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Table of Contents

 

Statement of Compliance -------------------------------------------- 2

Quality Assurance Statement----------------------------------------- 3

Signature of Approval ---------------------------------------------- 5

 

I )  Summary --------------------------------------------------------12

II)  Purpose --------------------------------------------------------16

A. General ----------------------------------------------------16

B. Protocol amendments ----------------------------------------17

      1. Processing experiment(#1): add line 40-3-2  ----------17

2. Processing experiment(#2): full-fat soybean flour

was prepared---------------------------------------17

3. Processing experiment(#3): toasted meal samples

were retoasted ------------------------------------17

4. Analytical experiment (#1) : add line 40-3-2 ---------17

5. Analytical experiment (#2) : the retoasted meal

was substituted for the initially-toasted meal

for the analytical assays -------------------------17

    

6. Analytical experiment (#3) : the enzymatic activity

of CP4 EPSPS and GUS were determined in pooled

seed extracts -------------------------------------17

7. Analytical experiment(#4) : one set of six seeds

from each plot was used for the ELISA expression

analysis, not two sets ----------------------------17

8. Analytical experiment(#5) : ELISA assays were

       performed on the seed pool extracts analyzed

by functional enzymatic assay ---------------------18

C. Report amendments ------------------------------------------18

 

III)  Justification of Test System ----------------------------------18

 

IV)   Materials and Methods -----------------------------------------19

A.  Field experiment -------------------------------------------19

1.  Lineage and history of seed used for field test------19

2.  Plot information  -----------------------------------19

3.  Sampling for analysis -------------------------------19

a.  Leaf tissue --------------------------------19    

i.  Initial leaf sampling  ------------19

ii. Monthly sampling  -----------------19

iii.Whole plant ----------------------20

b.  Seed tissue -------------------------------20

i.  Bulk seed ------------------------20

ii. Analytical seed ------------------20

4.  Field Monitoring ------------------------------------20

B.  Processing experiment --------------------------------------20

C.  Analytical experiment  -------------------------------------20

1.  GUS ELISA -------------------------------------------20

2.  CP4 EPSPS ELISA -------------------------------------21

3.  GUS enzymatic assay  --------------------------------21

4.  CP4 EPSPS enzymatic assay  --------------------------21

5.  Western blot assay for CP4 EPSPS and GUS ------------22

6.  Protein assay for extracts (Bio-Rad assay) ----------22

7.  Quality assays --------------------------------------22

a.  urease ----------------------------------------22

b.  sugar analysis by HPLC ------------------------22

c.  nitrogen solubility analysis ------------------22

d.  tryptophan-alkaline hydrolysis ----------------22

e.  moisture --------------------------------------22

f.  cysteine and methionine -----------------------22

g.  amino acids -----------------------------------23

h.  trypsin inhibition ----------------------------23

i.  ash -------------------------------------------23

j.  fatty acid profiles ---------------------------23

k.  fiber -----------------------------------------23

l.  fat -ether extraction -------------------------24

m.  total kjeldahl nitrogen-protein ---------------24

n.  phytic acid, by ion-exchange  -----------------24

o.  carbohydrates ---------------------------------24

p.  calories --------------------------------------24

q.  free and bound isoflavones --------------------24

r.  lectines --------------------------------------24

8.  Statistical analysis onb Soybean seed quality data----25

9.  Protein/aminoacids  in oil assays--------------------25

10.  Other circumstances pertaining to the study and

protocol and SOP deviations ----------------------25

a. Field experiment-------------------------------25

                 b. Processing experiment -------------------------25

c. Analytical experiment -------------------------26

 

V) Results and Discussion -------------------------------------------26

A.  Field Experiment ----- -------------------------------------26

1. Agronomic practices ----------------------------------26

2. Field monitoring  ------------------------------------26

3. Leaf and plant sampling ------------------------------26

4. Harvest ----------------------------------------------26

      B. Processing experiment --------------------------------------27

      C. Analytical experiment --------------------------------------27

              1. Levels of CP4 EPSPS and GUS proteins in Soybean

Leaves and seeds ----------------------------------27

           2. Quality analysis of raw Soybeans ---------------------28

                 a. Proximate analysis --------------------- -------29

                 b. Amino acid analysis ----------------------------29

                 c. Fatty acid analysis ----------------------------30

                 d. Isoflavone analysis ----------------------------30

                 e. Lectin analysis --------------------------------30

                 f. Trypsin inhibitor and urease -------------------31

           3. Proximate analysis of full-fat Soybean flour ---------32

           4. Quality analysis of toasted Soybean meal -------------32

                 a. Initial preparation of toasted Soybean meal ----33

                       i. Proximate analysis  ----------------------33

                       ii. Phytate  --------------------------------33

                          iii. Trypsin inhibitor,urease, and

      nitrogen solubility index --------------33

                 b. Retoasted Soybean meal -------------------------34

                       i. Proximate analysis  ----------------------34

                      

ii. Trypsin inhibitor, urease, and

     nitrogen solubility index ---------------34

iii. Laboratory cooking of Soybeans

     Samples to test T1 inactivation  --------35

iv. Phytate in retoasted meal ---------------36

v.  Stachyose and raffinose in retoasted

     meal -----------------------------------36

                       vi. Lectins in retoasted meal --------------36

                       vii. Isoflavones in retoasted meal ---------37

          

