ALFALFA SOMATIC EMBRYO PRODUCTION

Table of Contents

This file describes the procedures that are presently being used in alfalfa tissue culture to produce dry somatic embryos that may be used as artificial seeds.

Select one of the following topics to review the procedure in detail:

Sterilization and Induction

Suspension Culture

Sieving The Suspension

Development and Maturation

Drying

Germination

References

Sterilization and Induction

  1. Harvest petioles from shoots of donor plants. Petioles from young fully expanded leaves are the best; avoid selecting petioles from flowering shoots.
  2. Immerse petioles in 70% ethanol for 30-40 seconds and then in 25% commercial bleach (25 ml bleach + 75 ml water) for 20 minutes. Rinse petioles with three generous changes of sterile water.
  3. Place sterile petioles in Petri dish containing sterile water to keep the material hydrated until further processing.
  4. Cut petioles into 5-10 mm lengths; 8 mm is best, but depending on the experiment this may vary.
  5. Place 10 petioles on a Petri plate containing SH induction medium.
  6. An alternative explant that produces good callus growth is an immature embryo at the torpedo stage of development.
  7. Incubate the plates of induction medium at 25°C, 16 hour photoperiod, 75 µmol m-2s-1 PPFD for 14 to 21 days. Callus at this stage should be friable; the petiole should be completely overgrown with callus. If the callus is hard or not healthy in appearance, it should be discarded. This sometimes happens due to poor donor plant health, or poor choice of petioles.
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Suspension Culture

  1. Transfer petiole-derived callus from induction medium to 125 ml flasks containing 40 ml of B5 suspension medium, at a rate of approximately 1 gram of petiole-derived callus into each flask.
  2. Place flasks on shaker at 25C, 16 hour photoperiod 30 µmoles m-2s-1 PPFD for a period of 7 to 14 days. The suspension after 7 days will contain large clumps of callus, some green, globular somatic embryos, small proembryo clusters, and elongated single cells. The globular embryos developing in suspension are larger thanzygotic embryos and lack a normal suspensor.
  3. Subculturing the suspension may be done provided the large callus clumps are included in the transfer. If the suspension is subcultured too frequently, the small single cells begin to dominate the culture and its ability to form somatic embryos is lost.
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Sieving The Suspension

  1. Sieve the suspension through a 0.5 mm Nitex mesh and then through 0.224 mm mesh in succession. Wash the 0.5 mesh with 150 ml wash solution (consisting of macroelements and sucrose only of B5 suspension medium). Discard callus left on 0.5 mm mesh. An alternative method that will increase embryo numbers is to return the callus on the 0.5 mesh to fresh B5 suspension medium. Usually this will give another equivalent batch of embryos.
  2. Scoop about 1.2 g of cells collected on the 0.224 mm mesh and spread thinly and evenly on another disk of 0.224 mm mesh on top of BOi2Y development medium (this aids in subsequent transfer).
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Development and Maturation

  1. Incubate plates of somatic embryos spread on BOi2Y development medium at 25°C, 16 h photoperiod, and 35 µmol m-2s-1 PPFD for 7-10 days. During this time the somatic embryos appear initially as green dots that enlarge as the embryo develops through the globular, heart, torpedo and cotyledonary stages..
  2. Transfer mesh to BOi2Y maturation I medium. Incubate plates at 25C, 16 h photoperiod, and 75 µmol m-2s-1 PPFD for 10 days. During this period the somatic embryo accumulates dry weight, starch and storage proteins. The ratio of number of developing embryos to medium volume is critical because of competition for nutrients. Best results are often achieved if the embryos are transferred to fresh medium every 2-3 days; this is especially critical if there are 500-1000 embryos on a Petri plate.
  3. Transfer mesh to BOi2Y maturation medium II. Incubate as above for 3-5 days. During this stage, ABA induces the expression of desiccation tolerance in the somatic embryos. Fully mature embryos will lose their green colour and become yellow-brown; however, green embryos can be dried and remain viable.
Click here to view globular heart torpedo and cotylendary stages of embryo development

