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 25C, 16 hour photoperiod, 75 mol m2s-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. Click here to view picture of callus Go to next topic Return to Table of Contents

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 petiolederived callus into each flask. 2. Place flasks on shaker at 25C, 16 hour photoperiod 30 moles 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. veloping 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. Click here to view shaker and suspension cultures Go to next topic Return to Table of Contents

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).

Click here to see the setup for sieving Go to next topic Return to Table of Contents

Development and Maturation

1. Incubate plates of somatic embryos spread on BOi2Y development medium at 25C, 16 h photoperiod, and 35 mol 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 mol 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 Go to next topic Return to Table of Contents

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 Go to next topic Return to Table of Contents

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 Click here to review list of References Return to Table of Contents 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. 43:11991202. 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.) SpringerVerlag 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. Return to Table of Contents

Media Composition
SH Induction Medium

Components are given in mg/L. Macronutrients NH4H2PO4 KNO3 CaCl2 2H2O MgSO4 7H2O K2SO4 Micronutrients KI H3BO3 MnSO4 H2O ZnSO4 7H2O Na2MoO4 2H2O CuSo4 5H2O CoCl2 6H2O Na2 EDTA FeSO4 7H2O Amino Acids Proline Thioproline Vitamins Myo-inositol Nictonic acid Pyridoxine HCl Thiamine HCL Other 2,4-D Kinetin Sucrose Agar pH 5.8 300 2500 200 400 4350

1 5 10 1 0.1 0.2 0.1 20 15

288 53

200 5 0.5 5

1 0.2 30000 6000

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B5 Suspension Medium

Components are given in mg/L

Macronutrients KNO3 CaCl2 2H2O MgSO4 7H2O (NH4)2 SO4 NaH2PO4 H2O Micronutrients KI H3BO3 MnSO4 H2O ZnSO4 7H2O Na2MoO4 2H2O CuSo4 5H2O CoCl2 6H2O Na Fe EDTA Vitamins Myo-inositol Nictonic acid Pyridoxine HCl Thiamine HCL Other 2,4-D Sucrose pH 2500 150 250 134 150

0.75 3 10 2 0.25 0.025 0.025 43

100 1 1 10

1 20000 5.5

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BOi2Y development medium

Components are given in mg/L

Macronutrients NH4NO3 KCl KNO3 Ca(NO3)2 MgSO4 7H2O KH2PO4 Micronutrients KI H3BO3 MnSO4 H2O ZnSO4 7H2O 1000 65 1000 347 35 300

0.8 1.6 4.4 1.5

Na Fe EDTA Amino Acids Glycine Vitamins Myo-inositol Nictonic acid Pyridoxine HCl Thiamine HCL Other Sucrose Yeast Extract Agar pH

32

2 100 0.5 0.1 0.1

50000 2000 6000 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 Return to Table of Contents View another media composition

BOi2Y Maturation II medium

Components are given in mg/L

Macronutrients NH4NO3 KCl KNO3 Ca(NO3)2 MgSO4 7H2O KH2PO4 Micronutrients KI H3BO3 MnSO4 H2O ZnSO4 7H2O Na Fe EDTA Amino Acids Glycine Vitamins Myo-inositol Nictonic acid Pyridoxine HCl Thiamine HCL Other ABA Sucrose Yeast Extract Agar pH 1000 65 1000 347 35 300

0.8 1.6 4.4 1.5 32 2

100 0.5 0.1 0.1

5.3 50000 2000 6000 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