WO1990015526A1 - Tegument et methode de culture et de croissance de matiere organique - Google Patents

Tegument et methode de culture et de croissance de matiere organique Download PDF

Info

Publication number
WO1990015526A1
WO1990015526A1 PCT/US1990/002508 US9002508W WO9015526A1 WO 1990015526 A1 WO1990015526 A1 WO 1990015526A1 US 9002508 W US9002508 W US 9002508W WO 9015526 A1 WO9015526 A1 WO 9015526A1
Authority
WO
WIPO (PCT)
Prior art keywords
membrane
integument
mushroom
cellule
organic material
Prior art date
Application number
PCT/US1990/002508
Other languages
English (en)
Inventor
Malcolm Glen Kertz
Original Assignee
Agristar, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from PCT/US1989/002564 external-priority patent/WO1989012385A1/fr
Application filed by Agristar, Inc. filed Critical Agristar, Inc.
Publication of WO1990015526A1 publication Critical patent/WO1990015526A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/14—Bags
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/22—Transparent or translucent parts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/24—Gas permeable parts

Definitions

  • the present invention relates to a new and improved integument and method for culturing and growing organic material. More particularly, the invention relates to a new and improved integument and method for enhancing the growth and reproduction of living organic materials such as plant and animal cells including plant and mammalian tissue, seeds, plants, animals, bacteria and other microorganisms, and for preventing contamination from occurring during such growth and reproduction.
  • living organic materials such as plant and animal cells including plant and mammalian tissue, seeds, plants, animals, bacteria and other microorganisms
  • Tissue culturing is the process of growing cells in vitro and is used to grow both plant and animal cells. Tissue culturing technigues are commonly used in the early stages of the plant micropropagation process where it is desirable to rapidly produce plant cells. Improvements in tissue culturing techniques also have applications beyond the micropropagation of plants. Essentially the same culturing process is used to culture animal and even human tissue, such tissue being used in the fields of animal agriculture and human and veterinary medicine. Culturing of organic material other than plant and animal cells and tissue, such as bacteria, viruses and algaes, is also performed in vitro for both research and commercial purposes. Improvements in the procedures and apparatus used to reproduce and maintain these organisms would be beneficial, for example, to researchers and industry who require a large or steady supply of such material. Seed Germination
  • Seeds are normally germinated in soil.
  • the soil includes many biological contaminants which can contaminate the seed.
  • the seeds are exposed to the environment which if not conducive to growth, will cause the seed not to germinate or for the new plants produced by the seed to die. Also, without a controlled ambient environment, growth of the new plants may be restricted. Fungal and Bacterial Production
  • Mushroom production is a multi-step process and varies with the type of mushrooms being produced. For example, there are six primary steps in the production of the common table or Agaricus bisporus mushroom, namely (1) compost formation, (2) compost pasteurization, (3) spawning, (4) casing, (5) pinning, and (6) cropping. See P. Stamets, J. S. Chilton, The Mushroom Cultivator. Agarikon Press, Oly pia, Washington (1983) , for a general description of the mushroom production process.
  • spawn production is carried out in stoppered plastic or glass bottles. Glass bottles are reused in spawn production because of their expense. If grown in glass bottles, the spawn must be repackaged into non-breakable containers, such as plastic or paper bags, prior to shipment because of the chance of breakage during shipment which could leave hazardous broken glass in the spawn.
  • non-breakable containers such as plastic or paper bags
  • Another type of container is a bag with a porous membrane filter over one portion of the bag.
  • the porous filter allows gas exchange but also permits the entry of contaminants. Further, if spawn is grown in the filter bags, the spawn around the filter becomes desiccated due to moisture loss through the filter, is subject to microspore contamination, and does not grow uniformly. The spawn also tends to differentiate in growth character adjacent the filter area.
  • Conventional Growing Containers
  • Prior art culturing apparatus and processes have several deficiencies.
  • One of the primary problems is contamination. Any of a wide variety of micro ⁇ organisms, including viruses, bacteria, fungus, molds, yeast and single cell algae, can fill the living organic material during any of the various stages of growth and development.
  • the smallest of these biological contaminants are the viruses, the largest are the single cell algae.
  • a virus typically ranges in size from 0.1 to 0.45 micrometers although it is suspected that portions of the virus which are as small as 0.01 micrometers may separate from the virus and alone cause contamination.
  • Bacteria typically range in size from 5 to 100 micrometers, while fungi and molds are usually larger than 100 micrometers.
  • Yeast is larger than bacteria, with single cell algae, the largest of these biological contaminants, being larger than yeast.
  • the present invention includes a new integument and related process for culturing and growing living organic material.
  • the integument is made of a gas permeable, liquid impermeable membrane which allows gas exchange but seals out the biological contaminants in the ambient environment.
  • the membrane forms one or more cellules which contain the living organic material and media.
  • the cellule is sealed so as to completely enclose and seal off the living organic material from the ambient environment.
  • the integument is preferably made of a high density polyethylene, and more preferably of Chevron high density polyethylene 9650. The integument provides uniform gas exchange throughout the surface area of the integument and controls the moisture vapor transmission rate to inhibit desiccation of the living organic material.
  • the integument of the present invention is also liquid impermeable so that the nutrients or media, typically a liquid or semi-solid which sustains the living organic material's growth while in the integument, cannot escape and dry out.
  • the nutrients or media typically a liquid or semi-solid which sustains the living organic material's growth while in the integument, cannot escape and dry out.
  • a completely unexpected benefit of using the semipermeable integument is that the growth rates of the living organic material are dramatically increased. This increase is believed to occur because oxygen and carbon dioxide are available in greater quantities to the living organic material than prior art containers which impede gas exchange.
  • Figure 1 depicts a frontal view of the integument of the present invention
  • Figure 2 depicts a partial elevation cross-sectional view of the integument of Figure 1 taken along line 2-2 as shown in Figure- 1 with the material of the integument enlarged;
  • Figure 3 depicts a partial top view of the integument of Figure 1 with the material of the integument enlarged;
  • Figure 4 depicts a sample of living organic material being cultured and grown in the integument of Figure 1;
  • Figure 5 depicts the organic material from Stage 1 being multiplied during Stage 2 in a new integument of Figure 1;
  • Figure 6 depicts the growth of an individual organic material during Stage 3 in a new integument of Figure 1;
  • Figure 7 depicts an integument pack with individual cellules of the type shown in Figure 1;
  • Figure 8 depicts a perspective view of an alternative embodiment of the integument of Figures 1 and 7;
  • Figure 9 depicts a front elevation view of the integument of Figure 8 in the open position
  • Figure 10 depicts a side elevation view of the integument of Figure 9 in the open position
  • Figure 11 depicts a top view of the integument of Figure 9;
  • Figure 12 depicts a front elevation view of the integument of Figure 8 in the folded position
  • Figure 13 depicts a top view of the integument shown in Figure 12;
  • Figure 14 is a schematic drawing of a process for the manufacture of the high density polyethylene used for the membrane of the invention.
  • Figure 15 is a schematic view of the molecular structure of various polyethylenes
  • Figure 16 is a graph showing the moisture vapor transmission rate of three different Chevron polyethylenes versus film thickness
  • Figure 17 is a graph of the oxygen transmission rate of the three polyethylenes in Figure 16 versus fil .thickness.
  • Figure 18 is a graph of the carbon dioxide transmission rate of the three polyethylenes of Figures 16 and 17 versus film thickness;
  • Figure 19 is a front elevational view of an integument surrounded by a material handling guard
  • Figure 20 is an enlarged view of one corner of the open end of the integument/material handling guard shown in Figure 19;
  • Figure 21 is an enlarged view of one corner of the closed end of the integument/material handling guard shown in Figure 19;
  • Figure 22 is a sectional view of the integument/material handling guard of Figure 20 taken along the lines 22-22;
  • Figure 23 is a sectional view of the integument/material handling guard of Figure 21 taken along the lines 23-23. DESCRIPTION OF THE PREFERRED EMBODIMENT
  • integument 10 of the present invention for containing, culturing, and growing living organic material, such as plant and animal tissue and cells, plants, seeds, spores, animals, and microorganisms including bacteria, viruses, fungus, molds and single cell algae.
  • Integument 10 is made of a
  • membrane 12 which is a gas permeable, liquid and contaminant impermeable material.
  • membrane 12 of the integument 10 When sealed, membrane 12 of the integument 10 completely and entirely surrounds and encloses the living organic material from the ambient environment.
  • the integument 10 is made by folding membrane 12 over. at 14 such that two sides 16, 18 are formed. Sides 16, 18 are heat sealed at 24, 26 along the entire length thereof and adjacent to longitudinal edges 20, 22 of membrane 12 so as to form an envelope.
