Method for applying hydrogel coatings to embryonic plants

The invention is drawn to a method of applying a hydrogel coating to embryonic plants to improve early plant growth by controlling the amount of cross-linking. A dry coating of a water-soluble hydrogel in powder form is built up around individual embryonic plants. The powder-coated plants are introduced into a water bath which is free of polyvalent metal ions to partially hydrate the coatings. Rapidly thereafter and before the coatings are fully hydrated, the bath is modified by dispersing cross-linking polyvalent metal ions therein. The contact time of the coated plants in the modified bath is limited so that the polyvalent metal ions diffuse into the coatings and form an inwardly decreasing concentration gradient therein. The plants are separated from the modified bath so that the polyvalent metal ions are redistributed and equilibrated within the coatings. The resulting coatings are in a uniform partially cross-linked condition which promotes leaf emergence and plant vigor.

FIELD OF INVENTION 
The field of this invention is the use of hydrogel coatings on embryonic 
plants such as seeds and somatic embryos. 
BACKGROUND OF INVENTION 
Pelletizing small size seeds facilitates handling and planting. Various 
coating procedures have been used for this purpose. For example, it is 
known to coat plant seeds with hydrocolloids which hydrate to form gels, 
and thereby provide gel coatings around singulated seeds. Such coatings 
have been used particularly with pregerminated seeds, making it possible 
to handle and plant the pregerminated seeds without physical damage. This 
protective action can be of particular importance where the seeds have 
been germinated to the stage of radicle emergence. 
Prior art methods of applying hydrogel coatings to plant seeds are 
described in Redenbaugh et al., U.S. Pat. No. 4,715,143 and Nelson et al., 
U.S. Pat. No. 4,780,987. The hydrocolloid, such as sodium alginate, is 
formed into an aqueous solution, and individual seeds are coated with this 
solution. The coated seeds are contacted with an aqueous solution of a 
complexing or cross-linking reagent, such as calcium chloride, which 
provides calcium ions that react with the alginate to increase viscosity 
and reduce water solubility. 
As described in the cited Redenbaugh et al. patent (cols 4-5), the 
complexing agent may be mixed with the gel-forming solution and applied to 
the seeds or other living material to be encapsulated. Alternatively, as 
described in the paragraph bridging columns 4-5, a vibratory nozzle 
apparatus may be used to eject gel droplets containing the material to be 
encapsulated. These droplets are coated with a complexing agent. 
More commonly, as described in the Nelson et al. patent (cols 6-7), seeds 
are dispersed in a gel solution which is added drop-wise to the aqueous 
complexing reagent. Nelson et al. refers to a mechanized procedure using a 
vibratory nozzle which ejects seed-containing gel droplets from one source 
and coats the droplets with a complexing agent from another. 
Heretofore, as far as is known, cross-linked hydrogel coatings have not 
been applied to seeds by first building up a dry coating of hydrogel 
powder around the individual seeds. This type of coating is known for 
other purposes, as described for example in Sluis et al. U.S. Pat. No. 
4,658,539. As therein disclosed, pregerminated seeds are coated by a 
pilling procedure for purpose of incorporating a growth retarding agent in 
the coatings. 
The Kelco Division of Merck & Co., sells an alginate product under the name 
"ALGIN-TEX". Commercially available information concerning this product 
describes an "ALGIN-TEX System" to form cross-linked alginate matrices. As 
described in this "ALGIN-TEX" commercial brochure, an alginic polymer 
consists of units of alginic acid which have carboxyl side chains. The 
carboxyl groups are reactive with metal ions such as sodium to form sodium 
alginates. They are also reactive with polyvalent metal ions to form 
cross-links and thereby increase polymer chain lengths. As polyvalent 
metal ions are reacted with the alginic acid units, the viscosity of the 
solution increases because of the greater polymer chain lengths. When more 
fully cross-linked, insoluble fibers are formed which can precipitate out 
of solution. 
SUMMARY OF INVENTION 
This invention provides a method of applying hydrogel coatings to plant 
seeds and the like while avoiding or at least minimizing interference of 
the coatings with seed germination and resulting plant vigor. In prior art 
procedures using hydrogels, such as an alginate cross-linked with calcium 
ions, there has been little control over the extent of cross-linking of 
the hydrogel. When a liquid droplet containing a plant seed is contacted 
with an aqueous solution of calcium chloride, the calcium ions are 
available for reacting throughout the coatings. Such coatings become 
highly cross-linked and thereby essentially water-insoluble. Plants grown 
from pregerminated seeds encapsulated in this manner may exhibit reduced 
germination, reduced plant emergence, and reduced plant vigor. 
