Utility-high carbon dioxide and light quality and quantity in woody plant propagation

The present invention relates to the propagation of plants and plant tissue. In particular, the present invention provides a method for propagating woody plant material comprising culturing the material in the presence of carbon dioxide in excess of about 1000 .mu.l/l and pulses of filtered light. In another embodiment, the present invention relates to a propagation method comprising culturing the plant material in excess of about 7000 .mu.l/l of carbon dioxide and further exposing the plant material to pulses of filtered light.

FIELD OF THE INVENTION 
The present invention relates to the propagation of woody plants, plant 
cells and tissue cultures. In particular, the present invention provides a 
method for such propagation. 
Increasing the rate of photosynthesis in a plant, resulting in increased 
growth of plant material, is a goal having a significant economic outcome 
once realized. Economic benefits include increased rate of propagation, as 
well as improved efficiency of propagation by enhancing survival and early 
growth. These benefits are especially important for plants propagated from 
tissue culture. The culture conditions having an impact on growth and 
photosynthesis that are controllable include temperature, wavelength and 
intensity of light and period of exposure thereto, growth medium, and 
atmospheric gas concentrations. 
Varying the concentration of carbon dioxide has been explored. See Flygh et 
al., Ann.Sci.For., 46 suppl., 168s-170s (1989); Wittwer, in Carbon Dioxide 
Enrichment of Greenhouse Crops, Volume I. Status and CO.sub.2 Sources (H. 
Z. Enoch and B. A. Kimball, Eds., 1986; hereinafter) (hereinafter, "Enoch 
and Kimball"), pp. 3-15; Sionit and Kramer, in Enoch and Kimball, pp. 
70-85. For example, CO.sub.2 enrichment in vitro has been associated with 
growth responses such as increases in dry weight (Kozai et al., Symposium 
Florizel on Plant Micropropagation in Hort. Ind., pp. 135-141 (1987); 
Cournac et al., Plant Physiol., 97, 112-117 (1991); Fujiwara, et al., 
J.Agr.Met., 48 49-56 (1992)), plant height (Cournac et al., supra; 
Figueira et al., J.Amer.Soc.Hort.Sci., 116, 585-589 (1991)), fresh weight 
(Buddendorf-Joosten and Woltering, Sci.Hort. 65, 11-23 (1996)), or leaf 
area (Buddendorf-Joosten and Woltering, supra; Figuiera et al., supra). 
In woody plants it has been noted that increased CO.sub.2 concentrations 
increase the dry weight of, for example, conifer seedlings exposed to 
carbon dioxide concentrations of up to about 3500 .mu.l/l (about ten times 
the ambient concentration of CO.sub.2 which is about 350 .mu.l/l of 
CO.sub.2); beyond that point, no further benefit in dry weight increase 
has been noted relative to control plants. Flygh et al., supra. Other 
studies using woody plants have shown that CO.sub.2 concentrations at 
triple ambient levels or higher produced no greater increase in growth 
than a concentration of about double ambient, and in some experiments 
growth at 900 .mu.l/l or more was less than at 675 .mu.l/l of CO.sub.2. 
Sionit and Kramer, supra at 71. Prior studies conducted in greenhouses 
suggest that 1000 .mu.l/l CO.sub.2 is optimum for most plants. Enoch and 
Kimball, Carbon Dioxide Enrichment of Greenhouse Crops, Vol. 1 (CRC Press 
Inc., Boca Raton, Fla., 1986). The use of CO.sub.2 at greater than the 
1000 .mu.l/l level is considered unnecessary and is often detrimental to 
the growth of plants. Id. 
Varying the wavelength of light provided to plants and the duration of the 
plants exposure thereto has also been explored. For example, 
photomorphogenesis is a well-documented phenomenon that is affected by the 
wavelength of light to which the developing plant is exposed. See, for 
example, Eskins et al., J. Plant Physiol., 147, 709-713 (1996); Seibert et 
al., Plant Physiol., 56, 130-139 (1975). However, the effect of combining 
the wavelength and enhanced carbon dioxide levels used to accelerate 
growth of woody plants is not an area that has been explored previously. 
Nor has combining either or both of these factors to enhance tissue 
culture propagation. 
The present invention is directed to the use of ultrahigh CO.sub.2 levels 
and light quality and quantity in plant propagation. Propagation may 
either be by conventional means (seedlings or cuttings) or by plant tissue 
culture (micropropagation or somatic embryogenesis). These and other 
aspects of the inventions are set forth herein below. 
SUMMARY OF THE INVENTION 
The invention relates to a method for propagating woody plant material 
comprising exposing the plant material to a pulse of filtered light. The 
culturing can occur in vitro such as in tissue culture, or in non-aseptic 
conditions, such as in soil or soiless medium. The woody plant material 
can be tissue culture, seedlings, cuttings, somatic or zygotic embryos, or 
microshoots from tissue culture. Preferably, the material is from 
sweetgum, sycamore, oak, green ash, Douglas fir, Populus spp., Eucalyptus 
spp., Pinus spp., Acacia spp., Picea spp., Larix spp., Abies spp., or 
Gmelina trees. In one embodiment, the pulse of filtered light is 
substantially only red light. In another embodiment, the method further 
comprises the step of culturing the plant material in excess of a 
concentration of carbon dioxide of 1,000 .mu.l/l. Preferably, the 
concentration of carbon dioxide is greater than about 1,000 .mu.l/l to 
about 50,000 .mu.l/l. Still more preferably, the concentration ranges from 
about 7,500 .mu.l/l to about 30,000 .mu.l/l. In still another embodiment, 
the method further comprises the steps of introducing the plant material 
into a chamber, and introducing and removing a nutrient medium into and 
from the chamber. 
