Analogs of botanic seed

A novel analog to natural botanic seed, together with a process for producing this analog are provided. Plant meristematic tissue capable of producing an entire plant body, and generally from somatic sources, is encapsulated in a gel which protects the tissue during development, while allowing the meristem to grow and mature as normal plants. The chemical and mechanical characteristics of the gel can be varied to suit any plant or method of handling, while allowing inclusion of growth and development enhancing substances.

BACKGROUND OF THE INVENTION 
This invention relates to production of analogs of botanic seed wherein the 
genotype may be identical to the parental strain. 
The conventional techniques of crop improvement in agriculture involve a 
search for strains of plants which exhibit new and useful characteristics, 
or refine and improve on existing ones. The search has evolved from mere 
selection of a desirable parent plant to hybridization between parental 
strains which each exhibit desirable characteristics to, finally, 
crossbreeding between homozygous strains such that identical F.sub.1 
progeny will be produced in each subsequent crossbreeding. 
The conventional methods of maintaining genetic identity are well known and 
described in the literature. See, e.g. R. W. Allard "Principles of Plant 
Breeding," (John Wiley and Sons, Inc., 1960). The maintenance of purebred 
strains and the repeated crossbreeding to obtain F.sub.1 progeny are time 
consuming and labor intensive. 
An additional limitation on the sexual reproduction of parental strains has 
been the low seed productivity per plant. This often results from low 
vigour which is manifested by heavily inbred strains. Finally, only a 
relatively limited number of purebred lines may be produced, and this 
results in a decreased pool of genetic characteristics available for 
selection. 
It has been recognized that some of these difficulties may be overcome by 
vegetative propagation of the parental strain. See: W. C. Anderson and J. 
B. Carstens, "Tissue Culture Propagation of Broccoli, Brassica oleracea 
(Italica Group), for use in F.sub.1 Hybrid Seed Production," J. Amer. Soc. 
Hort. Sci., 102(1), pp. 69-73 (1977). This technique avoids the problem of 
the change in parental strain genetic characteristics through sexual 
reproduction. However, the sexual cross to produce F.sub.1 seed does not 
guarantee uniform progeny where there is chromosomal trait segregation in 
the parental strains. 
It has been suggested that a desirable species may be propagated 
vegetatively, and the somatic embryos or rooted plantlets produced thereby 
transferred to the field. However, this technique involves skilled labor 
in tissue culture laboratories, a transfer to a hot-house or nursery, and 
upon attaining sufficient acclimatization, a transplantation to the field. 
This procedure is costly and time consuming in comparison with the 
traditional methods of seeding. 
To overcome some of these difficulties, the technique of fluid drilling has 
been developed. Fluid drilling methods have been used with pregerminated 
seed, e.g., D. Gray, "Comparison of Fluid Drilling and Conventional 
Establishment Techniques on Seedling Emergence and Crop Uniformity in 
Lettuce," J. Hort. Science. 53:23-30 (1978), and it has been suggested 
that fluid drilling may be adaptable to transfer somatic embryos directly 
to the field. D. A. Evans and W. R. Sharp, "Application of Tissue Culture 
Technology in the Agricultural Industry," in Application of Plant Cell and 
Tissue Culture to Agriculture and Industry, D. T. Tomes et al., eds. 
(University of Guelph Press, pp. 212-13, 1982). However, fluid drilling 
technology is capital intensive and requires the purchase of machinery and 
the development of new techniques in the agricultural community, which has 
been historically resistant to such change. 
Thus, an object of this invention is to provide a technique whereby 
cultured plant tissue may be insulated from harmful conditions. 
Another object of this invention is to decrease the time for raising a 
mature or vigorous seedling from meristematic tissue, somatic embryos or 
tissue-cultured plants. 
Yet another object of the invention is to provide a medium to deliver the 
cultured plant tissue together with adjunctives facilitating seedling 
stand establishment. 
A further object of the invention is to reduce the amount of handling 
between the development of the cultured plant tissue and its planting in 
the field. 
A still further object of the invention is to reduce the need for special 
handling techniques and special technology during the development and 
growth of cultured plant tissue and, thus, overcome resistence to the 
introduction of new technology by adapting to existing methods of seed 
planting technology. 