  • the envelope shaped integument 10 includes a cellule 30 forming an expandable chamber for containing the living organic material and nutrients.
  • the cellule 30 has an approximate average volume of 50 ml for most types of living organic material. As can be appreciated, the size and volume of the chamber of cellule 30 can be varied to host the particular organic material to be contained therein. Thus, cellule 30 may be of various sizes.
  • the cellule 30 has at least initially, an open end 28 formed by the terminal edges 32, 34 of membrane 12. End 28 serves as a port of entry of cellule 30 for receiving the organic material and nutrients.
  • integument 10 may be made of two individual and separate pieces of material such as a base material and a frontal material.
  • the bottom of cellule 30 is formed by heat sealing the frontal material to the base material near the lower terminal edges thereof as distinguished from the fold at 14 where a single piece of material is used as described with respect to Figures 1-3.
  • Composite integuments may be formed to take advantage of the strength of one material and the permeability to oxygen and carbon dioxide of the other, as an example.
  • the integument 10 can also be made tubular and cut into predetermined lengths with one end closed by heat sealing.
  • the inventive membrane 12 of the integument 10 permits the living organic material to breathe by osmosis and air to diffuse through the semipermeable membrane 12.
  • the integument 10 will maintain the growth and development of living organic material therein without any artificial internal or external environment, i.e. pressures or gas concentrations.
  • the cellule 30 need not be inflated nor must the integument 10 be placed in an incubator to create an artificial environment to maintain life within the integument 10.
  • the gas mixture and pressure of the atmospheric ambient environment around the integument 10 tends to equalize with the gas mixture and pressure within the cellule 30 of integument 10 by gas and moisture vapor exchange through the interstices of membrane 12.
  • each wavelength of light necessary to sustain life pass through the integument.
  • individual wavelengths of light in the range of 400 to 750 nanometers are required by individual photosynthetic agents, such as the chlorophylls, to provide the reactions necessary for life and growth of plant tissue and plants.
  • the uniformity and light cross-linking of the molecular structure of the material for membrane 12 provides a pathway of lesser resistance for light.
  • the material of membrane 12 is normally translucent, the material may be opaque for the growing of mushroom spores which do not require light.
  • Polyethylene has been found to be the preferred material for membrane 12.
  • Polyethylene a thermal plastic resin, is melt formable and can be produced as a thermal plastic film.
  • Polyethylene film is pliable and collapsible such that it can be stored and shipped in rolls. Further, polyethylene is 12 inexpensive, as compared to glassware, such that it is disposable.
  • the material for membrane 12 is preferably between 1 and 2 mils thick, and it is most preferred that the material of membrane 12 have a thickness of 1.25 mils.
  • the reduced thickness of the material for membrane 12 and the uniformity of molecular structure formed in part by the extrusion process for the material for membrane 12 enhances gas transmission.
  • the approximate 1.25 mil thickness of the material for membrane 12 substantially enhances the amount of light and the various individual wavelengths of light which are received by the tissue culture. If the membrane material 12 is much thinner than 1.0 mil, handling the membrane 12 is made more difficult, as the opposing surfaces of the material of membrane 12 tend to adhere to each other when formed in such thin films.
  • Plant tissue includes any plant cell, undifferentiated plant tissue, differentiated plant tissue, pre-hardened plantlet or plant micropropagated or tissue cultured in a biological contaminant free environment during any of the stages of micropropagation or tissue culturing including initial tissue culturing, tissue culture multiplication, tissue differentiation or hardening.
  • plants is a broader term including plant tissue as defined above and further including hardened plants which have completed the hardening 13 stage and can grow in a natural environment where the plant is exposed to biological contaminants.
  • Media is the plural for "medium”.
  • tissue culturing medium generically refers to any sterile nutrient employed in the tissue culturing process including, but not limited to, minimal organic medium, multiplication medium, and pretransplant medium.
  • medium or “nutrient” means any nutrient for growing plants including artificial or synthetic medium, hydroponic nutrient medium, tissue culturing medium or soil.
  • the material for membrane 12 of integuments 10, 50 and 90 is critical to providing the desired environment for the plant tissue and plantlet during the first three stages of micropropagation and in particular enhancing growth by permitting optimum gas exchange and light transmission.
  • Gas exchange for example, is needed for the necessary biochemical actions required for culture growth. Understanding the role of the gases and gas exchange requires an explanation of the utilization of each gas individually.
  • Photosynthesis is the biochemical process where green plants convert carbon dioxide and water into complex carbohydrates in the presence of light of a given wave length and intensity for a given period of time. The process is affected by a number of environmental factors including quality of light, availability of water, availability of carbon dioxide, temperature, leaf age and chlorophyll content of the tissue. Photosynthesis is also referred to as a carbon dioxide fixation. The exact chemistry of the process is complex but in essence, chlorophyll in the presence of carbon dioxide, water and light converts the carbon dioxide and water into complex carbohydrates that are in turn converted into sugars and utilized by the plant as a food source.
  • respiration This process is essentially an oxidation reduction reaction where oxygen serves as the oxidizer to the carbohydrates and sugars formed during the process of photosynthesis. Again, the exact chemistry involved is very complicated. However, the end result is a release of chemical energy necessary for continued growth of the plant.
  • photosynthesis, or carbon dioxide fixation a number of environmental factors affect the uptake of oxygen for the respiratory process. These include temperature, light, tissue starvation, availability of oxygen and tissue age. While respiration is believed to take place at all times in plant tissue, there is a noted increase in this activity in the absence of light. This is believed to be a result of the decreased creb cycle activity in the absence of light.
  • Oxygen uptake for use in respiration varies from species to species and while no generally accepted range has been established for plants in ideal environmental conditions, uptake of up to 350 microliters to 1,480 microliters per gram of fresh tissue has been recorded. There has been no direct correlation of fresh weight to oxygen uptake. There is also a difference in oxygen uptake from tissue to tissue within a given plant. Woody tissue and starch storage organs have the lowest uptake, while root tips and other regions containing meristematic cells have the highest uptake rate. This can be directly related to the activity of growth in a given area of the plant where the most active areas require the greatest energy production and consume the greatest amount of oxygen. From this, it is clearly defined that , the presence of available carbon dioxide and oxygen is essential to the continued growth of green plant tissue.
  • FIG. 4 to 6 the integument 10 is shown in each of the first three stages of micropropagation of plant tissue.
  • the port of entry at open end 28 is heat sealed at 36 along the entire length thereof and adjacent to the terminal edges 32, 34 of membrane 12 to close and seal cellule 30 containing the plant tissue and medium therein.
  • the plant tissue is completely and entirely encapsulated from the ambient environment and sealed from biological contaminants in the ambient environment.
  • Figures 4 to 6 schematically illustrate the integuments 10A, B and C investing the plant tissue and medium in each of the first three stages of micropropagation.
  • Figure 4 depicts the meristematic tissue 38 from a parent plant or cultivar invested within integument 10A together with suitable medium 40 such as Murashige Minimal Organic Medium manufactured by Carolina Biological Supply Company.
  • Figure 5 illustrates the use of another integument 10B during the second stage of tissue culturing.
  • the initial tissue culture 42 from Stage 1, or a portion of such tissue is transferred to cellule 3OB containing a suitable Stage 2 medium 44 such as Murashige Shoot Multiplication Mediums A, B and C manufactured by Carolina Biological Supply Company.
  • Figure 6 shows an individual plantlet 46 grown in Stage 2 enclosed by another integument IOC and placed in a medium 48 such as Murashige Pretransplant Medium manufactured by Carolina Biological Supply Company to stimulate cell differentiation and the growth of individual plantlets such as 46, each plantlet 46 developing roots 47 and foliage 49.
  • a medium 48 such as Murashige Pretransplant Medium manufactured by Carolina Biological Supply Company to stimulate cell differentiation and the growth of individual plantlets such as 46, each plantlet 46 developing roots 47 and foliage 49.
  • Integument pack 50 is made of membrane 12 and is formed similarly to integument 10 of Figure 1. For most culture investments, the integument pack 50 has dimensions of approximately 12 inches wide and 6 inches high. Integument pack 50 is 17 formed by folding membrane 12 over at 52 so as to form sides 54, 56. As distinguished from integument 10, sides 54, 56 are heat sealed along the entire longitudinal length thereof at 58, 60, 62, 64, 66, 68 and 70 to form six individual cellules 72.
  • tissue samples 74 and media 76 are shown invested in each of the cellules 72.
