100  7.5,  lOx  37-  at  The sample was placed on ice and 1 1 of 15 M  45°C for 15 mm. dATP,  of  pH  room  3000 Ci/mmole)  Pont  Corp.,  and 4.5 l of a 0.5 unit solution  added.  polymerase  DNA  (NEN-Du  l o P-dATP 32 -  1.5  DTT,  This  the  was  solution  template/primer/label mix. a  In  petri  on  dish  (ddN/dN)  nucleotide/deoxynucleotide [(G mix:  18  l  1 mM ddG,  dTTP and 1 mM dCTP, ddA,  22  l  of  each  deionized water),  of  l  1.5  mix was  16 l 0.1 mM dGTP,  1 m dTTP  mM dGTP,  (T mix:  and  12.5 ,l 10 mM ddT,  the  dish  32 1 each of  1 mM  in  (A mix:  mM dCTP,  l  32 jl 1 mM dGTP,  26  97 ,il deionized water),  32 1 1 mM dCTP,  75 ,1 1 mM ddC,  32 1 each of 1 mM dGTP and 1 mM dTTP,  mM dCTP,  20 jl deionized water).  (3.5  was  added  to  each  of  23 jl 1 mM 111  1  1 0.1 mM dTTP,  l)  dideoxy  each  spotted  1 deionized water),  102 1  ice,  the  (C mix:  41 jll 0.1  The template/primer/label mix ddN/dN  spots,  and  the petri  55 dish placed onto a water bath at this,  the  petri  dish  was  37-42°C  placed  back  on  solution  [1 mM each of dGTP, dATP, dTTP,  and,  dish placed  the  Reactions  were  onto  stopped  the water  by  placeing  adding 5 jl formamide dye mix inN  EDTA  pH  8,  0.025%  bromophenol blue]  [98%  (w/v)  the (v/v)  5  1  of  l  chase  added to each spot  for  further  a  dish  petri  on  5  mm.  ice  and  deionized formamide,  cyanol  FF,  0.025%  10  (w/v)  The petri dish was placed on a  boiling water bath and the samples heated for 2 mm. 2 l)  Following  mm.  ice,  dCTP]  bath  xylene  to each spot.  for  Samples  (1-  7.5  ml  lOx  9.4  g/L  were then loaded onto sequencing gels. Sequencing  modified  TBE  gels  [162  EDTA.2H Na 0 2 ], water,  were  g/L  made  Tris  11.25 ml 40%  to give a  using  base, (w/v)  37.5  27  g/L  g  urea,  boric  acid,  acrylamide and 32 ml deionized  final urea concentration of 8 M and acrylamide  concentration of  6%.  The gel mix was  degassed for 30  filtered,  iuin and polymerized with 300 jl 10% ammonium persulfate and 15 jl TEMED.  Gels  were  electrophoresis  run  buffer.  at  2000  Following  V  using  lx  as  TBE  electrophoresis,  gels  the were  dried at 80°C for 1 hr and allowed to expose at -80°C using Kodak X-Omat RP film. coding  strand  Sequences for both strands were obtained and the sequence  compared  to  sequences  in  the  EMBL  bank using PC/GENE (IntelliGenetics Inc., Mountain View, 3.13.  CA).  GENOMIC DNA EXTRACTION, ELECTROPHORESIS AND BLOTTING  Spruce needles  (5 g) were ground in liquid nitrogen,  suspended  in 90 ml of extraction buffer [50 ml/L 1 M Tris-HC1 pH 8, 0.5  data  M  EDTA,  spermidine,  63.78 5  g/L  g/L  sorbitol,  spermine,  1  100 ml/L  g/L  PEG  8000,  10 ml/L 5  2-mercaptoethanol]  g/L and  56 filtered  through  12  layers  of  cheesecloth.  The  solution  was  filtered through 1 layer of Miracioth and 1 layer of 53 m nylon mesh,  centrifuged  and  Centrifuge. in  3.6  ml  CsC1—lauryl  lauryl  Ti70.1  rpm)  (400  solution  centrifuge  solution  l  and  of  a  10 mg/mi  (10 mm;  was  heated  [17.5  and  containing  8  4°C  CsC1  g  a  J2-2l  for  10  10 ml (w/v)  mm  at  60°C.  was  added and  at room temperature.  placed of  in 0.5%  solution)  ml  in  10 mM EDTA,  10,000 rpm)  removed  tube  at  the pellet resuspended  (50 inN Tris-HC1 pH 7.8,  the sample centrifuged clear  10,000  sarcosine  sarcosine)],  Ethidium bromide  The  mm;  The supernatant was removed,  sarcosine solution sodium  (20  at  the  light  bottom  CsC1  of  a  solution,  and the sample centrifuged as described previously for CsC1-EtBr gradient with  centrifugation.  isobutanol,  The  DNA  precipitated  band  as  was  removed,  extracted  previously  and  each restriction digest,  and,  described  resuspended in TE buffer. Five  of  following acetate  DNA were used  digestion,  and  ethanol  the as  for  DNA  was  described  precipitated previously.  with The  ammonium resulting  pellet was resuspended in 20 .tl TE buffer containing 5 l Ficoil dye mix,  and the DNA separated on a 1% agarose gel.  then rinsed 3 times  (10 mm  each)  followed by 3 rinses(10 mm then Corp.,  blotted  overnight  each)  onto  Arlington Heights,  with 0.5 M NaOH + 1.5 M NaC1, with 1 M ammonium acetate,  Hybond-N  IL)  The gel was  using  1  nylon  membrane  N ammonium  and  (Amersham  acetate.  The  blot was air dried and the DNA cross-linked to the membrane by UV exposure  for  10  mm.  Gene  copy  reconstructions  to  approximate  the number of vicilin gene copies were constructed using 115A.0  57  insert  assuming a  and  haploid DNA content  been described for white spruce 3.14.  [0.1%  isolated, —80°C  (v/v)]  protocol  the  frozen  until  nitrogen to (precooled tubes.  of  immediately  a  liquid  0.1 M LiC1,  sec,  then chloroform  phenol) After  liquid  500  l  that of  was  added  (250 l)  an  equal  to  each  centrifugation,  the  was  tubes using  (5 mm;  volume tube two  of  (15 mm;  .Ll water,  a  10 mM EDTA,  at  liquid  in  glass rod into  extraction  the  buffer  1%  (w/v)  and water  14,000 rpm).  followed by another 30 14,000 rpm),  extraction  the  1 vol 4°C,  (minus  buffer  from of  the water  back—extracted.  sample  phases  were allowed to precipitate overnight at  in 250  stored  precisely  (80°C)  added,  extraction were pooled and mixed with  centrifugation  ground  fitted  hot  and  were  The samples were mixed by vortexing for 30  After centrifugation  removed,  were  in Eppendorf  nitrogen)  Tissues  nitrogen  tissues  extracted  RNA was  (1989).  100 inN Tris-HC1 pH 8,  (1:1)] was added.  was  Total  al.  et  in  Frozen  fine powder  SDS  phase  Verwoerd  needed.  in  sec vortex.  (Dhillon 1987).  deionized water.  After grinding,  [phenol:  which has  pg,  for RNA work were made using diethylpyrocarbonate—  Solutions  using  8.5  ELECTROPHORESIS AND BLOTTING  RNA EXTRACTION,  treated  of  4  each  sample  M Lid.  RNA5  then collected by  The pellets were dissolved  0.1 vol of 3 N sodium acetate pH 5.2 was added  and the RNAs precipitated with 2 vol of 95% ethanol at -80°C for 30  mm.  ethanol, SDS.  After  centrifugation,  the  pellets  were  washed  dried and resuspended in TE buffer containing 0.1%  with (w/v)  58 The purity and integrity of samples was checked by agarose gel electrophoresis.  Two ,l of RITA sample was added to  buffer  0.1%  containing  (w/v)  SDS  and  consisting SDS.  Electrophoresis buffer consisted of lx TAE with 0.1%  (w/v)  SDS.  For RNA quantification, TE  (w/v)  buffer  and  agarose  and then applied to a gel (w/v)  with  1%  The  0.1%  il  of  l of TE  Ficoll dye mix.  1 l  samples were heated at 65°C for 5 mm  8  in  lx TAE  containing  1 l of sample was diluted to 100  quantified  spectrophotometrically  at  260  nm. For RNA electrophoresis and blotting,  et al.  (1988)  water,  25  l  formamide,  was used. of  l  lOx  propanesulfonic acid,  glycerol, 65°C  for  240  formaldehyde  37%  (w/v)  formaldehyde,  [225  ml  were  water,  g  4.1 ml 37% formaldehyde,  5 l of 10 mg/nil EtBr]  M  3-(N—morpholino)  1  pH 7 and  water,  100  l  and heated at  added,  fractionated 2.5  deionized  [750 ,l  10 mM EDTA,  100  bromophenol blue]  Samples  gels  (0.2  50 mM sodium acetate,  mm.  MOPS/EDTA buffer,  MOPS/EDTA buffer  jl  80 ,.Ll 10% 15  RITA samples were adjusted to 5 jl with  electrophoresis sample buffer  150  autoclaved),  the protocol of Fourney  on  agarose,  denaturing 25  ml  lOx  0.46 g iodoacetamide,  run overnight at 25 V,  using lx MOPS/EDTA  as the electrophoresis buffer. Following electrophoresis, NaOH in lx SSC by  2  rinses  [20x SSC  (20  mm  =  each)  gels were rinsed 20 mm  3 N NaC1, in  lOx  0.3 M Na 3 citrate], SSC.  They were  overnight onto Hybond-N nylon membrane using lOx SSC. allowed to  air  dry,  in 0.05 M followed  then blotted Blots were  and the RNA crosslinked to the membrane by  exposure to UV light for 5 mm.  59 3.15.  cDNA PROBE PRODUCTION ND HYBRIDIZATION TO BLOTS  All  probes  were  primer method. (25—50  ng)  was mixed with  lOx buffer  9.5  (NEN-Du and  incubated  2 p1 dNTP mix  Pont 1  p1  Corp.,  Klenow  overnight  at  375  p1  80°C for 30 mm, at  4°C.  The  resuspended  oligonucleotide  1 p1 of template cDNA  To this was added 2 p1  100 mM MgC1 ], 2  1 p1 1 mg/mi BSA,  [10 mM each of dGTP and dTTP],  Mississauga,  DNA  heated at  of deionized water,  polymerase  room  with  units).  (5  temperature.  1 p1 of  and  each  ON;  This  The  0.5 N EDTA,  1  probe  3000 was was  80 p1 deionized  coil carrier tRNA (2 mg/mi), 50 p1 7.5 M ammonium  3 p1 E.  acetate and  random  P-dATP) 32 2 p1 each of oc.-(  precipitated by addition of water,  p1  [900 mM HEPES pH 6.6,  p1 random hexamer primer,  Ci/mmol)  the  then snap cooled on ice.  100 mM DTT,  dCTP)  using  In a 1.5 p1 Eppendorf tube,  98°C for 10 mm,  1 p1  generated  95%  to the tube and  followed by centrifugation  pellet  in  ethanol  was  100 p1  washed with  TE buffer.  (15 mm;  ethanol,  One p1  incubation at  of  -  14,000 rpm)  vacuum  dried  and  labelled probe was  placed in a scintillation vial and the radioactivity determined. Probes were heated at 98°C for 5 mm  and then snap cooled on ice  immediately prior to their use. DNA  blots  were  pre-hybridized  overnight  approximately 30 ml of pre-hyb/hyb solution 3.6 M NaC1,  0.2 M 2 H Na . 4 0 7H PO ,  thymus DNA,  1%  (w/v)  BSA,  2%  =  2%  0.05%  (w/v) (w/v)  sodium  SDS, (w/v)  [5x SSPE  0.02 N EDTA),  polyvinylpyrrolidone,  Following pre-hybridization,  10%  (w/v)  68°C  (20x SSPE  in =  100—200 pg/mi calf  5x Denhardt’s solution  pyrophosphate,  at  (bOx Denhardt’s  2%  (w/v)  dextran  Ficoll), sulfate].  blots were hybridized at 68°C for 2  60 days  with  labelled  probe  (1-2  106  x  approximately 30 ml of hyb solution. (30 mm  each)  cpm/ml  of  solution)  Blots were washed 3  at 68°C with 2x SSC + 1%  (w/v)  in  times  SDS and allowed to  expose at -8 0°C with an intensifying screen using Kodak X-Omat AR film. RNA blots were pre—hybridized overnight at 68 C in pre-hyb/hyb 0  solution EDTA].  [0.5  M  Following  sodium this,  phosphate blots  pH  were  7.2,  7%  hybridized  (w/v) for  SDS, 22-24  1  itiN  hr  at  68°C in approximately 30 ml of hyb solution containing 1-2 x 106 cpm of labelled probe/mi of solution. were washed twice (w/v)  (20 mm  followed by a 20 mm  Blots were then allowed to expose at -80 C with an 0 screen using Kodak X-Omat AR film. blot was (w/v)  SDS  stripped with  blots  with a solution of lx SSC + 0.1%  each)  SDS at room temperature,  After hybridization,  several  250  wash at 68°C. intensifying  For reprobing of blots, ml  washes  of  boiling  each 0.1%  and the membrane was checked by overnight exposure to  ensure complete removal of probes.  Autoradiographs of RNA blots  were scanned at 595 nm in a DU—64 Spectrophotometer.  61 4.  4.1.  IDENTIFICATION  AND  RESULTS  CHARACTERIZATION  OF  ZYGOTIC  EMBRYO  STORAGE PROTEINS  The  storage  SDS-PAGE (Fig. 41,  proteins  analysis  1). 35,  of  of  interior  spruce  total  proteins  from  were  mature  identified seed  by  embryos  Prominent proteins with apparent molecular weights of 33,  proteins,  24,  22,  17  and  16  kD  with the exception of the  were 17 and  detected in megagametophyte tissue extracts determine  if  any  of  the  proteins  were  Similar  identified.  16 kD proteins,  were To  (data not shown).  storage  proteins,  protein profiles were examined during germination  (Fig.  embryo By  1A).  day 3 after sowing,  at which time radicle emergence had occurred,  the 41,  and 22 kD proteins were almost undetectable,  35,  indicating of  33,  24  rapid  storage  degradation  protein  during  mobilization.  germination, However,  proteins were still detected on day 9 suggesting  that  they  assumption  that  the  proteins,  protein  were 5  not  proteins  bodies  were  characteristic  the  17  and  of germination  storage missing isolated  proteins. by  day  from  solubilized and analyzed electrophoretically  The  35,  profile. 1B).  33,  24  and  22  kD  bands  were  the  storage  embryonic  mature  tissues, 41,  test  were  kD  1A),  (Fig.  To 3  16  (Fig.  prominent  in  Minor bands of 30 and 27.5 kD were also observed  1B). this (Fig.  