Summary
The goal is to illustrate that the model legume Medicago truncatula can be readily utilized to investigate the regulation of early plant embryogenesis to complement the non-legume Arabidopsis model. Pod morphology is linked to zygotic embryogenesis stages and a protocol to collect embryos using tissue culture is also provided.
Abstract
Early embryogenesis starting from a single cell zygote goes through rapid cell division and morphogenesis, and is morphologically characterized by pre-globular, globular, heart, torpedo and cotyledon stages. This progressive development is under the tight regulation of a complex molecular network. Harvesting sufficient early embryos at a similar stage of development is essential for investigating the cellular and molecular regulation of early embryogenesis. This is not straightforward since early embryogenesis undergoes rapid morphogenesis in a short while e.g. 8 days for Medicago truncatula to reach the early cotyledon stage. Here, we address the issue by two approaches. The first one establishes a linkage between embryo development and pod morphology in helping indicate the stage of the zygotic embryo. This is particularly based on the number of pod spirals and development of the spines. An alternative way to complement the in vivo studies is via culturing leaf explants to produce somatic embryos. The medium includes an unusual hormone combination - an auxin (1-naphthaleneacetic acid), a cytokinin (6-benzylaminopurine), abscisic acid and gibberellic acid. The different stages can be discerned growing out of the callus without dissection.
Introduction
Legumes are the third largest family of higher plants with approximately 20,000 species and the Leguminosae (or Fabaceae) family are second to cereals in area harvested and total production1. Soybean is the third largest cultivated crop. Grain legumes provide about one-third of dietary protein and one-third of vegetable oil for human consumption2. Legumes with their N2 fixing capacity also contribute to sustainable agricultural systems. Medicago truncatula, like soybean, stores protein and oil in the cotyledons of its seeds and is a genetic and genomic legume model with considerable genetic and genomic resources3,4. While M. truncatula has enabled advances in understanding the legume-rhizobium symbiosis4 it has been increasingly employed to study legume seed biology5-7 and embryogenesis8,9. Arabidopsis embryogenesis has been extensively studied10,11 but it is a non-legume and the details of embryogenesis are not identical to Medicago8,10. Zygotic embryogenesis in M. truncatula has interesting features, with a distinctive multicellular hypophysis, an endoployploid suspensor and basal transfer cell8.
Somatic embryogenesis (SE) is commonly used for regenerating plants12. In the legume model M. truncatula, the seed line Jemalong 2HA (2HA) has been developed from the parent Jemalong to have high rates of somatic embryogenesis13. The number of embryos produced has recently been substantively increased by adding both gibberellic acid (GA) and abscisic acid (ABA) to the long established medium14. In this case GA and ABA act synergistically, which is unusual given that GA and ABA usually act antagonistically14. The embryos produced from callus develop on the surface which allows the stage of embryogenesis to be readily determined visually and readily harvested. Having near isogenic lines that are embryogenic (2HA) and non-embryogenic (Jemalong) facilitates the investigation of somatic embryogenesis and having both in vivo and in vitro systems provides different experimental possibilities.
Understanding the cellular and molecular mechanisms of embryo development is essential for understanding seed and plant development. In legumes, as in other dicotyledons, it is the cotyledons of the embryo that store the products that are used for human nutrition. Early embryogenesis involves rapid cell division, and correct embryo patterning. In approximately 8 days after fertilization, the M. truncatula embryo reaches early cotyledon stages. The morphological characterization is not exactly indicated by days after fertilization in glasshouse conditions. Thus, an efficient standardized approach to indicate the stage of developing embryos is valuable in studying the genetic regulation of early zygotic embryogenesis.
In this paper, we provide two standardized protocols to collect developing embryos for biological studies of embryogenesis in the legume model M. truncatula. The first one is to collect zygotic embryos by associating embryogenesis and pod morphology while the second is somatic embryogenesis via culturing leaf explants to provide easily accessed large embryo numbers.
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Protocol
1. Zygotic Embryo Development
- Plant Material
- Grow the Medicago truncatula wild type Jemalong or its near isogenic, highly re-generable genotype Jemalong 2HA13 (known as 2HA) in a glasshouse with a 14 hr photoperiod and 23 °C/19 °C day/night temperature.
- Pierce the surface of the seed coat (with a 23 G needle) prior to sowing the seed so that water is allowed to enter the seed and soak in water overnight. Add enough water to fully cover the seed.