  • the plant tissue and media may be for any of the first three stages of micropropagation as represented in Figures 4 to 6.
  • the ports of entry at the upper end 78 have been heat sealed at 80 along the entire length of integument pack 50 to close cellules 72 after the tissue 74 and media 76 are inserted into the cellules 72.
  • An upper flap or band 82 may be formed at the upper ends 78 of membrane 12 for the purpose of suspending integument pack 50 in the vertical position.
  • Suitable connection means such as apertures 84, 86 may be provided through band 80 for attachment means such as drapery hooks or S-hooks to suspend integument pack 50 vertically.
  • the new and improved integument of the present invention permits the utilization of a new and improved process for micropropagation.
  • This process includes four stages as hereinafter described.
  • Stage l Initial Tissue Culturing
  • the cultivars or parent plants to be micropropagated are maintained under carefully controlled greenhouse conditions in an attempt to yield plant tissue which minimizes the growth of microorganisms and particularly any biological contaminants.
  • an area of the plant with meristematic (undifferentiated) tissue is identified, and a bulk sample, which includes the meristematic tissue, is removed from the parent plant. This area is usually where active growth takes place, such as at the tips of stems or at lateral buds (between the leaf apex and the connection to the stem) .
  • the meristematic tissue is excised from the bulk sample and transferred to the growing medium under a laminar flow hood which removes airborne contaminants.
  • a suitable media as distinguished from 10 ml in the prior art tissue culturing process
  • Murashige Minimal Organic Medium manufactured by Carolina Biological Supply Company
  • This medium is an agar-based substance containing all the required nutrients for tissue growth.
  • Integument pack 50, containing the media therein, is then rolled and sterilized in an autoclave. This procedure tends to close the open upper side 78 of cellules 72.
  • the integument packs 50 are unrolled and its cellules 72 opened one at a time prior to tissue placement.
  • a meristematic tissue sample typically a 0.2 to 1.0 mm cube, is then placed into an individual cellule 72 of integument pack 50, a single cellule being shown in Figure 4.
  • the ports of entry into cellules 72 again tend to immediately close, reducing the length of time that the samples are exposed to the environment and that contaminants can enter. Thereafter, the upper ends 78 of cellules 72 are heat sealed at 80, thereby forming a complete investment and envelope around the plant tissue. In this state, the plant tissue is completely impermeable to contaminants as distinguished from the prior art containers.
  • the integument packs 50 are then exposed to approximately 300-500 foot-candles of light during this first stage.
  • the inventive process Using the present inventive process, precise temperature and humidity conditions need not be maintained in the culture room.
  • atmosphere would be drawn into and expelled around the tops of the glass containers containing the tissue cultures, thereby increasing the risk of contaminations from airborne contaminants which had not been removed by the prior art air filtration system.
  • the 80% humidity level was typically maintained in the prior art in order to prevent the media from drying out through evaporation.
  • the inventive process as distinguished from the prior art process, can be carried out in an environment which does not require a sterile, filtered air-flow since each cellule 72 of the integument pack 50 is contaminant impermeable.
  • Stage 2 Tissue Culture Multiplication
  • Stage 2 the initial tissue culture resulting from Stage 1 is multiplied. Under the laminar flow hood, the cellules 72 of the integument packs 50 of Stage 1 are opened with a sharp sterilized knife and the tissue s ⁇ uitples, or portions thereof, are transferred to a second set of unused integument packs 50, an individual integument pack being shown in Figure 7. Multiplication of the tissue culture occurs by using a different media.
  • the media used for Stage 2 cultures differs from that used in Stage 1 culturing and includes hormones to induce rapid growth and multiplication of the tissue. Suitable Stage 2 media include Murashige Shoot Multiplication Mediums A, B, and C, available from the Carolina Biological Supply Company.
  • Stage 2 growth yields primarily non-differentiated tissue growth.
  • the cells in each tissue sample multiply rapidly during Stage 2 to form a cluster of primarily undifferen- tiated tissue cells, the size of which depends upon the plant variety.
  • the desired cell multiplication takes approximately 20 to 45 days, again depending upon the plant variety.
  • the integument packs containing the cultures are immersed in a solution of sodium hypochlorite, rinsed, returned to the laminar flow hood, opened, and the tissue is removed. The tissue is then subdivided by cutting into a number of small pieces, each of which will then be cultured. Each time the tissue samples are divided, the individual smaller tissue samples are inserted into cellules 72 of unused integument packs 50. All of these steps are performed in the laboratory under a laminar flow hood.
  • Stage 3 Differentiation and Plant Formation
  • the cellules 72 of the integument packs 50 of Stage 2 are opened and tissue samples therein are divided and transferred to a third set of unused integument packs 50 as shown in Figure 7.
  • a single plant tissue growing into a plantlet is shown disposed within each individual cellule 72 in Figure 7, during Stage 3, a plurality of plant tissues may be disposed within an individual cellule if desired. This may be done to save additional space.
  • the plantlets may be grown separately in the new integument packs 50, eliminating the need for plant separation and the damage associated with untangling roots and foliage of several individual plants.
  • Stage 3 the individual tissue samples grown in Stage 2 are placed in a media which stimulates cell differentiation and the growth of individual plantlets, each plantlet developing roots and foliage.
  • Suitable Stage 3 media includes Murashige Pre-Transplant Mediums, available from the Carolina Biological Supply Company.
  • the purpose of Stage 3 is to grow individual plantlets and prepare them for greenhouse culture.
  • the same size or a larger size integument pack 50 can be used.
  • the plants are still grown in the culture room during this phase of development, but they are placed under increased light conditions so as to promote photosynthesis and growth. Approximately 2000 foot-candles of light are provided.
  • the differentiation and growth process of Stage 3 requires between 20 and 45 days depending upon the plant variety.
  • the plantlets can be removed to the greenhouse to harden during the later portions of Stage 3 and need not be housed in a culture room for the entire Stage 3 period. This can significantly reduce the time normally required for the hardening process and reduce the size of the culture room.
  • Stage 3 Some commercial growers will purchase their plants upon completion of Stage 3. Many, however, will wait until the plants have completed Stage 4, the final production stage. If purchased at the end of Stage 3, the plants produced by the inventive process need not be immediately planted. They may be maintained for up to one month simply by keeping the plantlets in their integument packs under conditions of reasonable temperature and light. This is advantageous in commercial production where the
  • Stage 3 plants are sometime shipped directly to the grower, who may lack the time to plant them immediately.
  • the commercial grower since the plantlets have been removed from the sterile environment of the culture room, the commercial grower must immediately remove the plantlets from the shipping containers, rinse them to remove the media in which the contaminants can thrive, and then plant the plantlets immediately. Because the plantlets purchased by growers at the end of Stage 3 are shipped and maintained in the integument packs 50, they are contaminant impermeable and, therefore, without the danger of contamination.
  • the advantage in the new process is that the grower does not have to plant immediately. When the grower is ready to plant, he can simply slit the Stage 3 integument open, rinse and deposit the plantlet into the soil medium.
  • Stage 4 the plantlets are removed from the integument packs 50 of Stage 3 and are transferred to a greenhouse where they are individually planted in a soil medium.
  • the plant's tolerance to light must be increased so that the plant can adapt to its natural environment. This process is called “hardening" the plant.
  • the plant's tolerance to light is gradually increased in Stages 3 and 4.
  • Stage 4 the plants are exposed to up to 8,000 foot-candles in the greenhouse where growth and hardening is to take place. The exposure of the foliage of the plant directly to the atmosphere permits the plantlet to later grow in its natural environment without the protection of the integuments used in Stages 1 to 3.
  • the soil used in Stage 4 is typically a pre-sterilized peat moss mix. Depending upon the type of plant, most commercial plants remain in the greenhouse 30 to 90 days before they are shipped to the grower.
  • inventive process permits all stages of the micropropagation process to be less time consuming than their prior art counterparts, because the new and improved integuments are more easily and quickly handled. Thus, more tissue culture samples can be processed per day. Further, because the integuments consume less space than prior art containers, the costs associated with the culture room and the greenhouse are reduced.
  • FIG. 8 to 13 there is shown another embodiment of the integument of Figures 1 and 7 that is adapted and sized for the micropropagation of lettuce, spinach or other leafy vegetables.
  • the integument 90 for enclosing the tissue for a leafy vegetable is made of the same membrane 12 as that used for integument 10 or integument pack 50 as shown in Figures 1 and 7 respectively.
  • Integument 90 is extruded in tubular form having a circumference of approximately 24 inches.