In using the method of this invention, there is first formed a dry coating 
around individual seeds of a water-soluble hydrogel in powder form. A 
hydrogel is selected which is capable of cross-linking reaction with 
polyvalent metal ions. Next the dry-coated seeds are introduced into a 
water bath free of polyvalent metal ions to partially hydrate the 
coatings. Rapidly thereafter and before the coatings are fully hydrated, 
the bath is modified by dispersing cross-linking polyvalent metal ions 
therein. The contact time of the coated seeds with the modified bath is 
limited. The polyvalent metal ions diffuse into the coatings and form 
inwardly decreasing concentration gradients. The outer portions of the 
coating are thereby more highly cross-linked than the inner portions. The 
coated seeds are separated from the modified bath and held until the 
polyvalent metal ions redistribute and essentially equilibrate in the 
coatings. The resulting coatings throughout are in a partially 
cross-linked condition. 
The foregoing method provides a means for obtaining a predetermined degree 
of cross-linking, since the amount of cross-linking can be controlled by 
the contact time in the treatment baths. By using a short standardized 
hydration time prior to contracting with polyvalent metal ions, the ions 
will diffuse into the coatings as the coatings continue to hydrate. The 
diffusion is terminated at a predetermined time at which there is an 
inward concentration gradient of the calcium iotas within the coatings. 
For example, the outer portion of the coatings may then contain sufficient 
calcium to be essentially fully cross-linked and therefore insolubilized, 
while the inner portions contain less calcium and remain water-soluble. On 
separation of the seeds from the treating bath they contain a gradient 
distributed total amount of calcium ions. By holding the seeds, the higher 
concentration of the outer layers redistributes to an essentially uniform 
concentration throughout the coatings. The final coatings will have a 
controlled degree of water solubility. Preferably, no portion of the 
coatings remain cross-linked to water insolubility. 
In comparative studies of the method of this invention using variable 
contact times in the treating baths, it has been found that early growth 
of the seeds is improved by limiting the contact times. For example, in a 
preferred embodiment pregerminated powder-coated seeds are hydrated for 30 
seconds and then contacted with calcium ions for 5 to 6 minutes. When the 
contact time in the calcium ion-providing bath is increased to 15 minutes, 
the coated seeds exhibit lower plant emergence. Even more dramatically, 
the resulting plants have substantially reduced vigor, as photographically 
measured by the extent of early leaf growth. 
DETAILED DESCRIPTION 
When using the method of this invention, a gel material should be selected 
which has a cross-linking reaction with polyvalent metal ions. It is 
preferred that the hydrogel material prior to cross-linking is 
water-soluble, and that it be capable of cross-linking reaction to an 
insoluble condition, or at least to a condition of increased viscosity. A 
preferred hydrogel is an alginate polymer such as sodium alginate. Another 
natural gel material which can be employed is pectin, viz. as sodium 
pectate. If desired a mixture of hydrocolloids can be employed such as a 
mixture of alginate and pectinate. 
The polyvalent metal ions used for cross-linking should be non-toxic to 
plants. Calcium is preferred, and can be used in the form of its non-toxic 
water soluble salts. Calcium nitrate is especially desirable since the 
nitrate ion provides a fertilizer ingredient. Other water soluble salts of 
polyvalent metals can be used such as ferric chloride. Calcium ions are 
believed to be the most effective for forming cross-links with alginic 
molecules. 
The coating method of this invention is advantageous for both flower seeds 
and vegetable seeds, and especially for small size and/or pregerminated 
flower or vegetable seeds. In preferred embodiments, the seeds have been 
germinated to the stage of radicle emergence, and the hydrogel coatings 
provide protection for the emerged radicles. In commercial application, 
the plant seeds are germinated to the stage of emerged radicles, and the 
seeds are coated by the method of this invention, and then stored and 
shipped under refrigeration. The coated seeds may be planted in plug flats 
by commercial growers. 
The method is applicable to other embryonic plant materials besides seeds 
such as somatic embryos. It should be understood that the term "embryonic 
plants" is used herein generically for meristematic plant material capable 
of developing into an entire plant. 
In certain preferred embodiments, plant seeds are used and pregerminated to 
a stage where radicles have emerged in most of the seeds, for example, in 
90% or more of the seeds. In applying the method to such pregerminated 
seeds, it is not necessary to sort the seeds after germination to 
eliminate seeds that do not have emerged radicles. 
The method is particularly advantageous when used with seeds of very small 
size, such as seeds which are usually pelleted to facilitate handling. In 
marketing of flower seeds to commercial growers, it may also be desirable 
to pregerminate the small size seeds and then coat the seeds in accordance 
with the present invention. 