The invention further relates to culturing woody plant material in excess 
of a concentration of carbon dioxide of 7,000 .mu.l/l, preferably from 
about 7,000 .mu.l/l to about 50,000 .mu.l/l. In one embodiment, the method 
further comprises the step of exposing the plant material to a pulse of 
filtered light. In one embodiment, the filtered light comprises 
substantially red light. In another embodiment, the method further 
comprises the steps of introducing the plant material into a chamber, and 
introducing and removing the nutrient medium into and from the chamber. 
Preferably, the pH value of the medium is monitored and adjusted to 
maintain a pH value of between about 4 and about 6. Still more preferably, 
the plant material is exposed to about sixteen continuous hours of 
unfiltered light out of every twenty-four hours.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention relates to the propagation of plant material under 
conditions of carbon dioxide concentration that preferably exceeds 1000 
.mu.l/l, more preferably is from about 3500 .mu.l/l to about 50,000 
.mu.l/l, yet more preferably is from about 7000 .mu.l/l to about 30,000 
.mu.l/l, and even more preferably is from about 7500 .mu.l/l to about 
30,000 .mu.l/l. One embodiment of the present invention includes use of a 
carbon dioxide concentration of from about 10,000 .mu.l/l to about 25,000 
.mu.l/l. The carbon dioxide used in this invention can be provided from 
cylinders of carbon dioxide from commercial providers, such as Air 
Products Incorporated, mixed with ambient atmosphere as appropriate on 
site, or from cylinders of ambient atmosphere adjusted to the appropriate 
concentrations of carbon dioxide. The levels of carbon dioxide in the 
chamber of a bioreactor can be measured using a carbon dioxide sensor, 
such as a CO.sub.2 electrode (Diamond General, Ann Arbor, Mich.). 
Varying the carbon dioxide concentration under which the plant material is 
grown is accommodated by use of a chamber in which the plant material is 
grown and protected from other species that could outcompete the plant 
material of the plants of interest. Such a chamber can be constructed from 
any suitable material, such material being impassable or substantially 
impassable to microbes or aqueous solutions, but permitting passage of 
light energy without substantial filtering thereof except with respect to 
use of filtered light, such as to provide substantially red light, as 
further discussed below. Accordingly, a preferred material for 
construction of the chamber is any untinted transparent autoclavable 
material, such as, but not limited to, standard untinted clear 
polycarbonate (e.g., Biosafe.TM. Containers from Nalgene) or glass (e.g., 
Pyrex.RTM.). An alternative embodiment provides a chamber having 
transparent tinted polycarbonate or glass, such as, for example, a 
red-tinted such chamber. The chamber is preferably contacted with one or 
more suitable ports for accepting or venting gas, such as, for example, 
ambient atmosphere or ambient atmosphere supplemented with varying 
concentrations of carbon dioxide, such as supplementation in increments of 
2500 .mu.l/l from about 1000 .mu.l/l to at least about 50,000 .mu.l/l. 
The chamber is also constructed with one or more suitable ports for 
accepting or removing culture medium, which can be pumped into or removed 
from the chamber by means of mechanical pumps, such as a peristaltic pump, 
or by means of hydrostatic pressure. The volume of the chamber is 
preferably at least about twice the volume of the culture medium employed 
when introduced and resident in the chamber ("the resident medium 
volume"), wherein the additional volume not occupied by the plant material 
is predominantly situated above the plant, thereby providing "head room" 
for growth and maximal gas exchange of the plant material. More 
preferably, the chamber volume is at least about 4-5 times that of the 
resident medium volume; yet more preferably at least about 10 times; and 
even more preferably at least about 50 times. The resident medium volume, 
as herein defined, is at least the amount of culture medium required to 
submerge the inert substrate on which the plant material is supported in 
the chamber of the bioreactor, such that at least the bottom side of the 
plant material is in contact with the culture medium. Irrespective of this 
definition which is provided for determination of the volume of the 
chamber, in some embodiments, a greater proportion of the plant tissue is 
in contact with the culture medium, such that, for example, the plant 
material can be partially or entirely submerged in the culture medium. 
An embodiment of the bioreactor of the present invention is diagrammed in 
FIG. 1. As shown in FIG. 1, the bioreactor growth chamber 100 consists of 
a transparent base 101 and a transparent cover 102, between which is a 
silicone gasket 103. The chamber 100 includes ports 104 used as air vents 
for exchange of gases. Toward the bottom of the transparent base of the 
bioreactor is another port 105 that is attached to a peristaltic pump 106 
by silicon tubing 107, which in turn is attached to a medium reservoir 109 
having at least one air vent 110, which attachment is by means of another 
silicon tube 108. 
Plant material as used herein refers to any plant or portion thereof, 
including but not limited to whole plants, including cuttings and rooting 
cuttings thereof, seedlings, including cuttings and rooted cuttings 
thereof; tissue cultures, including cultures of roots, shoots, callus or 
other embryonic tissues, including somatic embryos, and the like, 
including any portions or explanted cultures thereof; and microshoots from 
tissue culture. Plant material used in the context of the present 
invention preferably is or is isolated from, but is not limited to, woody 
perennials, such as, for example, plants and tissue cultures of hardwoods 
and conifers. Preferred woody perennials include, but are not limited to 
sweetgum; sycamore; oak; green ash; cottonwood; loblolly, slash, or 
radiata pine; black, red, white, sitka, or interior spruce; European, 
Japanese or Western Larch; and Douglas Fir. More broadly, woody perennials 
preferably used in the context of the present invention include species of 
the following genera: Populus, Eucalyptus, Pinus, Acacia, Picea, Larix and 
Gmelina. Preferred conifers include Pinus spp., Picea spp., and Larix 
spp., including Douglas Fir. Although preferred embodiments discussed 
herein include application to the aforementioned trees, the present 
invention is intended for use with any plant material, including, without 
any intention of limitation, any herbaceous or woody perennial. 
