SOMATIC EMBRYOGENESIS:

THE DEVELOPMENT OF SYNTHETIC SEEDS

TABLE OF CONTENTS

Objective

Overview of Dry Synthetic Seed Production

Induction of Somatic Embryo Formation

Suspension Culture

Embryo Development and Maturation

Desiccation Tolerance

Germination and Seedling Vigour

Applications

References for this review article

Detailed procedures and media formulations
 

Objective

Our basic objective in developing synthetic seed technology is to develop a clonal propagation system that will enable the vegetative propagule to be stored for long periods of time and at the same time enable multiplication of the plant. This review is intended to give an overview of our progress in developing this technology. The experimental system that we are using is alfalfa (Medicago sativa L.) that is grown extensively in North America as a hay and silage crop for the dairy and beef industries. There are economic objectives in developing this technology in alfalfa which have been discussed in detail elsewhere (McKersie et al., 1989; McKersie and Bowley, 1993), but the technology that is being developed with this experimental system can also be applied to many other plant commodities. For reviews of the progress in other crops, the reader is referred to Redenbaugh (1993) and Bajaj (1994).

Synthetic or artificial seeds have been defined as somatic embryos engineered for use in the commercial propagation of plants (Gray and Purohit, 1991; Redenbaugh, 1993). Various forms of synthetic seeds have been envisioned over time. The first were simply hydrated somatic embryos produced from vegetative cells in plant tissue culture. These had the particular advantage of enabling rapid clonal multiplication of some plants, but the labour and therefore cost was high and the propagules were very delicate. This was partially overcome with the development of alginate capsules that encapsulated a single embryo in a protective coating enabling mechanised handling. These hydrated encapsulated embryos could only be stored using low temperatures for a few weeks (Redenbaugh et al., 1986; Fujii et al., 1989; 1992). The capability of prolonged storage was achieved when the somatic embryos could be dried to moisture contents less than 20% (McKersie et al., 1989). This is the current state of the technology. In the future, we will be developing coatings for these embryos that will provide protection and improve the ease of handling these propagules.

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Overview of dry synthetic seed production

The basic assumption that we made early in our research was that the natural development of a zygotic embryo was close to optimal and therefore if we wished to produce a synthetic version of a seed, we should force the somatic embryo to follow the same developmental program as the zygotic embryo in the seed. Figure 1 shows some of the key critical stages in this development. After pollination, a zygotic embryo of a dicotyledonous species develops through a series of morphological stages termed globular, heart and torpedo. Cotyledons develop and expand as the storage reserves of protein, starch and/or oil are deposited. At some time before the embryo achieves its maximum weight, it acquires the ability to tolerate drying. Then, the seed's vascular connections to the maternal plant are severed, it stops importing nutrients and it begins to lose water. Seeds of most crop plants can survive drying and can be stored for several years. Once they are hydrated, germination commences culminating in the emergence of a radicle and then the mobilization of the storage reserves by the seedling. The details of this complex process vary from species to species and the reader is referred elsewhere for a more thorough discussion (Bewley and Black, 1994).


Figure 1. Schematic representation of the major stages in zygotic embryo development from pollination to germination.

When we began our research in the mid 1980's, the plant tissue culture systems used to produce synthetic seeds mimicked only the very first part of zygotic embryo development. The embryos did not accumulate reserves, they did not develop tolerance of drying, and they could not be stored. They simply precociously germinated into a seedling - a process that could be slowed by low temperature but not stopped. A diagrammatic representation of the plant tissue culture that we subsequently developed in alfalfa is shown in figure 2.