5. Expression of CP4 EPSPS and GUS in retoasted

              Soybean meal---------------------------------37

                a. SDS-extractions and Western blot analysis --------37

            b. Expression of CP4 EPSPS and GUS in protein

fractions from non-denaturing extracts

and Western blot, ELISA,and enzymatic

activity assays ----------------------------38

i. GUS assays ------------------------------39

ii. CPE EPSPS assays -----------------------39

           6. Analysis of Soybean oil------------------------------40

        a. Fatty acid composition of refined, bleached,

@@deodorized oil --- -----------------[[-------40

 b. Determination of protein/amino acid levels

   @found in refined, bleached, deodorized oil---40

 

VI) Conclusions ----------------------------------------------------42

VII) References ----------------------------------------------------43

VIII) List of abbreviations-----------------------------------------50

 

 

 

 

 

 

 

 

             LIST OF TABLES

 

 

 

Table 1. Chemical treatments used for glyphosate-torelant

   Soybean field test -----------------------------------------52

Table 2. Summary of Disease and insect monitoring log.---------------52

Table 3. Plant tissuesampling log -----------------------------------53

Table 4. Yield of Soybean seeds from 1992 Puerto Rico field test-----53

Table 5. Puerto rico processing experiment shipping log -------------54

Table 6. Expression of CP4 EPSPS and GUS in Soybean plant tissues,

   determined by ELISA --------------------------------------- -55

Table 7. Puerto Rico analytical experiment shipping log -------------56

Table 8. Summary of Proximate analyses of Soybean seeds -------------56

Table 9. Summary of amino acid analyses of Soybean seeds ------------57

Table 10. Summary of fatty acid analyses of Soybean seeds -----------57

Table 11. Summary of isoflavones analyses of Soybean seeds ----------58

Table 12. Summary of lectin analyses of Soybean seeds ---------------58

Table 13. Summary of trypsin inhibitor and urease analyses

for Soybean seeds ---------------------------------------58

Table 14. Analysis of full-fat soybean meal -------------------------59

Table 15. Analysis of toasted meal ----------------------------------60

Table 16. Analysis of retoasted meal --------------------------------60

Table 17. Isoflavone analysis of retoasted meal ---------------------60

Table 18. Expression of CP4 EPSPS and GUS in retoasted Soybean

     meal by multiple SDS extractions using Western blots ----61

Table 19. Comparison of CP4 EPSPS and GUS expression in retoasted

     meal and seeds ------------------------------------------62

Table 20. Expression of CP4 EPSPS and GUS in desalted retoasted

Soybean meal extracts, and seed extracts,using a non-SDS

     buffer --------------------------------------------------63

Table 21. Fatty acid analysis of refined bleached deodorized oil ----63

Table 22. Summary of protein content of refined, bleached,

deodorized oil ------------------------------------------64

 

 

 

 

 

LIST OF FIGURES

 

Fig 1  Lineage of glyphosate-torelant Soybean lines 61-67-1

and 40-3-2  ----------------------------------------------64

 

 

 

 

LIST OF ATTACHMENTS

 

Att.1  Protocol for experiment 92-420-701 -----------------------66

Att.2 Protocol for experiment 92-419-712  -----------------------76

Att.3 Protocol for experiment 92-419-710 ------------------------93

Att.4 Protocol Amendments               ---------------------112

Att.5 Processing report from Texas A &M University -------------121

Att.6 GUS ELISA Validation data Summary  -----------------------143

Att.7 CP4 EPSPS ELISA Validation Data Summary ------------------144

Att.8 Method development and report of the determination of free

and bound isoflavones in soy products --------------------145

Att.9 Method development and report of the lection assay for

soybean seeds and meal -----------------------------------154

Att.10 Statistical analysis of CP4 EPSPS and GUS expression in

 Soybean seeds and leaves --------------------------------161

Att.11 Raw data sheets for quality analyses  -------------------170

Att.12 Statistical analysis of quality data for soybean seeds---211

Att.13 Analysis of trypsin inhibitor levels of retoasted

soybean samples after laboratory steaming ----------------216

Att.14 Determinations of Protein/Amino acids in Soybean Oil

by Amino acid Analysis -Data sheets ----------------------221

Att.15 Calculatiuons for Protein/Amiono acid in Soybean Oil

Recovery  ------------------------------------------------229

 

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