  • the tubular membrane 92 is folded into quarter panels 94, 95, 96, 97 and one eighth panels 98, 100 and 102, 104, best shown in Figures 8, 11 and 13.
  • One eighth panels 98, 100 and 102, 104 are formed by folding quarter panels 95 and 97 at 106 and 108, respectively.
  • Quarter panels 94, 95, 96 and 97 were formed by folding tubular membrane 92 into quarter lengths, at folds 110, 112, 114, 116.
  • Folds 106, 108 are directed inwardly as shown in Figure 13 and one end 118 of tubular membrane 92 is heat sealed at 120 in the folded position as shown in Figure 12 to produce a cellule 122 to house the leafy vegetable tissue and media.
  • the cellule 122 has a volume of approximately 1000 ml which can be varied according to the particular leafy vegetable plant tissue grown therein.
  • the other end 124 of tubular membrane 92 is initially left open as a port of entry 126 to receive the leafy vegetable tissue and media.
  • Cellule 122 preferably includes a foliage chamber 130 and a root chamber 132 with an open neck 134 therebetween, best shown in Figure 10.
  • Chambers 130, 132 and neck 134 are formed by heat sealing portions of one eighth panels 100 and 104 to quarter panel 96 at 136 and 138 and by heat sealing portions of one eighth panels 98 and 102 to quarter panel 94 at 140 and 142. Additionally, upon expanding integument 90, heat seals 136, 138 and 140, 142 create creases at 106, 108 and 146 shown in Figures 8 and 9 to form root chamber 132. 26
  • the foliage chamber 130 and root chamber 132 of cellule 122 permit a separation of the foliage from the root system during growth and more particularly to separate the foliage from the media.
  • a plantlet is positioned within cellule 122 such that the foliage grows within foliage chamber 130 and the root system extends from the foliage chamber 130 down through neck 134 and into the root chamber 132 where the media is disposed.
  • the root chamber 132 is sized to contain approximately 50 ml of media.
  • the reduced neck portion 134 tends to secure a mature plant in position within cellule 122 since the plant's roots will grow into a mass having a size larger than the cross sectional area of neck portion 134. This growth of root mass also acts to impede the flow of media into the foliage chamber 130.
  • the integument and method of the present invention may be used to germinate seeds in a sterile environment.
  • the following is an example of growing lettuce from seed using the integument packs 50 of the present invention.
  • the media Murashige Minimal Organic
  • sucrose sucrose
  • 8 grams of agar The final pH is adjusted to 5.5. 5 ml of the media is then placed into the cellules 72 of integument packs 50 depicted in Figure 7.
  • Black-seeded Simpson lettuce seeds are wrapped in gauze and surface sterilized by sonicating for ten minutes in a 10% sodium hypochlorite solution to which two drops of a wetting agent had been added.
  • the gauze packet is then removed and rinsed three times in sterile distilled water, with each rinse lasting for three minutes.
  • the gauze packet is then placed on a sterile work surface under a laminar flow hood.
  • the seeds are planted in the cellules 72 of integument packs 50 with one seed per cellule 72.
  • each cellule 72 After each cellule 72 is filled, it is sealed, labeled and placed in the culture room where it is checked daily for growth and contamination.
  • the culture room is maintained at 80°F with sixteen hours of light and light hours of darkness per twenty-four period.
  • the integument packs are checked daily for contamination and germination.
  • the integument and method of the present invention may also be used for the growth, isolation and storage of bacterial and fungal cultures.
  • a fungus substrate, inoculated with an inoculant culture can be stored, grown or transported in these integuments. Because the integument is sealed and contaminant impermeable, other molds, bacteria or fungus which could alter or damage the culture cannot enter.
  • Another advantage of the integuments of the invention relates to the fact that fungus grows best in the presence of water.
  • the integuments of the invention inhibit desiccation. Examples of fungal and bacterial production and culture storage are described below:
  • a semisolid Minimal Organic Media with 30 grams of sucrose and 8 grams agar was prepared. The final ph was adjusted to 5.5. 5 ml of the media was then placed into cellules 72 of integument packs 50 depicted in Figure 7. The integument packs 50 were then autoclaved for 15 minutes at 250°F at 15 p.s.i. They were then placed in the laminar flowhood and allowed to cool. Several cellules 72 of integument packs 50 were inoculated with Rhizoctonia solani. This was done by inserting a sterile inoculation loop into a pure culture of Rhizoctonia solani, removing it and then inserting it into a cellule 72 of integument pack 50.
  • the inoculant loop was placed in the center of the cellule and moved down until contact was made with the media. The loop was then resterilized and the process repeated until all cellules 72 of integument pack 50 had been inoculated.
  • the integument packs 50 were heat sealed, labeled and placed in the culture room * This process was repeated using pure cultures of Rhizopus stolonifer and Penicillium italicum on several integument packs 50.
  • integument packs were inoculated in the following manner.
  • the first, third and fifth cellule 72 of the integument pack 50 was inoculated with Rhizoctonia solani, Rhizopus stolonifer and Penicillium italicum respectively, leaving cellule 2 and 4 containing only sterile media.
  • the integument packs were sealed and each cellule containing an inoculant was labeled.
  • the integument packs were placed in the culture room.
  • microorganisms live and grow anaerobically, i.e. without oxygen. Although no such experiments have yet been conducted, the integument for culturing these microorganisms can be made from less permeable materials than the polyethylene of the preferred embodiment so as to preclude a gaseous interchange between the ambient environment and the microorganisms within the integument. Similarly, some microorganisms prefer or require the absence of light. When culturing such microorganisms, an opa»que material can be used for the membrane.
  • the integuments 10 of the invention can also be used for growing or storing insects or insect cultures.
  • An insect can be placed in the integument 10 and grown.
  • butterflies from the larvae and cocoon stage can be produced in integuments.
  • the integuments 10 of the invention retain moisture, which the insects need for growth and life, and also permit gas exchange.
  • the integuments are also relatively low in cost and unbreakable, unlike flasks or test tubes which are conventionally used for insect storage and growth.
  • insect cells and tissue can be cultured in the integuments 10 of the invention.
  • Compost Formation The production of compost for the Agaricus bisporus mushroom is initiated by mixing and wetting the ingredients.
  • Nitrogen supplements and gypsum are preferably spread over the top of the ingredients as they are mixed. The ingredients are then removed and preferably stacked in piles.
  • the nitrogen supplements increase nitrogen content, and the gypsum minimizes greasiness and also increases the flocculation of certain chemicals in the compost. When gypsum is added, it is generally at about 20 to 80 pounds per ton of dry ingredients.
  • Organic nitrogen supplements commonly include brewer's grain, seed meals of soybeans, peanuts, or cotton, and chicken manure, among others. Other inorganic nitrogen supplements can also be added to synthetic
  • compost including ammonium nitrate and urea.
  • the inorganic supplements provide microflora in the compost with a readily available source of nitrogen for their growth and reproduction.
  • compost pasteurization the compost is pasteurized and the ammonia which formed during compost formation is removed.
  • compost for growing oyster mushrooms would not require any nitrogen supplements, and no ammonia would be produced.
  • Spawn includes colonized mushroom mycelium growing in a substrate conducive to the particular type of mushroom being grown. Spawn can be produced by filling an integument 10 with the substrate, sterilizing the substrate and the integument, cooling the substrate, inoculating the substrate with mushroom culture, and colonizing the substrate to produce spawn. The spawn then can be grown in the integument or refrigerated for storage. After the substrate is colonized with mycelium, it can be grown further to produce mushrooms or, in the case of Agaricus bisporus, mixed with compost for mushroom production, as hereinafter described.
  • a grain such 33 as rye, millet or wheat
  • Agaricus bisporus is generally used as the substrate for spawn production of the table mushroom, Agaricus bisporus, although almost any grain may be used.
  • the grain is mixed with water and chalk.
  • the chalk maintains pH control and keeps the spawn from sticking together.
  • Gypsum may also be added.
  • the grain can be raw or pre-cooked. If one is producing Shiitake mushrooms, sawdust, wood plugs, small wooden dowels, or another cellulose containing material is substituted for grain as the substrate.
  • There are still other varieties which prefer a combination of grain and a wood material, such as sawdust, or other cellulose based substrates. These latter types of substrates can be used to grow mushrooms directly from spawn without the need for compost.
  • an integument 10 may be filled with the substrate and spawn produced therein.
  • a spawn production pack 200 shown in Figures 19-23, is used in spawn production.
  • Pack 200 is made from two inner membranes 210, 212, which are preferably the same material from which integument 10 is made.
  • Two outer membranes 214, 216 surround the inner membranes. The inner and outer membranes are all bonded together at three sides of their perimeters to form a seal 215 along the three sides.
  • an innoculation port 220 is formed at one side 201 of pack 200.
  • Port 220 is formed by bonding inner membrane 210 to outer membrane 214 at 217 and inner membrane 212 to " outer membrane 216 at 219.
  • Membranes 210, 212 are preferably of a high- density polyethylene material being no more than about 0.4 mils, in thickness, which is thinner than