Preferably, the culture conditions used preclude or largely preclude the 
introduction of fungal or bacterial species other than plant material of 
the plant of interest and any symbiants, if any, required for growth of 
the plant material; or if such fingal or bacterial species is introduced 
that could retard or overgrow the plant material of the plant of interest, 
then suitable conditions are used that will retard the growth of the 
undesirable fungal or bacterial species relative to that of the plant 
material of the plant of interest, 
The plant material is preferably placed into a chamber on an inert 
substrate, where the plant material can be exposed to light of varying 
wavelengths and intensities for defined periods of time. Medium containing 
nutrients sufficient for growth of the plant material is presented to the 
plant material by periodically immersing the inert substrate in the medium 
for a defined residence time as discussed below, thereby placing the plant 
material in contact with the medium, followed by the substantial removal 
of the medium from the inert substrate; the remainder of the time, i.e., 
between the residence times of immersion of the inert substrate in the 
nutrient medium, the plant material is in contact with the inert substrate 
and medium that is withheld by surface tension characteristics of the 
medium on the inert substrate. The inert substrate can be any suitable 
absorbent or non-absorbent material, and is preferably a non-absorbent 
material such as, but not limited to, glass, ceramic, stone, plastic; and 
the substrate can be any suitable shape or size, including spheres, cubes, 
or random shapes, each having an approximate longest dimension of length 
or diameter of, for example, from about 1 mm to about 5 mm. 
Airflow into the chamber is preferably controlled such that undesired 
microorganisms are not introduced and the carbon dioxide concentration is 
held at a preferred level. Accordingly, the airflow into or out of the 
chamber is screened preferably by a filter having a pore size that 
precludes or substantially precludes passage of a microbe, such as that of 
between about 0.2 .mu.m and 0.45 .mu.m pore sizes. Similarly, the 
introduction of medium into or out of the chamber preferably includes 
sterilization of same prior to entering the chamber and, upon recovery 
from the chamber, prior to reentry into the chamber. Such sterilization of 
the medium can be effected by any suitable method, such as, but not 
limited to filtering, exposure to ozone or ultraviolet light, or heating, 
such as in an autoclave. It is contemplated, however, that the chamber and 
medium as recited herein sufficiently retards the growth of undesirable 
microbes and that, in one embodiment, no sterilization between periods of 
introducing the nutrient medium into the bioreactor is required. 
In a first embodiment, the present invention relates to a method for 
propagating plant material comprising culturing the plant material in a 
concentration of carbon dioxide in excess of about 7000 .mu.l/l, more 
preferably, in excess of about 7500 .mu.l/l. Any plant material can be 
subjected to the inventive method, as noted above; however it is preferred 
to select plant material that is free or substantially free of 
contaminating bacteria or other microbes. Such selection can be effected 
using any method known in the art, such as, for example, incubating the 
plant material on standard bacterial and/or fungal growth plates, and 
selecting those specimens of plant material from which no or few 
deleterious bacteria or other microbes are detected on the growth plates. 
Preferred ranges of carbon dioxide concentrations used in the context of 
the present invention are from about 7000 .mu.l/l to about 50,000 .mu.l/l; 
and more preferably, from about 7500 .mu.l/l to about 30,000 .mu.l/l. 
Preferably, the first embodiment includes culturing the plant material in 
a concentration of carbon dioxide in excess of 7000 .mu.l/l of carbon 
dioxide and in the presence of substantially only red light applied for 
varying time intervals (from seconds to weeks) during a photoperiod of 
unfiltered light. The photoperiod of the unfiltered light can be for any 
suitable portion of a day, including continuous illumination. However, 
preferably, the exposure period is from about 12 to about 20 hours, more 
preferably from about 14 to about 18 hours, and yet more preferably about 
16 hours per day. This photoperiod is then interrupted by exposure to 
substantially red light for varying time intervals. 
The first embodiment preferably includes: (a) introducing the plant 
material into a chamber that includes the carbon dioxide; and (b) exposing 
the plant material to pulses of substantially only red light during the 
photoperiod of unfiltered light. It is further preferred that the first 
embodiment includes (c) introducing a nutrient medium into the chamber 
followed by (d) removing the nutrient medium from the chamber. Preferably, 
the nutrient medium has a pH of between about 4 and about 6. More 
preferably, the medium has a pH of from about 5 to about 6. The pH can be 
maintained via use of buffering agents known in the art or by measurements 
and adjustments over time using, for example, a pH titrant, such as an 
acid or base. Preferably, the medium is stored in a reservoir connected to 
the chamber by a conduit. The reservoir can have any suitable dimensions, 
and can be of any suitable shape, although generally, the reservoir will 
be a standard sterilizable container capable of holding at least about 500 
ml of liquid. The conduit is constructed from any suitable material, the 
suitability of which is determined by its flexibility, ability to be 
sterilized, and characteristic of not imparting material into the fluid 
being conducted by it. Such a suitable material includes polypropylene, 
polycarbonate, silicon rubber and the like. 