Figure 2. The alfalfa tissue culture system used to produce synthetic seeds

Briefly, petiole explants from greenhouse-grown plants are surface sterilized and cultured on SH medium (Schenk and Hildebrandt, 1972) containing 2,4-D, kinetin and many other nutrients. In simple terms, the 2,4-D activates the cell cycle of many cells in the petiole - those in the vascular cambium develop into a callus, whereas some subepidermal cells develop into a somatic embryo. The initial somatic embryos, which are only small dense cell clusters at this stage, are embedded in a callus mass of non-differentiated cells. To liberate these proembryonic structures, and to stimulate the formation of more embryos, the callus is dispersed in a liquid medium to form a suspension culture. Usually this medium is a modified B5 medium (Gamborg et al., 1968) containing 2,4-D but not kinetin, but other salt combinations have been effective as well. After 7 days, the suspension is sieved and a 224-500 mm fraction transferred to solid BOi2Y medium lacking 2,4-D (Bingham et al., 1975). On this medium the embryos develop through morphological stages that appear to be globular, heart and torpedo (Figure 3). Once the majority of embryos reach the torpedo stage (7-10 days after sieving) they are transferred to an enriched BOi2Y medium containing a high level of sucrose, nitrogen and sulphur to prevent precocious germination (Anandarajah and McKersie, 1990a) and to enable deposition of storage reserves (Lai and McKersie, 1994b). During this period, called maturation phase 1 in our experiments, the embryos rapidly accumulate fresh and dry weight, reaching 1-2 mg dry weight per embryo. To induce the acquisition of desiccation tolerance, called maturation phase II in our experiments, the somatic embryos are placed on a modified BOi2Y medium containing abscisic acid (ABA) for 3 days (Senaratna et al., 1989; 1990). Then they are removed from the medium, washed to remove sugar and other nutrients, and dried. The standard method of drying is to place the somatic embryos in a sealed chamber over a saturated salt solution designed to give specific relative humidifies (Senaratna et al., 1989; 1990). Daily for one week, the embryos are transferred to a progressively lower relative humidity chamber and finally are dried at ambient conditions. At this stage, the embryos have reached approximately 15% moisture and can be stored for a year or more with good viability. Subsequently, the embryos are germinated on moist filter paper (Lai et al., 1995). Each of these stages will be discussed in more detail in the following sections.

Figure 3. The morphological stages of somatic embryo development in alfalfa (Medicago sativa L.)

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Induction of Somatic Embryo Formation

Induction of somatic embryogenesis requires a change in the fate of a vegetative (somatic) cell. In most cases, an inductive treatment is required to initiate cell division and establish a new polarity in the somatic cell. In alfalfa, the inductive treatment is most commonly 2,4-D but other auxins such as 2,4,5-T are effective. The auxin response is quite complex (McKersie and Brown, 1996). Some auxins such as IAA and IBA are ineffective, and still others will stimulate the formation of embryos and callus but not somatic embryos (Table 1). Inorganic components in the medium such as potassium, and organic components such as proline can modulate the embryogenesis or callus response, but they can not replace auxin (Shetty and McKersie, 1993).
Table 1: The effects of auxin in the induction medium on callus and somatic embryo formation in Medicago sativa clone RL 34.

Somatic embryos were formed on SH medium containing auxin and then transferred to growth regulator-free B0i2Y development medium. Callus weight is the weight at the time of transfer from SH to B0i2Y media. Embryos were counted at 14 days on B0i2Y
Auxin
Callus/petiole (mg)
Embryos/petiole
Non-responsive:
indole-3-acetic acid (IAA)
0
0
indole-3-butyric acid (IBA)
0
0
Callus-Forming:
4-chlorophenoxyacetic acid (CPA)
48
0
naptholeneacetic acid (NAA)
37
0
Somatic embryo and callus-forming:
2,4-dichlorophenoxyacetic acid (2,4-D)
88
51
2,4,5-trichlorophenoxyacetic acid (2,4,5-T)
53
26