  • Membranes 210, 212 are made of the preferred high-density polyethylene material Chevron 9650 described hereinafter in great detail. Referring to the Chart on pages 46-47 hereof, such a material having a thickness of 0.50 mils has an oxygen transmission rate of 1143.5 (CC/100 IN.SQ./24 HRS.) and a carbon dioxide transmission rate of 2093.5 (CC/100 IN.SQ/24 HRS.).
  • a low density polyethylene, such as Chevron PE 5754 shown on the Chart, might be used for membranes 210, 212 to achieve an even greater gas exchange provided it will hold up during sterilization.
  • PE 5754 with a thickness of 0.625 mils actual guage has an oxygen transmission rate of 1556.5 (CC/100 IN.SQ./24 HRS.) which would achieve an even greater gas exchange.
  • the low-density polyethylene must, however, stand up under sterilization.
  • the thinner membrane of high density polyethylene is preferred because spawn production increases with greater gas exchange, as accomplished by the thinner membrane, and will withstand autoclaving. Spawn has a high biomass distributed throughout the substrate, and a high gas exchange through the membrane helps ensure gas exchange even in the spawn which is in the inner portions of the substrate.
  • Outer membranes layers 214, 216 have a multitude of holes 222 to facilitate gas exchange. Because spawn production generally takes place in two weeks or less, the increased moisture loss which results from use of a thinner membrane with better gas exchange is not problematic.
  • the outer membranes 214, 216 are formed of a polymer with greater thickness and rigidity than the inner membranes 210, 212 and preferably are made of the high-density polyethylene material Chevron 9650 with a thickness of between 2 and 3 mils.
  • the outer membranes protect the inner membranes from rupture and help maintain the shape of pack 200 even when it is filled with spawn. Also, by bonding the thin inner membranes to the respective thicker outer membranes at 217, 219, innoculation port 220 does not cling and close, as it otherwise would in the absence of the support provided by the outer membranes. Innoculation port 220 can be readily opened by the operator with only one hand, as is necessary to allow innoculation, because the innoculant is held in the operator's other hand.
  • a continuous sheet of integument 10 or pack 200 may be continuously filled with substrate by an automated filling system similar to that disclosed in U. S. Application Serial No. 278,681, filed December 1, 1988, and entitled "Automated System for Micropropagation and Culturing Organic Material", incorporated herein by reference.
  • the sheet of integuments 10 or packs 200 have one side 201 unsealed and open for the introduction of the substrate. A predetermined amount of substrate is introduced into each of the integuments or packs by the automatic filling system.
  • the integuments or packs make it easier to automate the substrate filling operation because the continuous sheet of integuments or packs can be dispensed from a roll and automatically filled. It is more difficult to automate the filling of individual filter bags.
  • the integuments or packs, filled with substrate, are then sterilized.
  • the substrate-filled integuments or packs are introduced into an autoclave where they are heated to a temperature of 250 ⁇ F under a 15 psi pressure. This step can also be automated.
  • the substrate-filled integuments or packs are then 36 removed from the autoclave and permitted to cool.
  • High-density polyethylene is preferred over low- density polyethylene because low-density polyethylene may melt or distend and distort during sterilization, particularly under heat such as in an autoclave.
  • the innoculant ports of the substrate-filled integuments or packs are then opened to receive mushroom culture to inoculate the substrate.
  • the opened substrate-filled integuments or packs can be inoculated by an automated inoculation system.
  • the automated inoculation system inoculates each substrate-filled integument or pack with a predetermined amount of mushroom culture.
  • the inoculated substrate-filled integument or pack is closed capturing air within the integument or pack and is then sealed by a heat sealer.
  • the substrate is incubated, preferably within about two degrees of 72°F, after the substrate is inoculated. Capturing air within the integument or pack at the time of sealing enhances the mixing of the substrate and inoculum.
  • the captured air provides additional open space within the integument for mixing. To accomplish mixing, the integument or pack is agitated while it is full of air.
  • the cultures grow in the substrate and form the thin thread-like cells which are the mushroom mycelium.
  • spawn for the Agaricus bisporus mushroom After spawn for the Agaricus bisporus mushroom is produced, it can be mixed with compost. Since compost is not a completely sterile substrate, there is no need to complete the growth of the mushrooms in a sterile integument.
  • the mushrooms can be grown directly on the spawn substrate, particularly for species other than Agaricus bisporus which do not require compost for growth.
  • the inoculated substrate-filled integument or pack may be pasteurized, for example, by chemical, steam, or heat treatment.
  • the sterilization and pasteurization of the substrate is advantageously carried out in the integument 10 or pack 200 of the invention. Thereafter, the substrate is inoculated and grown out in a sealed integument 10 or pack 200. Alternatively, the integument 10 or pack 200 could then be opened and the integument 10 or pack 200 could remain open during the oyster or straw mushroom growth phase.
  • the integument 10 or pack 200 can be kept sealed during the growth phase. In fact, it can be shipped while sealed so that it continues to grow during shipment. It can then be opened after arriving at the grower's location to complete the growing cycle. Alternatively, if it is a mushroom which grows in an open environment, the integument 10 or pack 200 can still remain sealed after inoculation and during shipment, so that the grower opens it to complete the growth cycle. Inasmuch as the integuments 10 or pack 200 are contaminant impermeable, mushroom species which heretofore could not be cultivated because of contamination problems during production, can now be produced.
  • the integuments 10 or packs 200 of the present invention are preferably used throughout the steps of spawning. As distinguished from growing the mycelium in conventional containers, i.e. , glass bottles or plastic filter bags, it is advantageous to produce spawn in the integuments 10 or packs 200 because they allow gas exchange all around the outer surface of the spawn. It has been found that spawn will grow faster when placed in an integument 10 or packs 200 which is larger than a glass container, due to the enhanced gas exchange. Further, there will not be non-uniform growth, as gas distribution around the spawn will be uniform. Further, unlike the problems associated with glass containers, there is no need for repackaging the spawn for shipment. Thus, labor is saved.
  • the compost is covered with a casing layer.
  • a casing layer As noted above, many species of mushrooms other than Agaricus bisporus do not require a casing layer. However, the straw mushroom, for example, produces at a faster rate if a casing layer is applied.
  • the casing layer's primary function is to induce sporophore production in quantity.
  • the casing layer also serves to protect the compost from desiccation, pests and diseases, and to provide physical support for the developing mushroom fruit bodies.
  • the casing material can be soil, preferably a clay or clay-loam subsoil. Sphagnum peat mixtures can also be used. Casing is usually done two weeks after spawning, at which time the compost is well colonized.
  • the casing can be applied to the substrate after inoculation.
  • the inoculated compost in the integument can be placed in an open container or an open integument 10 after casing.
  • the integuments 10 containing inoculated substrate can simply be cut open to serve 39 as open containers for the mushroom culture. No casing needs to be applied. Because the spawn is not mixed with compost, the mushrooms are produced directly on the substrate on which spawn production takes place, e.g. , on wood or a cellulose based substrate for oyster or Shiitake mushrooms.
  • Rhizomorphs which appear as thick strings, form, sequentially, mushroom initials, primordia, and pins. Initials develop as outgrowths on the rhizomorphs after the rhizomorphs have formed on the casing. Once an initial becomes visible, the structure is a pin. Pins grow into buttons, which ultimately become the mushroom fruit bodies.
  • Pins develop when the carbon dioxide content of room air is lowered to less than 0.08 pereent. This is done by introducing fresh air into the room. The relative humidity is kept high while the mycelium grows through the casing. For most species, pinning and fruit body development only takes place in the open environment, and cannot be done in a sealed integument.
  • the mushrooms are harvested in 3 to 5 day cycles. Each cycle is followed by a dormancy period of about 7 to 10 days in which there is no production. The cycle repeats itself, and harvesting can go on as long as the mushrooms continue to mature.
  • the integuments 10 are normally opened in the cropping phase in order to perform each harvesting cycle.
  • most types of mushrooms must be kept in an open environment throughout mushroom fruit body growth.
  • cauliflower mushrooms it is preferable from a labor savings standpoint if the integuments 10 are opened only once at the start of the harvest cycle, and then not resealed after each cycle.
  • the contents of the integument 10 can be kept sealed from the atmosphere even during the cropping phase. This can be accomplished by pushing ripened mushroom fruit bodies away from the substrate and towards the end of the integument 10, and creating a new seal along the integument 10 between the portion of the integument 10 housing these ripened mushrooms and the portion housing the substrate so that the integument 10 now effectively includes two sections sealed from each other. The section of the integument 10 containing the ripened mushrooms is opened, and these mushrooms are removed. The newly formed seal prevents exposing the substrate and the growing culture to the atmosphere once the integument 10 is opened, thereby preventing contamination. This arrangement is desirable for those mushrooms which fruit in a sealed environment, for example. Cauliflower mushrooms. Membrane Material 12