These proteins were also present in megagametophyte tissue  (data not shown). To determine if linkages,  extracts  individual proteins were joined by disulfide of  isolated  protein  bodies  made  under  non—  reducing conditions were examined by SDS—PAGE under non—reducing,  62  Coomassie-stained SDS-PAGE of embryo proteins. FIGURE 1. Total embryo protein changes during germination. Lane 1, A. mature embryos; Lane 2, 3 days after sowing; Lane 3, 6 days after 12 days after sowing; Lane 4, 9 days after sowing; Lane 5, Fifteen sowing; MW, molecular weight standards. jg protein was The 41, 35, applied to Lane 1 and 10 ,g protein to all others. well as the 17 and 33, 24 and 22 kD proteins (solid arrows), as 16 kD proteins (open arrows) are indicated. Proteins from mature embryos (Lane 1) and isolated protein B. Fifteen ,g bodies (Lane 2). NW, molecular weight standards. Arrows denote the 41, 35, 33, protein was applied to each lane. 30, 27.5, 24 and 22 kD proteins.  2 3  A  ,,  1 4  5  I—  MW  97.4  kD  B  1  b4  14.4  21.5  31.0  42.7  66.2  97.4  kD  64 and  then  reducing  conditions  (Fig.  2).  The  patterns  observed  under reducing conditions differed from those observed under non— reducing conditions. non—reducing  conditions  electrophoresis bands  of  35,  previously  The 55-57 kD protein doublet present under  under  33,  24  linked  reducing and  by  (Fig.  22  2B)  conditions  kD  (Figs.  disulfide  was  absent  and  replaced  2A  and  bonds.  It  2C),  was  doublet  (Fig.  non—reducing  2C).  The  conditions  minor  was  also  34  kD  protein  resolved  by  four  which  were  difficult  determine the specific association of the proteins kD  after  to  in the  55-57  observed  under  into  component  two  proteins of 14 and 22 kD when analysed under reducing conditions. The  14  kD protein was often not readily observed in the protein  body fraction and was kD  proteins.  less prominent than the minor 30  Apparently,  the  disulfide  linked  22  and 27.5  and  14  kD  complex is a very minor component of the protein body fraction. The  storage  proteins  identified  previously  according to their solubility characteristics.  were  classified  Isolated protein  bodies that were extracted with phosphate buffer or water yielded only the major 41 kD and minor 30 and 27.5 kD bands 3B).  3A and  The remaining proteins were only soluble in SDS-containing  buffer were  (Figs.  (Fig.  thus  3A)  or buffer containing 1 M NaCl  two major  protein bodies. cotyledonary  solubility  of  proteins  3B). in  There  isolated  Microscopic analysis of protein bodies from late  zygotic  staining regions  classes  (Fig.  (Fig.  embryos 4),  revealed  two  distinct  protein—  indicating the heterogenous nature of  the proteins within these organelles.  65  Coomassie-stained SDS-PAGE of zygotic embryo protein FIGURE 2. body extracts under reduced non—reduced (B), and two— (A), dimensional SDS—PAGE of non—reduced extract under non—reducing conditions followed by electrophoresis under reducing conditions (C). The 41, 35, 33, 30, 27.5, 24 and 22 kD proteins are indicated by open arrows (A). The 55-57 kD doublet present under non-reduced conditions is indicated by solid arrows (B). The molecular weights of non—reduced proteins and the corresponding proteins obtained by second dimension SDS-PAGE are indicated (C). Each sample contains 4 jtg protein.  .  4  F  41 55-57  Non—Reduced 35  1  C  -30 —27.5  —35 —33  —41  -22 ,-24  I’ll—  27.5 30  0.  e  0. C C)  m  0’  67  FIGURE 3. Coomassie-stained SDS-PAGE of protein body samples extracted under different conditions. A. Buffer-soluble (Lane 1) and SDS-soluble (Lane 2) proteins extracted from isolated protein bodies. A 10 l sample was applied to each lane. MW, molecular weight standards. B. Water-soluble (Lane 1), buffer—soluble (Lane 2), low salt soluble (Lane 3) and high salt-soluble (Lane 4) proteins extracted from isolated protein bodies. A 10 jl sample was applied to each lane. MW, molecular weight standards.  kD  97.4 66.2  42.7  MW  31.O  21.5  —  1  A  ,. ‘J  2 1 2  .  jzJ  3  —  B  I. L  4  r hflui  -  MW  .iii-i-  ,-  kO  97.4 66.2 42.7  31.0  21.5  14.4  a’  69  FIGURE 4. Light micrograph of a longitudinal cotyledon section from a late maturation stage zygotic embryo, stained by the periodic acid-Schiff’s technique and counter-stained with aniline blue black. Protein bodies (PB) containing light and dark staining zones are visible. N, nucleus. x 1100.  0  I  S  /  0  a  4. a  L  .4  *  ‘a z a  A  S  1  S Sr  j  71 Protein  body  samples  were  subjected  electrophoresis using both narrow pH pH  (3-10  ampholytes)  contained  the  ranges  largest  (5-8  (Fig.  number  of  to  two—dimensional  ampholytes)  5).  The  41  isoelectric  and broad kD  protein  variants,  with  approximately 10 or more proteins found across the range of the narrow  pH  gradient  gel.  It  distinct spots for the 35, different  gradients  33,  and  was  often  difficult  to  resolve  24 and 22 kD proteins and although  running  conditions  proteins were usually seen as smears.  were  tried,  these  The 35 and 33 kD protein  variants were  located primarily at the basic end of the  gradient gel.  However, the 24 and 22 kD proteins which were also  basic  variants  gradient gel located  were  (Fig.  towards  more  SB).  the  readily  observed  in  STORAGE  acidic  PROTEIN  broader  pH  The minor 30 and 27.5 kD proteins were region  of  the  gels  readily visualised in the narrow pH gradient gel 4.2.  the  narrow  ACCUMULATION  and (Fig.  DURING  were  more  5A). EMBRYO  ZYGOTIC  DEVELOPMENT  To time,  follow the appearance of the major storage proteins over embryos from Stage 2 to maturity were analyzed by one— and  two—dimensional summer of  electrophoresis.  Embryos  collected  1988 were used for two—dimensional  analysis  during  the  (Fig.  6),  and a more complete developmental sequence was obtained from the one-dimensional analysis 7).  of embryos collected during  Major accumulations of storage proteins,  kD protein, already  occurred in the Stage 4 embryo,  well  developed  proteins were detectable,  (Figs.  6  and  7).  1989  (Fig.  especially the 41  when cotyledons were The  albeit at low levels,  other  storage  at much earlier  72  FIGURE 5. Silver-stained two-dimensional electrophoretograms of zygotic embryo protein body extract examined using pH 5-8 ampholytes (A) or pH 3-10 ampholytes (B). The acidic (+) and basic (-) ends of the gel are indicated. A total of 12 ug protein was analyzed for each pH gradient.  r)3  IEF  +  —  —  4—  1  —  kD 427  —31.0  —21.5  A  —42.7  —l  I;  74  Silver-stained two-dimensional electrophoretograms of FIGURE 6. 3 representative zygotic embryo stages collected during the Proteins were separated using pH 5—8 ampholytes, summer of 1988. and the representative portions of the gels containing the major Acidic (+) and basic storage proteins (boxed regions) are shown. (—) ends of the gels are indicated.  rg  IEF +  -  Stage 2  ii  Stage 4-1  -1  S  Mature  SDS— ,PAGE  76  FIGURE 7. Coomassie-stained SDS-PAGE of developmental stage changes in total protein during EK1O zygotic embryogenesis. Zygotic embryos collected during the summer of 1989, ranging in development from Stage 2 to mature seed embryos (Stage 4, August 28 collection) are shown. Molecular weight standard locations are shown on the left. Twelve pg total protein was loaded in each lane.  21.5—  31.0—  42.7—  66.2—.  97.4—  kO  Stage Stage 2 3A  —  -,.  1.  Stage Stage 38 3—4  b-  —  ..4b  27  iP  20  JN  4I  .4  4  .  ..  -  —  -..  .-  —.  .— -.-*  *  -  AU 15  --.q  •*••• .—..  8  .  —.——,————.. —. —  1  I  23  *1  —I--I  28  rr-’ZE.W  -  -  —  25  Stage 4 18  ——-.————a—. -—  11  JL  78 stages of development, storage proteins.  suggesting the differential regulation of  Although the 35,  33,  24 and 22 kD proteins did  not show major accumulations until cotyledonary development, they were  detected  summary  of  at  the  low  levels  in  developmental  stage  embryos  2  appearance  of  (Fig.  the  major  6).  A  storage  proteins in 1988 collection embryos is presented in Table 2. 4.3.  IDENTIFICATION  STORAGE  PROTEINS  AND  AND  CHARACTERIZATION  COMPARISON  WITH  OF  SOMATIC  ZYGOTIC  EMBRYO  EMBRYO STORAGE  PROTEINS  Somatic embryos were matured in the presence of various levels of ABA  (0-60 jIM)  obtained (Fig. of  structures  production extensive  did  period. M.  8).  The level of ABA affected the number and type  that  developed.  shooty  structures  chlorophyll  stimulated that  of  and an ABA-dependent developmental profile was  the not  Low or  germinate  of  late  levels  precocious  development.  production  ABA  Higher  precociously  germinants  ABA  cotyledonary during  favoured with  concentrations somatic  the  embryos  experimental  The optimal level of ABA for line W29 was between 40-60  Based on morphological characteristics described by Buchholz  and Stiemert  (1945),  proceeded through  somatic embryos differentiated on 40 M ABA  embryogenesis  conifer zygotic embryos embryos were  larger  (Fig.  in  9).  in girth but  a manner  similar to  that  of  The late cotyledonary somatic similar  in  length and overall  appearance to zygotic embryos after 9 weeks of maturation on ABA (Fig.  9).  These  embryos  were  used  for  the  identification  characterization of somatic embryo storage proteins.  and  79  TABLE 2. The presence (+) or absence (—) of various storage proteins during zygotic embryo development, 1988 collection. Storage protein ( kD)  Embryo developmental stage  2  3  3—4  4—1 (JL13)  41  -  —  -  -  35 33 24 22  +  + + + +  + + + +  + + + +  + + +  4—2 (JL27)  + + + + +  4—3 (AU24)  + + + + +  Mature  + + + + +  80  FIGURE 8. Abscisic acid-dependent developmental profile of genotype W29, showing the types and numbers of structures obtained with maturation using different ABA concentrations. ME, late cotyledonary somatic embryo; PE, precociously germinating somatic embryo; SH, shooty structure. The bar graphs represent quantitative data for the cultures depicted below.  500  -  ME  450 PE  400 SH (J] LJ  350  100  -  -  50 00  40  20 ABA (tM)  60  82  FIGURE 9. Examples of somatic embryo developmental stages. PE, proembryo; G, globular embryo; R, round head torpedo embryo; F, flat head torpedo embryo; EC, early cotyledonary embryo; LC, late cotyledonary embryo; ZE, zygotic embryo.  1mm  84 analysis  SDS-PAGE proteins were as well  of  similar in  total  embryo  proteins  revealed  zygotic and somatic embryos  that  (Fig.  10),  in the isolated protein bodies of both embryo types.  as  Total protein gels suggested that there were differences between storage  protein  levels  of  the  two  However,  the relative levels of 35,  embryo  types  (Fig.  10).  24 and 22 kD proteins in  33,  although  isolated protein bodies were similar from both sources,  the level of 41 kD protein was greater in somatic embryo protein bodies  (Fig.  10).  An analysis of the somatic embryo protein body fraction under reducing  and  differences  non—reducing  between  the  two  conditions treatments  distinct  revealed  (Fig.  similar  11),  those observed for zygotic embryo storage proteins  (Fig.  to  2).  The somatic embryo storage proteins were characterized further Isolated somatic embryo protein body  using solubility criteria. preparations kD  proteins,  with  solubilization  the  (Fig.  remaining  12).  These  proteins  embryo  two—dimensional gradients  and  27.5  requiring  SDS  for  (Fig.  protein  body  electrophoresis were  13)  similar  41,  similar  results were  obtained for zygotic embryo storage proteins Somatic  30  contained phosphate buffer-soluble  samples using to  (Fig.  which  those  those  3).  were  narrow  to  analysed  and  observed  broad for  by pH  zygotic  embryo storage proteins. A  comparison  of  the  steady  state  storage  protein  levels  between zygotic and W29 somatic embryos by scanning densitometry of total protein profiles revealed some differences between the proportion  of  major  storage  proteins  in  the  two  embryo  types  85  FIGURE 10. Coomassie-stained SDS-PAGE of zygotic embryo (Lane 1), zygotic protein body (Lane 2), 9-week ABA somatic embryo extracts. (Lane 3) and somatic embryo protein body (Lane 4) Arrows indicate the 41, 35, 33, 30, 27.5, 24 and 22 kD proteins. Lanes 1 and 3 contain 15 protein and lanes 2 and 4 contain 5 protein. MW, molecular weight standards.  MW  kD  3  2  1 --  4  —.