Patent Application
20160120133
Tissue culture is used for plant propagation and genetic transformation. Tissue culture propagation, sometimes referred to as micropropagation, is the process of growing new plants from plant tissue that has been extracted from a parent plant. Horticulturists favor micropropagation as a growing method because it provides relatively high production efficiency, cleanliness and greater uniformity of plants. The process results in mass production of plants having certain desirable characteristics as substantially all of the plants produced are genetically identical to and have the desirable traits of the parent plant.
With genetic transformation, foreign DNA is introduced into a plant's genome by plasmids during co-culture, for instance via an Agrobaterium vector or particle bombardment. After selecting for successfully transformed tissue, cells regenerate shoots and root. Alternatively, transformed tissue may form bipolar embryos and then develop into plants. The transformed plantlets must be handled much like micropropagated plantlets to be brought to sexual maturity and used in breeding programs.
Micropropagation is generally described according to separate phases each of which includes growth stages. Phase I generally includes those stages in which the plant tissue is primarily heterotrophic. Stage I comprises initiation, in which explanted donor tissue is initiated in a growing media. Stage II comprises a multiplication phase in which nutrients and hormones are provided to enable rapid cell division and substantial growth of multiple plants from the explant.
In genetic transformation, Stage I explants such as immature seed embryos, or young leaf tissue are disinfested and introduced to a tissue culture medium, as in micropropagtion. In stage II of transformation, the tissues are induced to callus during co-culture with the vector, e.g., Agrobacterium, containing the foreign DNA. Following, the transformed cells are selected by transfer to a medium that would be toxic to any plant cells that did not integrate the foreign DNA. Regeneration and propagation of stage II tissues results in shoots that contain the successfully introduced foreign DNA.
In these early stages for both systems, it is very important to keep pathogens and biological pests from infesting the culture. Accordingly, the culture is generally held in an environment that shields the maturing plant from pathogens while also facilitating rapid and vigorous growth. In the first two stages of growth there are high metabolic requirements for energy consumption, but the plant tissue is not generally capable of carrying out adequate photosynthesis to meet this high demand for energy. Thus, these stages are accomplished heterotrophically. During Phase I, the plant tissue is typically exposed to adequate light intensity to signal chlorophyll development and organic carbon is obtained from sugar such as sucrose that is provided in a growth media.
In Stage III during the second phase of development, leaves and shoots expand and the plantlet becomes more photoautotrophic. During the latter phase the plant tissue derives energy when exposed to light, gases, water and essential nutrients through the process of photosynthesis. In Stage IV, the plant can be matured, often in a greenhouse, and the plant may begin to take on larger amounts of light and heat, developing roots that will be needed for transfer, for instance to a field, greenhouse or the natural environment.
The delicate state of the plant tissue, particularly in the early stages, has led to difficulties in successfully carrying out micropropagation on a large scale. For instance, the nutrients required during the early stages of growth are easily targeted by microorganisms that can destroy the young plantlets. Moreover, transplanting the plantlets from one growth media to another and/or from one growth environment to another between stages will often damage the developing plant tissue, leading to slower growth and development or even plant destruction.
One particularly difficult transition is when rooted plantlets are removed from gelled medium that is often utilized in early stage development as the soft roots are easily broken. In addition, the roots must be rinsed free of sugar-containing medium so they do not rot in soil, and the process of planting rooted plantlets in soil is tedious. Often the roots are cut off of tissue-cultured plantlets and the plantlets (micro-cuttings) are forced to root in soil under mist in a shaded greenhouse. Although this is damaging to the plantlet, economics forces require the use of micro-cuttings to be a preferred practice.
Attempts have been made to mitigate such problems. For instance, systems have been designed for maintaining the developing plant tissue in an isolated environment during early stage development. Unfortunately, many of these systems utilize hard plastic or glass containers for developing the plantlets, and there are disadvantages with the use of such containers as they are relatively heavy, and therefore typically are very costly to ship from the laboratory to a greenhouse. Further, these types of containers must be re-used many times to make their use economically viable. Re-use requires that the containers be shipped back to the laboratory as well as washed and sterilized before re-use, adding to their expense. Storage also presents a problem, as the containers are not compressible (and sometimes not even stackable) which requires a large volume of space for storage. Moreover, none of these rigid systems integrate the tissue-cultured plantlet with the transitions necessary for stage IV autotrophic growth.