In the first step of the method, the seeds to be coated, such as 
pregerminated flower seeds, are provided with a substantially dry coating 
of water-soluble hydrogel material in powder form. For example, as 
indicated above, sodium alginate or sodium pectinate powders can be used. 
The dry coating can be built up according to known pelletizing procedures. 
For example, the seeds may be placed in a rotary pelleting pan, and 
sprayed with a fine mist of water as the seeds rotate. The water may 
contain an adhesive agent. 
A small amount of the hydrogel powder is added to form a coating layer 
around the individual seeds. This process can be repeated a series of time 
with intermittent sprayings of water and addition of hydrogel powder. In 
this way the seed pellets are built up to a desirable screen size. The 
coated seeds may be screened to remove oversize and undersize pellets. The 
size-selected coated seeds are introduced into a room temperature (e.g. 
20.degree.-25.degree. C. ) water bath to partially hydrate the hydrogel 
coatings. The bath may contain a low concentration of a fertilizer 
ingredient such as potassium nitrate, but it is free of polyvalent metal 
or other substance reactive with the hydrogel. It is desired to limit the 
extent of hydration. The contact time in the non-reactive water bath 
should be shorter than that required to fully hydrate the seed coatings. A 
preferred initial contact time is 30 seconds. This may be varied somewhat, 
for example, by using from 15 to 45 seconds. Preferably the hydration is 
for less than 60 seconds and more than 10 seconds. 
When the coatings are partially hydrated, a solution of complexing agent is 
dispersed in the bath containing the seeds. For example, a solution of 
calcium nitrate can be added. The added solution can be fairly 
concentrated, such as 5 to 10% w/v CaNO.sub.3. 
These treatment operations are carried out with gentle stirring of the 
seeds in the bath. Stirring is preferably started at the time the seeds 
are introduced into the initial water bath and continued throughout the 
contacting. 
To control the amount of complexing agent introduced into the gel coatings, 
the contacting is preferably carefully timed. For example, using an 
initial hydration of 30 seconds, the reactions contacting with the 
modified solution is carried out in 5 to 6 minutes. With shorter times, 
less complexing agent will diffuse into the coatings. As the contact time 
is extended the coatings will absorb more of the complexing agent. It is 
desired to form a decreasing concentration from the outside to the insides 
of the coatings. The reaction contacting should not therefore be permitted 
to proceed until coatings are saturated with the complexing agent and 
thereby insolubilized. 
A reaction contacting time of around 5 to 6 minutes has been found 
desirable but somewhat shorter or longer contacting times can be used, for 
example, from 3 minutes to 9 minutes. If the concentration of the 
complexing agent in the contacting solution is increased then the contact 
time can be reduced, or conversely, if the concentration is reduced, the 
contacting time can be extended. 
The reaction contacting is terminated by removing the coated seeds from the 
reaction bath. The removed seeds are further separated from the residual 
treating solution by drainage, such as on a screen, and the seeds may be 
washed with water on the separating screen. 
The coatings on the seeds is in gel form. After internal redistribution of 
the complexing agent, the coatings throughout will be only partially 
cross-linked. The seeds can be planted shortly after completion of the 
coating process. Redistribution and equilibration of the complexing agent 
will occur even after the seeds are planted. However, the seeds will 
usually be held for a substantial periods. In commercial processing, it 
may be preferred to store the seeds in sealed containers under 
refrigeration, which can be distributed to commercial growers. 
To facilitate handling, the coated seeds may be dusted with an anti-caking 
powder such as talc. It is desired to have the seeds in free-flowing 
condition. Also, to absorb free moisture, a drying powder may be used.

ILLUSTRATIVE EXAMPLE 
Dry Process 
Plant seeds, which may be pregerminated, are pelletized using sodium 
alginate powder. The pelletizing can be carried out in a rotating pan 
employing an airbrush for applying a water mist to the rotating seeds. The 
applied spray may contain a pelletizing agent such as "VANGEL", viz 3% w/v 
of VANGEL. This pelletizing agent is a smectite clay sold by R.I. 
Vanderbilt Company of Norwalk, Conn. The rotating pan is turned on and a 
speed selected. Preferably a slower speed is used. The airbrush is held 
over the seed with the nozzle pointed toward the center of the rotating 
seeds. The water mist is sprayed until the seeds appear slightly wet. When 
the seeds begin to clump as they roll around in the pan, spraying is 
terminated. The addition of the alginate powder is started, using a small 
amount at a time, such as 0.40 to 0.45 ounces of powder per ounce of 
seeds. Mist spraying and addiction of alginate powder is repeated as often 
as necessary to build up the seeds to a desired pellet size. For example, 
a pellet size of 10 (A.S.T.M.E.) can be used. The pelletized seeds can be 
screened to remove oversize and undersize pellets. The oversize pellets 
are discarded and the undersize can be returned to the coating pan. 