-  97.4  I  66.2  4  42.7 1  I__I___  l4  — r — Ii  j  LZ  21.5 14.4  —  -  em -1_._____—  —  87  FIGURE 11. Coomassie-stained SDS-PAGE of somatic embryo protein body extracts under reduced (A), non—reduced (B), and two— dimensional SDS—PAGE of non—reduced extract under non—reducing conditions followed by electrophoresis under reducing conditions (C). The 41, 35, 33, 30, 27.5, 24 and 22 kD proteins are indicated by open arrows (A). The 55-57 kD doublet present under non-reduced conditions is indicated by the solid arrow (B). The molecular weights of non—reduced proteins and the corresponding proteins obtained by second dimension SDS-PAGE are indicated (C). Each sample contains 5 jg protein.  I  w  L I.4  _z 0 0  o. C, Il I  I  g  UI •bJ  )U1 UI  89  FIGURE 12. Coomassie-stained SDS-PAGE of somatic embryo protein body samples extracted under different conditions. Buffer— soluble (Lane 1) and SDS-soluble (Lane 2) proteins extracted from isolated protein body samples are shown. A 10 ,l sample was applied to each lane. NW, molecular weight standards.  cjo  kD  MW  97.4 66.2  42.7  31.0  21.5  144I  1  2  91  FIGURE 13. Silver-stained two-dimensional electrophoretogram of somatic embryo protein body extract examined using pH 5-8 ampholytes (A) or pH 3-10 ampholytes (B). The acidic (+) and basic (-) ends of the gel are indicated. A total of 20 jg protein was analyzed for each pH gradient. MW, molecular weight standards.  cia  IEF  +  MW kD 42.7  —  .rwI:H  -  *  II  j 111  I  A  411’! 1  B  I.  21.5  1SDS  IPAGE  93 (Table ABA,  3).  Somatic  embryos  of  genotype W29,  after  weeks  9  on  contained more of the major storage proteins as a percentage  of total protein than did mature zygotic embryos  (Table 3).  To determine the extent to which storage proteins accumulated in other  somatic embryo genotypes,  cotyledonary  (7 weeks on ABA)  the protein profiles  (Fig.  from EK1O W29  14).  Somatic  embryos  seed showed variable  embryos  containing  late  somatic embryos of uniform length  were compared with those from genetically related seed EK1O)  of  of  levels  the highest  several of  levels  (PG118 or  genotypes  derived  storage proteins, (Fig.  14A).  with  Embryos  from two different genotypes derived from PG11B seed had storage protein  levels  similar to  slightly higher l4B).  than  However,  the  storage  each other, levels  in  proteins  which were  PG118  zygotic  accumulated  to  similar to embryos a  or  (Fig.  significant  degree in late cotyledonary embryos of each genotype. 4.4.  STORAGE  PROTEIN  ACCUMULATION  IN  SOMATIC  EMBRYOS  AND  COMPARISON WITH ZYGOTIC EMBRYOS  A  comparison  cotyledonary  of  (Stage  early  4-9)  W29  cotyledonary  somatic embryos  electrophoresis revealed a differential proteins  somatic  embryos,  However,  during earlier embryogenic stages,  the  41  development. kD  kD proteins were detected at was  not.  proteins (Fig.  In  addition,  occurred  16).  during  the the  protein  low  was  later  of  late  late  the 35, but the  of  The differential appearance of the  the major  cotyledonary  abundant  accumulation stages  and  by two-dimensional  In  levels,  major  3—2)  appearance  storage  during  (Stage  (Fig.  33, 41  of  embryo  15).  24 and 22 kD protein  all  storage  maturation  41 kD versus the  94  TABLE 3. Major storage protein distribution in EK1O zygotic and W29 somatic embryos as determined by densitometry. Mean ± SE.  Storage protein ( kD)  Zygotic embryo  Somatic embryo  % of total protein  % of total protein  41 35 + 33 24+22  24±3 5 ± 3 12±2  32±1 13 ± 1 13±2  Total protein  41 ± 4  58 ± 1  95  FIGURE 14. Coomassie—stained SDS-PAGE of total proteins for two zygotic embryo genotypes and different somatic embryo genotypes derived from them. Protein profiles of the different somatic embryo genotypes derived from (A) EK1O (W29, W74, W76, W77) and (B)PG118 (W46, W70) seed are indicated. Each lane contains 7.5 MW, molecular weight standards. g protein.  14.4  21.5  31.0  42.7  97.4 66.2  kD  -  -:--  —  MW W76  A  —  .-  W77 W74 W29  I  EK 10  W70 W46  B  PG 118  -  MW  14.4  21.5  31.0  42.7  97.4 66.2  kD  0•  97  FIGURE 15. Silver-stained two dimensional electrophoretograms of total proteins from stage 4-9 (B) somatic (A) and stage 3-2 embryos differentiated on 40 ,.M ABA. Proteins were separated using pH 3-10 ampholytes, and the representative portions of the gels containing the major storage proteins (boxed regions) are (+) and basic (-) regions of the gels are shown. Acidic indicated.  4  +  I 1-  0 C  99  Coomassie-stained SDS-PAGE of developmental stage FIGURE 16. changes in total somatic embryo protein of genotype W29 matured on 40 ,M ABA. Storage proteins are indicated by arrows. G, globular embryos; R, round head torpedo embryos; F, flat head embryos; LC, late torpedo embryos; EC, early cotyledonary cotyledonary embryos. The range from early cotyledonary to late cotyledonary includes the range of embryos from the onset of Stage 3 development to 9 weeks on ABA as described in Table 1. Each lane contains 15 jg protein. MW, molecular weight standards.  I00  G R  FIEC  +LCj MW  97.4 66.2 --  -  ._.  ._ p1,4  e  .1  42.7  31.0  21.5  ——  .  — 14.4  101 35,  33,  24  and 22 kD proteins during the early stages of embryo  maturation was similar in zygotic and somatic embryos. Polyclonal antibodies were made against the 41 and 24 + 22 lcD storage 18)  proteins,  showed  zygotic  that  and  analysis  by  these  proteins  accumulated  embryos,  although  there  immunoblotting  were  in  (Figs.  17  and  somatic  and  between  the  both  differences  accumulation patterns of the two embryo types.  Quantification of  immunoblots by densitometry indicated that the  41 kD protein  somatic  embryos  showed  an  initial  accumulation  rapid  in  which  continued during the 6—week period of cotyledon/embryo maturation in our differentiation protocol there  also  was  protein over a remained 17).  an  initial,  The  rapid  3—week period,  relatively  constant  accumulation  of  (Fig.  17).  zygotic  In  accumulation  or  the  increased 24  +  22  only  was  a  prolonged period  in  and  a  more  embryos proteins  (Fig.  18).  actually  accumulation  shorter The  accumulation  relative  declined  41  during  the  abundance the  of  later  (Fig.  displayed There  embryos. somatic  period  kD  levels  slightly  proteins  kD  zygotic  rapid,  the  but thereafter the protein  similar differences between somatic and of  of  embryos,  in  the  the  24  stages  embryos, zygotic +  of  22  kD  embryo  maturation in the zygotic samples. 4.5.  IDENTIFICATION AND CHARACTERIZATION OF A CDNA ENCODING THE  41. ]cD STORAGE PROTEIN Comparison of storage protein gene expression at the molecular level in zygotic and somatic embryos required the use of storage protein  cDNA  expression  probes.  vector  pUC  A  cDNA 18,  library was using  mRNA  constructed extracted  with  from  the late  102  Relative quantification of 41 kD protein immunoblots FIGURE 17. during zygotic and somatic embryo development. Immunoblot of zygotic embryo cotyledonary stages UPPER PANEL: ND, not detected on scan. collected during 1989. Immunoblot of somatic embryo developmental stages LOWER PANEL: The developmental stages from early to late on 40 jM ABA. ND, not detected on scan. cotyledonary are described in Table 1.  o3  006  ZE-4lkD  0.05  0.04  0.03  -  >  0.02  0.01  I 0  —  0.5  I  2  I —  IHI  I —  —  —  — a. — — a —  3  4 5 6 7 9 8 WEEKS OF COTY1EDON DEVELOPMENT  EARLY COTYLtDONARY  —  —  10  10.7  LATE  COWLEDONARY  0.06  0.05  0.04  0.03  0.02  0.01  0 —  —.  — — —  :  1.4 1.7 2.1 2.4 2.9 3.3 4.1 4.6 5.6 6.6 WEEKS OF COTYLEDON DEVELOPIIENT EARLY COTYLEDONARY  LATE COrILEDONARY  104  FIGURE 18. Relative quantification of 24 + 22 kD protein immunoblots during zygotic and somatic embryo development. UPPER PANEL: Immunoblot of zygotic embryo cotyledonary stages collected during 1989. ND, not detected on scan. LOWER PANEL: Immunoblot of somatic embryo developmental stages on 40 jM ABA. The developmental stages from early to late cotyledonary are described in Table 1. ND, not detected on scan.  ‘Os  0.024 0.022 0.020 0.018 0.016 0.014 0.012 0.010 0.008 0.006 0.004 0.002  3 4 5 6 7 8 WEEKS OF COTYLEDON DEVELOPMENT  EARLY COTYLEDONARY  LATE COTYLEDONARY  SE-24+22kD 0.22 0.20 0.18  0.16 0.14 a.  0.12 010  0.08 0.06 0.04 0.02 ND. 1 EARLY  rnrnFiFiITTi 1.4 1.7 2.1 2.4 2.9 3.3 4.1 4.6 5.6 6.6  WEEKS OF COTYLEDON DEVELOPIIENT LATE  COTYLEDONARY  COTYLEDONARY  106 cotyledonary screened  somatic  using  storage  embryos.  polyclonal  protein.  A  identifed  and  insert  approximately  of  portion  A  antibodies  positive  characterized 1.7  of  against  clone,  This  kb,  it  for the 41 kD protein class.  purified  designated  further. making  library  the  41  clone  was  contained  1406  bp,  personal communication).  large enough to code  (Fig.  19),  with a reading frame  acids which ran from the ATG start codon at base 9  of  51,835.  The  (Dr.  Sequencing of the 115A.0  encoding a putative precursor protein of  codon at base 1406.  an  Other clones have been identified  clone revealed a 1633 bp insert of  kD  115A.0  by cross hybridization with 115A..0 and are of similar size Craig Newton,  was  466  amino  to the stop  This protein had a predicted molecular mass  putative  polypeptide  was  compared  with  known  protein sequences in the EMBL data bank and showed a high degree of similarity to several angiosperm vicilin—type storage protein sequences  (Fig.  20).  It was concluded that the 115A.0 cDNA was a  spruce storage protein cDNA. The predicted molecular mass of the putative storage protein was  slightly  mature  higher  protein,  sequencing  of  as  the  than  the  apparent  determined protein  by  suggested  molecular  SDS-PAGE. that  the  mass  of  the  Amino-terminal mature  protein  started at the glycine residue which corresponded to amino acid 48  (Dr.  Craig  Newton,  personal  communication),  with  the  preceeding amino acid region serving as a signal sequence which was  co—translationally  the  potential  first 18  cleaved.  hydrophobic  signal  Computer sequence  analysis  to  indicated  identify that  the  amino acid residues could serve as the signal sequence  107  FIGURE 19. Sequence analysis of the spruce 115A.0 cDNA clone. The nucleotide sequence of the spruce 115A.0 cDNA described in the Results is shown. The deduced amino acid sequence is given in single—letter code. The N—terminal amino acid sequence displaying almost complete duplication is underlined, and the potential N—terminal amino acid residue of the mature protein is indicated by the solid triangle.  108  GCATCATCATGGTTTTCGCTTCTTTACTTATGATTCTTCTTGCAATCTCCTCCTCCTCGGCTGCCCTCACCGAG N V S L S S S A ALT F A L MILL A IS E  74  CCACTAGCCAGCACGGCCAATCCAACCTCCTCCTCCTCGGCTGCCCTCACTGAGCCACTATCCAGCACGGCCAAT P LAST SS A N PT S S SS A ALT E P L TAN CCAGGAGTTTTTCCTGAATATCTCGGCCGAGGCCGAGGGAGACGAGAAGAAGAGCGAGAGGAGAATCCATACGTA 224 PG V F P L G R G RE E ER E EN P E Y R G R Y V A —  TTCCACAGTGACAGCTTCAGGACCAGAGCATCATCTGAAGCTGGTGAAATCAGAGCTCTGCCGAACTTTGGGGAG F H SD R A L P N F GE SF R T S SEA GE IRA GTCTCTGAACTTCTTGAAGGGATTAGAAAATTCAGAGTTACCTGCATTGAAATGAAACCCAATACAGTGATGCTC 374 VS EL LEG IRK FR VT CI EM K P N TV ML CCTCACTATATTGATGCGACATGGATCTTATATGTTACTAGAGGAAGAGGCTACATAGCCTATGTGCACCAGAAT PH V H Q N Y IDA T WILY VT R G R G Y IA Y GAGCTGGTTAMAGAAAGTTGGAGGAAGGAGATGTATTCGGTGTTCCAAGTGGTCATACATTTTATCTCGTTAAC 524 EL V GD V F G VP S G H T F Y LV N KR K LEE AACGATGACCATAGCACCCTTCGCATTGCCAGTCTCCTGCGTCCCGTGTCTACGATCCCAGGAGAATATCAGCCC ND D V ST I PG H ST L RI A S L L R P E Y Q P TTCTACGTTGCGGGAGGTCGGAATCCTCAGAGTGTTTACTCTGCCTTTAGCGATGATGTTCTCGAGGCTGCATTC 674 F Y VA G G RN P Q S V Y S A F SD DV LEA A F AATACGAACGTACAGCAGCTTGAACGTATTTTCGGTGGACACAAAAGCGGAGTCATAATCCACGCAAATGAAGAA NT N V Q Q L ER IF G G H KS G VII H A NE E CAGATTAGAGAAATGATGAGGAAACGGGGATTATCAGCAGGATCCATGTCTGCACCTGAGCACCCCAAGCCTTTC Q IRE M MR KR G L SAGS N SAP E H P K P F  824  AACCTTCGGAACCAGAAGCCAGATTTCGAGAACGAAATGGCAGGTTTACTATTGCTGGTCCCAAAAATTATCCT N L RN Q K PD F EN ENGR F TI AG P K NY P TTTCTAGACGCGCTCGACGTTTCTGTTGGGCTTGCCGATTTGAATCCTGGATCCATGACAGCCCCATCTCTCAAC TAPS FL D AL DV S V GLAD L N PG SM L N  974  TCGAAATCAACGTCAATCGGCATTGTTACGAATGGGGAAGGAAGGATTGAGATGGCATGCCCGCACCTTGGTCAA S K L G Q ST SI G IV TN GE G RI EM AC PH CATAGCTGGTCTAGTCCGCGTGAGAGAGGCGACCAAGATATTACTTACCAGAGAGTCTGGGCAAAGCTGAGGACC 1124 H SW S S PR ERG D Q DI T Y Q R V WA K L RT GGCAGCGTTTATATTGTTCCTGCTGGTCATCCAATCACGGAGATAGCTTCAACAAACAGCCGCCTGCAAATCTTG G S SR V Y IV PA G H PIT El A S TN L Q IL