Other systems have been designed that utilize flexible polymeric containers for early stage micropropagation. Problems still exist with these systems, however. For instance, gelled media utilized in the containers may lose integrity or liquid media in the base of the container can pool and waterlog developing root systems. Also, moving the plantlets from one container to another so as to provide the desired environment as the plantlets mature can damage or destroy the developing tissues. Moreover, in order to prevent infection of the developing plantlets, all of the components of a propagation system should be sterilized, and materials that could be otherwise useful in forming a flexible container system often do not hold up well to sterilization procedures. In addition, the flexibility of such containers leads to difficulties in storing and transporting the plants, for instance necessitating specially designed storage racks to secure the containers and the plants in the proper orientation.
What is needed in the plant propagation industry is a method and system for producing plants in a manner that addresses such problems. For instance, a sterilizable system that provides for light transmission and a steady nutrient supply throughout early stage development without the need for transplanting of the developing plant tissue to multiple successive culturing containers is desired. Furthermore, a system and method of safely transporting large numbers of plants easily, reliably, and at a minimum cost is needed. Additionally, what are needed are systems that can better integrate the laboratory with the greenhouse nursery.
According to one embodiment, disclosed is a system for plant development. The system can include a container and a support within the container. The container can include a base having a flat outer surface such that the container is self-standing. The container can also have a flexible wall that is semi-permeable. The entire container, e.g. the container wall and the base can be impermeable to biological microorganisms. The support can be formed of a rigid or semi-rigid material of fixed dimension that can support developing plant tissue.
A method of plant development is also described. The method can include sterilizing the plant development system. Beneficially, the sterilization can take place with the support held in the container, which can protect the support from damage during sterilization and prevent deformation of the container while maintaining the overall stability of the system. A method can also include placing plant material in or on the support, the plant material including totipotent plant cells, and sealing the container with the plant material held within the container and in or on the support. Following, the system can be maintained under conditions conducive to the growth and development of the plant material held within the system.
A full and enabling disclosure, including the best mode thereof, to one of ordinary skill in the art, is set forth more particularly in the remainder of the specification, including reference to the accompanying Figures, in which:
Reference will now be made in detail to various embodiments of the presently disclosed subject matter, one or more examples of which are set forth below. Each embodiment is provided by way of explanation, not limitation, of the subject matter. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present disclosure without departing from the scope or spirit of the disclosure. For instance, features illustrated or described as part of one embodiment, may be used in another embodiment to yield a still further embodiment. Thus, it is intended that the present disclosure cover such modifications and variations as come within the scope of the appended claims and their equivalents.
As used herein, the term “heterotrophic” generally refers to plant tissue, including cultured somatic embryos, that is incapable or at most weakly capable of photosynthesis. Heterotrophic plant material requires an extraneous source of carbon such as sucrose that is provided in a growth medium to provide energy and maintain normal growth and development at a desired rate.
As used herein, the term “autotrophic” with regard to plant material generally refers to plant tissue that is capable of photosynthesis. As a result, external energy-supplying compounds are not required in order for autotrophic plant material to sustain a normal growth rate.
As utilized herein, the term “callus” generally refers to a mass of unorganized and undifferentiated totipotent plant cells which, at least at a macroscopic level, are either unconnected or loosely connected, generally arising from culturing of an explant.
As utilized herein, the term “explant” generally refers to a piece of plant tissue excised from a donor plant for culturing in vitro as the source of cultured plant tissues.
As utilized herein, the term “plantlet” generally refers to a small plant with a shoot and root pole, but more immature than a seedling. A plantlet is usually heterotrophic, but may also be autotrophic.
As utilized herein, the term “somatic embryo” generally refers to a plant embryonic structure arising from an explanted somatic tissue, zygotic embryo or other totipotent plant tissue.
As utilized herein, the term “zygotic embryo” generally refers to a plant embryo that has developed directly from the zygote produced from the sexual fusion of gametes. For example, the embryo found in a seed is a zygotic embryo.
As utilized herein, the term “bud” generally refers to an organized mass of various plant tissues from which a shoot or flower will develop.