  • Polyethylene is the preferred material for membrane 12.
  • Linear polyethylene is preferred over branched polyethylene.
  • Linear polyethylene is distinguished from branched polyethylene by the method of manufacture in that linear polyethylene is polymerized in reactors maintained at pressures far lower than those required for branched polyethylene.
  • Linear polyethylene encompasses ultra-low-density. 41 linear low-density, high-density, high-molecular- weight high-density, and ultrahigh-molecular-weight polyethylene.
  • Linear polyethylene density varies with the quantity of comonomer used with ethylene. The comonomer forms short-chain branches along the ethylene backbone. The greater the quantity of comonomer the lower the density" of the polymer.
  • Polyethylene resins are characterized by their density.
  • Linear low-density polyethylene has a density generally between 0.916 and 0.940 grams per cubic centimeter.
  • High-density polyethylene is characterized by densities in the range of 0.940 to 0.965, with 0.962 grams per cubic centimeter as the practical upper limit.
  • Polyethylene resins are also characterized by a particular resin's molecular weight which is an inverse function of its melt index.
  • melt index is a basic physical characteristic of a particular polyethylene resin.
  • Most commercial high-density polyethylene resins have molecular weights in the range of 50,000 to 250,000.
  • High-density polyethylenes are available with molecular weight distributions ranging from narrow to broad Molecular weight distribution is generally expressed by the measurement of the resin's ability to flow under controlled conditions, i.e. its melt index.
  • High-density polyethylene resins have melt indexes ranging from 0.04 to 75 grams per 10 minutes.
  • High-density polyethylene is preferred over low- density polyethylene for membrane 12 in the culturing and growing of living organic material.
  • High-density polyethylene is a highly crystalline, lightweight thermoplastic.
  • High-density polyethylene is semipermeable. Its principle characteristics are stiffness, good environmental stress-crack resistance, chemical resistance, toughness (even at low temperatures) , dielectric properties, low- temperature properties, and relatively high softening temperature.
  • the particle form slurry reactor method is the most widely used and includes a supported transition metal catalyst slurried in a diluent that is circulated in a closed- loop reactor at a low pressure and low temperature. Ethylene, which is dissolved in the diluent, reacts with the catalyst to form solid high-density polyethylene particles. The paxticles are dried, combined with additives, and extruded as pellets.
  • High-density polyethylene can be processed by all melt forming methods, including extrusion, injection molding, rotational molding, blow molding, and powder coating.
  • the primary property affecting processing is melt index.
  • the largest amount of high-density polyethylene is consumed in blow molding applications.
  • High-density polyethylene resins with a melt index in the 0.1 to 1.0 range generally are used. See Modern Plastics Encyclopedia, published by McGraw-Hill, Inc. (1988) incorporated herein by reference.
  • the high density polyethylene at a thickness of 1.25 mils forms a molecular structure during the extrusion process which is especially useful as membrane 12 for integuments 10, 50, 90.
  • the high density polyethylene is made from linear crystalline polymers of suitable molecular weight with high tensile strength and extension modulus, a high degree of symmetry, strong intermolecular forces and a 43 controlled degree of cross-linking between layers.
  • the cross-links between adjacent layers of polymers are introduced to prevent the polymeric chains from slipping under applied stress. See Figure 15.
  • the lightly cross-linked adjacent uniform layers of polymers of the high density polyethylene for membrane 12 form interstices therebetween which allow the preferred diffusion and osmosis therethrough for the desirable gas exchange between the ambient environment and the living organic material. These interstices are smaller than .01 micrometers so as to preclude the passage therethrough of even the smallest microorganisms, such as viruses. It also provides rigidity to facilitate the transfer and handling of the organic material.
  • the membrane material 12 have a permeability to C0 2 of from 200 to 1190 cc/100 sq. in./24 hours at 1 atm. and a permeability to 0 2 of from 100 to 424 cc/100 sq. in./24 hours at 1 atm.
  • Chevron HiD-9650 the preferred material for membrane 12, in the preferred 1.25 mil thickness, has a permeability to C0 of approximately 450 cc/100 sq. in./24 hours at 1 atm. and a permeability to 0 2 of approximately 190 cc/100 sq. in./24 hrs. at 1 atm.
  • membrane 12 Other materials which have the desired gas permeability and contaminant impermeability are also available for membrane 12.
  • certain low density polyethylene is suitable and even allows greater gas permeability than the preferred high density polyethylene; however, such low density polyethylene cannot withstand the high temperatures of the autoclave (250° at 15 p.s.i. for example) and the material melts or is otherwise deformed in the - process. Accordingly, if such materials are used for membrane 12 they must be sterilized through other means.
  • the moisture vapor transmission rate is an important factor in the selection of materials for membrane 12. It is preferred that membrane 12 have a MVTR of from 0.2 to 0.684 gm/100 sq. in./24 hrs. at 1 atm.
  • Membrane 12 of other materials, thicknesses, and permeabilities can be used depending upon the length of time the living organic material is to be grown in that integument.
  • other polymeric materials may have a greater permeability than the preferred high density polyethylene, if the permeability is too great, water within the integument 10 vaporizes and passes through membrane 12 and out of the integument.
  • the preferred high density polyethylene for membrane 12 is, high-density polyethylene No. HiD-9650, manufactured by the Chevron Chemical Company of Orange, Texas.
  • Hi-D 9650 resin is a dry, fluffy powder as it exits the reaction process. This powder is run through a compounding extruder where it is melted and mixed with appropriate additives. As the 9650 polymer exits the compounding extruder, it is formed into round circular pellets which are subsequently packaged and shipped to customers for the manufacture of film through an extrusion process.
  • Chevron has been manufacturing and selling Chevron Hi-D 9650 since approximately 1984. Its prior uses include the manufacture of notion and millinery bags and as barrier packaging.
  • the Hi-D 9650 resin includes the following nominal properties:
  • Hi-D 9650 polyethylene film is manufactured from the Hi-D 9650 resin using the blown film extrusion process which is standard in the industry.
  • Hi-D 9650 blown film having a nominal film thickness of 1.0 mils includes the following nominal properties:
  • Chevron Hi-D 9650 is a high density, linear polyethylene copolymer resin made by the Phillip's loop reactor slurry process as depicted in Figure 14. Like all polyethylenes, Chevron Hi-D 9650 is a polymer of ethylene gas. To create Chevron Hi-D 9650, the ethylene gas can be reacted as a mixture with a comonomer, such as hexene or butene; hydrogen as a polymerization terminator; and a catalyst initiator. The reaction results in a long chain, slightly branched molecule of ethylene and the comonomer as depicted in Figure 15. The density of Hi-D 9650 is achieved by the amount of comonomer that is reacted with the ethylene. The greater amount of the comonomer, the lower the density of the resulting polyethylene.
  • the recommended commercial processing conditions for converting Hi-D 9650 resin to a film with a nominal film thickness of 1.0 mil includes a melt temperature of 430°p and a die gap of greater than 0.035 inches. A smaller die gap may be used.
  • GAUGE MILS.
  • GAUGE MILS.
  • the thinner the membrane the greater its transmission rate for these gases.
  • the low density material (Chevron PE-5754) has the highest transmission rate for each of these gases.
  • the preferred membrane 12 made from Chevron's HiD-9650 has a low moisture vapor transmission rate which is lower than the other materials tested.
  • a high moisture vapor transmission rate is acceptable for spawn production in pack 200. Because this higher MVTR is acceptable, a low density 48 polyethylene can be used as the inner membrane of pack 200.
  • Integument packs 50 were then placed in the culture area of the laboratory. At thirty day intervals, each integument pack 50 was weighed, and an average weight of an integument was calculated for each of the fifty samples of the same membrane material and the same mil thickness. This data was recorded and is reproduced in the above table. After one hundred eighty days of the experiment, the thirty day average weight was subtracted from the last recorded weight and the difference was recorded in the above table below the one hundred eighty day weight. As can be seen, the preferred membrane 12 of Chevron high density polyethylene HiD-9650 enabled plant material to grow at greater rates and for longer periods of time than the other materials tested.
  • the above examples demonstrate that the integument and method of the present invention yields dramatic improvements in the culturing and growing of living organic material. These same improvements will follow irrespective of whether the plants cultured are horticultural, agricultural or even aquatic in variety. It is believed that the invention will yield dramatic improvements in animal and human tissue culturing as well.
  • the integument and method described herein may be used for culturing microorganisms, such as viruses, single celled algae, fungus and bacteria, and in pre ⁇ venting the cultured microorganisms from escaping from the integument and contaminating or infecting others.