TGGTTTGATCTTAATACCCGCGGCAATGAGAGACAATTCCTGGCAGGAAAGAACAATGTGCTTAACACGTTGGAG 1274 W F N N V L NT L E DL NT R G ER Q FLAG K N AGGGAGATCAGGCAGATATCCTTCAACGTACCACGTGGGGAAGAGATTGAAGAAGTGTTGCAGGCACAAAAGGAT RE I R Q 1SF N VP R GEE I E E V L Q A Q K D CAAGTGATCCTCAGAGGCCCCCAACGACGAAGCCGGGACGAGGCGAGGAGCTCTTCTTAGATCCATGTCATCATT 1424 Q VI L R G P Q R R SR DEARS S S * GCAGATCGCATTATGGACGACATGACAAGAGTTTCTCCACGTTCACTCTTAATATGTAGTTAAGAATAAGCTATC CATAAATGTGTTCGAAGATGAACTCTTTCTGTTTAAATGAATTATGTATGAGTCTAACAAAGCTATCGTTGGGCT 1574 CCTCTTTCTACTTCAATGCAATGAAACGCAGGTCTTCTCTTAAAAAAAAAAAAAAAAAA  109  20. Amino acid sequence comparison of 115A.0 with other angiosperm vicilin—type storage protein sequences. The figure compares the amino acid sequence of spruce 115A.0 with those of the cotton vicilin (alpha-globulin A) precursor, the broad bean vicilin precursor (BRDBN) and the soybean 13-conglycinin alpha chain precursor (SOYBN). Sequences were aligned to maximize identities. Positions in the alignment that are perfectly conserved are boxed. Positions that are well conserved are indicated by asterisks. FIGURE  110 IS- SSS-----AALTEP FGLLCSAKDFPGR--—-RSEDDPQQRY CLSS -MRARFPLL- VVFtJ.SVSVSFG 1AYWEKQNPSHNKCLRSCNSEKDSYR  FA4ILN----VRNKSVFVjLj4FsLFL  I  -  *  -  -  -  -  *  *  *  115A. 0 COTTON BRDBN SOYBN  SSSAALTE RSETQLKEEQQRDGEDPQR-  S LASTANPT EDCRKRCQLETPGQTEQDKCED  NQACHARCNLLKVEE-—EEECEEGQIPRPRPQHPERERQQHGEKEEDEGEQPRPF  PLSSTANPGVFPEYL RRLRPHCEQSCREQYEKQQQQQPDKQFKECQQ  RYQDCRQHCQQEE  115A. 0 COTTON SOYBN  115A. 0 COTTON  PFPRPRQPHQEEEHEQKEEHEWHRKEEKHGGKGSEEEQDEREHPRPHQPHQKEEE  SOYBN  GRGRGRREEEREE RCQWQEQRPERKQQCVKECREQYQEDPWKGERENKWREEEEEESD-EGEQQQR*I RSDQLjNI KHEWQ-HKQEKHQ- -GKESEEEEEDQDEDEEQDXESQESEGSESQREPRRMKNIJ  115A, 0 COTTON BRDBN SOYBN  1l4EP  II 5A. o COTTON BRDBN SOYBN  EM  FLPjQ  J*  • FI14SKJF TLFKNQ\1$  YITWILYVT1RGYIAYVHQNELVKRKLEDVFG’HTFY HcDAEKIYVVT4RGTVTFVTHENKESYNVVrVVVR*STVY QDA)FILVVLcAILTVLLPNDRNSFSLEIDTIK$jr *  *  ***  L  NREIIA  *  *  *  ***  **  *  **  II 5A. 0 COTTON BRDBN SOYBN  1N-QFQ4FPAGQENPQfLRIjFR  GVIIUANEiRE —HKS D\l4F14VQQLERIF -44RQSHRRQQGQGMFRKASQIjRA EIjLE?IVFl4ISEQLDELP FZ’41)JYKEI EKVLLEEHGKEKYHR1KDRRQRGQEENVIVKISRKIIEE E N E N YEtCFEEINKVLF---GREEGQQcj-EERLQ----ESVIVEISKKbRE **  ****  **  **  *  ***  *  *  *  .***  **  *  VGLADIMTLcSTSIGIVTEGRIACPHLGQHS--WSSPRE--R VVAFEIN4 I VNYVEILLISRAIVIVTVN$(GDFjEVGQRNENQQGLREEYDEEKE **  *  ***  **  ***  *  **  **  QGEEEIRK__QVQNYAPGDVLVIPAGfrT__AIKASSt4t44LVGI*EN **  *  *  **  **  *  *  *  **  *  **  *  **  *  **  *  1DEARSSS FDERRGSNNPLSPFLDFARLF EIGSQEIKDHLYSILG--SF— EEGNKGRKGPLSSILR--AFY *  **  *  ****  115A.0 COTTON BRDI3N SOYBN  *  *****•***  *  *  **  *  115A. o COTTON 8RDBN SOYBN  *  EI-KVLNTLEREIRQINVPRGEEIEEVLQAQKDQV—ILRGPQRRS I-KNVRQWDRQAKELNF3VE-SRLVDEVFNNNPQESYFVSGRORRG SQIHKPVKELFtPGS-AQEVDTLLENQKQ-SHFANAQPRER NLJSKUJVISQIPSQVQEL1PRS-AKDI ENLIKSQSE—SYFVDAQPQQK *  II 5A. 0 COTTON BRDEN SOYBN  **  *  GDQDIT  **  115A.O COTTON BRDBN SOYBN  ***  *  **  **  I 15A. 0 COTTON BRDBN SOYBN  *  F(FFEITPKRNP—I4DLNIF SRjPtY SRIjJt Y(IJCLFEIT-QRNP—JDLDVF  *  **  **  *  Q1j)FFTIAGPKNYPF-)ALDVS  N N N N  MNRKRGLSAGSMSAPEHPK LSQG---ATSPRGKGSEGY L-NKNAKSSSKKSTSSESE L-SKHAKSSSRETI SSEDK  ***  -  I I 5A. 0 COTTON BRDJ3N SOYBN  115A. 0 COTTON BRDBN SOYBN  111 (MVFASLLMILLAISSSSA/ALT). signal  sequence  duplication  was  at  the  Interestingly,  contained amino  in  an  terminus  part  of  almost (Fig.  the  potential  complete  19)  The  sequence predicted  molecular weight of the protein encoded by amino acids 48 through 466  was  mature  approximately protein,  processing  47  kD,  suggesting  might  still that  occur.  To  slightly further  test  larger  than  the  post—translational  this  assumption,  late  cotyledonary somatic embryos were labelled for 4 hours with 35_ methionine, profiles  and then chased  (Fig.  labelled  proteins  lower panel) 3-4  kD  larger  almost  upper  with  panel)  41  kD  24  hours.  and  Total  protein  immunoprecipitation  polyclonal  antibodies  (Fig.  than  the  mature  protein.  not evident until  complete  by  8  at  hours  least  of  Processing 4  chase  hours (Fig.  of  the  chase,  and  21).  Another  immunoprecipitation that did not  during the  (Fig.  size  21,  of  protein was detected by  this  of  revealed a precursor protein that was approximately  precursor was was  21,  for up to  chase period were  observed  in  21).  total  change  Since no major proteins labelled  protein profiles,  of it  was concluded that this is a cross—reacting protein. Two-dimensional  protein  analysis  suggested  that  the  41  kD  protein class consisted of several isoelectric variants, possibly encoded by a multi-gene family DNA  was  blotted  digested and  conditions digest  and  with  probed (Fig.  with  22).  (Figs.  several 115A.O  5 and 13).  different cDNA  Numerous  under  bands  copy reconstruction standards  of approximately 10-15 copies per genome  Spruce genomic  restriction  enzymes,  moderately-stringent  were  observed  suggested (Fig.  22).  the  in  each  presence  112  FIGURE 21. Pulse:chase-labelling of late cotyledonary somatic embryos differentiated on 40 M ABA. Embryos were labelled for 4 hours with S-methionine 35 (0 time), then chased with cold methionine for up to 24 hours. UPPER PANEL: Total labelled protein profile. Open arrow denotes the potential precursor to the mature 41 kD protein (closed arrow). LOWER PANEL: Labelled proteins from the above treatments inuuunoprecipitated with 41 kD antibody. The disappearance of the precursor protein (open arrow) and the appearance of the mature protein (closed arrow) is visible.  “3  kD  0  2  Chase IhrI 4 16 8  24  99.4— 68.0— 43.0—  1  29.0—  18.4—  V.  iiii  — ——  14.3—  E. 43 .o_v  —  A  114  FIGURE 22. DNA gel blot analysis of spruce DNA probed with 115A.0 cDNA. Lane 1 contains lambda Hind III molecular weight standards. Lanes 2 through 5 contain 5 g of spruce DNA digested with Hind III (Lane 2), Barn HI (Lane 3), Eco RI (Lane 4) or Xba I (Lane 5). Lanes 6 through 10 contain gene copy number reconstructions at 0.8 copy per haploid genome (Lane 6), 1.6 copies (Lane 7), 2.4 copies (Lane 8), 4 copies (Lane 9) or 8 copies (Lane 10). Gene copy reconstructions were made using 115A..0 cDNA, and a haploid DNA content of 8.5 pg.  p  0)  I  II  !43M  I  4  I I  0  CD  -.4  0)  01  c)  F)  -  0•  116 During the course isolated  and  angiosperm personal  of the above work,  sequenced.  The  legumin—type  storage  communication).  designated  XI5H,  results  spruce  precursor  cDNAs were also  revealed  proteins  The  encoded  other  similarity  (Dr.  Craig  legumin—type proteins  to  Newton,  sequences,  with  conserved  proteolytic processing  sites which would yield disulf ide-linked  35-33  proteins  and  24—22  communication), this study.  kD  similar  Thus,  to  (Dr.  the  storage  Newton,  proteins  personal  identified  in  probes were available to study the expression  of the spruce vicilin-type (41 kD) kD)  Craig  and legumin-type  (35-33,  24-22  storage proteins.  4.6.  RNA  PROTEIN  GEL  BLOT  ANALYSIS  TRANSCRIPTS  IN  OF  ZYGOTIC  AND  LEGUMIN EMBRYOS  AND  VICILIN  STORAGE  SOMATIC  EMBRYOS  DIFFERENTIATED ON 40 LM AND 10 jtM ABA  Analysis of zygotic embryos collected during 1991 revealed, described  previously,  differential  that  accumulation  storage  the  patterns,  during cotyledon development,  proteins  accumulated  to  as  displayed levels  high  and reached steady state levels 1—  1½ months prior to mature seed shed  (Fig.  23).  Analysis of the  mRNA levels for the spruce vicilin and legumin proteins revealed that,  although  differential detectable  different  accumulation at  development reaching  the  the  (Fig.  high  same 23).  levels  protein  patterns, time, The  during  torpedo messages  cotyledon  classes  their stage  displayed  messages (stage  accumulated development,  were 2),  of  rapidly, and  then  declined rapidly to low levels,  Scanning densitometry of RNA gel  blots  to  (using  values  normalized  constant  rRNA)  was  used  to  117  FIGURE 23. Changes in total proteins and storage protein mRNA during zygotic embryo development. UPPER PANEL: Coomassie-stained SDS-PAGE of total proteins from zygotic embryos collected during 1991, ranging in development from proembryos (PE) to mature seed embryos (Stage 4, September 4). proteins are The spruce vicilin (V) and legumin (L) indicated. Each lane contains 12 g protein. MW, molecular weight standards. LOWER PANEL: RNA gel blot analysis of total RNA from the same zygotic embryo developmental stages described above, probed with spruce vicilin (115A.O), legumin (XI5H) or yeast 18S rRNA cDNA.  Stage 4 AU JL SE 3..4 9 16 23 30 6 13 22 28 4  Stage PE2 kD  MW  ---:  97.4 66.2 45.0  Iv JL  31.0 •  I,  21.5  JL  14.4  Stage PE2 33-4  Stage 4 94  Viciliri  Legumin rRNA  119 compare changes in levels of the mRNAs during development. low  in  loading  inadequate required  the  supply  use  proembryo  of  of  and  Stage  zygotic material  correction  normalize these values. the normalized values,  values  2  from  (on  lanes,  these  the  caused  early  order  of  The by  stages, 68x)  to  This increased the error probability for so only samples from Stage 3 onwards were  normalized.  Densitometry revealed that the leguinin mRNA reached  high  levels  earlier  mRNA  (Table  4).  in  The  cotyledon  vicilin  development remained  mRNA  than  the  slightly  vicilin  higher  in  Stage 4 embryos during the period between July 16 to July 30, but then both mRNA classes declined to around 1% or less of maximal levels  (Table 4).  A different genotype research because of a  (W70)  loss  was used during this phase of the  in vigour of genotype W29.  Somatic  embryos of W70 were similar in general appearance to those of W29 although, higher  by  9 weeks on 40 M ABA,  chlorophyll  content  (Table  those of genotype W70 had a 5),  but  had  not  precociously  germinated. The W70 embryos which were matured on 40 jM ABA displayed a similar  trend  in  storage  for genotype W29. by  SDS-PAGE  (Fig.  in  24).  ABA  (Fig.  accumulation  that  observed  early  cotyledonary proteins  stages,  accumulated  prior to during  the vicilin cotyledonary  and accumulation was still evident after 9 weeks on  24).  RNA  gel  blots  revealed  that  legumin mRNAs were detectable at the same time stage embryos  to  Legumin protein accumulations were detectable  All  development,  protein  (Fig.  both  vicilin  and  in round torpedo  24), similar to the observations with zygotic  120  TABLE 4. Developmental changes in storage protein mRNAs in zygotic embryos as determined by scanning densitometry of RNA gel blots.  Developmental Stage  Storage protein mRNA level of maximal hybridization)  (%  Stage 3 Stage 3—4 Stage 4 (JL Stage 4 (JL Stage 4 (JL Stage 4 (JL Stage 4 (AU Stage 4 (AU Stage 4 (AU Mature  9) 16) 23) 30) 6) 13) 28)  Vicilin  Legumin  72 % 96 % 100 % 93 % 32 % 7 % 1 % 1 % 1 % 2%  100 % 50 % 59 % 39 % 11 % 1 % < 1 % < 1 % < 1 % <1%  121  TABLE 5. Chlorophyll content (mg/g FW) of Stage 4-6 and 9 week ABA somatic embryos of genotypes W29 and W70. Mean ± SE.  Genotype  W29 W70  Stage of embryo development  4-6  9 weeks ABA  0.014 ± 0.006 0.019 ± 0.001  0.011 ± 0.001 0.098 ± 0.030  122  FIGURE 24. Changes in total proteins and storage protein mRNA during somatic embryo development on 40 M ABA. UPPER PANEL: Coomassie-stained SDS-PAGE of total proteins from somatic embryos matured up to 9 weeks on ABA. Developmental stages are as described for Fig. 16. The vicilin (V) and legumin (L) proteins are indicated. Each lane contains 12 g protein. NW, molecular weight standards. LOWER PANEL: RNA gel blot analysis of total RNA from the same• somatic embryo developmental stages described above, probed with spruce vicilin (115A.0), legumin (XI5H) or yeast lBS rRNA cDNA.  kD 97.4 66.2  MWPE G  RF  ii —  45.0 I  31.0  21.5 14.4  PE G R  F(EC  +LC)  124 increased  Both  development,  but declined during the later stages.  were  analyzed  classes  during  embryos.  nRNA  densitometry  by  as  cotyledon  RNA gel blots  previously.  described  The  legumin mRNA reached high levels earlier in cotyledon development than  the  vicilin  with  zygotic  their peak large  as  after  9  mRNA  (Table  embryos.  levels  (Fig. for  observed weeks  While  on  24,  6), both  Table  zygotic  ABA,  similar  (Fig.  similar  embryos  manner  to  stage.  However,  (after  Stage  exhibited  which matured  those  j.M  the  germination.  