As utilized herein, the term “meristem” generally refers to a group of undifferentiated plant cells that divide to form more meristematic cells as well as somewhat differentiated cells capable of elongation and further development into plant organs and structures.
As utilized herein, the term “seedling” generally refers to a plant developed from a germinating seed to autotrophic growth.
As utilized herein, the term “embling” generally refers to a plantlet grown from a somatic embryo that is sufficiently developed for transplantation into soil.
As utilized herein, the term “rigid” refers to a material is not flexible.
As utilized herein, the term “semi-rigid” refers to a material that exhibits an amount of flexibility in that a surface of the material can deform from the original shape (e.g., planar) by an amount without cracking or breaking. For instance, upon deformation of a surface of a semi-rigid material from the original shape to an angle of greater than about 10°, 15°, or 20° from the original orientation of the surface, the semi-rigid material can crack or break. However, at a lower deformation angle, the material will not crack or break.
The present disclosure is generally directed to a plant propagation system that can protect developing plant tissue from microorganisms while supporting the tissue so as to allow for growth and development of a shoot and root without the need to transplant the plantlet in the early stages of development. The system is an inexpensive, single-use, multi-component system that can be assembled prior to sterilization and then sterilized in a single step that prevents damage and deformation of the components of the system. Moreover, the system can be efficiently shipped and stored without damage to system components both prior to and following location of developing plant tissue within the system. For example, the system can be utilized to transport large quantities of developing plants while preventing damage and maintaining sterility of the plantlets held in the system. As such, the system can be used to facilitate transportation of plant material to any desired location. For instance, some material is prohibited from international commerce due to accidental inclusions of biological contaminants with plant tissue or media. As plants held in the system can be maintained free from biotic contaminants and held on sterile substrata, quarantine regulations can be satisfied with plants transported internationally by use of the disclosed systems.
The system includes a flexible, self-standing container and a support that can be held within the container during one or more of sterilization, storage, shipping, and use (i.e., plant development) of the system. These two components can together provide many benefits to the system. For instance, the self-standing container can protect the support held inside from damage during sterilization, storage, shipping, and during plant development. Meanwhile, the support can provide an excellent growth and development medium for the plant tissue held in the system, can prevent deformation of the container during sterilization, shipping, etc., and can improve stability of the system during shipping, storage, and growth of plants. Moreover, the support can maintain the developing plant tissue prior to, during, and following development of the root structure, and the developing plant can be transplanted following the desired level of maturation in conjunction with (without removal from) the support, so as to prevent damage to the young plant at transplant. The development period for the plant within the system can include a period of growth under natural light when the plantlet or embling is protected from both microorganisms and desiccation. For example, a somatic embryo can be located in a system, developed into a photosynthetic seedling, and transplanted without ever being removed from the support.
Container 10 can include fold lines 20, 20′ on each side 14a, 14b, as well as a fold line 24 on the base. Fold lines 20, 20′, 24 can allow for convenient storage of the systems when not in use as the container can be folded and stacked with other containers prior to combination with the support of the system.
In one embodiment, container 10 can be formed from a single polymeric member, for instance an extruded tubular polymeric member that can be sealed at one end, for instance along fold line 24 via, e.g., a heat seal or by use of an adhesive. Following, the sealed end of the extrudate can be folded to form gussets 76, 78 according to known methodology. The gussets 76, 78 can then be adhered to the base 28, for instance by a heat seal or alternatively by use of a suitable adhesive. When an adhesive is used in formation of the container 10, the adhesive should be one that can withstand the sterilization procedures, described at more length below, and one that will not damage plant material held in the container via, e.g., leaching of the adhesive into growth media held in the container during plant development.