The medium used preferably includes nutrients that foster growth of an 
explanted plant tissue, such as, for example, the macro- and 
micronutrients set forth in Murashige & Skoog, Physiol. Plant., 15, 
473-497 (1962), which are hereinafter referred to as "MS salts." MS salts 
used in the context of the present invention include suitable 
concentrations of ammonium nitrate, boric acid, calcium chloride, cobalt 
chloride, cupric sulfate, Na.sub.2 -EDTA, ferrous sulfate, magnesium 
sulfate, manganese sulfate, molybdic acid, potassium iodide, potassium 
nitrate, potassium phosphate monobasic, sodium nitrate, sodium phosphate 
monobasic and zinc sulfate. Minimal MS salts used in the context of the 
present invention preferably include the following as the recited 
concentration: NH.sub.4 NO.sub.3 (1650 mg/l); KNO.sub.3 (1900 mg/l); 
CaCl.sub.2 2H.sub.2 O (440 mg/l); MgSO.sub.4 7H.sub.2 O (370 mg/l); 
KH.sub.2 PO.sub.4 (170 mg/l); KI (0.83 mg/l); H.sub.3 BO.sub.3 (6.3 mg/l); 
MnSO.sub.4 4H.sub.2 O (22.3 mg/l); ZnSO.sub.4 7H.sub.2 O (8.6 mg/l); 
Na.sub.2 MoO.sub.4 2H.sub.2 O (0.25 mg/l); CuSO.sub.4 5H.sub.2 O (0.025 
mg/l); CoSO.sub.4 6H.sub.2 O (0.025 mg/l); Na.sub.2 EDTA (37.3 mg/l); 
FeSO47H2O (27.8 mg/l). Additionally, other components can include glycine, 
glutamine, myo-inositol, nicotinic acid, pyridoxine HCl, sucrose, and 
thiamine, for example. Such a medium can also include components to cause 
or foster differentiation or dedifferentiation of the explanted tissues 
being propagated in the chamber. Such components include, but are not 
limited to, auxins, cytokinins and abscisic acid. As noted, the medium 
preferably also is adjusted to a suitable pH range that is preferably 
between about 4 and about 6. In a preferred embodiment, the nutrient 
medium includes suitable buffering agents for maintained the preferred pH 
range. Suitable buffering agents preferably have a pKa between about 4.5 
and about 5.5, and include, but are not limited to, citric acid, 
N-morpholino-ethansulfonic acid, potassium hydrogen phthalate, and benzoic 
acid. 
The chamber in which the plant material is being incubated can be held 
static with respect to the medium provided to the chamber. In such an 
embodiment of the invention, no additional medium is added to the 
reservoir while incubating the plant tissue. Alternatively and preferably, 
the aforementioned steps (c) and (d) are repeated, such that the residual 
medium left on the inert substrate and plant material after the medium is 
removed from the chamber is refreshed periodically. Preferably, the 
infusion or introduction of medium and removal of same thereafter occurs 
at about four (4) intervals during a 24 hour period (further described 
below), with a residence time of the medium in the chamber of between 
about one (1) and one hundred twenty (120) minutes, more preferably 
between about fifteen (15) and sixty (60) minutes, yet more preferably 
between about fifteen (15) and thirty (30) minutes. 
The present method preferably further includes: (e) monitoring the pH value 
of the medium; and (f) adjusting the pH of the medium to maintain the pH 
between about 4 and about 6. Adjustment of the medium in response to the 
aforementioned measurements is accomplished by addition of suitable 
quantities of titrant, i.e., acid or base, as appropriate. 
After removal of the nutrient medium from the chamber, an amount of medium 
remains in the chamber due to surface tension characteristics of and 
entrainment in the inert substrate, accordingly, the removal is referred 
to herein as "substantial" removal. The amount remaining preferably does 
not substantially impede atmospheric contact with the plant material such 
that adverse effects of impeding transpiration is preferably minimized. To 
the extent that availability of the medium becomes growth limiting in the 
intervals between flooding the chamber with medium, then the intervals are 
shortened accordingly to increase the rate of introducing medium into the 
chamber. The removal of the nutrient medium allows maximal contact of the 
cultured plant material with the preferably heightened carbon dioxide 
concentration of the atmosphere maintained in the chamber. 
The medium can contain agents to prevent or retard the growth of bacteria 
or fungae, such as an antibiotic or antimycotic. Suitable antibiotics 
include those that retard or prevent the growth of bacteria including, but 
not limited to carbenecillin, gentamycin, and streptomycin. Suitable 
antimycotics include those that retard or prevent the growth of yeasts, 
including but not limited to miconazole (Sigma Chemical Company, St. 
Louis, Mo.; Cat No. M3512). The antibiotics or antimycetics are included 
in the medium preferably at a concentration range of from about 25 mg/l to 
about 1,000 mg/l; more preferably from about 100 mg/l to about 750 mg/l; 
yet more preferably from about 350 mg/l to about 600 mg/l; most 
preferably, at about 500 mg/l. 
The plant material contained in the chamber is exposed to unfiltered light 
continually or for photoperiods that are preferably from about twelve (12) 
to about twenty (20), more preferably from about fourteen (14) to about 
eighteen (18), and yet more preferably about sixteen (16) continuous or 
interrupted hours out of every twenty-four (24) hours. Unfiltered light 
includes natural light, and light from artificial sources which includes 
all or substantially all of the wavelengths of natural light necessary for 
plant growth. Pulses of filtered light are included in this exposure, 
which filtered light can be provided by use of suitable filters applied to 
a suitable light source. Filters are available from commercial sources, 
such as Edmund Scientific Company, Barrington, N.J. Various light filters 
can be used to provide filtered light in the yellow, red, orange, green, 
blue, indigo and violet ranges of the visible light spectrum. Because of 
the typically imprecise filtering of inexpensive filters that are useful 
in the context of the present invention, it is contemplated that, for 
example, when red light is applied in the present method, that such red 
light is substantially only red light. By substantially only red light, it 
is intended that at least about 10% of the visible spectrum that is in the 
red range is included, more preferably, about 20% of the red range is 
included, yet more preferably about 50% of the red range is included. 