A final object of the invention is to provide a large scale, economical 
method to clone superior plants or hybrid plants. 
DISCLOSURE OF INVENTION 
Briefly, in accordance with the invention, analogs of botanic seed are 
created by encapsulating totipotent meristematic tissue in a gel which 
permits germination and development. 
The invention is based in part on the recognition that botanic seed 
contains meristematic tissue which has the potential to differentiate to 
produce an entire plant body. Included also are various accessory 
structures which promote the development and survival of the plant. 
The invention also includes a recognition that totipotent meristematic 
tissue may be isolated from many sources and the accessory structures may 
be selectively included, or improved upon, in encapsulated meristematic 
tissue. 
Further, many of the advantageous properties of the seed coat and accessory 
structures may be recreated by the gel used to encapsulate the 
meristematic tissue. The gel can cushion the meristem from mechanical 
stress. The gel can accept and hold various adjunctives which provide 
nutrition or enhance development. The gel can allow germination inhibitors 
to diffuse away at a controlled rate, thus allowing preselected 
germination time. The outer surface of the gel can be treated to harden it 
or alter its permeability. 
In accordance with one aspect of the invention, meristematic tissue is 
isolated by inducing the formation of somatic embryos. These embryos are 
then encapsulated in a gel which permits development. 
In accordance with another aspect of the invention, meristematic tissue is 
isolated from somatic sources and, having the potential to differentiate 
to produce an entire plant body, is encapsulated without somatic 
embryogenesis being induced. 
In accordance with another aspect of the invention, meristematic tissue is 
isolated from zygotic or germ line sources, and encapsulated in a gel 
which will permit development. 
The invention is particularly advantageous in creating analogs to botanic 
seed which promote the delivery of superior clones or hybrids to the field 
using traditional planting methods.

BEST MODE FOR CARRYING OUT THE INVENTION 
Selection of Meristematic Tissue 
Botanic seed is a means which has evolved to deliver the progeny of plants 
to sites which are suitable for development and growth. The essential 
element of botanic seed is the meristematic tissue which differentiates to 
form an entire plant body. Also included are various other accessory 
structures which provide nutrition or protection to the developing embryo. 
Cultured plant tissue may be derived from numerous sources, including 
somatic tissue, zygotic tissue or germ line tissue. Regardless of its 
source, the tissue must pass through a meristem stage in order to undergo 
organogenesis and develop into a regenerated plant body. 
Plant meristematic tissues are units of organization. Some meristematic 
tissues have the capacity to produce an entire plant body; others produce 
only selected tissues. Those which produce an entire plant body are termed 
totipotent. 
It is an inherent property of an embryo to recapitulate ontogeny. This 
capacity resides in the meristem, and the other structures in a seed or 
embryo are accessory to this meristematic tissue. 
Those tissue sources which are not ordinarily involved in reproduction may, 
under appropriate circumstances or inducement, form meristematic tissue. 
As a first step in the production of encapsulated somatic embryos, crop 
strains must be selected which are capable of somatic embryogenesis. For a 
representative list of such species see D. A. Evans and D. R. Sharp, 
"Application of Tissue Culture Technology in the Agricultural Industry," 
in Application of Plant Cell and Tissue Culture to Agriculture and 
Industry, D. T. Tomes et al., editors, (University of Guelph Press, page 
214, 1982), which is incorporated herein by reference. Further species may 
be shown capable of somatic embryogenesis with further experimentation and 
refinement of technique. 
Once the appropriate strain is selected, preparation of somatic embryos may 
proceed by any of numerous known techniques. For example, in alfalfa, see 
K. A. Walker and S. J. Sato, "Morphogenesis in Callus Tissue of Medicago 
Sativa: The Role of Ammonium Ion in Somatic Embryogenesis," Plant Cell 
Tiss. Org. Cult. 1:109-121 (1981), which is incorporated herein by 
reference. For other techniques known to the art see "Plant Tissue and 
Cell Culture", H. E. Street, ed., Univ. of Calif. Press (1977). 
The somatic tissue of certain other species are able to undergo shoot 
organogenesis without the intermediate formation of somatic embryos. See, 
T. Murashige, "Plant Propagation Through Tissue Culture, Ann. Rev. Plant 
Physiol. 25:135-46 (1974). Tissue from these plants may be encapsulated 
without the preliminary embryogenesis step, and mature plants grown 
therefrom. 