At least one of the container walls can be formed of a polymeric material that is semi-permeable while being impermeable to biological contaminants. More specifically, and with reference to
The wall(s) of the container can be liquid impermeable while allowing for respiration of the developing plant tissue held within the container. For example, in one embodiment, at least one wall of the container 10 can have a permeability to carbon dioxide (CO2) that is equal to or greater than about 100 cubic centimeters (cc) per 100 square inches (in2) per 24 hours (h) at 1 atmosphere (atm) pressure or greater. For example, the CO2 permeability can be from about 200 to about 1200 cc/100 in2/24 hours at 1 atm. The container wall(s) can have a permeability to oxygen (O2) of equal to or greater than about 100 cc/100 in2/24 hours at 1 atm, for instance from about 100 to about 450 cc/100 in2/24 hours at 1 atm. The moisture vapor transmission rate (MVTR) of the container (i.e., at least one wall of the container) can generally be equal to or less than about 1 gram (g)/100 in2/24 h at 1 atm. For example, the MVTR can be from about 0.2 g/100 in2/24 hours at 1 atm to about 0.7 g/100 in2/24 hours at 1 atm.
In addition, in order to shield the interior of the container from pathogenic organisms, the walls 12a, 12b, 14a, 14b, and base 28 can have a low porosity, so as to block pathogenic microorganisms from entry. For example, the walls 12a, 12b, 14a, 14b, and base 28 can generally have a pore size of about 0.2 micrometers (μm) or less, about 0.1 μm or less, or about 0.05 μm or less, for preventing the passage of pathogenic microorganisms.
The thickness of the walls 12a, 12b, 14a, 14b and base 28 can vary, but can generally be from about 1.0 mil to about 4.0 mils, for instance about 2.0 mils. If the membrane material is much thinner than about 1.0 mil, handling of the container may be more difficult, as the opposing walls 12a, 12b, 14a, 14b may adhere to each other. Of course, this may vary depending upon the specific material used to form the container 10 and in one embodiment the wall(s) may be thinner than about 1.0 mil. Beneficially, the relatively thin, translucent walls of a container 10 can provide excellent clarity and permit viewing of a tissue sample enclosed in the container, for instance by the laboratory staff. For example, the walls of a container may be thin and translucent as compared to wall materials of many prior art containers such as glass containers. The thicker structure of many previously known containers often hinders visibility, which can affect decision making regarding plant care as well as complicating the storage and transport of these heavier, bulkier containers.
The walls 12a, 12b, 14a, 14b of the container 10 can be translucent so as to allow the passage and diffusion therethrough of light rays having at least the wavelengths of from about 400 nanometers (nm) to about 750 nm. Wavelengths in this range are required by individual photosynthetic agents, such as the chlorophylls, in green tissue plants to provide the reactions necessary for life and growth.
The materials used to form the container 10 can also be capable of withstanding a sterilization procedure. For instance, the container 10 can be resistant to high temperature and high pressure sterilizing treatments such as autoclave treatment conditions including subjection to high pressure saturated steam at temperatures of from about 120° C. to about 140° C. and at a pressure of from about 15 pounds per square inch (psi) to about 30 (psi).
In general, any polymeric material that meets the desired characteristics including permeability and ability to withstand the heat and pressure of a sterilization procedure can be utilized in forming the container 10. By way of example, a high density polyethylene or a polypropylene can be utilized to form the container 10. For instance, a polyethylene having a density of from about 0.93 g/cm3 to about 0.97 g/cm3 can be utilized in forming the container. In one embodiment, at least one wall of the container can be formed of biaxially oriented polypropylene.
A biaxially oriented polypropylene is stretched in both the machine and cross directions so as to increase the strength and clarity of the material. According to one embodiment, a sequential biaxial orienting method can form a film that can be utilized in forming a container 10. By way of example, pellets or chips of a polypropylene resin can be supplied to an extruder and then heated and melted at a temperature of about 170° C. to about 320° C. The melt is extruded from a die and then cooled and solidified, for instance on a metal drum held at a temperature of from about 60° C. to about 140° C. to obtain a cast raw sheet with β-form crystals. Next, the cast raw sheet is made to pass between rolls rotating at different rates while maintaining the temperature of the cast raw sheet from about 100° C. to about 160° C. to stretch the cast raw sheet in the flow direction and gain, e.g., a three- to seven-fold length increase. After that, the resultant sheet is cooled. Following, the cooled sheet is directed to a tenter, and stretched in the width direction to gain, e.g., a three- to eleven-fold width increase while keeping the temperature of the sheet at about 150° C. or more. Finally, the resultant biaxially oriented sheet is relaxed and subjected to thermal fixing, followed by winding and further processing, for instance to form the container 10.