- Sow 3 seeds in each 15 cm diameter pot (total of 10 pots) in potting mixture (coarse sand, perlite, coir-peat (1:1:1) plus 5 g of Osmocote Exact Standard: slow release fertilizer) and transfer to the glasshouse. Retain the healthiest seedling after germination so there is 1 plant per pot and surround by a trellis for the plant stems to climb.
- Grow the Medicago truncatula wild type Jemalong or its near isogenic, highly re-generable genotype Jemalong 2HA13 (known as 2HA) in a glasshouse with a 14 hr photoperiod and 23 °C/19 °C day/night temperature.
- Harvesting Pods
- Collect pods from the wild type Jemalong or 2HA to obtain the appropriate early embryo stages as first described9.
NOTE: The different pod stages are shown in Figure 1 and the corresponding embryo stages are shown in Figure 2. - Check the different pod stages at harvest using criteria as per Table 1. Measure the time elapsed from stage 6 to help separate stages 6 and 7 (Table 2).
- Collect pods from the wild type Jemalong or 2HA to obtain the appropriate early embryo stages as first described9.
- Dissecting ovules from pods and checking the embryo stage
- Isolate ovules from pods using fine forceps alone or a stereo dissecting microscope and forceps as necessary. If required the embryo stage can be quickly checked using a standard compound microscope (Figure 3B).
- Prepare modified Hoyer's solution containing 7.5 g gum Arabic, 100 g chloral hydrate, 5 ml glycerol, 60 ml deionized distilled water. Dissolve by stirring for 3-5 hr or overnight.
- For more refined microscopy clear the dissected ovules in modified Hoyer’s solution15,16. Carefully pick up the intact ovules and place in a small volume of Hoyer’s solution (enough to cover the ovule) at room temperature until clear.
- View the samples with differential interference contrast (DIC) optics. Capture the images iwth a digital camera9 (Figure 2).
- Obtain the most uniform ovules from the central region of the pod and accumulate the required number. An example is shown in Figure 3A. Collect the ovules on ice and store at required temperature (-80 °C for nucleic acids) for further analysis.
NOTE: For details on analysis of histological sections, transmission electron microscopy, and in situ hybridization please see papers8,9.
2. Somatic Embryo Development In Vitro
- Use an M. truncatula cultivar that is able to readily form embryos in culture. For this protocol for leaf explants use 2HA plants13,17 that are 2-4 months old.
- Take leaflets from the latest nearly fully expanded trifoliate leaf of an elongating stem.
- Keep the trifoliate leaves on moist paper toweling in a culture pot to avoid dehydration.
NOTE: Once the trifoliate leaves are harvested they need to be utilized with minimal delay. - Preparation of the culture medium
- Use the P4 10:4:1:5 medium in agar (0.8% w:v) in 9 cm Petri dishes.
NOTE: The P4 10:4:1:5 medium consists of the P4 basal medium18 plus 10 μM 1-naphthaleneacetic acid (NAA), 4 μM 6-benzylaminopurine (BAP), 1 μM abscisic acid (ABA) and 5 μM gibberellic acid (GA). The use of GA + ABA accelerates embryo formation and causes substantive increases in embryo number14. - Make up the the P4 basal medium in 1 L; consisting of major salts (make calcium up separately), minor salts and vitamins (Table 3), chelated iron (make up separately), casamino acids (casein hydrolysate), 30 g sucrose, 8 g agar.
- Store stock solutions of major salts, calcium, casamino acids, minor salts and vitamins at -20 °C in suitable aliquots. Store chelated iron at 4 °C.
- Make up the chelated iron (200x) by dissolving 7.44 g of Na2EDTA•2H2O in approximately 900 ml of deionized distilled water while stirring, then bring the solution to 98-99 °C while adding 1.853 g of FeSO4•7H2O. Finally make up to 1 L when the ingredients are dissolved. Store in amber colored bottles at 4 °C.
- Make up the P4 basal medium with the stock solutions as in Table 4. The hormones in the medium are 10 μM NAA, 4 μM BAP, 1 μM ABA, 5 μM GA (see Table 4 and Specific Materials Table). Add the NAA and BAP prior to autoclaving and add ABA and GA after autoclaving and cooling to about 55 °C, using filter sterilization with a 0.22 µm filter attached to a syringe. Mix well by gentle swirling.
- Adjust the media to pH 5.8 with 1 M KOH prior to autoclaving. Autoclave at 121 °C for 20 min.
- After autoclaving add the filter sterilized ABA and GA, and pour approximately 25 ml of media into sterile 9 cm Petri dishes in a laminar flow hood or biohazard cabinet. Cool and let the agar set with the lid off. Then put on the lid and make sure there is no condensate on the lid. Label as required and store at 4 °C (in a cardboard box to prevent lid condensate).