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Drying

  1. Wash the somatic embryos from the screen with sterile (if desired) de-ionized water. If the dry embryos are to be germinated on nutrient medium, it is essential that they remain sterile during drying.
  2. Transfer the embryos to a sterile piece of germination paper, filter paper or blotting paper to absorb excess moisture.
  3. Spread the loose embryos in a thin layer in a sterile Petri plate. Do not seal the plates; leave open or secure the top loosely with two pieces of labelling tape. Place in air for 2 days. The rate of drying is critical. Large batches of embryos may dry too slowly and lose viability or desiccation tolerance as a result. In other cases, slow drying through a sequence of progressively lower relative humidities aids in the acquisition of desiccation tolerance; presumably because it allows immature embryos to complete their maturation.
  4. Dry somatic embryos weigh approximately 1-2 mg each. They lack the testa and endosperm associated with true seeds, and only have rudimentary cotyledons.
Click here to view a close-up of dry somatic embryos

Click here to view a comparison of alfalfa seeds and dry somatic embryos

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Germination

  1. After drying is complete, store the embryos in a desiccator in a dark, cool place (in cold room) over a saturated K2CO3 solution to control relative humidity. Embryos have remained viable in this state for over two years. Immature embryos or embryos with minimal desiccation tolerance however will lose viability with time in storage.
  2. To germinate, place the dry embryos on agar medium containing 1/2 MS salts and 1% sucrose (if they were kept sterile during drying).
  3. An alternative is to place the dry embryo on moist filter paper as in standard seed germination, or the embryo can be planted directly into a peat plug or soil in the greenhouse. Direct greenhouse planting usually reduces emergence by about 50%, but this varies from batch to batch.
This is the last topic in the methods section.
Click here to view an alfalfa seedling from a dry somatic embryo

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Additional information

References

The following are selected references that will provide more detail about the development of this somatic embryogenesis system and about somatic embryo development in alfalfa
Anandarajah, K. and B.D. McKersie. 1990. Enhanced vigour of dry somatic embryos of Medicago sativa L. with increased sucrose. Plant Science 71:261-266.

Anandarajah, K. and B.D. McKersie. 1990. Manipulating the desiccation tolerance and vigor of dry somatic embryos of Medicago sativa L. with sucrose, heat shock and abscisic acid. Plant Cell Reports. 9:451-455.

Anandarajah, K. and B.D. McKersie. 1992. The influence of plating density, sucrose and light during development on the germination and vigour of Medicago sativa L. somatic embryos after desiccation. Seed Sci. Res. 2:133-140.

Bowley, S.R., G.A. Kielly, K. Anandarajah, B.D. McKersie and T. Senaratna. 1993. Field Evaluation following two cycles of backcross transfer of somatic embryogenesis to commercial alfalfa germplasm. Can. J. Plant Sci. 73:131-137.

Kepczynski, J., B.D. McKersie and D.C.W. Brown. 1992. Requirement of ethylene for growth of callus and somatic embryogenesis in Medicago sativa L. J. Exptl Bot. 43:1199-1202.

Lai, F. -M., and B.D. McKersie. 1993. Effect of nutrition on maturation of alfalfa (Medicago sativa L.) somatic embryos. Plant Sci. 91:87-85

Lai, F. -M., and B.D. McKersie. 1994. Scale-up of somatic embryogenesis in alfalfa (Medicago sativa L.) I Subculture and indirect secondary somatic embryogenesis. Plant Cell Tissue Organ Culture 37:151-158.

Lai, F.-M. and B.D. McKersie. 1994. Regulation of starch accumulation in alfalfa (Medicago sativa L.) somatic embryos. Plant Sci. 100:211-219.

Lai, F.-M. and B.D. McKersie. 1994. Regulation of storage protein synthesis by nitrogen and sulfur nutrients in alfalfa (Medicago sativa L.) somatic embryos. Plant Science. 103:209-221.