As an alternative, zygotic embryos may be used when for example the species 
is incapable of somatic embryogenesis. These zygotic embryos may be grown 
in culture or suspension, and then may be encapsulated with or without 
their seed coat and other accessory structures. 
In certain wide crosses, a fertile embryo is formed but the endosperm fails 
to develop and the embryo then dies. Thus the cross appears sterile, but 
viable progeny can be obtained by isolating the embryo from the aborted 
ovuli. The zygotic embryos may be separated from their seed coat and then 
encapsulated with additives which will enhance their growth and viability. 
See for example M. Monnier, "Culture of Zygotic Embryos," Frontiers of 
Plant Tissue Culture, T. A. Thorpe, ed. (The International Association for 
Plant Tissue Culture, University of Calgary, Alberta, Canada pp. 277-80, 
1978). 
Encapsulation Media 
It has been recognized that the germination and development of seeds may be 
enhanced by coating them with various materials. For example, it has been 
reported that coating seeds with Super Slurper (trade name) will result in 
a water-absorbent reservoir which improves the germination rate in arid 
conditions. 
It has also been suggested that encapsulation of somatic embryos may be 
accomplished. 
It has also been demonstrated that perishable foods may be preserved by 
coating them with a complexed carbohydrate, e.g. Earle U.S. Pat. No. 
3,395,024. 
The meristematic tissue may be enclosed in any of numerous media which 
provide an appropriate encapsulation matrix, hereafter termed "gel". In 
general, a gel must allow meristem or embryo respiration by permitting 
diffusion of gases. The gel should provide an environment strong enough to 
resist external abrasion and adverse forces, yet pliable enough to allow 
the growth of the embryo and its germination at the appropriate time. It 
may be desirable to use various gels in combination, either as a mixture 
or in layers, to achieve the desired results. 
Gels which have been found useful for encapsulating meristematic tissue 
include sodium alginate, guar gum, carrageenan with locust bean gum, and 
sodium alginate with gelatin. Other suitable gels include, but are not 
limited to: 
TABLE 1 
______________________________________ 
Gel Agents 
______________________________________ 
I. Natural Polymers 
A. Ionic bonds (requires complexing agents) 
Furcellaran 
Pectin 
Hypnean 
Dextran 
Tamarind 
Guar Gum 
B. Hydrophobic Interactions 
Amylose 
Agar 
Agarose 
Agar with Gelatin 
Gelatin 
Starch 
Amylopectin 
Cornhull Gum 
Starch Arabogalactan 
Gum Ghatti 
Gum Karagan 
Ti Gum 
Gum Tragacanth 
Wheat Gum 
Chitin 
Dextrin 
II. Chemically Modified Natural Polymers 
A. Ionic bonds (requires a complexing agent) 
Ethyl Succinylated Cellulose 
Succinylated Zein 
B. Hydrophobic Interactions 
Methylcellulose 
Hydroxyethyl Cellulose 
C. Covalent Bonds 
Gelatin with Glutaraldehyde 
III. Synthetic Polymers 
A. Covalent Bonds 
Polyacrylamide 
B. Hydrophobic Interactions 
Polyethylene Glycol 
Polyvinylpyrrolidone 
Polyoxyethylene 
Hydrophilic Urethane 
Polyvinylacetate 
Vinyl Resins 
Hydron (hydroxyethylmethacrylate) 
2-methyl-5-vinylpyridine-methylacrylate- 
methacrylic acid 
C. Ionic Bonds 
sodium poly(styrene sulfonate) with poly(vinyl 
methyl pyridinium) chloride 
sodium poly(styrene sulfonate) with poly(vinyl 
benzyl trimethyl ammonium) chloride 
strongly acidic polyanion with strongly basic 
polycation 
IV. Stabilizing Compounds 
1. Trade Names 
Super Slurper 
Viterra 
Laponite 
Gelrite 
SeaKem 
SeaPlaque 
SeaPrep 
IsoGel 
2. Organic Compounds 
Methylan Clear Wallpaper Paste 
Lactose 
Wax 
Protein Colloids 
3. Inorganic Compounds 
a. Clay 
b. Compounds that adhere by means of a 
water-soluble plastic such as methylcel: 
Fly Ash 
Feldspar 
Celrite 
Bentonite 
Vermiculite 
Diatomacous Earth 
Lime 
Calcium Carbonate 
______________________________________ 
Selecting Optimum Gels 
A gel chosen for encapsulation would ideally include the following 
characteristics (although the invention may be practiced in other modes): 
1. A compliance adequate to protect and cushion the meristem; 
2. The interior material would have solubility characteristics such that it 
can accept and contain adjunctives, including but not limited to aqueous 
or hydrophobic substances; 
3. An outer surface to provide a protective barrier to mechanical stress, 
facilitate handling and maintain meristem viability; 
4. Sufficient gel strength to maintain capsule integrity, but still allow 
the meristem to break out during germination. 