The container can optionally include a vent in a wall. For example, container 200 illustrated in
In the embodiment of
Alternatively, a vent may be open throughout use of the device with no closure or may have a separate closure. For instance, in the embodiment illustrated in
Referring again to
To affix the membrane vent 210 to the container, a slice or hole can be formed in the container and the membrane vent can be affixed to the container over the hole via, e.g., a heat seal, a suitable adhesive, etc.
In one embodiment, support 300 can be a polymeric foam that can be utilized as a substrate for plant growth. This is not a requirement, however, and support 300 may be formed of other materials that can provide a rigid or semi-rigid support of fixed dimension. For instance, support may be formed of a natural material that can include a natural binder or that is held together with a natural or synthetic binder. Examples of natural materials include mineral wool materials, such as rockwool or glass wool or Jiffy® Preforma®, which is formed of a natural material adhered with a synthetic polymer.
Support 300 can be a composite that can include an open-celled foam composition in conjunction with other materials. For instance, support 300 can be at least in part formed of a foam made from a phenolic, polyurethane, latex, urea-formaldehyde, or polyisocyanurate-based homopolymer or copolymer, with phenolic-based foam being utilized in one embodiment. The foam or natural material may be used in conjunction with one or more filler materials, such as peat. Materials for use as a support 300 are available on the retail market, for instance from Smithers-Oasis Company of Kent, Ohio. By way of example, Oasis® Horticubes® XL seed propagation medium can be utilized to form support 300. A foam support can be preferred in one embodiment as use of a foam support can make transplanting the grown plant easier, as a section of foam (e.g., a foam cube) can be easily broken off from the remainder of the support and the resulting element can then be directly transplanted into the soil. Other support materials may require that the support material be separated from the roots before transplanting, which can cause root damage and can make transplanting more difficult. A foam support such as the Horticubes® XL foam product breaks apart, allowing the roots to be washed of the growth medium without damage to the roots.
In one embodiment, the support can be pre-formed and then placed within the container. Alternatively, the support can be formed in situ. For instance, the support can be a porous polymeric material that can be cast as a liquid (e.g., a solution or dispersion) and then can cure within the container to form the support having fixed dimensions within the container.
The support 300 can have dimension stability and be self-supporting and rigid or semi-rigid. The material used as support 300 can be absorbent, but can also have water holding capacity, in that it releases water as well as absorbs it, making water available to the plant. It can be capable of withstanding conditions encountered during sterilization, such as by autoclaving. One advantage of a foam material is that it can cause minimal or no damage to the plant roots when the plant is removed from the material. Moreover, a foam material has a cellular structure providing air porosity that aids in the exchange of oxygen to the developing root structure. The support 300 may also be capable of being segmented into individual cells 310, as hereinafter described.
A support 300 can be designed with a plurality of individual cells 310, each of which can support a developing plant. The cells can be separable from one another, which can aid in transplant of the plantlets following initial development utilizing the disclosed system. For instance, in the illustrated embodiment of
As shown in
Following assembly, the entire system can be sterilized, for instance by use of an autoclave, according to standard practice. By way of example, an autoclave sterilization process may be utilized during which the autoclave may reach a temperature of about 250° F. (about 120° C.) at a pressure of about 15 psi.
An aqueous growth medium can be included in the system either prior to or following sterilization. Addition of the aqueous growth medium prior to sterilization may be preferable in some embodiments, as the sterilized system can be stored and shipped ready for use, and the end user need only insert the desired plant tissue (e.g., a callus, a microcutting, meristemic cells, etc.) into an opening 406 of the support 400 and close the top of the container. The top of the container 410 can be held in a closed arrangement either with a tight seal or a loose closure. For instance, the container top can be closed with a heat seal for a tight seal or more loosely, as with a clip, for a loose seal.