- Use the P4 10:4:1:5 medium in agar (0.8% w:v) in 9 cm Petri dishes.
- Sterilization of Leaf Tissue
- Work in a UV-sterilized laminar flow hood or biohazard cabinet.
- Place leaves into the mesh ball of a previously autoclaved spring tea infuser.
- Immerse tea infuser containing the leaves in 70% (v:v) ethanol in a culture pot for 30 sec.
NOTE: For all steps in this procedure use 250 ml screw cap polycarbonate culture pots that are autoclaved. - Drain and then transfer and immerse the leaf tissue in 1:8 (v:v) diluted bleach (0.5% chlorine) for 10 min. Swirl gently from time to time.
- Drain the bleach and transfer the tea infuser in a culture pot containing sterile deionized distilled water. Gently swirl and drain excess water. Repeat one more time with a fresh culture pot of sterile deionized distilled water.
- Remove leaves from the tea infuser with sterile forceps to a fresh culture pot of sterile deionized distilled water. Screw on the sterile cap and rinse by inverting and swirling. Leave leaves floating on the sterile water until ready to prepare explants.
- Preparing and Plating Explants
- Using sterile techniques, trim individual sterilized leaflets from the edge with a scalpel, and cut the remaining rectangle into two or three rectangular explants (8-10 x 3-5 mm) with the mid-vein in the center of each explant (Figure 5A).
NOTE: The size of the explant is quite important in initiating the first callusing. Carry out the cutting on sterile lids from take-away food containers. - Plate 6 explants on agar-solidified culture medium in 9 cm diameter Petri dishes (Figure 5B). Plate the explants abaxial side down to maintain usual leaf orientation.
- Seal the Petri dishes with Parafilm (Figure 5C) stretched around the dish. The tissue is now ready for incubation.
- Using sterile techniques, trim individual sterilized leaflets from the edge with a scalpel, and cut the remaining rectangle into two or three rectangular explants (8-10 x 3-5 mm) with the mid-vein in the center of each explant (Figure 5A).
- Tissue Incubation
- Incubate the plates in the dark at 28 °C in a controlled temperature room or growth cabinet throughout the culture period.
NOTE: The explants will initiate dedifferentiation, undergo cell division, proliferation (callus formation) and embryogenesis (differentiation). - Subculture the differentiating explants after 3 weeks on to fresh medium. At this subculture transfer the whole explant using a sterile stainless steel spatula and forceps, in a laminar flow hood or a biohazard cabinet. As callusing occurs and the callus exceeds the size of the largest one in Figure 5C, transfer 50% of the individual callus when subculturing to fresh medium.
- Observe the first embryos in about 4 weeks. While there is a degree of synchrony, collect different embryo stages over a 4-8 week incubation period (Figure 6A,B). Collect under a dissecting microscope and process according to experimental objectives.
- Subculture every 3 weeks and remove embryos periodically so that the explants will continue to produce embryos for 3 months.
- Incubate the plates in the dark at 28 °C in a controlled temperature room or growth cabinet throughout the culture period.
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Representative Results
For zygotic embryogenesis different pod structures corresponding to the different embryo stages are shown in Figure 1A-F while the different embryo stages are shown in Figure 2A-F. By selecting pods at the same stage, samples of ovules that are quite uniform can be obtained (Figure 3A). By using RT-qPCR embryo specific genes can be readily detected and time course studies evaluated9. Some additional dissection will allow for further enrichment of the embryo (Figure 3B). Processing for in situ hybridization strategies can be readily carried out as well as any complementary studies involving light (Figure 4A, B) and electron microscopy8. Of particular value in this system is the distinctiveness of the hypophysis and suspensor (Figure 4A, B).
For somatic embryogenesis the cutting of the initial explant from the individual folioles is shown in Figure 5A. The explants are then placed abaxial side up in close contact with the agar (Figure 5B). After callus formation embryos start to appear after 3-4 weeks and by 7 weeks there are numerous embryos at the cotyledon stage (Figure 5C). By following the plates through between weeks 4 and 7 embryos at discrete stages can be located (Figure 6A, B). The different embryo stages can be readily observed and simply picked off for analysis. This can be quite a quick way to gain certain types of information. For example Figure 7 illustrates how embryos at the early cotyledon stage showed expression of one type of MtOLEOSIN gene but not another. Time courses studies of the gene of interest can then be examined using zygotic or somatic embryos. A particular advantage of the somatic embryo system is that transformation of the callus can be carried out without regenerating a whole plant and gene expression at different stages of embryogenesis visualized using GUS (β-glucuronidase) or fluorescent labels. An example is shown in Figure 6C.