A very important complication is plant genetics. As in many other plants, relatively few alfalfa plants from a population will respond in culture to 2,4-D and produce a somatic embryo (for a review of genetic effects in plant tissue cultures see Henry et al. (1994)). Somatic embryogenesis is a genetic trait that is sexually heritable in alfalfa and encoded by two independent, dominant genes (Reisch and Bingham, 1980; Hernandez-Fernandez and Christie, 1989; Kielly and Bowley, 1992). One plant from the cultivar Rangelander called RL34 is highly embryogenic and genetically stable during in vitro culture (McKersie et al., 1989). However, those are its only redeeming characteristics because it is susceptible to most diseases, lacks vigour and has low seed yields. Screening for regeneration ability showed that few plants adapted to Northeastern North America were capable of forming somatic embryos in culture (Atanassov and Brown, 1984). Therefore, a recurrent selection plant breeding program was established to transfer these two genes for embryogenesis from RL34 into adapted germplasm. RL34 was crossed with a single plant selected from a commercial cultivar adapted to our region and the progeny were screened for the ability to form somatic embryos. Selected plants in the F1 were then crossed with a second plant selected from another commercial cultivar to produce BC1 seed. This modified version of the backcross was used to avoid inbreeding depression which is common in alfalfa. The BC1 generation was selected for embryogenesis and the crossing and screening repeated to the BC4 generation. This approach was used to produce 7 distinctly different populations that contained the genes from somatic embryogenesis at a relatively high frequency. Field evaluation of these plants has identified individuals with good forage and seed yields and with good combining ability with other genotypes (Kielly and Bowley, 1992; Bowley et al., 1993).

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Suspension Culture

In suspension, proembryogenic cell clusters form and can be separated from the single cells and the larger clumps of callus by sequentially sieving through nylon membranes of 500 and 224 mm pore size. The suspension, however, can not be maintained in this embryogenic state. If the 224-500 mm embryogenic fraction is placed back into suspension, the single cell population increases, the embryogenic cell clusters disperse and they become a progressively smaller proportion of the culture (Lai and McKersie, 1994c). Identifying techniques to stabilize these embryogenic cultures is critical for the scale-up of this technology.

The organic and inorganic components of the liquid suspension culture medium have been precisely defined (McKersie and Brown, 1996). Also, physical factors including inoculant density (weight of callus added to 40 ml of liquid suspension) are also critical and show precise optimal values (McKersie and Brown, 1996).

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Embryo Development and Maturation

To allow development of somatic embryos from a sieved suspension culture, the fraction collected on the 224 mm screen is spread in a thin layer on BOi2Y development medium lacking growth regulators. At approximately 4 days after sieving, green globular somatic embryos appear, enlarge, and develop through the heart stage into torpedo shaped embryos by days 7 - 10. Once at the torpedo stage, somatic embryos in culture typically germinate precociously into a seedling (Anandarajah and McKersie, 1990b). If the embryos germinate, there is insufficient time to accumulate storage reserves or acquire desiccation tolerance, and the embryo does not become quiescent. Imposing an osmotic stress on the embryos by including 5-6% sucrose in the medium, instead of the normal 3%, prevents precocious germination and maintains embryogenic development.

Subsequently the embryos accumulate storage proteins and carbohydrates (Lai et al., 1992; Lai and McKersie, 1993; 1994ab). Storage protein accumulation is facilitated by the inclusion of an organic nitrogen source, glutamine, and an inorganic sulphur source, potassium sulphate.

The final stage of maturation is achieved by transferring the embryos to a medium containing ABA. This growth regulator apparently triggers a process leading to the expression of desiccation tolerance (Senaratna et al., 1990). Other physical treatments including cold, heat, osmotic or nutrient stress can elicit a similar response, presumably because they stimulate the endogenous synthesis of ABA (McKersie et al., 1990).

On normal development medium, the somatic embryos fail to accumulate the 2S and 11S storage proteins found in zygotic embryos. The cause is primarily nutrient deprivation. To understand this, recognize that the embryogenic potential of this tissue culture system is exceedingly high. As many as 900 somatic embryos may be developing on a single Petri plate containing 25-30 ml of medium. There is simply insufficient N and S in the standard BOi2Y medium to support 900 embryos growing to over 1 mg dry weight at 40% protein. Consequently, the maturation phase 1 medium contains a large amount of both glutamine and sulphate. If the nutrient requirements of the embryo are satisfied, by either addition of these nutrients (Lai et al., 1992; Lai and McKersie, 1994ab) or by the frequent transfer to fresh media (Lai and McKersie, 1993), the somatic embryos will accumulate storage proteins, albeit at approximately 50% of the amount found in a zygotic embryo. The proportion of the storage reserves in the embryo that accumulate as starch or as protein is regulated by the carbon:nitrogen ratio in the medium (Lai and McKersie, 1994a). As might be expected, a high carbon:nitrogen ratio increases starch relative to protein accumulation.