A growth medium can generally be any suitable medium as is known in the art, with preferred media depending upon the plants to be developed within the system as well as the growing conditions to be utilized during the time the plants are held in the system. By way of example, a growth media can include, without limitation, one or more of NH4NO3, KNO3, H3BO3, KH2PO4, KI, Na2MoO4.H2O, CoCl2.6H2O, CaCl2.2H2O, MgSO4.7H2O, MnSO4.H2O, ZnSO4.7H2O, CuSO4.5H2O, pyridoxine nicotinic acid, glycine, sucrose, and so forth. Plant growth media as are known in the art can be utilized such as, for example, micropropagation medium as described by Murashige and Skoog (MS 1962), supplemented with 3% sucrose, organic components as described by Linsmaeier and Skoog (1965), with 1 μM of a plant growth regulator such as meta-topolin. A plant growth media can be at a pH of between about 5 and about 7, for instance at pH 5.7. Of course, desirable pH of a media can depend upon the specific plant tissue to be developed by use of the system.
A medium containing mineral nutrients can be utilized in promoting the growth of the heterotrophic plants, but is not necessary in all cases. In addition, the nutrients and carbon and energy source may be mixed in dry powder or particulate form and thereafter water can be added to form the aqueous medium. The term aqueous solution or medium thus encompasses a solution formed by adding water to a support that contains particulate nutrients and other materials as well as a solution formed by mixing such materials with water and applying the formed mixture to the support.
The aqueous medium may also include one or more plant growth hormones to stimulate growth and development of plant structures, such as shoots or roots, from the plant tissue supplied to the system. While somatic embryos may have sufficiently developed rudimentary shoot and root so as to not require growth hormones in the medium, other types of heterotrophic plant material may not, such as micropropagated adventitious meristematic tissue, buds, or microcuttings. Hence, depending upon the particular type and state of development of the heterotrophic plant material embedded in the support, the addition of plant hormones such as auxins and cytokinins may be advantageous.
The amount of aqueous media included in a system can vary depending upon the length of time the system is to be utilized, the type of plant to be developed by the system, the particular growing conditions to be utilized, etc. For example, aqueous media can be included in a system in an amount of from about 0.3 milliliters per cubic centimeter of support (mL/cm3) to about 1 mL/cm3, for instance from about 0.4 mL/cm3 to about 0.9 mL/cm3. Of course, higher or lower amounts are likewise encompassed herein, depending upon the nature of the development process.
To utilize the system, a plant material such as vegetative buds, bulbets, miocrotubers, transformed tissue, or somatic embryos can be placed within the container, for instance when the plant tissue is capable of forming shoots, but ill-suited for autotrophic growth. The plant material to be developed in the system can be a somatic embryo or developed from a somatic embryon, but the system is not limited to development of only somatic embryos, and the heterotrophic plant material may be any viable unit of living plant material containing totipotent cells capable of growing under controlled conditions into a complete autotrophic plant possessing normal roots and shoots. One source of such heterotrophic plant material is a liquid culture of plant somatic embryos that can be derived from explanted zygotic embryos of a source plant. This process, such as described by Durzan and Gupta, Plant Science 52:229-235 (1987), involves several culture steps involving different gel and liquid media containing mineral nutrients, organic compounds to supply carbon and energy, specific plant hormones, and water. Other sources of suitable heterotrophic plant material are cultured meristematic tissue, explanted zygotic embryos, cultured bud tissues, totipotent callus tissues, and the like, produced by any of a number of currently practiced plant propagation techniques including micropropagation techniques, somatic embryogenesis, plant regeneration, genetic transformation, and so forth.
After the plant tissue has been placed in the system, the container can be sealed, for instance with a heat seal as illustrated in
The pressure inside the sealed container 501 can increase as compared to the external pressure upon folding over the top 502 and sealing the system. The amount of folding (i.e., the decrease in internal volume of the container) can be utilized to control the pressure increase in the system.
In one embodiment, illustrated in
The initial growth and development stages of the plant tissue can be carried out in a laboratory setting, for instance under a predetermined growth schedule and with controlled lighting sources. The systems can optionally be utilized for growth and development of the plant material within a greenhouse. For example, initial growth and development can be carried out in a laboratory setting, and the system can be moved to a greenhouse when the developing plant material is strong enough and further development can then be carried out in the greenhouse. Beneficially, the container of the system is water impermeable and as such the exterior of the container may be intermittently misted without diluting the growth media held in the container. Upon misting, the evaporation of water from the container's exterior surface can help to control the interior temperature of the system (for instance when the containers are utilized in a greenhouse).