Figure 1: Pod morphology and embryo development stages. Pods at different stages of development with embryos at discrete stages. Stages 2-7 indicated. (A) and (B) are stages 2 and 3 very early and early pre-globular (C) stage 4 early globular (D) stage 5 late globular (E) stage 6 heart and (F) stage 7 late torpedo. Bar is 2 mm. Stage I is flowering (not shown).
Figure 2: Embryo development stages. Cleared embryos at different development stages. (A) stage 3 early pre-globular, (B) stage 4 early globular, (C) stage 5 late globular, (D) stage 6 heart, and (E) stage 7 late torpedo. Bar is 60 µm.
Figure 3: Isolated ovules. (A) Group of ovules with stage 5 embryos. (B) Ovule viewed under standard compound microscope to show embryo which can be excised at “hook” end. Bar is 1 mm (A) and 200 µm (B).
Figure 4: Morphology of the early embryo. (A) Section through very early embryo showing embryo proper (four cells), hypophysis (next four cells), suspensor (next four cells which have large vacuoles) and relationship to ovule tissues. (B) Section through early torpedo stage embryo (E) with prominent suspensor (S). Stained with toluidine blue. Bar is 10 µm (A) and 50 µm (B).
Figure 5: Culturing explants to produce somatic embryos. (A) Trifoliate leaf showing where explants from foliole are taken. (B) Explants plated on agar. (C) Somatic embryos produced from explants after 7 weeks culture. Bar is10 mm
Figure 6: Somatic embryo stages and identifying the location of gene expression using transformation and GUS. (A) Somatic embryos at globular (G), heart (H) and torpedo (T) stages. (B) Somatic embryos at early cotyledon stage. (C) Globular stage somatic embryo showing strong GUS expression for the MtWOX9 gene in embryo proper (E) and decreasing staining in hypophysis (H) and suspensor (S). Bar (A, B) is 100 µm. Bar (C) is 50 µm.
Figure 7: Gene expression duing embryogenesis in vitro using the quantitative polymerase chain reaction (qPCR). Gene expression of OLEOSIN3 (A) and OLEOSIN4 (B) in early cotyledon stage embryos excised from embryos from embryogenic callus. Expression is relative to leaf. Standard errors indicated.
| Stage number | Embryo stage | Pod stage |
| Stage 1 | Flower at anthesis | |
| Stage 2 | Very early pre-globular embryo | Pod with one or two spirals |
| Stage 3 | Early pre-globular embryo | Pod with three complete spirals |
| Stage 4 | Early globular | Pod with five complete spirals and spines not visible |
| Stage 5 | Late globular | Pod with six spirals and immature spines not exceeding pod width |
| Stage 6 | Heart stage | Pod with six spirals and elongated maturing spines exceeding pod width |
| Stage 7 | Torpedo stage | Pod with six spirals, mature thicker longer spines and increased girth |
Table 1: Criteria for harvest of pod stages. Morphological criteria used in identifying pod stages corresponding to defined embryo stages.
| Flower Stage 1 (S1) | Pod Stage 2 (S2) | Pod Stage 3 (S3) | Pod Stage 4 (S4) | Pod Stage 5 (S5) | Pod Stage 6 (S6) | Pod Stage 7 (S7) | |
| Days | 0 | 1.5-2 | 0.5-1 | 0.5 | 0.5-1 | 1 | 1-1.5 |
Table 2. Time course for pod collection. The time from the previous embryo development stage indicated in days.
| Major salts/L | Minor salts/L | Vitamins/L | |||
| mg | mg | mg | |||
| KNO3 | 1875 | MnSO4•H2O | 10 | Myo-Inositol | 100 |
| NH4NO3 | 600 | H3BO3 | 3 | Thiamine HCl | 10 |
| KH2PO4 | 131 | ZnSO4•7H2O | 2 | Nicotinic acid | 1 |
| KCL | 225 | KI | 0.75 | Pyridoxine HCl | 1 |
| MgSO4•7H2O | 225 | Na2MoO4•2H2O | 0.25 | ||
| Calcium separate | CuSO4•5H2O | 0.025 | |||
| CaCl2•2H2O | 300 | CoCl2•6H2O | 0.025 | ||
| Chelated iron separate | |||||
| FeSO4•7H2O | 9.267 | ||||
| Na2EDTA•2H2O | 37.2 | ||||
Table 3: P4 Medium. The components of the P4 medium.
| Component | Amount/L |
| 10 x major salts | 100 ml |
| 100 x calcium | 10 ml |
| 1,000 x minor salts | 1 ml |
| 200 x iron | 5 ml |
| 100 x casamino acids | 10 ml |
| 1,000 x vitamins | 1 ml |
| Sucrose | 30 g |
| Agar | 8 g |
| Hormones | |
| 1,000 µM NAA | 10 ml |
| 1,000 µM BAP | 4 ml |
| 1,000 µM ABA | 1 ml |
| 1,000 µM GA | 5 ml |
Table 4: Making up the P4 medium with hormones. Making up the P4 medium with hormones from stock solutions.