Preferably, at least about 50% of the light intensity used to expose the 
plant material is in the red range; more preferably, at least about 75%, 
yet more preferably at least about 80%. Pulses of filtered light can vary 
in intensity, from, for example, about three hundred foot-candles to in 
excess of ten thousand foot candles. The effects of the invention are 
preferably achieved with shorter pulses, for example, less than one 
second, of high-intensity light, for example, greater than 1,000 foot 
candles, or longer pulses, for example, about two (2) weeks, of low 
intensity light, for example, 500 foot candles. One skilled in the art can 
determine without undue experimentation the optimal duration of pulses of 
a particular light intensity and wave length. Repeated pulses can occur at 
regular intervals or at irregular intervals. 
In a second embodiment, the present invention preferably relates to a 
method for propagating plant material by exposure to concentrations of 
carbon dioxide in excess of 1000 .mu.l/l. The concentration of the carbon 
dioxide preferably ranges from in excess of 1000 .mu.l/l to about 50,000 
.mu.l/l; more preferably from about 3500 .mu.l/l to about 50,000 .mu.l/l; 
yet more preferably from about 7000 .mu.l/l to about 50,000 .mu.l/l; even 
more preferably from about 7500 .mu.l/l to about 30,000 .mu.l/l. 
The second embodiment further includes exposing the plant material to 
filtered light applied for varying time intervals (seconds to weeks) 
during a photoperiod of unfiltered light. 
The second embodiment is implemented preferably in the context of the 
chamber recited above with respect to the first embodiment. The container 
can accommodate a wide variety of plant material including but not limited 
to (a) the plant material in a chamber as recited above with respect to 
the first embodiment, (b) plant tissue cultures on semi-solid or liquid 
media, and (c) cuttings, microcuttings, or seedlings in soil or soilless 
media under non-aseptic conditions. 
Preferably, the second embodiment relates to a method for propagating plant 
material including introducing the plant material into a chamber and 
therein culturing the plant material in excess of 1000 .mu.l/l 
concentration of carbon dioxide, and exposing the plant material to pulses 
of substantially only red light during a photoperiod of unfiltered light. 
EXAMPLE 1 
This example sets forth methods used for obtaining various plant tissues 
for use in illustrating the present invention. 
Plant cultures and media used in illustrating the present invention 
included the following: Seeds of carrot (Daucus carota L. cv. `Danver's 
Half Long`), kale (Brassica oleracea L. cv. unknown), lettuce (Lactuca 
sativa L. cv. `Grand Rapids Lettuce`), radish (Raphanus sativus L. cv. 
`Scarlet Globe`), tomato (Lycopersicum esculentum L. cv. `Cherry Red`), 
loblolly pine (Pinus taeda) and thyme were surface sterilized in a 2.6% 
sodium hypochlorite solution (containing 2 drops of Tween-20 emulsifier 
per 100 ml solution) for 20 minutes and placed on the surface of basal 
medium ("BM"). Two seeds were cultured per vessel. Stock plantlets of 
citrus (Citrus macrophylla L. cv. unknown) were maintained as 
proliferating axillary buds on BM as source of shoots. A single 2-cm long 
shoot was cultured per vessel. The BM consisted of MS salts (Murashigi & 
Skoog, supra) plus (per liter): 0.5 mg thiamine HCl, 100 mg i-inositol, 
and 10 g agar (Difco Laboratories, Detroit, Mich.). BM with 0 and 30 g 
liter.sup.-1 sucrose was tested. The pH was adjusted to 5.7.+-.0.1 with 
0.1 N HCl or NaOH before the addition of agar, then melted and dispensed 
in 25-ml aliquots into 25 150 mm borosilicate glass culture tubes and 
capped with transparent polypropylene closures (Sigma Chemical Co., St. 
Louis, Mo.). Medium was autoclaved for 15 minutes at 1.05 kg cm.sup.2 at 
121.degree. C. and agar medium was then slanted at a 45.degree. angle 
while cooling. 
Sweetgum (Liquidambar styraciflua) shoot cultures were also used in 
illustrating the present invention, and were established from mature trees 
by the method of Sutter & Barker, Plant Cell, Tissue and Organ Culture, 5, 
13-21 (1985). 
EXAMPLE 2 
This example presents plant tissue culture experiments conducted to 
investigate the effects on growth of plant tissue of varying 
concentrations of carbon dioxide in the atmosphere and sucrose in the 
medium, using a carbon dioxide flow system. 
A CO.sub.2 flow testing chamber 201 was constructed from a 94.5-liter 
transparent polycarbonate Carb-X tote box and lid (Consolidated Plastics, 
Twinsburg, Ohio) (45 cm width.times.65 cm length.times.37.5 cm depth; 
94.5-liter capacity) (FIG. 2). A silicone tape gasket (112 cm 
long.times.6.3 mm wide.times.3.2 mm thick) (Furon, New Haven, Conn.) was 
attached to the lid. The box was modified by mounting three polypropylene 
spigots 202 to allow for the inflow and evaction of gases. Two 0.45 .mu.m 
air vents (Gelman Science, Ann Arbor, Mich.) were attached to two of these 
spigots 202 with silicone tubing to 1.6-mm inner diameter female barbed 
fittings (Ark-Plas Products, Flippin, Ark.). The box and lid were clamped 
with 12 equally spaced stationary binding clips (50 mm long). The CO.sub.2 
testing chamber was attached to a water reservoir 203 with silicon rubber 
tubing 209. The water reservoir 203 consisted of a 2.25-liter 
polycarbonate bottle containing 1.5-liter distilled water. Carbon dioxide 
was provided by gas cylinder 204 (National Welding Supply Company, Inc., 
Bloomington, Ill.) rated 99.8% pure and was mixed with room air flow 
produced by an aquarium pump 205 (Whisper 1000, Carolina Biological Supply 
Company, Burlington, N.C.) with a flow meter 206 (Cole Parmer Instrument 
Co., Niles, Ill.) to provide 350, 750, 1,500, 3,000, 10,000, 30,000, and 
50,000 .mu.L liters.sup.-1. The CO.sup.2 gas cylinder 204 was connected to 
a flow regulator 207 and a solenoid valve 208; all interconnections 
between components were effected using silicon tubes 209-212, as shown in 
FIG. 2. Carbon dioxide ranges above 10,000 .mu.L liter.sup.-1 were 
adjusted using a Model #3000 LIRA infrared Gas Analyzer (Mine Safety 
Appliances Company, Pittsburgh, Penn.) and CO.sub.2 ranges.gtoreq.3,000 
.mu.L liter.sup.-1 adjusted with the aid of a LI-6262 Li-Cor CO.sub.2 
/H.sub.2 O infrared gas analyzer (Li-Cor, Inc., Lincoln, Neb.). The 
CO.sub.2 and air streams were added at about 1,500 ml min.sup.-1 for 16 
hours photoperiod. Control cultures were given a stream of room air 
generated by the aquarium pump 205 and hydrated with the water reservoir 
203. In flow experiments, air flow rates were adjusted with gang value and 
flow meters to 250, 500, 1,000, 1,500 and 2,000 ml min.sup.-1. 