Once developed into a small plant capable of surviving in soil, the plant can be transplanted. For instance, the container can be opened, and an individual cell can be broken off from the other cells of the system. The cell, which carries a young plant, may be voided of organic compounds by rinsing, and can then be transplanted in its entirety, with no need for separation of the young plant from the support, which can increase likelihood of the plants long-term survival.
The present disclosure may be further understood with reference to the examples, below.
Aseptic cultures of Echeveria ‘Black Prince’were maintained in vitro on the standard micropropagation medium Murashige and Skoog (MS 1962), solidified with agar, and supplemented with 3% sucrose, Linsmaeier and Skoog (1965) organic components, with 1 μM of the plant growth regulator meta-topolin, at pH 5.7. Oasis® Horticubes® XL foam blocks of 11.75 cm×7.75 cm×2.5 cm were top grooved 2 cm deep to form 35 cells of 0.5 cm×0.7 cm. Flat-bottom gusseted containers made of clear, biaxially oriented polypropylene (BOPP; 2 mil thickness) film were used. Each 12 cm×8 cm×18 cm container was fitted with an Oasis® Horticubes® XL foam block similar to that illustrated in
Each system included one block of foam inserted into one container and infused with liquid nutrient medium (as above, without the agar solidifying agent). Each system was autoclave sterilized and then cooled with a paper clip lightly closing the container by rolling the top of the container over. The support used in the example was Oasis® Horticubes® XL, a phenolic foam growing medium with three volumes of liquid medium (100, 150, and 200 ml); and the inclusion or omission of four 1 cm ventilation patches on the containers; with two vessel system replicates for each treatment factor combination.
Microcuttings of Echeveria ‘Black Prince’were aseptically inserted into scored cells of the foam blocks. The containers were sealed with an impulse sealer three times across the top (0.5 cm between each seal). Then the sealed portion was rolled downward effectively decreasing the volume of the vessel and forcing its inflation. A paper clip held the fold in place, maintaining the 3-dimensional confirmation. Plantlets were grown in a culture room with 40 μmol cool white fluorescent light, 16 hr days, at 24° C. for 6 weeks.
In the greenhouse, the containers were cut open, the liquid medium was removed by soaking in water, each plant was evaluated (diameter, number of divisions, presence of hyperhydric tissue, etc.), and the foams cells were separated and planted. Greenhouse medium was a Fafard 3b soilless mix (peat, pine bark, perlite; Fafard Inc., Anderson S.C.) supplemented with 30% additional perlite. A rooted plantlet in a saturated Oasis® Horticubes® XL plug was planted in greenhouse medium in 10″×20″ greenhouse flats with 1206 cells (72 cells per flat). Plants were grown for 5 weeks in December and January under full sun, hand irrigated with 60 ppm-N 20-10-20 Peat Lite Special water soluble fertilizer (Peter's Co., Allentown Pa.). Following greenhouse culture, plants were evaluated (survival, diameter, number of divisions).
The experiment was a completely randomized design. ANOVA was conducted on all quantitative data for the full factorial of media volume×ventilation×Oasis® Horticubes® foam type. Data presented are as significant when prob. >F≦0.05.
Laboratory nutrient medium containing sucrose and other organic compounds was rapidly removed by rinsing the Oasis® Horticubes® XL growing medium, as shown in
Following 5 weeks of greenhouse growth in full sun, non-hyperhydric plants survived at a rate of 95%, where the hyperhydric plants survived at a rate of 40%-70% (95% confidence interval). The greenhouse growth ratios are shown in
While the present subject matter has been described in detail with respect to specific embodiments thereof, it will be appreciated that those skilled in the art, upon attaining an understanding of the foregoing may readily produce alterations to, variations of, and equivalents to such embodiments. Accordingly, the scope of the present disclosure is by way of example rather than by way of limitation, and the subject disclosure does not preclude inclusion of such modifications, variations and/or additions to the present subject matter as would be readily apparent to one of ordinary skill in the art.
The present application claims filing benefit of U.S. Provisional Patent Application Ser. No. 61/830,216 having a filing date of Jun. 3, 2013, which is incorporated herein in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2014/040631 | 6/3/2014 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61830216 | Jun 2013 | US |