Glutamine plays a regulatory as well as a nutritive role in somatic embryo maturation. When included in the medium, glutamine is converted to 5-oxoproline by autoclaving (Lai et al., 1992; Lai and McKersie, 1994b). Other amino acids cannot substitute for glutamine, but 5-oxoproline can mimic the glutamine effect. Whether 5-oxoproline is simply a convenient form of N for transport, or whether 5-oxoproline has a regulating role in metabolism is unknown.

Because nutrition plays such a critical role in embryo maturation, physical factors that influence nutrition such as plating density (the number of somatic embryos per Petri plate) must also be considered (Anandarajah and McKersie, 1990ab; 1992). Light intensity (optimal at 75 mmol m-2s-1) is also critical at this stage of the culture process (Anandarajah and McKersie, 1992). Therefore, practices, such as stacking Petri plates, which create variable physical environments should be avoided.

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Desiccation tolerance

Desiccation tolerance is a characteristic of somatic embryos that must be induced and therefore requires a pretreatment with ABA or stress to elicit the desired response. The type of pretreatment used, the duration for which it is applied and the stage of embryo that is treated are critical factors (Senaratna et al., 1989; 1990; Lecouteux et al., 1993). In our hands, three days on a medium containing 20 mm ABA is sufficient to induce tolerance, but chilling requires almost 3 weeks (Table 2). The rate of drying has secondary importance. If the embryos are immature, slow drying over one week is optimal, but if there are large numbers of fully mature embryos, rapid drying in a laminar flow bench is preferable (Senaratna et al., 1989; 1990).
Table 2. Survival (%) of alfalfa somatic embryos given different inductive treatments for different durations after desiccation to 15% moisture. Data from Lecouteux et al. (1993). 
Days
ABA
Sucrose
Chilling
3
99
99
36
12
95
47
56
19
10
0
87

Desiccation tolerance is a quantitative characteristic not a qualitative one and embryos can have varying degrees of tolerance. This is especially true among different species, but is also evident with different inductive treatments (McKersie et al., 1994). Drying the somatic embryos promotes metabolic changes particularly in sugar metabolism (Horobowicz et al., 1995). Before drying, the alfalfa somatic embryos contained high amounts of sucrose, glucose and fructose (Table 3). After drying, the amounts of glucose and fructose were very low, and stachyose had increased proportionately. Raffinose was also present. Given the importance of these sugars in desiccation tolerance in seeds (Leprince et al., 1993), these changes might be quite significant.
Table 3. Concentration of soluble sugars (mg/g dw) in alfalfa somatic embryos before and after fast drying. Data from Horobowicz et al. (1995).
Sugar
Before
After
Fructose
13.5
0.4
Glucose
5.0
0.4
Sucrose
84.5
48.0
Raffinose
14.7
9.2
Stachyose
16.6
10.9

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Germination and Seedling Vigour

Dry somatic embryos lack the vigour normally associated with seedlings from normal seeds. The reason for this is not obvious, although there are several possibilities. The dry somatic embryos may lack storage proteins or some other critical component required after germination by the seedling . Storage protein levels have been increased with some improvement in vigour (Lai et al., 1992; Lai and McKersie, 1993; 1994ab). Somatic embryos store starch and sucrose, whereas seeds store a hemicellulose in the cell walls of the endosperm called galactomannan (McCleary and Matheson, 1974; 1976). We thought initially that perhaps this carbohydrate was not available to the germinating somatic embryo, but this is not correct because the starch and sucrose reserves in the dry somatic embryos are rapidly depleted after imbibition (Lai et al., 1995). In some instances, there may be injury to the somatic embryo if the proper drying procedure is not used (Lecouteux et al., 1993).

In comparison to seeds, water uptake during imbibition of dry somatic embryos is quite rapid. Because the somatic embryo lacks a testa, there is no barrier to water uptake. Imbibitional injury is therefore another possible cause of poor seedling vigour in the synthetic seeds. Prehydration of the somatic embryos in a moist atmosphere (100% relative humidity generated in a sealed chamber over water) for 24 h improves the vigour of some but not all batches of somatic embryos.

The most probable cause of poor seedling vigour is abnormal apical or plumule development. In germinating somatic embryos, we usually refer to two separate processes; germination is defined as the process culminating in the emergence of the radicle, whereas conversion is defined as the emergence of the radicle and the shoot (Lai et al., 1995). Rarely does a somatic embryo form a shoot without roots, but root emergence without plumule emergence is common. Therefore, although germination rates of seeds and zygotic embryos are quite similar (Figure 4), conversion rates are much lower in dry somatic embryos.

Figure 4. Germination and conversion frequencies (%) of dry alfalfa somatic embryos and seeds. Data from Lai et al. (1995). 

Although the storage protein and starch reserves are rapidly hydrolysed following inhibition, the germination of dry somatic embryos on a nutrient medium greatly improves seedling development (Lai and McKersie, 1995). A nutrient medium of 1% sucrose or ½ strength MS salts added individually is not dramatically better than water. However, when the dry somatic embryos were germinated a ½ strength MS salts with 1% sucrose, the fresh weight of the seedlings from somatic embryos was 2-2½ times that of those germinated on water or MS salts or 1% sucrose individually (Table 4).
Table 4. Vigour of seedlings from dry somatic embryos germinated on nutrient media containing sucrose and MS salts.

Data from Lai and McKersie (1995).
Medium
Seedling fresh weight (mg)
Water
30
1% Sucrose
35
½ MS salts 
40
1% Sucrose and ½ MS salts 
81

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Applications

By combining the benefits of a vegetative propagation system with the capability of long-term storage and with clonal multiplication, synthetic seeds have many diverse applications in agriculture (Gray and Purohit, 1991; Redenbaugh et al., 1991; Redenbaugh, 1993). The application of this particular tissue culture system for the production of hybrids in alfalfa has been discussed in detail elsewhere (McKersie et al., 1989; McKersie and Bowley, 1993). A similar system could be developed in other crops although the specific commercial applications will undoubtedly differ. In plant breeding programs of cross pollinating species, this technology might provide an alternative way to store germplasm instead of a field or greenhouse nursery. Synthetic seeds also offer the opportunity to store genetically heterozygous plants or other plants with unique gene combination that can not be maintained by conventional seed production due to genetic recombination that occurs at each generation of seed increase.

Many plants are sterile and do not set seed. Somatic embryogenesis would in many instances be preferable to making cuttings as a means to propagate these plants. Other plants, particularly some tropical species, produce recalcitrant seeds that can not be dried. Therefore, long term storage of these species in seed banks is not currently possible. Somatic embryos might be an alternative that would enable long-term storage of these species, but the success of the somatic embryo approach will depend on the cause of the seed's recalcitrance. If its inability to tolerate drying is due to a genetic trait that makes the seed unable to respond to the inductive signals that trigger the expression of desiccation tolerance (e.g. they lack ABA receptors), then the synthetic seed approach will not work. However, if the inability is due to a genetic trait that prevents the plant from producing the required signal (e.g. block in ABA synthesis), then the synthetic seed approach might work.

Synthetic seed technology may also have application in the short term storage of propagules to enable the synchronized planting of commodities in a greenhouse. One of the limitations of current micropropagation procedures is that the tissue culture facility and the greenhouse production facility must be physically linked; as well their production must be synchronized to meet peak market demands. Synthetic seeds could be a means to uncouple this linkage enabling the micropropagation to be done year round at a site distance from the production facility.

As stated earlier, the exact application of synthetic seeds will vary from species to species. In self-pollinated crops that currently have good seed production systems, synthetic seeds will not have any practical applications, but in cross pollinating species, especially those where seed production is difficult and expensive, synthetic seeds offer many advantages and opportunities.

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References

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