Carrot, kale, radish and tomato seeds (two per 25.times.150 mm tube) and 
citrus microshoots were planted in BM containing 0 or 3.0% sucrose and 
grown under 350, 750, 1,500, 3,000, 10,000, 30,000 and 50,000 .mu.l 
liter.sup.-1 CO.sub.2 within 94-liter transparent containers as shown in 
FIG. 2. Cultures were grown in a culture room maintained at 25.degree. 
C..+-.1.degree. C. and employed a photoperiod of 16 hr light/8 hr. dark. 
Light was supplied by a combination of fluorescent tubes (Coolwhite), 
metal-halide and incandescent lights at a photosynthetic photon flux 
density (PPFD) of 260 .mu.E m.sup.-2 s.sup.-1 at the vessel periphery. 
Ten to twenty replicates were planted originally, and experiments were 
repeated at least twice. After 8 weeks of incubation, data on culture 
fresh weight, shoot height, leaf number, leaf length, leaf width, root 
number, and root length were recorded and analyzed with 
Student-Newman-Keuls multiple range test (P&lt;0.1) when appropriate. Fresh 
weight data are reported in FIG. 3. Columns in the same sucrose 
concentration with the same letter in FIG. 3 were not significantly 
different. 
For radish and citrus, increasing the CO.sub.2 concentration to 1,500 
.mu.L/l was beneficial to growth regardless of the sucrose concentration. 
For carrot, kale and tomato, high concentrations of CO.sub.2 aided growth, 
but the optimum concentration of CO.sub.2 was dependent on the sucrose 
concentration. Optimum concentration of CO.sub.2 appeared to exist where 
above or below this concentration less growth (i.e. fresh weight) occurs. 
For example, citrus shoots grown in 0% sucrose exhibit maximum growth at 
the 10,000 .mu.L liter.sup.-1 CO.sub.2 level while kale seedlings grown in 
0% sucrose exhibit maximum growth at the 30,000 .mu.L liter.sup.-1 
CO.sub.2 level. The optimum CO.sub.2 level varied somewhat among species 
and media employed but generally 3,000 to 30,000 .mu.L liter.sup.-1 
CO.sub.2 levels were found to give the largest fresh weight increases 
(FIG. 3). Carrot exhibited maximum fresh weight increase of 9.5-fold on BM 
without sucrose with 30,000 .mu.L liter.sup.-1 CO.sub.2 ; while on BM with 
sucrose, a maximum fresh weight of only 1.7-fold occurred with 10,000 
.mu.L liter.sup.-1 CO.sub.2. Similarly, kale plantlets exhibited their 
maximum fresh weight response, a 6.5-fold increase, on BM without sucrose 
on 30,000 .mu.L liter.sup.-1 CO.sub.2 ; while on BM with sucrose only a 
1.7-fold increase in fresh weight occurred on 3,000 .mu.L liter.sup.-1 
CO.sub.2. Citrus shoots exhibited a maximum fresh weight increase of 
4.7-fold on BM without sucrose with 10,000 .mu.L liter.sup.-1 CO.sub.2 but 
on BM with sucrose only a maximum of 1.3-fold increase with 10,000 .mu.L 
liter.sup.-1 CO.sub.2. Radish plantlets exhibited a maximum fresh weight 
increase of 6.3-fold on BM without sucrose with 3,000 .mu.L liter.sup.-1 
CO.sub.2. Tomato plantlets exhibited maximum fresh weight increase of 0.8 
fold on BM without sucrose with 3,000 .mu.L liter.sup.-1 CO.sub.2 and a 
1.2-fold on BM with sucrose with 10,000 .mu.L liter.sup.-1 CO.sub.2. 
EXAMPLE 3 
This example illustrates in influence of carbon dioxide treatments on the 
growth of sweetgum shoot cultures. 
Sweetgum shoot cultures were grown in agar medium (containing 3% sucrose) 
as set forth in Example 1, in the presence of 350 .mu.l/l or 10,000 .mu.l 
carbon dioxide using the carbon dioxide delivery mechanism diagrammed in 
FIG. 2. After 8 weeks of growth under the specified carbon dioxide 
concentration, the sweetgum cultures were measured with respect to (1) 
leaf length in millimeters per culture, (2) fresh weight in grams per 
culture, (3) shoot length in millimeters per culture, and (4) number of 
leaves per culture, which data are presented graphically in FIG. 4. 
As seen in FIG. 4, each parameter measured is significantly greater for the 
high carbon dioxide exposed culture compared to the ambient atmosphere 
control. Both leaf length and shoot length doubled, number of leaves and 
fresh weight increased by nearly two-thirds and one-third, respectively. 
Exposure to high concentration CO.sub.2 clearly benefited growth. 