10  ABA  as  Table  were  and,  4)  present  at  (Table 6). M  up  embryos  ABA developed the  to  accumulated  elongation,  These  in  a  mid-cotyledon  standards  used  for  and  chlorophyll  characteristic  differences  later stage embryos on  length/morphological j.M ABA,  on  after  once the cotyledons had overgrown the shoot apex  4-2),  classification of  1-6).  40  hypocotyl/cotyledon  precocious  on 40  on  23,  classes  mRNA  declined  made  their decline was not  6),  approximately 50% of their maximal levels Somatic  observations  classes  mRNA  embryos  both  to  prohibited  of the  10 ).LM ABA based on the non—germinating  embryos  and they were thus classified as germinants  (Germ  Embryos from both 10 M and 40 iM ABA were collected for  analysis after the same duration of ABA exposure.  Germ 1 samples  were precociously germinating embryos collected at the same time as Stage 4—3 precocious embryos  embryos on 40 M ABA and Germ 6 were more advanced  germinants (8  weeks).  collected The  at  the  same  intermediate  time  as  stages  developmental size continuum of precocious embryos.  Stage formed  4—8 a  125  TABLE 6. Developmental changes in storage protein mRNAs somatic embryos differentiated on 40 M ABA as determined scanning densitometry of RNA gel blots.  Developmental Stage  Storage protein mRNA level  (% of maximal hybridization) Vicilin  Proembryo Globular Round head Flat head Stage 3—1 Stage 3—2 Stage 3—3 Stage 3—4 Stage 4—1 Stage 4—2 Stage 4—3 Stage 4—4 Stage 4—5 Stage 4—6 Stage 4—7 Stage 4—8 Stage 4—9  0 0 8 36 62 84 76 80 50 86 100 69 73 60 61 46 51  % % % % % % % % * % % % % % % * *  Legumin  0 0 7 29 66 100 63 55 50 64 67 40 46 41 47 41 50  % % % % % % % % % % % % * % % * *  in by  126 embryos  Somatic  on  10  M  ABA  also  displayed  differential  storage protein accumulation during the early stages of cotyledon development. (Fig.  25),  stage  in  Legumin accumulation was detected prior to vicilin  although these proteins occurred at a cotyledon  However,  the  germination  development  storage  than  protein  commenced  (Fig.  in  40  ,M  levels  declined  and  not  25)  did  slightly later ABA as  embryos.  precocious the  reach  levels  found in 40 jM ABA somatic embryos.  RNA gel blots revealed that  both vicilin  detectable  stage  embryos  cotyledon  6  (Fig.  25).  were  Both  and  mRNA  declined  classes as  in  flat  torpedo  increased  precocious  during  germination  However, very low levels were still detectable at the  stage,  started  legumin mRNA5  development  commenced. Germ  and  (Fig.  several 25).  weeks  after  precocious  Densitometric  analysis  germination  of  RNA  gel  had  blots  revealed that the legumin mRNA increased earlier than the vicilin mRNA  (Table  embryos and  7),  similar  to  somatic embryos  the  pattern  on 40  observed  uM ABA.  for  Comparison  zygotic of  10  M  ABA samples to a control sample from 40 M ABA somatic embryos on the same blots revealed that the maximum vicilin and legumin mRNA levels  attained in the 10 ,LM ABA samples were approximately 70%  of the maximum levels found in 40 jM ABA somatic embryos. 4.7.  ANALYSIS  OF  STORAGE PROTEIN EXPRESSION  IN SOMATIC  EMBRYOS  IN RESPONSE TO OSMOTIC STRESS Early cotyledonary ABA  were  cultured  regulators,  40  pM  (Stage 3-4)  for ABA,  2  weeks 15%  embryos differentiated on 40 pM on  medium  mannitol  biosynthetic inhibitor fluridone,  or  containing mannitol  no  plus  growth the  ABA  to determine if osmotic stress-  127  FIGURE 25. changes in total proteins and storage protein mRNA during somatic embryo development on 10 jM ABA. UPPER PANEL: Coomassie-stained SDS-PAGE of total proteins from somatic embryos matured up to 8 weeks on ABA. Somatic embryos were collected after the same duration of total ABA exposure as those on 40 ).LM ABA. Embryos were classified by the same developmental stages as those on 40 jtM ABA until after mid— cotyledon development (Stage 4-2), after which they began to precociously germinate (Germ 1). Precocious germinants were collected at increasing stages of development (Germ 1—Germ 6; see text). The vicilin (V) and legumin (L) proteins are indicated. weight Each lane contains 12 protein. MW, molecular jg standards. LOWER PANEL: RNA gel blot analysis of total RNA from the same somatic embryo developmental stages described above, probed with spruce vicilin (115A.0), legumin (XI5H) or yeast 18S rRNA cDNA.  kD  MWPE G  R •. —  97.4  GERM 1  FIEC  GERM 6 A  .  ,...  66.2  -  -  .  •-•.4-  -.h  • -  45.0  31.0  ...  21.5 IL 14.4  --  PEGR FIEC  -  GERM  —  —..  r  1 GERM  Legumin  129  TABLE 7. Developmental changes in storage protein mRNAs somatic embryos differentiated on 10 jM ABA as determined scanning densitometry of RNA gel blots.  Developmental Stage  Storage protein mRNA level  (% of maximal hybridization)  Proembryo Globular Round head Flat head Stage 3—1 Stage 3—2 Stage 3—3 Stage 3—4 Stage 4—1 Stage 4—2 Germi Germ2 Germ3 Germ4 Germ5 Germ6  Vicilin  Legumin  0 % 0 % 0 % 5 % 15 % 23 % 82 % 100 % 88 % 75 % 41% 9% 1% 1% 1% 3%  0 % 0 % 0 % 12 % 47 % 60 % 81 % 95 % 100 % 68 % 67% 14% 2% 2% 2% 2%  in by  130  FIGURE 26. Effects of culture on media containing no growth regulators, 40 M ABA, 15% inannitol or fluridone on somatic embryo development. Somatic embryos were matured on 40 M ABA until early cotyledonary stage (EC; Stage 3-4), after which they were cultured for a further 2 weeks on media containing no growth regulators (GRF), 40 jM ABA (ABA), 50 mg/L fluridone (50 FL), 15% mannitol (MAN), mannitol + 50 mg/L fluridone (MAN + 50 FL), mannitol + 10 mg/L fluridone (MAN + 10 FL) or mannitol + 50 mg/L fluridone + 40 ,M ABA (MAN + 50 FL + ABA).  %31  -J Li.  —  2mm  z  -J Li.  LL  0  0<  It) ++-J  zzu <<0  z  132 induced storage protein accumulation was mediated via ABA. 2  weeks  on  fluridone), although  growth  regulator—free  embryos  those  appearance.  had  precociously  cultured  These  medium  with  results  ABA  or  15%  mannitol  fluridone  indicated  did  not  or  germinated were  that  inhibitory to embryo survival or growth. M  (with  After without  (Fig.  26),  bleached  fluridone  was  in not  Embryos cultured on 40  germinate  precociously,  and  embryos cultured on mannitol were smaller in size than those on 40 M ABA (Fig. fluridone size,  or  (Fig.  Protein  Embryos cultured on mannitol, mannitol plus  mannitol  although  appearance  26).  those  plus  fluridone  exposed  to  and  ABA  fluridone  were were  similar  in  bleached  in  26).  profile  differences  between  the  various  treatments  after the 2 week culture period were analyzed by SDS-PAGE 27).  (Fig.  Early cotyledonary embryos collected prior to the start of  the  treatments  27).  After  2  contained weeks  on  low  levels  growth  of  storage  regulator-free  proteins medium  (Fig.  (with  or  without fluridone), no storage protein accumulation was observed, and the low levels initially present in the embryos had declined (Fig. did  27). those  Embryos on 40 exposed  to  15%  ABA accumulated storage protein, mannitol,  although  slightly less in the mannitol-treated embryos cultured levels  of  on  mannitol  storage  plus  proteins  50  mg/L  (Fig.  fluridone  compared to  accumulation 27).  was  Embryos  contained  the mannitol  as  lower  treatment,  while embryos on mannitol plus 10 mg/L fluridone or mannitol plus 50 mg/L fluridone and 40 J.LM ABA displayed higher storage protein  133  FIGURE 27. Changes in total proteins and storage protein mRNA in somatic embryos exposed to no growth regulators, 40 j&M ABA, 15% mannitol or fluridone. UPPER PANEL: Coomassie-stained SDS-PAGE of total proteins from somatic embryos. Embryos were treated as described for Fig. 26. The vicilin (V) and legumin (L) proteins are indicated. Each lane contains 12 g protein. LOWER PANEL: RNA gel blot analysis of total RNA from the same somatic embryo treatments as above, probed with spruce vicilin (115A. 0), legumin (XI5H) or yeast 18S rRNA cDNA.  AN+ 5OFL+ABA  MAN+1OFL  MAN+5OFL  -  C.)  I  O1-1  irrn  kti in1  ‘  ø)(Ø  4 (71  t’aut  Ii 7 flU  -  MAN+5OFL+ ABA  MAN+1OFL  MAN+5OFL  MAN  5OFL  ABA  GRF  EC  135 levels  than  those  treatment (Fig.  above  explants,  regulator—free exposed  to  fluridone.  mannitol  plus  storage protein mRNAs were  treatments.  initial  the  Vicilin declined  treatment,  growth  and  legumin  to  very  and  regulator-free  levels  in  undetectable conditions  for the  present  were  mRNA5  low  were  the in  growth embryos  50  with  in  mg/L  Storage protein mRNAs were present at high levels in  exposed to high levels of fluridone  low fluridone levels levels, 27).  mannitol  plus  legumin  mRNA  However, 50  mg/L  levels  fluridone treatment.  to  the  cultured  fluridone similar  to  and  ABA  the  on  Embryos  exposed to  but embryos  mannitol—only  embryos  27).  while on mannitol  contained higher  (10 mg/L)  similar  (Fig.  (50 mg/L)  had reduced storage protein mRNA levels,  (Fig.  fluridone  out  carried  embryos cultured on 40 j.M ABA or 15% mannitol  mRNA  mg/L  50  27).  of  Analysis  in  and  storage protein ABA  treatments  medium  containing  contained  mannitol  vicilin  plus  50  and Lug/L  The addition of ABA to this treatment did  not enhance storage protein transcript levels.  136 5.  5.1.  DISCUSSION  INTERIOR SPRUCE STORAGE PROTEINS  This study showed that the major storage proteins of interior spruce were a buffer-soluble 41 kD matrix protein and high saltsoluble disulf ide-linked crystalloid proteins of 35-33 kD and 2422  kD.  Minor proteins of  Storage  proteins  of  30  and  similar  27.5 kD were  molecular  also  weight,  identified.  solubility  and  disulf ide—linkage characteristics have been described from other conifers such as white spruce  Pinus species  Green 1990), et al.  1991),  (Gif ford and Tolley 1989, Misra and  (Gif ford 1988)  and similar disuif ide-linked proteins were found in  the non—coniferous gymnosperm Ginkgo biloba 1989).  In  (Green  and Douglas fir  addition  disuif ide-linked  to  proteins  these  proteins,  of  kD  22  and  14  “small-dimer” protein found in Ginkgo biloba  (Jensen and Berthold there  were  2  kD,  similar  minor to  the  (Jensen and Berthold  1989) The results of this present study differ somewhat from those of  Misra  and  crystalloid  Green  storage  (1990,  1991),  protein.  who  However,  described  this  may  a  42  have  been  artifact arising from incomplete extraction procedures. that  antibodies  cross—reacted 1991)  raised  with  against  the  matrix  the 42  kD  42  kD  (Misra  an  The fact  crystalloid  protein  kD  protein  and  Green  supports this conclusion.  The spruce  solubility 35-33  kD  and  and  disulf ide-linkage  24-22  characteristics  kD proteins were  similar to  of  those  the of  angiosperm ll-12S globulin legumin-type storage proteins, whereas the  41  kD  protein  was  similar  to  angiosperm  albumin  or  7S  137 globulin  vicilin-type  proteins  (Higgins  1984,  Shotwell  and  Larkins 1989).  The identification and characterization of 2 cDNA  classes  are  that  development  revealed  highly  expressed  during  further  similarity  to  type and legumin-type storage proteins Newton,  personal  spruce  storage  communication),  proteins  are  spruce  angiosperm  embryo vicilin—  (this study and Dr.  suggesting  homologous  to  that  these  the  two  Craig major  angiosperm  storage protein classes. Further similarities between interior spruce storage proteins and  angiosperm  storage  proteins  included  isoelectric  heterogeneity for each protein class, revealed by two—dimensional electrophoresis, of multi-gene vicilin  suggesting that both protein groups were members  families.  using  DNA  gel  This was blots  substantiated  which  indicated  for the  the  spruce  presence  of  approximately 10-15 gene copies, a similar result to reports that angiosperm  storage  (Harada et al.  proteins  1989,  are  encoded  Higgins 1984,  by  multi—gene  Nielsen et al.  1989,  families Shotwell  and Larkins 1989). The relatively simple mixture of storage protein  body  samples  proteolytic synthesis.  processing In  (legumin—type) processing,  and  patterns  angiosperms, storage  embryos  both  proteins  proteins in spruce  suggested  that  did  not  7S  (vicilin-type)  can  occur  show  complex  during  their  and  liS  post—translational  apart from co-translational signal sequence cleavage,  to yield mature proteins Angiosperm  uS  proteins.  