Landscapes

  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Sustainable Development (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Clinical Laboratory Science (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Tégument (10) et procédé y relatif pour la culture et la croissance de matière organique vivante (38). Ledit tégument (10) comprend une cellule (30) fabriquée dans une membrane (12) perméable au gaz, mais imperméable aux liquides et aux contaminants, qui isole et protège entièrement la culture (42) des contaminants biologiques de l'environnement. Cette membrane (12) permet un échange gazeux entre la matière organique vivante (38) et l'environnement pour favoriser une meilleure croissance et prévenir toute contamination.
PCT/US1990/002508 1989-06-13 1990-05-04 Tegument et methode de culture et de croissance de matiere organique WO1990015526A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US36532389A 1989-06-13 1989-06-13
US365,323 1989-06-13
PCT/US1989/002564 WO1989012385A1 (fr) 1988-06-14 1989-06-13 Tegument et procede de mise en culture et de croissance de matiere organique
AUPCT/US89/02564 1989-06-13

Publications (1)

Publication Number Publication Date
WO1990015526A1 true WO1990015526A1 (fr) 1990-12-27

Family

ID=26779742

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1990/002508 WO1990015526A1 (fr) 1989-06-13 1990-05-04 Tegument et methode de culture et de croissance de matiere organique

Country Status (2)

Country Link
AU (1) AU5727490A (fr)
WO (1) WO1990015526A1 (fr)

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993024013A2 (fr) * 1992-05-27 1993-12-09 Ecoscience Corporation Procede pour le stockage de cultures de champignons et de conidies
US5989898A (en) * 1992-05-27 1999-11-23 Ecoscience Corporation Method for storing fungal conidia
WO2006006166A2 (fr) 2004-07-14 2006-01-19 Glusense, Ltd. Capteurs et sources electriques implantables
US8012500B2 (en) 2000-01-01 2011-09-06 Beta-O2 Technologies Ltd. Implantable device
US8043271B2 (en) 2008-11-26 2011-10-25 Beta 02 Technologies Ltd. Apparatus for transportation of oxygen to implanted cells
US8444630B2 (en) 2000-01-12 2013-05-21 Beta-O2 Technologies Ltd. Oxygen supply for cell transplant and vascularization
US8821431B2 (en) 2007-09-07 2014-09-02 Beta O2 Technologies Ltd. Air gap for supporting cells
US9037205B2 (en) 2011-06-30 2015-05-19 Glusense, Ltd Implantable optical glucose sensing
US9446168B2 (en) 2010-06-07 2016-09-20 Beta-O2 Technologies Ltd. Multiple-layer immune barrier for donor cells
US9540630B2 (en) 2008-09-17 2017-01-10 Beta O2 Technologies Ltd. Optimization of alginate encapsulation of islets for transplantation
US10358629B2 (en) 2006-03-08 2019-07-23 Kwalata Trading Limited Regulating stem cells
US10575765B2 (en) 2014-10-13 2020-03-03 Glusense Ltd. Analyte-sensing device
US10871487B2 (en) 2016-04-20 2020-12-22 Glusense Ltd. FRET-based glucose-detection molecules
CN113875586A (zh) * 2021-09-28 2022-01-04 金华市农业科学研究院(浙江省农业机械研究院) 一种桃胚无菌自动培养装置及方法