EXAMPLE 4 
This example sets forth results of an experiment that tested the effect of 
varying wavelengths of light on the growth of loblolly pine seedlings. 
The effect of red, blue, green, yellow, orange, and white or natural light 
was tested on 200 mm high loblolly pine seedlings. The results are shown 
in Table 1, wherein R stands for red filter, Y stands for yellow filter, O 
stands for orange filter, N stands for natural sunlight, B stands for blue 
light; G stands for green filter, S stands for l shade cloth and D stands 
for dark conditions only. These filtered light sources were tested for 
various durations and combinations to determine their optimum 
effectiveness (see Table 1). No beneficial difference in growth of these 
seedlings was observed for any of the tests conducted except for those 
seedlings subjected to treatment #21 (exposure to red filtered light for 4 
weeks followed by natural light). Continuous exposure to any other light 
filters did not give any better results than filter alterations (i.e. 
filter treatment followed by natural light). 
TABLE 1 
______________________________________ 
Filtered light regimens used with loblolly pine seedlings. 
Week # Original 2 4 6 8 12 # 
______________________________________ 
1 O N N N N N 10 
2 O O N N N N 10 
3 O O O N N N 10 
4 O O O O N N 10 
5 O O O O O O 10 
6 Y N N N N N 10 
7 Y Y N N N N 10 
8 Y Y Y N N N 10 
9 Y Y Y Y Y Y 10 
10 G N N N N N 10 
11 G G N N N N 10 
12 G G G N N N 10 
13 G G G G G G 10 
14 B N N N N N 10 
15 B B N N N N 10 
16 B B B N N N 10 
17 B B B B N N 10 
18 B B B B B N 10 
19 B B B B B B 10 
20 R N N N N N 10 
21 R R N N N N 10 
22 R R R N N N 10 
23 R R R R N N 10 
24 R R R R R N 10 
25 R R R R R R 10 
26 N S N S N S 10 
27 S N S N S N 10 
28 S N N N N N 10 
29 D N N N N N 10 
30 D D N N N N 10 
31 D N D N D N 10 
32 N R N R N R 10 
33 N B N B N B 10 
34 N O N O N O 10 
35 N Y N Y N Y 10 
36 N N N N N N 10 
______________________________________ 
EXAMPLE 5 
This example sets forth results of an experiment that tested the effect of 
ultra-high carbon dioxide levels on growth of loblolly pine seedlings. 
The flow-through CO.sub.2 system of FIG. 2 set forth in Example 2 was used 
to glow loblolly pine seedlings in soil under ultra-high carbon dioxide 
concentrations, with the variation that low humidity air was used and the 
air within the chamber was stirred with miniature electrical fans 
positioned in the CO.sub.2 chamber. Using the lowered humidity CO.sub.2 
chambers, pine seedlings were grown in several ultra-high CO.sub.2 
environments and exhibited substantially better results than without high 
CO.sub.2. Pine seedlings, .about.50-55 mm in height, were grown in 350 and 
10,000 .mu.L CO.sub.2 liter.sup.-1 for 45 days. Results of this experiment 
are presented in FIGS. 5 and 6. The benefit of the CO.sub.2 appears to be 
strongest after 30 days of treatment, as shown in FIG. 4, where the curves 
indicate increasing growth with respect to needles per plant and shoot 
length. The upper panel of FIG. 5 is a graph of shoot length measured in 
millimeters over time of the enhanced carbon dioxide treatment; the lower 
panel of FIG. 5 is a graph of needles per plant over the same time course. 
As shown in FIG. 6, which is a series of bar graphs comparing the 
influence of null versus ultra-high levels (0 versus 1%; 1% is equivalent 
to 10,000 .mu.l/l carbon dioxide) of carbon dioxide on various growth 
parameters of pine seedlings after 45 days of treatment, fresh weight and 
roots/plant increase dramatically, 223.7% and 285%, respectively, when 
grown in the 10,000 .mu.L CO.sub.2 liter.sup.-1 (i.e., 1% CO.sub.2) 
environment. In addition, number of needles/plant (38.3%), needle length 
(18.7%), root length (32.2%), and shoot length (59.6%) also increased by 
the percentages noted parenthetically. 
EXAMPLE 6 
This example sets forth results of an experiment that tested the effect of 
combining ultra-high carbon dioxide levels with varying the wavelength of 
light on growth of loblolly pine seedlings. 
Using the carbon dioxide flow chamber described in Example 5, several 
ultra-high levels of CO.sub.2 were employed with and without use of red 
filter on .about.85 mm high pine seedlings. The red filter was employed 
since it was found to stimulate growth in the older 200 mm tall seedlings. 
The results are portrayed in FIG. 7, where the upper panel shows the 
influence of CO.sub.2 on shoot length and the lower panel shows the 
influence of CO.sub.2 on axillary shooting, where the diagonally lined 
bars represent the results from inclusion of the aforementioned red filter 
and the blank bars represent the results from inclusion of normal light. 
As shown in FIG. 7, the high concentration of CO.sub.2 stimulated pine 
shoot length for all ultra-high CO.sub.2 levels tested. Red light 
similarly stimulated shoot length growth and in every case a synergistic 
response was found coupling red light and ultra-high CO.sub.2 
concentrations. Further, axillary shooting from the relatively small pine 
plantlets was substantially enhanced using 50,000 .mu.L CO.sub.2 
liter.sup.-1 and a red filter. 
EXAMPLE 7 
This example illustrates the response of sweetgum sterile shoot cultures to 
various culture environments. 