Encapsulation with Selected Gel 
Once the gel has been chosen, there are numerous parameters which influence 
the characteristics previously mentioned. 
A sodium alginate solution, for example, will form a gel when a complexing 
agent is added. Calcium chloride (CaCl.sub.2) is generally used, however, 
lanthanum chloride, ferric chloride, cobaltous chloride, calcium nitrate 
and calcium hydroxide are also acceptable, as are other compounds 
generally with multivalant cations. 
A chosen gel will have a range of concentrations usable in working the 
invention. A concentration should be chosen to optimize ease of handling, 
gelling time, strength of gel and coating thickness around the 
meristematic tissue. If the gel is too dilute, the tissue may settle 
during gel formation and produce an uneven encapsulation. 
The sodium alginate may be prepared in a concentration of 1 to 10% w(in 
grams)/v(in milliliters) in water, more usually 2 to 10% and ideally from 
3 to 5%. 
The meristematic tissue to be encapsulated may then be added to the sodium 
alginate solution at a concentration of 1 to 50 meristems per milliliter, 
more usually from 5 to 20 meristems per milliliter. This concentration 
will vary as the appropriate size of meristematic tissue varies with 
species, source and stage of development. 
The dispersed meristematic tissue in gel solution may then be added 
dropwise to the complexing agent. Alternatively, the gel solution and 
complexing agent may be mixed by any of numerous techniques known to the 
art. These may include droplet formation and agent addition as a one step 
process by a vibrating nozzle which ejects a gel droplet from one source 
and coats the droplet with complexing agent from another. 
The calcium chloride (or other complexing agent) may be made up in solution 
at a concentration of 1 to 1,000 millimolar, more usually 20 to 500 
millimolar and ideally from 50 to 100 millimolar. Other complexing agents 
will have different preferred concentration ranges. 
The time for gel formation and the temperature of the gelling solutions are 
interrelated parameters, for selected concentrations of gel and complexing 
agent. The temperature should be chosen so as to avoid damage to the 
meristematic tissue, usually in the range of 1.degree. to 50.degree. C., 
more usually 10.degree. to 40.degree. C., and preferably at 20.degree. to 
40.degree. C. 
Within the range of acceptable temperatures, a particular value may be 
chosen to give the shortest possible gelling time consistent with complete 
gel formation. Typically, the gel will form immediately, but the 
complexation takes much longer. For a solution of sodium alginate at a 
concentration of 3.2 grams per 100 milliliters H.sub.2 O, calcium chloride 
solution concentration of 50 millimolar and 25.degree. C. reaction 
temperature, adequate gelling is obtained in 5 to 120 minutes, more often 
10 to 90 minutes and is usually sufficiently complete in 30 to 60 minutes. 
The gel characteristics described above are modifiable for each gel, but 
are determined generally by the concentration parameters and chemical 
properties of the gel. 
Hardening Capsules 
Subsequent to encapsulation, it may be desirable to increase the rigidity 
of the outer surface of the gel matrix, through numerous techniques known 
to the art. In this manner, a softer gel may be used for the encapsulation 
and inclusion of appropriate additives, and in the outer surface 
resistence to abrasion and penetration may be increased with no loss of 
meristem viability. 
The encapsulated meristematic tissue may be subjected to a partial 
desiccation, which results in a more rigid outer surface. 
Alternatively, the encapsulated meristematic tissue formed of a selected 
gel may again be coated with a thin layer of a more rigid gel, or may be 
encased in a gelatin capsule available through commercial sources. 