The precursor  proteins  (Müntz 1989, are  Shotwell and Larkins 1989).  synthesized  protein  forms  an  as  large  intrachain  precursor disulfide  138 linkage  and  is  proteolytically  processed  at  either  point to yield two disulf ide-linked proteins, proteolytically processed linked proteins and  Larkins  and  a  1989).  proteins may  also  at two  short,  Further occur  sites  free  processing  (Dure  1989,  two  disulfide  polypeptide  of  the  Raynal  single  or the precursor is  to yield  linker  a  et  (Shotwell  disuif ide-linked al.  1987).  The  spruce legumin cDNA sequence revealed a similar single conserved proteolytic  cleavage  site  (Dr.  Craig  Newton,  personal  communication). Pulse:chase labelling revealed that the mature interior spruce vicilin protein arose from the processing of a precursor protein 3-4  kD  of the type  larger,  suggesting that extensive proteolytic processing  spruce vicilin did not occur.  proteins,  such  phaseolin  (Shotwell  extensive removal. from  as  13-conglycinin  angiosperm vicilin—  (Meinke  et  al.  1981)  Larkins  1989)  also  do  proteolytic  processing  apart  from  signal  In  some  cotton  and  Some  contrast, (Dure  1989)  angiosperm vicilins,  and pea  (Spencer  extensive post—translational processing to  et  not  such  al.  and  undergo sequence as  1983)  those  undergo  form complex protein  patterns. 5.2.  ZYGOTIC EMBRYO STORAGE PROTEIN EXPRESSION The  results  legumin-type thus of  of  this  proteins  present  were  study  detectable  indicate that before  the  indicating differential accumulation patterns.  legumin  proteins  stage embryos, cotyledonary  were  detected  in  the  spruce  vicilin-type, Low  pre—cotyledonary  levels  torpedo—  whereas the vicilin protein was only detected in embryos,  although  both  classes  displayed  major  139 accumulations levels  well  during before  cotyledon  mature  development  seed  shed,  as  and  water  with maturation drying began  (data not shown).  similar  described  to  different  that  commonly  storage  displayed  protein  different  classes  accumulation  for  1987),  while  cruciferin soybean,  in  rapeseed  within  (Crouch and Sussex  1981,  associated  This pattern was  the  patterns.  napin  loss  peak  angiosperms,  where  same  In  synthesis and accumulation precedes that of al.  reached  embryo  pea,  legumin  accumulates  vicilin  (Boulter et  earlier  Murphy et al.  1989)  than  and  in  13—conglycinin proteins are detectable prior to glycinin  (Meinke et al.  1981).  Storage protein accumulation occurs during the cell expansion phase,  after  cell  (Bewley  and  storage  proteins  maturation  division and prior  Black  1985). during  drying  angiosperms.  is  The  storage  proteins  storage  protein  in  pea  legumin  major  cotyledon similar  presence  of  expression  accumulation  development  of  and  spruce  prior  these  observations  low  levels  of  embryos  occurred  Domoney et al.  accumulated  drying and desiccation  to  torpedo—stage  development/expansion. some  The  to  prior  indicated prior  (1980) to  spruce  to with  legumin  that  to  some  cotyledon  also reported that  the  expansion  phase.  Recent work has shown that storage protein mRNAs only accumulate in  cells  lacking  mitotic  activity  (Hauxwell  et  al.  1990),  so  spruce storage protein expression in pre—cotyledonary embryos may reflect  expression  in  early stage embryos. appearance  of  spruce  non—dividing,  expanding  cells  of  these  It is also possible that the differential legumins  in  pre—cotyledonary  embryos  140 reflected between  differences  the  primarily storage  in  embryonic  expressed  protein  glycinin  storage  axis in  and  patterns  compared  expression  cotyledons,  cotyledonary  expression  accumulation  protein  to  with  cells. in  patterns  the  vicilins  Organ—specific  soybean  showed  13—conglycinin  in  little  embryonic  axes, but prominent levels of both storage proteins in cotyledons (Meinke et al.  1981).  The pattern of storage protein accumulation described in this study does not agree with most of the results reported recently for  white  spruce  (Misra  and  Green  1991).  While  these  workers  also reported accumulations of some crystalloid proteins prior to the accumulation of a 42 kD protein, they found that the 35-34 kD crystalloids  accumulated  before  24-23  crystalloids.  kD  In  contrast, the present results indicated that the 35-33 kD and 2422 kD crystalloids displayed the same temporal pattern. solubility, the the  spruce  Based on  disulf ide-linkage and cDNA sequence characteristics, 35-33  angiosperm  kD and 24-22 legumins  kD proteins  and,  since  appear homologous to the  disulf ide-linked  angiosperm legumin proteins show concurrent accumulation patterns (Higgins  1984,  Larkins 1989),  Meinke  et  al.  1981,  MUntz  1989,  Shotwell  and  the results of this present study agree with the  angiosperm data.  Misra and Green  (1991)  also reported that the  major accumulation of the 42 kD protein occurred between the last two  collection dates  attainment development contrasted  (Aug.  29  and mature  of maximum dry weight in with  which those  maturation of  this  seed),  well  after the  and during the period of drying present  occurs. study,  These where  seed  results greatest  141  protein  levels  seed shed.  were  attained  at  least  with  angiosperms.  Misra and Green by  these  (1991)  workers  antibody  prior  to mature  of  the  similar to  discrepancies  between  to  carry It  was  those  protein  their also  were  and  to  the  Green  compare  so that  analyzed  Misra  and  they did not include data on morphological development, samples  of  difficult  extractions  because  protein  with  out  (1991)  the  results  Some  (data not shown),  and this work arise from the methods used  production.  present  month  This maximum coincided with steady state dry weight  and the onset of maturation drying results  1  related  only  to  collection  date. Analysis of interior spruce zygotic embryos revealed that both vicilin  and  embryos,  legumin  even  messages  though  accumulation patterns. during  cotyledon  the  detectable  were  storage  proteins  Both the mRNA5  development,  with  declining prior to that of vicilin.  torpedo  in  stage  different  displayed  increased to peak levels  legumin  mRNA  peaking  and  However, both dropped to low  levels during the last month or so of zygotic seed development, coincident with the drying phase.  The simultaneous appearance of  both  have  storage  ordinate  protein  regulation.  mRNAs  may  Most  been  angiosperm  indicative  embryos  of  which  co show  differential storage protein accumulation also show differential appearance classes days  al.  of  storage  displaying  temporal  (Boulter et al. 1986,  protein  Yang et al.  1987, 1990),  mRNA5,  differences  with in  the  different  appearance  Finkelstein et al.  1985,  although Meinke et al.  by  mRNA  a  few  walling et (1981)  noted  that soybean 7S and uS storage protein mRNAs were detectable at  142 the same time during development.  It is possible that the spruce  vicilin  display  and  patterns, the  legumin  mRNA5  do  different  appearance  but these were not detected due to the time  zygotic  embryo  collections.  More  frequent,  frame of  meticulous  collections during the early stages of zygotic embryo development are required to determine if different appearance patterns exist for the various spruce storage protein mRNAs. Angiosperm  storage  protein  mRNAs  appear  well  after  fertilization and during early embryo development (Boulter et al. 1987,  Finkeistein et al.  1989),  in  (1991)  levels  of  crystalloid  following  accumulation levels  of  in  of  of  may  embryo  eastern  white  and Misra and Green  protein  study.  transient  megagametophytes induction  tissue  in  fertilization, this  However, recent work  described the appearance of legumin—like  megagametophyte  following fertilization,  just  1989, Nielsen et al.  as did those in spruce (this study).  by Kamalay et al. mRNAs  1985, Harada et al.  be  (1991)  white  spruce  well  before  These  storage associated  development.  results  reported low  their suggest  visible that  expression  fertilization  Recent  just  megagametophytes  protein with  pine  work  with  or  low in the  rapeseed  microspore—derived embryos revealed that the heat shock treatment used to induce embryogenesis also caused the transient appearance of  storage  protein  (napin)  mRNA  (Boutilier  et  al.  1991),  spruce  storage  providing further support for this hypothesis. The  different  accumulation  patterns  for  the  protein mRNAs were similar to results described for angiosperms. In pea,  vicilin mRNA and protein showed peak accumulations prior  143 to those for legumin (Boulter et al. in  rapeseed,  napin  mRNA  and  1987, Yang et al.  protein  showed  peak  al.  1985).  transcripts  The accumulation of  during  transcriptional also play  a  Harada  al.  et  development  level,  role  although  (Delisle  1989,  is  and  Nielsen  angiosperm  primarily  differences  Crouch  et  al.  protein accumulation patterns, their  mRNA5  suggest,  by  regulated  et  While  at  the  stability  mRNA  Evans  1989).  transcriptional rates were not performed,  Finkelstein  storage protein  in  1989,  and,  accumulations  prior to those of cruciferin (Crouch and Sussex 1981, et  1990)  al.  1984,  analyses  the different storage  and the accumulation patterns  analogy  of  with  other  systems,  of  that  regulation in spruce may also be primarily at the transcriptional level,  although other factors may also be involved.  protein  mRNAs  vicilin  protein  suggesting may  also  well  are  that  be  synthesized, 1985).  becomes  developmental soybean  then As  more  In  same  early  later  than  the  changes exist.  or  accumulation degraded  in  legumin  vicilin mRNA and  the  rapidly  development  and  the  proteins, regulation detectable  proteins  proceeds,  accumulates,  similar  is  but  are  (Shuttuck-Eidens  type  of  the  and  protein  indicating  post—translational A  stage,  post—translational  soybean,  embryo  stable  vicilin  the  translational  protein but  at  accumulates  involved.  before  Beachy  present  Both storage  that  regulation mechanism  may  for be  associated with spruce vicilin accumulation. 5.3.  SOMATIC EMBRYO STORAGE PROTEINS This  study  revealed  that  interior  spruce  accumulated the same major storage proteins as  somatic  embryos  zygotic embryos,  144 based  on  molecular  characteristics, et  al.  weight,  solubility  and  and migration in two—dimensional  (1990)  also  reported  that  Norway  reported  that  white  spruce  proteins  were  absent  or were  not  somatic  and  zygotic  embryos  highly  contain  expressed.  the  same  al.  1987,  results the  Stuart  al.  1988,  suggest that the tissue  regions  of  information, proteins somatic  et  storage  leading  as  found  to  in  Tewes  the  zygotic  embryo—derived  et  genes  al.  of  embryos.  This  is  to  as  does  the is  their  Shoemaker  1991).  encoding  expression  material  (somatic and  1989,  culture process  protein  embryo  Work with  proteins  zygotic counterparts (Crouch 1982, Krochko et al. et  and  although some of the major  angiosperm systems has also shown that non—zygotic microspore—derived)  Hakman  while Joy IV et al.  somatic  total protein profiles were similar,  gels.  spruce  zygotic embryos contained similar proteins, (1991)  disuif ide-linkage  These  not alter structural  same  storage  important  be  used  if for  biotechnological applications. The  levels  of  storage  proteins  that  accumulated  in  spruce  somatic embryos in the presence of 40 M ABA were similar to or higher than those found in mature zygotic embryos. in our  lab  (Cyr et al.  1991)  Recent work  has confirmed that somatic embryo  storage protein levels may differ from those of zygotic embryos. These  results  are  in  contrast  to  the  angiosperm  data,  where  storage proteins were significantly lower in non—zygotic embryos (Crouch 1982, et  al.  Krochko et al.  1988,  proportions  are  Taylor  et  altered  in  1989,  al.  Shoemaker et al.  1990).  alfalfa  Also,  somatic  1987,  storage  embryos  Stuart protein  (Krochko  et  145  al.  