Wet Process 
Ion free water, such as distilled water, can be used to form a room 
temperature (20.degree.-25.degree. C. ) coating bath. The bath may contain 
0.5% w/v KNO.sub.3 as a fertilizer. A modifying solution is prepared 
comprising an aqueous solution of calcium nitrate. This solution may 
contain 7.5% w/v CaNO.sub.3, and can be prepared by dissolving 70g calcium 
nitrate per liter of water. For modifying the bath 100 ml of this solution 
is added per 2 liters of bath. 
The coating bath is provided with a stirring device which can create a 
vortex when operated at low speeds. With the stirrer in operation, the dry 
coated seeds are introduced into the vortex of the water bath. After 30 
seconds, the calcium nitrate modifier solution is added. Stirring is 
continued and the seeds are permitted to remain in the bath for 5 to 6 
additional minutes. The coated seeds are then removed from the complexing 
solution. For example, the coating bath may be poured into a screen box 
with a small mesh size for retaining the seeds. The seeds can be rinsed 
with water while in the screen box, and excess water vacuumed off until 
the coated seed surfaces appear dry. The seeds are then placed in 
containers. The addition of small amounts of drying or anti-caking powder 
can be used. 
Comparative Example 
The small size of impatiens seeds makes it desirable to pelletize the seeds 
to facilitate handling and planting. This study was conducted with four 
varieties of commercial impatiens seeds which are marketed by the Ball 
Seed Company of West Chicago, Illinois. These varieties are sold under the 
names "SUPPER ELFIN BLUSH", "SUPPER ELFIN SALMON", "SUPPER ELFIN ORANGE", 
and "SUPPER ELFIN VELVET". These varieties normally exhibit somewhat 
different growth characteristics. 
Each variety of impatiens was pregerminated by a standard pregerminating 
procedure, for example, using overnight pregermination in water columns 
with an incubation temperature of about 25.degree. C. The pregerminated 
seeds were recovered and externally dewatered by centrifugation. More than 
90% of the pregerminated seeds of each of the four varieties had emerged 
radicles. These pregerminated seeds were used without separating seeds 
without radicles. 
Two batches of each of the varieties were coated as described in the 
foregoing Illustrative Example. Identical conditions were used except that 
one batch of the coated seeds was held in the modified bath containing the 
calcium ion for 5 minutes and a second batch for 15 minutes. 
After recovery of the seeds, the resulting "gelballs" were sown in plug 
flats, which were held under standard growth conditions of temperature, 
light and watering for promoting growth. As a control, uncoated 
pregerminated seeds of each of the four varieties was sown in plug flats 
and grown under the same conditions. All flats were visually and 
photographically examined after seven days. 
The data is summarized below in Table A. 
Leaf emergence was determined by the percent of the plants displaying 
dicotyledons. The photographs of the plug trays were processed to obtain a 
"Vigor Index". Machine vision image analysis was used. The image analysis 
equipment measured the total surface area of the leaves of the seedlings 
in each plug flat. A standard deviation in the leaf area of the seedlings 
was determined by a computer program. The Vigor Index of the seedlings was 
determined by dividing the total surface area of the leaves by the 
standard deviation of the leaves multiplied by the leaf emergence 
percentage. This method of plant vigor comparison is described more 
particularly in my co-pending application Ser. No. 08/407,411, filed Mar. 
17, 1995. 
Other Examples 
Commercial sodium pectinate powder is substituted in a similar amount for 
the alginate powder of the Illustrative Example set out above. In another 
alternative embodiment, moist somatic embryos are processed by the coating 
method of this invention. The procedure is otherwise the same as with 
respect to moist pregerminated seeds. Other examples and variations will 
occur to those skilled in the growth of flower and vegetable seeds. 
TABLE A 
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Comparism of Early Plant Growth of Pregerminated 
Coated Varieties of Impatiens 
Leaf Growth Area 
Variety Leaf Emergence (%) 
as Vigor Index 
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Super Elfin Blush 
Control-uncoated 
98 446 
Coated-5 min. 
96 442 
Coated-15 min. 
91 287 
Super Elfin Salmon 
Control-uncoated 
98 354 
Coated-5 min. 
94 326 
Coated-15 min. 
69 116 
Super Elfin Orange 
Control-uncoated 
96 472 
Coated-5 min. 
97 448 
Coated-15 min. 
82 233 
Super Elfin Velvet 
Control-uncoated 
97 510 
Coated-5 min. 
88 270 
Coated-15 min. 
66 133 
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