The surface may also be hardened by treatment with various chemical agents 
known to the art, which increase the gel surface breaking resistance. 
Such techniques include, in part: 
TABLE 2 
______________________________________ 
Capsule Coating Compounds 
______________________________________ 
I. Coacervation 
Gelatin and Gum Arabic 
Lecithin and Cephalin with Cellulose Nitrate 
Paraffin Oil with Cellulose Nitrate 
II. Interfacial Polymerization 
Sebacoyl Chloride with Hexanediamine 
III. Tannic Acids 
Persimmon Tannin 
Chinese Gallotannin 
IV. Poly Amino Acids 
Polyornithine 
Polylysine 
Polycitrulline 
Polyarginine 
Polyhistidine 
Polyasparagine 
Polyglutamine 
Combination of Poly-L-Amino acids 
V. Glutaraldehyde 
Glutaraldehyde with gelatin 
VI. Gelatin capsules 
______________________________________ 
In utilizing the means of hardening the outer surface of the gel capsule, 
care must be taken to avoid damage to the meristematic tissue. For 
example, crosslinking the gel matrix with glutaraldehyde will provide 
surface strength, but if the glutaraldehyde is applied for an extended 
period of time, it will penetrate the gel completely and damage the 
meristematic tissue. This time period will vary with the gel material, the 
thickness of the gel coat and the temperature of the solutions. 
It has been noted that certain of the above-mentioned treatments, e.g. 
polylysine, will change the water retention characteristics of the gel, 
but not the breaking strength. Thus by appropriate combination of 
treatments, most gel characteristics may be adjusted to their desired 
values. 
Further Modifications 
In agricultural applications, it is generally preferred that harvesting be 
accomplished in a brief period of time and in the appropriate season. 
Therefore, either before or during the gelling process, it may be 
desirable to synchronize the germination of the meristems or embryos 
through techniques known to the art, such as the use of mitotic blockers 
or sizing through sieves, so that any given batch of encapsulated 
meristems or somatic embryos will germinate at approximately the same 
time. 
While the gel solution is being prepared, it may be desirable and 
appropriate to include certain adjunctives which will not interfere with 
the gel formation, but which may provide germination control, nutrition, 
disease resistance, pest resistance, nitrogen fixation capability, 
herbicide capability, or compounds which enhance embryogenesis or 
organogenesis. For example, abscisic acid, or high concentrations of 
sucrose, will inhibit germination. These adjunctives may be encapsulated 
with the meristematic tissue, and germination will occur only when the 
adjunctive has been removed by diffusion or otherwise. 
Subsequent to encapsulation, it may be desirable to store the encapsulated 
meristematic tissues, transport them to the field, hothouse, or the 
nursery, and treat them in a manner consistent with botanic seed. 
Planting these encapsulated meristematic tissues may be accomplished in the 
nursery or hothouse for species unable to tolerate the ambient climatic 
conditions without some period of acclimatization. Alternatively, for more 
hardy species, the encapsulated meristems may be planted directly in the 
field through numerous techniques employed in the art for botanic seed. 
Experimental 
In order to demonstrate the invention, the following experiments were 
carried out with a variety of meristematic tissue material, and gel media. 
All quantities labeled percent (%) are grams per 100 milliliters, unless 
otherwise indicated. 
EXAMPLE A 
(Alfalfa somatic embryo) 
1. Encapsulation with sodium alginate 
Callus from alfalfa, Medicago sativa L. strain RA-3, is induced to form 
somatic embryos by a 3-4 day exposure to Shenk and Hildebrandt (SH) medium 
(Can. J. Bot. 50:199-204, 1972), supplemented with 50 micromolar 
2,4-dichlorophenoxyacetic acid (2,4-D) and 5 micromolar kinetin. The 
tissue is then transferred to a 2,4-D- and kinetin-free SH regeneration 
medium. This procedure is explained in detail in: K. A. Walker, et al. 
"The Hormonal Control of Organ Formation in Callus of Medicago sativa L. 