Lai, F.-M., C.G. Lecouteax, and B.D. McKersie. 1995. Germination and Conversion of Alfalfa (Medicago sativa L.) Seeds and Desiccated Somatic Embryos. I. Mobilization of Storage Reserves. J. Plant Physiol. 145:507-513.

Lai, F. -M., T. Senaratna and B.D. McKersie. 1992. Glutamine enhances storage protein synthesis in Medicago sativa L. somatic embryos. Plant Sci. 87:69-77.

Lecouteux, C., F.-M. Lai, and B.D. McKersie. 1993. Maturation of alfalfa (Medicago sativa L.) somatic embryos by absisic acid, sucrose and chilling stress. Plant Sci. 94:207-213.

Leprince, O., B.D. McKersie, and G.A.F. Hendry. 1993. The mechanisms of desiccation tolerance in developing seeds. Seed Sci. Res. 3:231-246.

McKersie, B.D. 1995. Somatic embryogenesis in alfalfa. A model for the development of dry artificial seed technology. IN Seed Development and Germination. J Kigel and G Galili (eds). Marcel Dekker, NY. Chap. 31: pp.833-846.

McKersie, B.D. and S.R. Bowley. 1993. Synthetic Seeds of Alfalfa. IN: K. Redenbaugh (ed.) Synseeds: Application of Synthetic Seeds to Crop Improvement. CRC Press. Chapter 14: 231-255.

McKersie, B.D., T. Senaratna and S.R. Bowley. 1990. Drying Somatic Embryos for Use as Artificial Seed. Proc. of the Plant Growth Regulator Society. Aug. 5-9, St. Paul, MN. 17:199-207.

McKersie, B.D., T. Senaratna, S.R. Bowley, D.C.W. Brown and J.D. Bewley. 1989. Application of artificial seed technology in the production of hybrid alfalfa (Medicago sativa L.). In Vitro Cell Develop. Biol. 25:1183-1188.

McKersie, B.D., S. Van Acker, and F. Lai. 1994. Maturation and Desiccation of Somatic Embryos IN: Biotechnology in Agriculture and Forestry Vol. 30. YPS Bajaj (ed.) Springer-Verlag pp.152-169.

Senaratna, T., B.D. McKersie and S.R. Bowley. 1989. Desiccation tolerance of alfalfa (Medicago sativa L.) somatic embryos. Influence of Abscisic acid, stress pretreatments and drying rates. Plant Science 65:253-259.

Senaratna, T., B.D. McKersie and S.R. Bowley. 1990. Artificial seeds of alfalfa: induction of desiccation tolerance in somatic embryos. In Vitro Cell and Develop. Biol. 26:85-90.

Shetty, K. and B.D.McKersie. 1993. Proline, thioproline and potassium mediated stimulation of somatic embryogenesis in alfalfa (Medicago sativa L). Plant Science 88: 185193.

Van Acker, S. and B.D. McKersie. 1994. Desiccation tolerance in somatic embryos. IN: Biotechnological Applications of Plant Culture. P.D. Shargool and T.T. Ngo eds. CRC Press pp.129-150.

Theses on alfalfa somatic embryogenesis at Crop Science, University of Guelph

Karin Schneider; M.Sc.: The effect of polyamines and their biosynthetic precursors on somatic embryogenesis in Medicago sativa L. 1989

Susan Van Acker; M.Sc. A comparison of desiccation tolerance in zygotic and somatic embryos of alfalfa (Medicago sativa). 1992.

Yangling Zhang; M.Sc. Induction and maturation of alfalfa somatic embryos. 1992.

Barron Mertens. M.Sc. Induction and utilization of somatic embryos of alfalfa (Medicago sativa). 1993.

Fang-ming Lai. Ph.D. Maturation and conversion of alfalfa (Medicago sativa) somatic embryos. 1994.

Kasia Napierala. M.Sc. Induction of somatic embryogenesis in suspension cultures of Medicago sativa. 1995.

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Media Composition

SH Induction Medium
                          
Components are given in mg/L.              