Stuart et al.  1989,  levels  alfalfa  in  translation  a  of  is believed to be  storage  (Pramanik et  studies  al.  However,  ABA  due to  during  inability to be  1991).  utilized  mRNAs  protein  reflection of their  polysomes  angiosperm  The alteration of storage protein  embryos  somatic  efficiency  development, into  1988).  early  incorporated  none  of  somatic  during  low  these embryo  differentiation, with the exception of Taylor et al.  (1990),  used  maturation.  short  a  Since  embryo  Croissant—Sych  low ABA  of  induces  ABA  zygotic  pulse  storage  tissues and  1988,  Finkelstein  et  translation  (Finkelstein  somatic  embryos  observed  proteins  al.  (1990)  proteins, Recently, embryos some  in  quantitative  Joy IV et al.  major  significantly as  Mascarenhas  1985,  storage fact  the  that on  levels  high  levels  comparisons  spruce M  Hakman  et  embryos storage  of  were  ABA  storage  of  somatic  spruce  abundant  40  study.  in this  protein  not  made.  reported that white spruce somatic  (1991)  proteins  the  Norway  contained  ABA  although  contained  that  1985,  continuously  embryos  somatic  noted  on  1985),  explain  Beachy  enhance  may  al.  and  cultured  in  and  Bray  Eisenberg  and  et  accumulation  1986,  differentiated  could  also  differentiated  and  were  maturation  during  1985)  embryo  during  protein  (Barratt  Bopp  al.  level  who  lower  levels  identified  these workers used low levels of ABA  by  (10 suM)  of  total  SDS-PAGE.  proteins However,  compared to the 40  M level used here. The analysis of different somatic embryo genotypes that  differences  in  storage  protein  levels  indicated  occurred  among  cotyledonary somatic embryos after 7 weeks of maturation on 40 ,.LM  146 ABA,  although all genotypes accumulated significant levels.  variations  observed  between  the  different  genotypes  could  The have  reflected effects of the differentiation protocol, which may have to  be  optimized  for  each  embryogenic  line  in  similar storage protein levels in each line. the  variations  from  the  may  have  individual  induction,  seed  reflected embryos  to  obtain  On the other hand,  genetic used  order  differences  for  arising  embryogenic  tissue  since it is known that genotype—dependent differences  in storage protein accumulation can occur (Higgins 1984). 5.4.  DEVELOPMENTAL  EXPRESSION  OF  STORAGE  PROTEINS  IN  SOMATIC  EMBRYOS ON 40 JLM AND 10 jLM ABA Spruce  somatic  differential  storage  those described for appeared  earlier  primarily  cultured  protein  on  development,  cotyledon  40  accumulation  zygotic embryos.  in  during  embryos  The  but  M  ABA  patterns  displayed similar  to  legumin-type proteins  both  development.  types The  accumulated  appearance  of  vicilin and legumin mRNAs in torpedo stage somatic embryos on 40 M  ABA  was  also  zygotic embryos.  similar  to  their  developmental  appearance  in  Also, the developmental accumulation pattern of  storage protein mRNA5 in somatic embryos on 40 ).LM ABA was similar to  that  in  zygotic  embryos,  with  legumin  starting to decline prior to vicilin mRNA.  mRNA  peaking  and  All of these results  suggest that the patterns of storage protein gene  induction and  accumulation were similar between the two embryo types,  although  total storage protein mRNA levels were not compared between them. These  results  angiosperm  are  different  non—zygotic  from  embryos,  those  which  commonly show  reported  for  temporally—altered  147 storage  protein  development  expression,  than  in  Shoemaker et al. during  their  1987).  in  accumulations  zygotic  However,  differentiation.  maturation  with  counterparts  zygotic  (Crouch  embryos  more  normal  (Ackerson  1981,  protein  Roberts et al.  gene  developmental  expression  exogenous ABA, more  normal  1990a),  expression  which,  1982,  in  as well  as  may  be  due  and  Kamada and  induces  storage  these  altered  angiosperms,  patterns  embryo  l984ab)  developing somatic embryos (this study, Ammirato 1974, Harada  in  these studies did not use ABA  Since ABA promotes  excised  earlier  to  the  lack  of  if it were supplied would help to promote  patterns  of  gene  expression  in  these  embryos.  Support for this proposal comes from a recent study by Wilen et al.  (1990)  using  microspore—derived  rapeseed  These  embryos.  workers found that exposure of embryos to a 48 hour pulse of ABA at  different  stages  developmental  of  induction  development  of  napin  promoted  the  cruciferin  and  correct  transcripts.  They also obtained levels of storage protein mRNA similar to that observed  in  equivalent  developmental  stage  accumulation  zygotic  patterns  embryos,  of  the  although  storage  the  proteins  themselves were not characterized. Apart from the similarities described above for spruce zygotic and  somatic  embryos,  there  protein gene expression.  were  also  in  storage  Storage proteins accumulated gradually  and continuously for a prolonged period were still  differences  in somatic embryos,  and  increasing after 9 weeks of maturation on 40 ).LM ABA.  High  levels  this  period,  of  storage protein mRNA5 were  such  that  after  9  weeks  on  also ABA,  observed during they  were  still  148 present  50%  at  marked  of to  contrast  displayed  a  their  more  maximal  that  levels.  observed  rapid and  for  did  not  increase  approximately accumulation  or  1% of  and  storage  of  and  embryos,  in  which  storage protein  of  declined  maximal  proteins  was  after which protein levels  levels  mRNA  less  zygotic  transient period  and storage protein mRNA accumulation,  pattern  This  levels. the  continuous  The levels  high  to  rapidly,  of  their  transcripts may reflect the constant exposure of somatic embryos to  high  embryos  levels remain  of  ABA  fully  throughout  hydrated  Also,  maturation.  during  maturation  and,  embryos  quiescence Black  desiccate  during  1985).  later  Storage  and  stages  enter of  protein  seed  a  of  period  development  synthesis  and  message  al.  1985,  Galau  et  al.  (Dure and Galau 1981, Kermode  1987,  et  al.  on  metabolic and  (Bewley  generally decline during the maturation drying and stage of embryo development  based  In contrast,  storage protein accumulation, metabolically active. zygotic  somatic  levels  desiccation  Finkelstein et Recent  1989).  results in our lab have indicated that storage protein transcript levels also decline rapidly in spruce somatic embryos exposed to an  artificial  drying  regime  (Dr.  David  Cyr,  personal  communication) that is used to mimic normal seed/embryo drying. Spruce somatic embryos differentiated on low levels ABA  also  accumulated  some  storage  protein.  (10 jIM)  of  differential  The  storage protein accumulation patterns observed in zygotic embryos and somatic embryos exposed to 40 M ABA were also observed on 10 M  ABA.  detected  Furthermore, by  torpedo  both stage  vicilin in  these  and  legumin  embryos,  as  mRNA5 were  were the  149 differential accumulation patterns, prior to vicilin.  This was similar to the pattern observed with  zygotic  embryos  maximal  storage protein mRNA  less  than  and somatic embryos on  those  attributed  to  with legumin mRNA increasing  for  the  40  levels  J.LM  lower  ABA,  attained on  ABA  ABA  40  somatic  level  although the M ABA were  10  embryos.  used.  This  Similar  ABA  is  dose-  dependence has been shown for storage protein gene expression in cultured 1989) ABA  zygotic  and  non-zygotic  treatments  levels.  resulting  similar  regulation  of  the  regardless  in  normal the  al. al.  somatic  storage  and  on  high and  suggesting pattern  although  transcript or  gene  ABA  ABA  low  differential  that  of  Crouch  with higher  protein  induction  developmental used,  Delisle embryos,  embryos  proteins,  level  1985, 1990)  higher  developmental  storage  of  et  (Wilen et  Interestingly,  displayed  maintain  (Finkelstein  did  ABA  could  expression affect  the  quantitative expression of storage protein genes in similar ways to its action in microspore-derived embryos  (Wilen et al.  1990).  The fact that both high and low ABA-treated somatic embryos did not  exhibit  development,  storage protein although  proembryo stage, as  manifested by  they  transcripts had  been  until  exposed  torpedo to  ABA  stage since  of the  suggested that the competence to respond to ABA, storage protein mRNA  until torpedo stage.  induction,  did not  occur  The differences between the relative levels  of vicilin and legumin transcripts during their accumulation in early cotyledonary embryos were not as great on  high  levels  of  stimulatory effect  ABA. of high  This levels  may  be  in somatic embryos  attributed  to  a  more  of ABA on vicilin transcript  150 levels.  The preferential enhancement by ABA of vicilin proteins  and transcripts  over  other  (Bray and Beachy 1985,  storage proteins has been documented  Schroeder 1984).  In contrast to the pattern observed in somatic embryos on 40 jM ABA, they  embryos on 10 uM ABA began to germinate precociously once  had  reached mid-cotyledon  parallelled protein  by  a  levels,  decline  so  that  stage  storage  in  of  development.  protein  storage proteins  mRNAs  did  not  This was  and  accumulate  the high levels observed in 40 ,M ABA somatic embryos. Joy  IV  embryos contain  et  al.  that  (1991) were  storage  counterparts. and  prevent  reported  differentiated proteins  While  at  low ABA  precocious  effective by of  also  that  on  the  low  white (10  level  germination  the mid-cotyledonary  the  ABA  somatic did  their  embryo  initially, stage,  sensitivity to ABA occurred as  M)  it  to  Recently,  spruce  of  could maintain  storage  not  zygotic  development  was  no  longer loss  suggesting that a  embryo matured.  Loss  of  ABA sensitivity has been reported to occur during zygotic embryo maturation 1985, in  (Eisenberg  Kermode et al.  sensitivity  and  Mascarenhas  1985,  Finkelstein  1989, Rivin and Grudt 1991),  has  been  attributed  to  drying  decline  storage protein transcripts commonly  late  in  zygotic  Harada et al.  and  desiccation  embryogeny 1989,  (this  study,  Nielsen et al.  partially reflect changes due to ABA sensitivity.  (Kermode  et  al.  caused 1989).  observed  Finkelstein  et  1989, Walling et al. lowered ABA  al.  and this change  changes  maturation  et  levels  al.  by The  during 1985,  1986)  may  and reduced  151 While  changes  desiccation,  work  in  ABA  with  undergo desiccation,  sensitivity  maize  occur  viviparous  in  mutants,  response which  do  has shown that embryos also undergo a  of ABA sensitivity during maturation without desiccation and  Grudt  spruce  1991).  These  results  somatic embryos on  developmental  changes  maturation,  and  present  the  to not loss  (Rivin  results  with  10 M ABA indicate that there may be  in  regardless  ABA  sensitivity  of  drying,  that  occur  allowing  during  precocious  germination. Spruce somatic embryos that germinated precociously on 10 jM ABA  still  even  contained  after  low  levels  several weeks  and partially—dried  of  of  storage protein  germination.  Both  transcripts,  zygotic  somatic embryos have undetectable  embryos  levels  of  storage protein transcripts within days of germination (Dr. David Cyr,  personal communication).  It has been suggested that drying  or desiccation is required to switch from an embryo maturation to a  germinative program  1989). drying  Furthermore, treatment  germinative 1991). during  and  spruce  prior  to  and Bewley somatic  embryos  germination  post-germinative  Kermode et  1989,  given  a  exhibit much more  growth  (Roberts  et  al.  al.  partial normal l990b,  The role of embryo developmental stage on gene expression precocious  Crouch  (1984).  placed  on  cotyledon  germination  but  media  little  development  embryos  was  Mid-cotyledon  germination  development  These  (Kermode  stage  hypocotyl  to  as  express  by  rapeseed  germinated  formation  continued  explored  Finkeistein zygotic  abnormally,  elongation opposed storage  to  and  embryos  with  root  and  secondary  leaf  formation.  protein  genes  and  152 accumulate  storage  proteins  germination.  