Cultured in Vitro," Am. J. Bot. 65:654-659 (1978); K. A. Walker, et al., 
"Organogenesis in Callus Tissue of Medicago sativa, The Temporal 
Separation of Induction Process from Differentiation Processes," Plant 
Sci. Lett. 16:23-30 (1979); and K. A. Walker and S. J. Sato (1981) as 
cited elsewhere herein. These articles are incorporated herein by 
reference. 
Somatic embryos form over a period of one to three weeks. At this point, 
the cultured somatic embryos may be synchronized, or be encapsulated 
directly. 
The somatic embryos were adjusted to a concentration of 10 embryos per 
milliliter of sterile 3.2% sodium alginate at 25.degree. C. The mixture 
was stirred into a slurry and then dispensed dropwise from a 5 milliliter 
Pipettman pipette with sterile tip into 500 milliliters of 50 millimolar 
calcium chloride at 25.degree. C. At these concentrations, capsules form 
immediately, but the complete complexation takes 30 to 60 minutes. At this 
point, the calcium chloride solution is poured off and the capsules are 
washed twice in SH liquid medium. Using this technique, 50% of the 
capsules contain embryos. The capsules were then cultured on SH medium in 
a room with 16 hours of light per day in order to promote germination. 
Using the above protocol, germination rates of 56 to 70% were achieved. 
Subsequently, some of these plants were raised under greenhouse 
conditions. 
1.a As an alternative complexing agent, 8 to 80 millimolar ferric chloride 
(FeCl.sub.3) can replace calcium chloride in the protocol of A.1. 
1.b As an alternative complexing agent 10 to 100 millimolar lanthanum 
chloride may replace calcium chloride in the protocol of A.1. 
1.c As an alternative germination method, the encapsulated somatic embryos 
may be inserted directly into a plug of peat composition (an Isocyanate 
and polyalcohol pre-polymer combined with water and peat. Available from 
Castle & Cook Techniculture, Inc.). 
2. Encapsulation with sodium alginate plus gelatin 
The experimental protocol A.1 was duplicated, substituting a mixture of 2% 
sodium alginate plus 5% gelatin for the 3.2% sodium alginate. 
3. Encapsulation with carrageenan plus locust bean gum 
The experimental protocol of A.1 was duplicated, using 0.25 to 0.8% 
carrageenan plus 0.4 to 1.0% locust bean gum instead of sodium alginate, 
and 50 to 500 millimolar ammonium chloride (NH.sub.4 Cl) instead of 
calcium chloride. 
3.a As an alternative, 100 to 500 millimolar potassium chloride (KCl) can 
replace ammonium chloride. 
4. Encapsulation with guar gum 
The experimental protocol A.1 was duplicated, substituting 2% guar gum for 
sodium alginate and substituting 10 to 120 millimolar sodium tetraborate 
for calcium chloride. 
EXAMPLE B 
(Celery somatic embryo) 
Callus was induced from cotyledons and hypocotyls from two week old celery 
plants (Apium graveolens L., strains Utah Tall 5270, 5275, Calmario, and 
Golden Self Blanching) on SH medium containing 0.5 to 25 micromolar 2,4-D 
and 3 micromolar kinetin. Somatic embryos formed one to three months after 
transfer to SH medium containing 25 millimolar ammonium nitrate. The 
somatic embryos were then encapsulated as in protocol A.1. The 
encapsulated somatic embryos were germinated on SH medium (half strength) 
with 25 micromolar gibberellic acid (GA3) and 0.25 micromolar 
napthaleneacetic acid (NAA), and plants were produced. 
EXAMPLE C 
(Brassica oleracea L.) 
1. Organogenetic shoots 
Cauliflower seeds (Brassica oleracea L. strain Monarch 73M) were germinated 
under sterile conditions on agar water. Eight days after germination, 
hypocotyl segments were placed on SH medium with 1 micromolar 
para-chlorophenoxyacetic acid (pCPA) and 10 micromolar kinetin to induce 
callus. After three to four weeks, the callus was transferred to SH medium 
with 10 micromolar indole acetic acid (IAA) and 3 micromolar kinetin and 
meristematic tissue formed. Some of the tissues formed adventitious 
shoots. The shoots were encapsulated as described in protocol A.1. and 
placed on SH medium with 20 millimolar ammonium nitrate and 3% w/v 
sucrose. The shoots emerged from the capsule and roots developed producing 
complete plants. 