Macronutrients                
NH4H2PO4          300        
KNO3            2500       
CaCl2 2H2O       200        
MgSO4 7H2O       400        
K2SO4           4350       

Micronutrients                
KI                 1          
H3BO3              5          
MnSO4 H2O         10         
ZnSO4 7H2O         1          
Na2MoO4 2H2O       0.1        
CuSo4 5H2O         0.2        
CoCl2 6H2O         0.1        
Na2 EDTA          20         
FeSO4 7H2O        15         

Amino Acids                   
Proline          288        
Thioproline       53         

Vitamins                      
Myo-inositol      200        
Nictonic acid       5          
Pyridoxine HCl      0.5        
Thiamine HCL        5          

Other                         
2,4-D               1          
Kinetin             0.2        
Sucrose         30000      
Agar             6000      
pH      5.8
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B5 Suspension Medium
Components are given in mg/L 
                            
Macronutrients                 
KNO3              2500        
CaCl2 2H2O         150         
MgSO4 7H2O         250         
(NH4)2 SO4         134         
NaH2PO4 H2O        150         

Micronutrients                 
KI                 0.75        
H3BO3              3           
MnSO4 H2O          10          
ZnSO4 7H2O         2           
Na2MoO4 2H2O       0.25        
CuSo4 5H2O         0.025       
CoCl2 6H2O         0.025       
Na Fe EDTA         43          

Vitamins                       
Myo-inositol     100         
Nictonic acid      1           
Pyridoxine HCl     1           
Thiamine HCL       10          

Other                          
2,4-D              1           
Sucrose        20000       
pH                 5.5         
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BOi2Y development medium
Components are given in mg/L 
Macronutrients                                        
NH4NO3                         1000                  
KCl                              65                    
KNO3                           1000                  
Ca(NO3)2                        347                   
MgSO4 7H2O                       35                    
KH2PO4                          300                   

Micronutrients                                        
KI                              0.8                   
H3BO3                           1.6                   
MnSO4 H2O                       4.4                   
ZnSO4 7H2O                      1.5                   
Na Fe EDTA                     32                    

Amino Acids                                           
Glycine                         2                     

Vitamins                                              
Myo-inositol                  100                   
Nictonic acid                   0.5                   
Pyridoxine HCl                  0.1                   
Thiamine HCL                    0.1                   

Other                                                 
Sucrose                       50000                 
Yeast Extract                  2000                 
Agar                           6000                 
pH                              5.8                   
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BOi2Y Maturation I medium
Components are given in mg/L
 

Macronutrients
NH4NO3 1000
KCl 65
KNO3 1000
Ca(NO3)2 347
MgSO4 7H2O 35
KH2PO4 300
K2 SO4 4350

Micronutrients
KI 0.8
H3BO3 1.6
MnSO4 H2O 4.4
ZnSO4 7H2O 1.5
Na Fe EDTA 32

Amino Acids
Glycine 2

Vitamins
Myo-inositol 100
Nictonic acid 0.5
Pyridoxine HCl 0.1
Thiamine HCL 0.1

Other
Sucrose 50000
Yeast Extract 2000
Agar 6000
pH 5.8

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BOi2Y Maturation II medium
Components are given in mg/L 
Macronutrients                     
NH4NO3             1000            
KCl                  65              
KNO3               1000            
Ca(NO3)2            347             
MgSO4 7H2O           35              
KH2PO4              300             

Micronutrients                     
KI                  0.8             
H3BO3               1.6             
MnSO4 H2O           4.4             
ZnSO4 7H2O          1.5             
Na Fe EDTA         32              

Amino Acids                        
Glycine            2               

Vitamins                           
Myo-inositol       100             
Nictonic acid        0.5             
Pyridoxine HCl       0.1             
Thiamine HCL         0.1             

Other                              
ABA                    5.3             
Sucrose            50000           
Yeast Extract       2000            
Agar                6000            
pH                     5.8             
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Composition of Media used for alfalfa somatic embryogenesis

SH induction medium

B5 suspension medium

Development medium

Maturation phase I medium
 

Maturation phase II medium