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Discussion
The protocols described are relatively straight forward and allow investigation of legume embryogenesis with all the contemporary cell and molecular techniques. We recognize that there are advantages and disadvantages of both in vivo and in vitro approaches. Both allow more focus on early embryogenesis compared to culture of immature seeds19.
In the case of in vivo studies what is described is predominantly the isolation of the ovule from the pod which is suitable for many embryo studies. It is of course possible to further enrich for embryo tissue by slicing off the “hook” area (Figure 3B). It is difficult to isolate the very earliest stage of embryo development as during disection the embryo is often lost as it is not well attached to the adjacent tissue, making the use of ovules advantageous. Using pod morphology eliminates the tagging of flowers and having a time course regime for each flower. In addition there is potential variability in developmental timing methods unless the environment is very tightly controlled. Optimum plant growth is important so that pod formation does not occur under stress conditions. Become familiar with pod development and slight adjustments can be made to define morphological stages to match specific embryo development stages.
In the case of somatic embryogenesis, it has to be recognized that the embryos are derived asexually and not from a zygote. Also, the nutritional source is different to the zygotic embryo. In case of the somatic embryo, it is the culture medium and surrounding callus, and in the case of the zygotic embryo it is the endosperm. This means the suspensor is much better developed in the case of the zygotic embryo (Figure 4A, B). The particular value of the M. truncatula system is the large number of embryos in response to the unusual four hormone regime. If the large numbers don’t occur then checking the details of making up the hormones and their addition to the medium should be checked. As with any culture methodology sterility of the explant tissue without damage (make small adjustments to timing of ethanol and hypochlorite sterilization times if this is a problem) is important together with the care in maintaining sterility during all preparative steps.
What we have found is that it is valuable to complement in vivo and in vitro processes. An example from our own studies is the investigation of the transcription factor MtSERF1 (SOMATIC EMBRYO RELATED FACTOR 1) which is an ethylene response transcription factor gene. MtSERF1 was first discovered in somatic embryos using RNAi, promoter-GUS fusions and in-situ hybridisations; and then shown to be expressed in zygotic embryogenesis20.
The study of legume embryogenesis is important for human nutrition, and using M. truncatula embryos together with the cellular and molecular technologies now available can enhance this area. Insight can be obtained into how embryo size and hence yield can be optimized together with the required carbohydrate, oil and protein composition. These determinants are largely set in train in early embryogenesis8,9.
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Disclosures
The authors declare that they have no competing interests.
Acknowledgments
This research was supported by the Australian Research Council grant CEO348212 and the University of Newcastle. The assistance of Dr. Sam Zhang is acknowledged.
Materials
| Name | Company | Catalog Number | Comments |
| P4 medium | Sigma-Aldrich | Use Sigma-Aldrich Chemicals or other analytical grade supplier | |
| Major salts | |||
| Minor salts | |||
| Vitamins | |||
| Agar | Bacto Laboratories | 214010 | Bacto agar |
| Plant hormones | |||
| 1-Naphthaleneacetic acid | Sigma-Aldrich | N0640 | Dissolve in small amount of 1 M NaOH |
| Abscisic acid | Sigma-Aldrich | A1049 | Dissolve in small amount of 1 M NaOH |
| 6-Benzylaminopurine | Sigma-Aldrich | B3274 | Dissolve in MQ water with heating and few drops 1N HCl |
| Gibberellic Acid | Sigma-Aldrich | G7645 | Dissolve in small amount of ethanol |
| Equipment | |||
| Stereo dissecting microscope | Leica | MZFLIII | Or similar |
| Light microscope | Zeiss | Axiophot | Or similar, with suitable optics |
| Digital camera | Zeiss | AxioCam HRc | Or similar |
| Sterilising leaves | |||
| 250 mL screw cap polycarbonate container with polypropylene lid | SARSTEDT | 75.9922.519 | Autoclavable |
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