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0471947A1 (fr) * 1990-06-29 1992-02-26 Sekisui Chemical Co., Ltd. Sac de culture

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3184395A (en) * 1962-10-01 1965-05-18 John H Brewer Apparatus and method for culturing aerobic microorganisms
US3955317A (en) * 1974-01-28 1976-05-11 The British Petroleum Company Limited Method of growing plant cells
US4063383A (en) * 1975-04-18 1977-12-20 H. J. Heinz Company Limited Production of mushroom spawn
US4118890A (en) * 1977-02-16 1978-10-10 Shore William S Plant package
US4311477A (en) * 1979-12-26 1982-01-19 Polyplastics Company, Ltd. Bag for cultivating mushrooms
US4400910A (en) * 1980-04-25 1983-08-30 Koninklijke Emballage Industrie Van Leer B.V. Method for protecting plants during transportation by packaging and article
US4463522A (en) * 1979-07-26 1984-08-07 Eckhart Lindemann System and apparatus for plant tissue culture propagation
US4769945A (en) * 1986-01-31 1988-09-13 Kirin Brewery Co., Ltd. Delivery unit of plant tissue

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3184395A (en) * 1962-10-01 1965-05-18 John H Brewer Apparatus and method for culturing aerobic microorganisms
US3955317A (en) * 1974-01-28 1976-05-11 The British Petroleum Company Limited Method of growing plant cells
US4063383A (en) * 1975-04-18 1977-12-20 H. J. Heinz Company Limited Production of mushroom spawn
US4118890A (en) * 1977-02-16 1978-10-10 Shore William S Plant package
US4463522A (en) * 1979-07-26 1984-08-07 Eckhart Lindemann System and apparatus for plant tissue culture propagation
US4311477A (en) * 1979-12-26 1982-01-19 Polyplastics Company, Ltd. Bag for cultivating mushrooms
US4400910A (en) * 1980-04-25 1983-08-30 Koninklijke Emballage Industrie Van Leer B.V. Method for protecting plants during transportation by packaging and article
US4769945A (en) * 1986-01-31 1988-09-13 Kirin Brewery Co., Ltd. Delivery unit of plant tissue

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
"Chevron Chemical Company Technical Data Sheet", published February 1986, by CHEVRON CHEMICAL COMPANY (HOUSTON, TEXAS, USA), see entire document. *
BIOTECHNOLOGY LETTERS, Volume 7, Number 7, issued July 1985, (Kew, England), KYBAL et al.: "A device for cultivation of plant and animal cells", pages 467-470, see especially page 468. *
EXPERIMENTAL CELL RESEARCH, Volume 84, issued January 1974, (San Diego, California, USA), JENSEN et al.: "Comparative growth characteristics of VERO cells on gas-permeable and conventional supports", pages 271-281, see especially pages 272-274. *

Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993024013A3 (fr) * 1992-05-27 1994-01-20 Ecoscience Corp Procede pour le stockage de cultures de champignons et de conidies
US5679362A (en) * 1992-05-27 1997-10-21 Ecoscience Corporation Packaged fungal culture stable to long term storage
US5989898A (en) * 1992-05-27 1999-11-23 Ecoscience Corporation Method for storing fungal conidia
WO1993024013A2 (fr) * 1992-05-27 1993-12-09 Ecoscience Corporation Procede pour le stockage de cultures de champignons et de conidies
US8012500B2 (en) 2000-01-01 2011-09-06 Beta-O2 Technologies Ltd. Implantable device
US8444630B2 (en) 2000-01-12 2013-05-21 Beta-O2 Technologies Ltd. Oxygen supply for cell transplant and vascularization
EP2532356A1 (fr) 2004-07-14 2012-12-12 Glusense Ltd. Sources d'alimentation implantables et capteurs
US7951357B2 (en) 2004-07-14 2011-05-31 Glusense Ltd. Implantable power sources and sensors
WO2006006166A2 (fr) 2004-07-14 2006-01-19 Glusense, Ltd. Capteurs et sources electriques implantables
US10358629B2 (en) 2006-03-08 2019-07-23 Kwalata Trading Limited Regulating stem cells
US8821431B2 (en) 2007-09-07 2014-09-02 Beta O2 Technologies Ltd. Air gap for supporting cells
US9463083B2 (en) 2007-09-07 2016-10-11 Beta-O2 Technologies Ltd. Air gap for supporting cells
US9540630B2 (en) 2008-09-17 2017-01-10 Beta O2 Technologies Ltd. Optimization of alginate encapsulation of islets for transplantation
US8043271B2 (en) 2008-11-26 2011-10-25 Beta 02 Technologies Ltd. Apparatus for transportation of oxygen to implanted cells
US9446168B2 (en) 2010-06-07 2016-09-20 Beta-O2 Technologies Ltd. Multiple-layer immune barrier for donor cells
US9037205B2 (en) 2011-06-30 2015-05-19 Glusense, Ltd Implantable optical glucose sensing
US10575765B2 (en) 2014-10-13 2020-03-03 Glusense Ltd. Analyte-sensing device
US10871487B2 (en) 2016-04-20 2020-12-22 Glusense Ltd. FRET-based glucose-detection molecules
CN113875586A (zh) * 2021-09-28 2022-01-04 金华市农业科学研究院(浙江省农业机械研究院) 一种桃胚无菌自动培养装置及方法

Also Published As

Publication number Publication date
AU5727490A (en) 1991-01-08

Similar Documents

Publication Publication Date Title
AU625573B2 (en) Integument and method for culturing organic material
US5171683A (en) Integument and method for micropropagation and tissue culturing
US4127965A (en) Method for growing wood mushrooms
WO1990015526A1 (fr) Tegument et methode de culture et de croissance de matiere organique
CA2069338C (fr) Methode et appareil pour la culture de plantes autotropes a partir de materiel vegetal heteroptrope
US9095099B2 (en) Container for cultivating biological materials
EP0418323B1 (fr) Tegument et procede de mise en culture et de croissance de matiere organique
US4977702A (en) Process for growing Pleurotus
KR20200012367A (ko) 꽃송이 버섯 재배방법
KR100897522B1 (ko) 느타리버섯 재배용 침공 멀칭비닐백 및 이를 이용한버섯재배방법
US20160120133A1 (en) System for Plant Development
US6306645B1 (en) Container for explant culture, method of using same, and process, mold and apparatus of making same
US20180206427A1 (en) Plant propagation system and method
WO1990010381A1 (fr) Procede de production de bacs de culture remplis de microcultures de vegetaux, et dispositif de mise en ×uvre dudit procede
JP4060773B2 (ja) きのこの人工栽培方法
WO1990015527A1 (fr) Tegument comportant des receptacles internes qui peuvent etre dechires
JP2699145B2 (ja) 成形パック入り種菌
CN1048010A (zh) 生长以及保存与运输用容器
JPH08294326A (ja) きのこ類の栽培方法
JPH0662662A (ja) 茸類の人工栽培方法
JPS60137213A (ja) 茸用培地の製造方法
JPH02156826A (ja) 植物の栽培方法およびその装置
JPH1175593A (ja) 植物培養法および容器
CN112088718A (zh) 米林灵芝菌种的一种制备方法
JPS59173021A (ja) きのこ類栽培のためにたい肥包装体に菌糸体を植えつける方法及びこれに用いる複合物

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AT AU BB BG BR CA CH DE DE DK ES FI GB HU JP KP KR LK LU MC MG MW NL NO RO SD SE SU US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE BF BJ CF CG CH CM DE DK ES FR GA GB IT LU ML MR NL SE SN TD TG

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

NENP Non-entry into the national phase

Ref country code: CA