2. Somatic embryos 
Somatic embryos with a distinct radicle and a distinct apical tip were 
produced from meristematic tissue from callus in the same manner as 
described for adventitious shoots in C.1. These embryos were encapsulated 
as in protocol A.1. and plants were produced. 
EXAMPLE D 
(lettuce) 
1. Organogenetic shoots 
Seeds of lettuce (Lactuca sativa L. strain Arctic King) were germinated on 
agar water. Hypocotyl segments were placed on SH medium with 0.5 
micromolar NAA, 2.5 micromolar kinetin, and 10 mm ammonium nitrate for the 
production of callus. After subculture, meristematic tissue was produced 
from the callus and adventitious shoots from part of the meristematic 
tissue. These shoots were encapsulated as in protocol A.1. When the 
capsules were placed on SH medium with 0.5 micromolar NAA and 2.5 
micromolar kinetin the shoots emerged, roots formed, and complete plants 
were produced. 
2. Lettuce somatic embryos 
Somatic embryos with a distinct radicle and a distinct apical tip were 
produced from meristematic tissue from callus in the same manner as 
described for adventitious shoots in D.1. These embryos were encapsulated 
as in protocol A.1. and plants were produced. 
EXAMPLE E 
(gopher plant organogenetic shoots) 
Leaf mesophyll protoplasts were isolated from gopher plant, Euphorbia 
lathyris L. and callus produced according to techniques known to the art. 
Meristematic shoots were produced by placing the callus on Murashige and 
Skoog medium ("A Revised Medium for Rapid Growth and Bioassays with 
Tobacco Tissue Cultures." Physiol. Plant. 15: 473-97, 1962) containing in 
milligram per liter quantities: inositol (100), sodium phosphate (170), 
nicotinic acid (1), pyridoxine. HCl (1), thiamine.HCl (10) and sucrose 
(20,000) plus 0.4 nanomolar picloram and 20 micromolar 6-(gamma, 
gamma-dimethylallylamino)-purine. 
The shoots were encapsulated as described in protocol A.1. 
EXAMPLE F 
(wild mustard zygotic embryo) 
Flower pods, two to four weeks after pollination, were removed from 
Brassica campestris L. plants growing in the wild. Zygotic embryos were 
dissected from the ovules contained within the flower pods and 
encapsulated as in protocol A.1. The embryos germinated when placed on an 
appropriate medium (see, M. Monnier, 1975, as cited elsewhere herein). 
Germination was inhibited for at least one month when embryos were 
encapsulated with 12% sucrose and placed on medium containing 12% 
(w/v)sucrose. Germination suppression was overcome by placing encapsulated 
embryos on medium containing 2% (w/v) sucrose and plants were obtained. 
Abscisic acid (10.sup.-6 M) was also used to suppress and control 
germination. 
EXAMPLE G 
(Brassica oleracea L. zygotic embryo) 
Zygotic embryos were excised from Brassica oleracea L. (variety PHW, Ccc-1) 
flowers and encapsulated as in protocol A.1. Plants were obtained on 
medium containing 2% (w/v) sucrose. 
EXAMPLE H 
(hard outer covering) 
1. Brassica campestris L. zygotic embryos were encapsulated as in protocol 
F. The capsules were allowed to dry down over a solution of sulfuric acid 
for one week. At that time alginate had dried around the embryo and had a 
breaking strength of greater than 60 kg/cm.sup.2 (compared to a breaking 
strength of 7-15 Kg/cm.sup.2 for newly encapsulated embryos). The dried 
capsules were placed on a medium with 2% w/v sucrose (see protocol F.), 
the embryos germinated, and plants were recovered. 
2. Alfalfa somatic embryos were encapsulated as in protocol A.1. The 
capsules were coated with poly-L-amino acids for one hour to produce an 
outer covering with specific pore size. Polyamino acids used were 
poly-L-lysine (MW 4,000, 60,000, and 150,000) and poly-L-proline (MW 
30,000) at 0.1% and 0.02% w/v for each molecular weight. The germination 
and embryo emergence frequency was 44%. 
Although the foregoing invention has been described in some detail by way 
of illustration and example for purposes of clarity of understanding, it 
will be obvious to one skilled in the art that certain changes and 
modifications may be practiced within the scope of the appended claims.