Older,  normal—looking  seedlings,  still  detectable  weeks.  This  maturation  and  was  during  the  stage  4  weeks  embryos  protein  storage  proteins.  protein  degradation  to  the  continued  normally  transcript  during  retention  These  of  programs  expressing  results  both  simultaneously.  took  several  synthesis  and  indicating concurrent expression of Only mature, dry,  germination,  and  rapid  suggested  required to switch developmental programs, capable  were  storage  responded  storage  transcripts  protein  embryo developmental and germinative programs. embryos  into  storage  attributed  precocious  germinated  but  turnover of storage proteins,  seed  of  embryo  no  degradation  of  that  drying  was  and that embryos were  maturation  Kriz et al.  with  and  germination  also reported that  (1990)  precociously germinating embryos of viviparous mutants, which did not  undergo  desiccation,  exhibited  prolonged  expression  of  storage protein transcripts and an incomplete switch from embryo development to germination,  further indicating the importance of  drying  embryo  for  the  gene expression. with  spruce  germinated, transcripts,  change  embryos  still  suggesting  germination programs, or  developmental  to  germinative  These results are similar to the observations  somatic but  from  partially-dried  on  contain  10 low  simultaneous  iM  ABA,  levels  which of  appear  storage  expression  of  have  protein  embryo  and  in contrast to germinants of normal embryos somatic  embryos  (Dr.  David  Cyr,  personal  communication). It was suggested that somatic embryos on 10 M ABA lost their sensitivity to ABA and germinated precociously.  However,  somatic  153 embryos After  on  9  40  M  ABA  weeks  on  ABA,  germinated, ABA will  although  eventually  Dunstan  al.  et  did  not  these  display embryos  such had  prolonged culture lead to their  1991),  not  on  suggesting  that  evident yet  these  germination  change.  precociously  high  (data  they  developmental change in ABA sensitivity. btM ABA  an  levels not  also  of  shown,  exhibit  a  Somatic embryos on 40  displayed a decline in storage protein transcript levels  by 9 weeks of maturation.  This decline may be  indicative of a  change  in ABA  suggesting that  the  embryos  are,  least  at  to  switch  into  sensitivity,  the  molecular  level,  starting  at a  precocious germination mode even though protein levels are still high.  Further  studies  using  germination-specific  required to determine if this is the case. probes has  to determine the  important  state of  implications  probes  are  The use of molecular  embryo development/germination  in  determining  the  appropriate  developmental stage at which maturing somatic embryos  should be  removed for partial drying and artificial seed production. 5.5.  OSMOTIC STRESS AND STORAGE PROTEIN GENE EXPRESSION Early  ABA  or  cotyledonary  15%  mannitol  spruce  for  2  somatic weeks  embryos  matured  cultured  into  well  on  40  LM  developed  cotyledonary stage embryos which did not precociously germinate,  although  embryos  promotion  and/or  on  15%  mannitol  enhancement  of  has been reported for angiosperm and  conifer  (Lu  Tremblay 1991a) on  ABA  or  and  Thorpe  systems.  mannitol  were somatic (Litz  1987,  high  embryo  1986,  Roberts  In this study,  contained  smaller  in  size.  This  differentiation  Nadel et al.  1991,  1989)  Tremblay  and  somatic embryos matured  levels  of  storage  protein  154 transcripts and storage proteins,  although storage protein levels  in the mannitol-treated embryos were not as high as treated embryos. an  osmotic  This  confirms previous  stress—induced  spruce somatic embryos  accumulation  (Roberts  1991)  in the ABA-  results that of  indicated  proteins  storage  and agrees with angiosperm  data showing the same pattern in cultured embryo tissues 1986, 1990)  Finkelstein and Crouch 1986, Goffner et al. and  developing  in  microspore-derived  Xu et al.  1990,  embryos  (Barratt  (Wilen  et  al.  1990) Osmotic stress—induced storage protein accumulations have been attributed stress  to  stimulation  a  (Barratt 1986,  although  others  directly  associated  Finkeistein 1990).  and  suggested  have  both  endogenous  Rivin and Grudt  with  Crouch  Since  of  the  1986,  ABA  1991,  that  and  (Finkelstein and Crouch 1986,  levels  (Barratt  al.  et  osmoticum  by  Wilen et al.  endogenous  response Goffner  ABA  ABA et  1990,  1990), is  al. Xu  the  not 1989,  et  al.  inhibit  water  uptake  Schopfer and Plachy  1984),  it has  been suggested that alteration in embryo cell osmotic potential, and  not  ABA,  response. induced  is  the  primary  effector  of  the  storage  protein  To study the potential role of ABA in osmotic stress— storage  protein  gene  expression  in  spruce,  somatic embryos were exposed to the ABA biosynthetic fluridone,  during  osmotic  stress.  This  study  developing inhibitor,  indicated  that  somatic embryos exposed to high levels of fluridone contained low levels of storage protein transcripts and did not accumulate high levels of storage proteins.  Furthermore,  in  stress,  combination  with  osmotic  lower fluridone levels  which  should  have  allowed  155 more  endogenous  storage These  protein results  protein  gene  via ABA. medium  ABA  transcripts suggested  expression  However,  plus  storage  biosynthesis,  high  protein  and  that in  displayed storage  osmotic  spruce  levels  higher  protein  accumulation. storage  stress—induced  somatic  of  embryos  was  mediated  while embryos matured on mannitol—containirig levels  of  fluridone  levels  compared  to  and  ABA  those  contained  on  higher  mannitol  plus  fluridone, the storage protein transcript levels did not increase substantially.  This  suggested  that  exogenous  could  ABA  not stimulate the fluridone-induced inhibition of storage protein transcript levels under the conditions used here.  The inability  of exogenous ABA to stimulate storage protein transcript  levels  may have  to the  highly  negative  medium. was  been due  to  little uptake  osmotic  of  potential  applied ABA,  of  the  mannitol-containing  The possible inhibition of ABA uptake by osmotic stress  suggested by the results of Bray and Beachy  workers  noted  that  low  levels  stimulated endogenous ABA levels enhanced  these  levels.  from  the  0.5-3%  osmoticum  of  at  the  These  (1985).  (0.5—3%  and exogenous ABA  However,  concentration tested (10%), those  due  sucrose)  applications  highest  sucrose  endogenous ABA levels were lower than  sucrose  treatments,and  exogenous  ABA  application did not enhance these levels. If ABA uptake was low due to the high osmoticum used study, observed  this  could  between  potentially storage  Finkelstein et al.  (1985)  cultured  ABA  on  low  account  protein  and  for  the  discrepancy  transcript  reported that excised zygotic did  not  accumulate  in this  storage  levels. embryos protein  156 transcripts to the levels found in seed embryos, proteins  reach  did  their  enhanced translation.  normal  although storage  suggesting  levels,  that  ABA  It is possible that the ABA level to which  the spruce somatic embryos were exposed during the inannitol plus fluridone and ABA culture was not  induce a major  sufficient to  increase in steady state storage protein transcript enhanced  translation  of  the  message  to  allow  levels,  storage  but  protein  accumulation. It  inability of ABA to completely  is also possible that the  restore storage protein transcript levels in the presence of high levels  of  fluridone  may  effect of fluridone, embryo  survival  or  have  been  due  to  some  other  indirect  although fluridone exposure did not inhibit growth.  Interestingly,  Fong  et  al.  (1983)  noted that exogenous ABA could only partially reverse fluridone— induced vivipary in maize embryos. While this present study did not measure endogenous ABA levels during development, inhibit  ABA  biosynthesis  (Barratt et al. via  the  Zeevaart  1991),  to  suppression is  response  to  pathway  endogenous  and  ABA  levels  ABA is synthesized  Horgan  1991,  Rock  inhibits phytoene desaturation  and in  (Zeevaart and  inhibiting ABA biosynthesis  The bleached appearance of the somatic embryos was  (Fong et al.  the  (Parry  and fluridone  fluridone  possible  reduces  and  Bray and Beachy 1985).  thereby  Creelman 1988).  It  1989,  carotenoid  this pathway,  exposed  other studies have shown that fluridone does  that  indicative 1983),  stress  carotenoid  and therefore,  alterations  osmotic  of  in  could  ABA biosynthesis.  endogenous have  biosynthetic  ABA  occurred,  pools but  in  other  157 studies using fluridone have shown that endogenous ABA levels do decline,  and  the  two  week  period  of  culture  during  this  experiment should have allowed depletion of endogenous ABA pools without replenishment via biosynthesis. 5.6.  CONCLUDING STATEMENT  This spruce  study  showed that both  expressed  the  same  zygotic  storage  and  somatic  proteins.  embryos  These  of  proteins  appeared homologous to known angiosperm storage protein classes, based  on  solubility,  characteristics.  The  displayed differential to  the  differential  angiosperm  disulfide  storage  two  linkage  different  and  storage  accumulation patterns. accumulation  proteins.  cDNA  patterns Spruce  sequence  protein  classes  This was  similar  often  reported  storage  protein  for gene  expression was regulated by ABA and while somatic embryos matured on high or induction zygotic ABA  low  of  were  embryos,  only those  capable  levels  of ABA displayed a  storage protein  embryos,  germination. to  levels  of  transcripts embryos  prolonged  similar  although  to  or  zygotic  embryos.  was  These  of  more  than  without  levels This  of  results  ABA were  finding  suggested  well  levels  to of  precocious  storage proteins found  than  in  zygotic  transcript  that  that,  observed  using  and in  storage  somatic embryos that matured  developmentally  bodes  biotechnological applications.  levels  prolonged  proteins  on high  storage protein  protein gene expression as a marker,  embryos.  their  cultured  development  higher  the period  accumulation  high  and  This allowed the accumulation of  protein  on  similar developmental  for  similar  their  to  zygotic  utilization  in  158 Somatic embryos must exhibit vigorous germinative growth  if they are to be useful.  somatic  embryos  subject  mimics  normal  seed/embryo  expression  from  perform  much  Roberts  et  somatic  embryos  germination, somatic  an  is  to  than  1990b). at  the  on  artificial  not  dried  appropriate to  high  optimize ABA  the  to  is known that which  switches  gene  germinative  mode,  (Parrott  et  removal  and  time,  and post—  regime,  and  developmental if  It  drying  desiccation  Therefore,  critical  embryos  an  embryo  better  al.  germination  prior  to  al.  drying  did  of  precocious While  plantlet recovery.  levels  1988,  germinate  not  precociously during the culture period used here, they eventually did,  indicating  an apparent  loss  sensitivity during the  of ABA  later stages of somatic embryo maturation. had  a  prolonged  indicated  a  period  delay  in  of  storage  precocious  Although the embryos  protein  accumulation  germination,  the  that  decline  of  storage protein mRNAs may have indicated the onset of loss of ABA sensitivity and precocious germination.  Further  studies,  using  embryo developmental and germination—specific probes are required to confirm this, but molecular markers clearly have the potential to gauge the maturity of somatic embryos and could indicate the onset become  of  precocious  apparent.  germination  This  would  well  before  facilitate the  protein  removal  of  embryos for partial drying and artificial seed production.  changes somatic  159 6.  LITERATURE CITED  Ackerson, R.C. 1984a. Regulation of soybean abscisic acid. J. Exp. Bot. 35(152): 403—413.  embryogenesis  by  Ackerson, R.C. 1984b. Abscisic acid and precocious germination in soybeans. J. Exp. Bot. 35(152): 414—421. Anuuirato, P.V. abscisic acid on the 1974. The effects of development of somatic embryos from cells of caraway (Carum carvi L.) Bot. Gaz. 135: 328—337. 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