Sweetgum shoot cultures were prepared in accordance with Example 1 and 
grown in presence of based medium, i.e., the minimal MS salts set forth 
above. Growth was measured with respect to fresh weights of cultured 
tissue and shoots per cultures. The cultures were grown (1) on solid agar; 
(2) in liquid media; (3) in the bioreactor of the present invention, 
wherein the cultured tissue was placed on glass beads and soaked for 15 
minutes each time with the MS salts once, twice or four times per day; (4) 
in the bioreactor just recited with exposure of air only; or (5) in the 
bioreactor just recited with exposure of a 10,000 .mu.l/l concentration of 
carbon dioxide. 
The results are presented in FIG. 8, which is two bar graphs displaying the 
fresh weights and shoots/culture as a function of the procedure used to 
grow the cultures. Best growth responses were obtained using 15 minute 
soakings 4 times daily within the bioreactor coupled to periodic CO.sub.2 
aeration treatments. Worst growth, in terms of fresh weight and shoot 
number, was obtained in continuous liquid medium (second column on graphs 
of FIG. 8). Experiments were repeated at least 2 times and a 
representative replication is presented. Media was replaced every 4 weeks. 
Mean separation by Student-Newman-Keuls multiple range test. Columns with 
the same letter on top were not significantly different. 
Within the continuous liquid system, cultures quickly browned and died and 
did not exhibit any desirable growth responses at all. If we compare 
growth obtained with the agar medium (first column in FIG. 8 graphs) as 
our control standard cultures, cultures grown in the bioreactor can be 
seen to be superior regardless of the number of soakings administered 
(FIG. 8). Increasing the number of soakings from once daily to 4 times 
daily doubles the fresh weight and number of shoots produced per culture. 
Fresh weights and number of shoots/culture increased 10.9-fold when 
cultures were grown in the bioreactor and soaked 4 times daily compared to 
culture chamber atmosphere using charcoal filtered air (i.e. 350) or 
10,000 .mu.l/l liter.sup.-1 CO.sub.2 enhanced sweetgum culture growth. For 
example, culture fresh weight increased 11.9-fold and 16.3-fold, 
respectively, using the bioreactor with periodic air and CO.sub.2 
flushing, compared to growth obtained from sweetgum grown on agar medium. 
EXAMPLE 8 
FIGS. 9-12 present data from experiments designed to illustrate the 
influence of various light filters with or without the benefit of 
supplemental enrichment with 10,000 .mu.l/l liter.sup.-1 CO.sub.2. The 
light filters used were yellow, fire red, orange, light blue, dark blue, 
cherry red and blue green, and were purchased from ROSCO Corporation, Port 
Chester, N.Y. The plant cultures were prepared and grown in accordance 
with Example 1. 
Lettuce and thyme tissue cultures fresh weights increased dramatically when 
filters were supplemented with 10,000 .mu.l/l liter.sup.-1 CO.sub.2 
whether 3% sucrose was included in the medium or not. Various filters have 
different effects on growth depending on the species tested and 
supplementation with CO.sub.2. For example, with lettuce grown on basal 
medium (i.e., minimal MS salts recited above) with 3% sucrose, the fire 
red filter allowed for only modest growth when compared to control (i.e. 
no filter) but when supplemented with 10,000 .mu.l/l liter.sup.-1 CO.sub.2 
maximum fresh weights were obtained that compared favorably to all other 
treatments. Also, carrot grown under the blue-green filter expressed 
enhanced callusing when enhanced CO.sub.2 was administered. 
Filters were also found to influence pine seed germination as shown in FIG. 
12. Loblolly pine seeds were found to exhibit enhanced germination on 
blue-green filters compared to control treatments. It is of interest to 
note that while pine vegetation growth was promoted by red light, 
germination was promoted by blue-green light. Germination and vegetative 
growth are different physiological processes and accordingly respond 
differently to the stimulus provided by light coupled with CO.sub.2. This 
result, as well as the observation that lettuce and thyme each have a 
preferred wavelength/CO.sub.2 combination for maximum growth, illustrates 
that for each species (and physiological process under investigation) a 
variety of wavelengths must be tested empirically to find the optimum. 
Such testing is easily performed by one skilled in the art with readily 
available filters. 
EXAMPLE 9 
This example illustrates the response of sweetgum microshoots on soilless 
medium. 
Sweetgum shoot cultures were prepared by the method of Sutter & Barker in 
accordance with Example 1 and grown in the bioreactor of the present 
invention. Sweetgum microshoots were harvested at about 1 and 2 cm in 
length, dipped in a commercial rooting powder (0.37% IBA) and set in a 
soilless medium of equal parts peat:vermiculite and perlite. CO.sub.2 
concentrations of 350, 1,500, 3,000, 10,000, and 30,000 .mu.l liter.sup.-1 
CO.sub.2 were provided by means of the apparatus shown in FIG. 2. The 
results are present in Table 2. Rooting and survival were optimized at a 
CO.sub.2 concentration of 10,000 .mu.l/l. 
TABLE 2 
______________________________________ 
Influence of Various levels of Carbon Dioxide levels on Percent "Rooting 
and Survival of Sweetgum Shoots ex vitro. Shoots were grown in soil for 
4 weeks before data was taken.* 
CO.sub.2 
Concentrations Shoot length (cm) 
(.mu.L liter.sup.-1 CO.sub.2 
1 2 
______________________________________ 
350 50 a 63 a 
1,500 50 a 63 a 
3,000 63 a 69 a 
10,000 92 b 94 b 
30,000 63 a 63 a 
______________________________________ 
*Treatments sharing the same letter in the same column are not 
significantly different using the Fisher's Exact test (P &lt; 0.5). 
While this invention has been described with an emphasis upon preferred 
embodiments, it will be obvious to those of ordinary skill in the art that 
variations in the preferred devices and methods may be used and that it is 
intended that the invention may be practiced otherwise than as 
specifically described herein. Accordingly, this invention includes all 
modifications encompassed within the spirit and scope of the invention as 
defined by the claims that follow: