Classifications

• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
  • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
  • A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
  • A01G31/00—Soilless cultivation, e.g. hydroponics
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
  • A01G7/00—Botany in general
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
  • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
  • A01H4/001—Culture apparatus for tissue culture

Definitions

• This invention relates generally to the field of the botanical sciences, and more particularly to a systemic plant graft interface and support system.
• a plant is broadly defined as any member of the group of living organisms which, although characterized by irritability or excitability, is typically lacking in locomotor movement or rapid motor response. Plants may be divided into two classes: vascular and non-vascular plants. Non-vascular plants, e.g., bryophytes and algae, do not have vascular tissue which enables the circulation of nutrients and sap within the organism. This class of plants is not within the subject matter of this invention, and will not be discussed further herein.
• Vascular plants are plants which have evolved physiologically complex conducting systems, and the efficiency of these vascular systems has led to tremendous ecological diversification. These plants range from grasses to large trees, and provide vegetables, fruit, flowers, wood and various photosynthates as products of plant growth and reproduction.
• Vascular tissue is comprised principally of xylem and phloem.
• Xylem is the principal water and mineral-conducting tissue in vascular plants, and is a complex tissue composed of non-living, lignified tracheids, vessels and fibers and their associated living parenchyma cells.
• Xylem also may provide mechanical support, especially in plants with secondary xylem, i.e., wood.
• Phloem tissue is that part of the vascular system which transports the elaborated food materials (photosynthates) from the leaves.
• the phloem consists typically of sieve tubes, and companion cells. Transportation of the phloem is typically from leaves to roots.
• Xylem tissue forms vascular paths along the long axis of the plant stems from roots to the tips of the aerial, i.e., above-ground, portions of the plant. From these longitudinal pathways extend radial xylem tissue, termed xylem rays, which conduct water and water nutrient solutions to the leaves, shoots and other surface portions of the plant. The phloem tissue forms a distinct pathway which surrounds the xylem.
• the stems that is, the above-ground portions of vascular plants, vary from plants less than 1.2cm in height to forest giants towering 100
• stems have essentially similar vascular systems.
• the growing tip of any young stem has cells which are initially very similar, but which differentiate to form the plant subsystems as the stem grows.
• the cells which are nearest the center of the stem become xylem cells and those towards the circumference of the stem become phloem cells.
• the vascular tissues occur in the form of separate small bundles with the phloem surrounding the xylem tissue.
• fruit-bearing and other vascular plants are grown by placing a seed, sprout or other natural plant portion in fertile soil or hydroponic medium and allowing roots to form and extend to provide anchorage, and absorption of water and minerals which are conducted by the vascular system to the aerial portions of the plant which, in turn, provide the desired plant products.
• shoot is used herein to indicate any aerial stem of a vascular plant rather than the commonly-employed reference to a young growing branch or twig with leaves.
• hydroponic methods have inherent difficulties because plants which have evolved to be rooted in a nutrient soil are required to root in an unnatural medium. For these reasons, most plants which are hydroponically grown are of the smaller, rapidly-maturing variety and large-scale hydroponic production of fruits and vegetables has not been economically feasible.
• micropropagation has posed unique problems for many tissues such as woody fruits, tropicals and ornamental plants due to the need to modulate or change nutrients over time.
• Multiple transfers of propagating tissues from one growth medium to another as the plant matures have thus been required to prepare the plant for growth in a natural environment.
• the in vitro propagation of plants has provided advantages, especially in the regeneration of plants from protoplast cells
• many potential advantages of culture propagation have not been realized in practice due to the difficulty of culturing plant cells or tissues to mature plants.
• many species are resistant to micropropagation techniques. While I do not wish to be bound to any particular theory, this failure may be due to the fact that the growth media comprise an artificial interface for plant growth which fails to adequately support the developing plant.
• the present invention furnishes a solution to many of the problems noted above with respect to the propagation and growth of whole plants and affords advantages not previously envisioned by workers in the plant sciences by providing an artifical replacement for the vascular and support functions normally provided by the root system of a plant.
• the invention includes a method for the growth of a vascular plant shoot by providing a synthetic, biocompatible interface for the direct exchange of nutrient fluid with the shoot's living vascular tissue, thus forming a plant which comprises an aerial portion which grows and produces plant products in the absence of roots.
• the synthetic vascular interface may be adapted to support the branch or other shoot growing therefrom to permit the manipulation of plant tissue at a systemic level.
• the development period from the juvenile state to the mature cropproducing state may be as long as three to seven years.
• fruit trees do not produce fruit, i.e., ripened ovaries, until a nearly mature root and trunk system is produced, and substantial fruit yields from such trees do not occur until the trunks and branches are of sufficient thickness and metabolic maturity to support the fruit.
• the apparatus and method of the present invention allow the growth of branches without the need for roots or any main stem (trunk), the period of time for the growth and maturation of roots and trunks is unnecessary and only the time for the growth of the product-bearing branches, potentially a single season, is required.
• substantial space savings are achieved.
• the systemic interface of the invention includes an artificial and biocompatible xylem means, preferably a perforate surface such as that formed by a cross-section of a plurality of parallel hollow fibers, and means for applying a flow of nutrient solution to the xylem means such as by applying a flow of such solution to the perforate surface.
• the apparatus further includes absorption means which form an artificial and biocompatible phloem system.
• the apparatus is employed in a method for growing a plant or plant portion, which includes placing a totipotent plant cell, that is, a cell capable of growing and developing into an entire
• a growing rootless vascular plant which comprises a shoot having a periderm and containing xylem and phloem tissue within the vascular system of the shoot, the shoot growing from a systemic interface including an artificial xylem means mated to and permitting fluid communication with the xylem tissue of the shoot, and an artificial phloem means mated to and permitting fluid communication with the phloem of the shoot.
• the plant includes retaining means abutting the periderm of the shoot and adapted to support the shoot on the systemic interface and maintain the fluid communication of the interface with the xylem and phloem tissue, and means for providing a nutrient solution to the shoot.
• the vascular plant thus described grows in the absence of roots.
• the systemic interface is used to form a culture carousel for the in vitro growth of cells on tissue to form shoots, and provides a means for the support of the growing shoots in a manner which offers substantial advantages over existing agar techniques. Means are also provided for the modification of the humidity within the culture vessel while maintaining sterility by the sequential removal of relatively impermeable portions of the vessel.
• FIG. 1 is an exploded side elevational view in cross-section, of an in vitro tissue culture carousel according to the present invention
• FIG. 2 is a partially exploded cross-sectional side elevational perspective view of a systemic plant interface for the growth of field plants in the absence of roots;
• FIG. 3 is a perspective view of an artificial xylem interface employed in Fig. 2;
• FIG. 4 is a perspective view of a portion of the artificial xylem interface described with respect to Figs. 3 and 5;
• FIG. 5 is a perspective view of a xylem interface disc described in more detail in FIG. 1;
• FIG. 6 is a perspective view of a limb retaining means described with respect to Fig. 2;
• FIG. 7(A) is an exploded perspective view of an alternative embodiment of the systemic plant interface adapted for use in vitro or in field application;
• FIG. 7(B) is an exploded perspective view of the xylem-phloem interface and limb retention apparatus of the interface shown in Fig. 7(A);
• FIG. 7(C) is a cross-sectional view of a rootless shoot growing in the interface shown in Fig. 7(A);
• FIG. 8 is a schematic cross-sectional view of hollow fiber configurations.
• Fig. 9(A) is a schematic cross-sectional end view of a chamber for propagating plant cells or explant tissue to form viable shoots;
• Fig. 9(B) is a schematic cross-sectional side view of the propagation chamber shown in Fig. 9(A);
• Fig. 9(C) is a schematic top view of the shoot propagation chamber
• Fig. 10 is a schematic cross-sectional side view of the hollow fiber configuration employed in the embodiments set forth in Figs. 9 and 11;
• Fig. 11(A) is a schematic cross-sectional end view of a plant growth chamber useful in forming roots on established shoots in communication with the artificial plant interface, so that transplanting to a natural environment is facilitated;
• Fig. 11(B) is a schematic cross-sectional side view of the chamber shown in Fig. 11(A);
• Fig. 12 is a cross-sectional side view of a transferrable xylem interface which facilitates transfer of an established shoot to hardening environments
• Fig. 13 is a schematic cross-sectional view of a plant growth cartridge, for example, a rooting cartridge, which employs the xylem interface shown in Fig. 12.
• a plant growth cartridge for example, a rooting cartridge, which employs the xylem interface shown in Fig. 12.
• the invention proceeds by providing a factitious interface which serves the support and nutrient functions of natural plant roots.
• the words factitious or artificial, as used herein, are meant to convey the meaning of being made by art or science, in distinction from what is produced by nature.
• Factitious xylem and phloem interfaces are provided which allow growth of callus tissue which is fed with selected hormones to prohibit root formation and encourage the growth of the desired aerial plant portions.
• callus cells grow under the influence of appropriate plant hormones, they gradually change in form. Those cells which are nearest the center of the stem differentiate into xylem cells, the water- and mineral-conducting tissues, and those toward the circumference mature as phloem cells, the principal food-conducting tissue of the plant.
• the vascular system and its associated ground tissue is commonly referred to as the stele of the plant.
• each stele type includes one or more internal xylem channel surrounded by phloem tissue.
• the artificial xylem interface includes a biocompatible surface having perforations of a size and shape which approximate the xylem lumens in the target plant's vascular system.
• the xylem interface is formed from a plurality of parallel hollow fibers which are bundled longitudinally to present a porous surface and permit nutrient solution to pass through the lumens of the hollow fibers.
• Hollow fibers which have been found to be effective are those sold by the Celanese Company of Charlotte, North Carolina under the trademark Cel-Guard M.H.F. and by Mitsubishi Rayon Division of Tokyo under the trademark K.P.F.-200.
• xylem interface for the growth of a ficus branch may be constructed of hollow polypropylene fibers having a lumen diameter of from 30 to 65 microns. Both ends of the hollow fiber are sealed with a biocompatible material such as urethane so that the nutrient solution passes only through the lumens of the hollow fibers rather than between the fibers.
• a biocompatible material such as urethane
• Medithane 3905 by the Hexcel Corporation has been found to be effective for this purpose.
• the wall fibers are constructed to have a porosity which is sufficent to allow oxygenation of the nutrient solution by passage of air or oxygen around the outside of the fibers under a positive pressure (referred to herein as membraneoxygenation). Alternatively, oxygen may be sparged through the solution to permit bubble oxygenation of the nutrient fluid.
• an artificial phloem means which mates and permits fluid communication with the developing phloem tissue of the shoot.
• the artificial phloem means comprises a porous sieve material which will mate with the developing phloem tissue and allow the removal of sap from the developing shoot.
• a biocompatible open-cell sponge is disposed around the callus tissue and functions as a phloem interface by permitting the return of excess feeding solution and phloem vascular sap. As the limb develops from callus tissue, the phloem tissue grows into the open-cell material.
• the phloem interface means is biocompatible (i.e., will sustain the growth of living plant tissue) it becomes unitary with the natural phloem tissue in the sense that it allows the natural phloem cells to interface with the artificial phloem means so that phloem juices flow from natural phloem tissue to the interface.
• the artifical phloem is porous to the extent that phloem juices may be conducted away from the natural phloem tissue.
• neointimal material which mates with and drains fluid from the natural phloem is preferred for the artificial phloem, and a polyvinyl acetyl or dacron-velour sponge sold by Americal Corporation under the trademark Merocel has been found to be particularly advantageous.
• This material is biocompatible and the plant tissue grows naturally into the open cells.
• neointima as used herein is used to convey the meaning of an artificial material into which living tissue will grow naturally.
• a neointimal sponge is preferred as the phloem interface.
• any cell or explant capable of growing and developing into an entire plant organism may be employed with the systemic plant interface.
• Many isolated cells of a mature plant are totipotent, but those which lack cytoplasm and nucleus are not.
• mature tracheids, fibers and sclereids are not totipotent cells.
• Protoplasts are specifically included within the definition of totipotent plant isolates.
• isolates from the apical meristem tissue are preferred.
• the meristem is a localized tissue in which rapid mitotic cell divisions occur.
• the apical meristem is that group of meristemic cells at the tip of a shoot from which all the tissues of the mature plant axis originate.
• totipotent plant tissue is cultivated in an artificial culture medium, unorganized cell multiplication may occur to form a wound-like tissue referred to as callus, or adventitious shoots may form directly from the explant.
• a totipotent explant contains the genetic instructions necessary for the development of a whole plant, the proliferation induced by the present invention must be directed toward the formation of the aerial plant portions rather than roots. For this reason it is preferred that the enclosure or other apparatus surrounding the growing plant isolate be translucent or transparent, since actinic (plant growth stimulating) radiation may have an inhibitory effect on root growth.
• growth or nutrient media or various growth solutions may be employed to stimulate or inhibit certain directions of cell, tissue and organ growth.
• growth media or growth solutions include mineral salts and vitamins necessary for plant growth, generally containing combined nitrogen, potassium, phosphorous, calcium, sulphur and magnesium with traces of iron, boron, zinc, copper and various organic substances which stimulate the growth of shoots, branches or other desired aerial plant portions while inhibiting root growth.
• the growth media employed in the various steps hereinafter described may be any medium understood by those skilled in the art to provide hormones and nutrients necessary for such growth.
• hormone refers to an organic compound, either natural or synthetic, that modifies or controls one or more specific morphological, physiological or biochemical processes within the plant.
• auxins promote growth along a longitudinal axis in plants, and are known to induce flowering in some species
• one of the principal uses of synthetic auxins has been to promote the formation of roots on stem cuttings and accordingly the use of auxins at concentrations which stimulate root growth in the nutrient solutions should be avoided.
• root-forming hormones such as indolebutyric acid and 3-aminopyridine should also be avoided.
• an auxin-to-cytokinin ratio of less than 1-to-1 is advantageous in promoting the formation of shoots over roots.
• Zeatin riboside and gibberellic acid are also known to induce shoot formation over root production.
• the need for means to inhibit the formation of roots as the plant isolate matures may vary depending on the particular species. Accordingly, the preferred transparent or translucent housing for the artificial plant interface may not be necessary in all instances. For example, root formation in many woody species does not occur spontaneously even under appropriate culture conditions.
• tissue culture media Due to the nature of the growth solutions (tissue culture media) which facilitate the development of contaminating organisms, it is necessary to maintain the tissues, media containers and associated apparatus under sterile conditions to avoid growth of bacteria, virus and fungi which would otherwise overwhelm the plants. Accordingly, the appropriate cells or tissues are isolated under clean room conditions. With respect to cells, the apical meristem of selected limbs of targeted species is isolated and introduced into a sterile solution of cellulase and glucose. The enzyme induces the dissociation of the tissue into individual cells, which are isolated and inoculated into appropriate growth media to stimulate callus formation.
• the callus tissue is placed on a perforate surface having a plurality of apertures which are of an appropriate size and shape to mate to and permit fluid communication with the xylem tissue of the target plant, that is, the xylem tissue which will develop from the cell culture.
• the xylem-mating perforate surface is comprised of the transverse cross-section of a plurality of lumenous fibers, the cross-section thus presenting a surface upon which the callus or other plant tissue can grow to form mating natural xylem tissue having vascular pathways which communicate directly with the lumens of the fibers.
• the perforate surface, and thus the hollow fibers should be biocompatible so that growth of and bonding to the xylem tissue is encouraged.
• the fibers be bonded one to another by a biocompatible material. This material then provides an aseptic seal around each hollow fiber and functions as an interface substrate to the vascular system of the developing plant.
• a biocompatible material provides an aseptic seal around each hollow fiber and functions as an interface substrate to the vascular system of the developing plant.
• the hollow fibers can be produced in various diameters, and with various lumen sizes or shapes depending upon the vascular system of the target plant. Generally, it has been found that a perforate surface which approximates the transverse cross-section of a haplostele provides superior results with most plants.
• a haplostele is a cylindrical protostele, i.e., a solid column of vascular tissue with the xylem centrally located and surrounded by phloem, having a smooth margin in transection.
• stele may be employed for a variety of plants, and transections of various types of stele are shown in Figure 8 wherein the xylem tissue is shown as a darkened area, the phloem in white, and the central pith denoted by cross-hatching.
• the actinostele is similar to the haplostele but the xylem passageway is arranged with radiating star-shaped rays.
• the plectostele is shown to be a protostele which further includes bands of phloem across the xylem passageway when viewed in cross-section.
• Dictyostele and eustele are seen to comprise separate vascular bundles, each termed a meristele, and including both xylem and phloem. These vascular bundles are disposed around a central pith.
• the amphiphloic siphonostele is seen to have a central pith area and a xylem cylinder with phloem both interior and exterior to the xylem, as is the ectophloic siphonostele,
• the atactostele is seen to consist of vascular bundles scattered throughout the parenchyma tissue of the stem. This type of stele is characteristic of monocotyledons. In all of these steles, all or a substantial part of the vascular phloem is in fluid communication with the external phloem cells.
• the outer cylindrical size of the hollow fiber membranes of the invention are of a size and diameter which comports with the dimensions of the xylem tissue of the mature plant.
• hollow fiber transections having a lumen diameter of less than 100 microns, preferably 30 to 65 microns, and an overall cylindrical diameter of 150 microns or less are preferred.
• the hollow fibers can be made from a variety of biocompatible materials and by methods which are known in the art. Generally, the hollow fibers are made by forming the desired shape in a polymeric tube having a diameter of 1 or 2 cm or less and a length of about 1 meter, and then drawing or extruding this rod to the appropriate diametral dimension. Any desired porosity may be generated by chemical leaching, gamma radiation or other known means.
• the explant tissue is placed on the artificial xylem surface under sterile conditions, A nutrient solution is then presented to the callus tissue either by capillary action, i.e., by natural flow through the lumens, or if the fibers are of sufficient length to restrict such natural flow a low pressure solution feed, e.g., two pounds per square inch, may be applied to overcome the fluid friction in the lumens.
• a low pressure solution feed e.g., two pounds per square inch
• the natural xylem tissue mates to the artificial xylem surface, and the natural phloem tissue begins to grow into the artificial phloem means as the callus expands radially, thus ensuring fluid communication between the natural phloem tissue and the porous phloem means of the graft interface.
• Natural capillary flow in the phloem sponge aids the natural translocation within the growing plant portion.
• draining means may be associated with the phloem sponge to assist in the removal and/or recirculation of fluid from. the plant phloem.
• Fig. 1 illustrates a tissue culture carousel 10, which includes legs 12 and a supporting member 14.
• the carousel 10 includes, in the member 14, a plurality of apertures 16 having annular flanges 18 as shown.
• capillary sponges 20 Disposed in the apertures 16, and resting on the flanges 18, are capillary sponges 20 which extend sufficiently below the member 14 to contact a growth or nutrient solution 22 which is dispersed in a cup member 24 having flanges 26 which are adapted to mate and seal with the flanges 13 on the carousel 10 by means which are not specifically described by a particular reference numeral.
• discs 28a and 28b Resting directly above, and in abutting contact with the capillary sponge 20, there are seen discs 28a and 28b, respectively, on the left and right of the Figure. These discs will be described in more detail with respect to Fig. 5, but briefly comprise longitudinal hollow fibers disposed vertically with respect to Fig. 1. It will be apparent from the Figure that when the carousel 10 is lowered to mate with the cup member 24 as described, and the capillary sponges 20 are immersed in the solution 22, the solution will be drawn toward the discs 28 by capillary action, which will continue the flow of the growth solution 22 to the top surface 30 of the discs 28.
• the carousel 10 is seen to further include upper arms 32, terminating in flanges 34, which laterally enclose a biocompatible, neointima phloem sponge 36. Disposed in the sponge 36 and centered above the discs 28 are phloem sponge apertures, as shown by the reference numeral 38 above the disc 28a. It will be apparent from the drawing that the solution 22, having previously been described as flowing to the top surface 30 of the disc 28 by capillary action, will similarly be drawn into the sponge 36. Excess nutrient solution and fluids transported by the natural phloem tissue of the growing shoot may drain through the ports 39 in the carousel 10 into receiving means not specifically shown.
• shoot tip or callus tissue 40 has been placed within the aperture 38, and directly upon the disc 28a.
• the tissue 40 has been formed from a totipotent plant isolate.
• a cover 42 including a flange 44 at the lower edge thereof, may be lowered so that the flange 44 is brought into mating contact with the flange 34 of the carousel 10.
• the cover 42 may be fixedly attached to the carousel 10 by means which are not specifically shown.
• a flange 46 and annular threads 48 are seen to secure a series of transparent caps 50, 52 and 54 which maintain the humidity formed by evaporation of the nutrient solution 22 within a cavity 56 which is formed above the carousel 10.
• the cap 50 is nonpermeable by moisture but as the callus 40 grows radially and longitudinally to form a shoot the cap 54 is removed and the partially permeable cap 52 and the permeable cap 54 left in place, so as to begin the adjustment of the developing shoot to ambient humidity. As shoot development continues, the cap 52 is removed and the more permeable cap 54 remains until the hardening process is completed. The developing plant can thus be adjusted to ambient humidity without disrupting the sterility of the cavity 56 or requiring transfer of the growing shoot from one container to another.
• a shoot 58 is shown to have developed over a period of about eight weeks from the initial explant tissue such as that designated by the reference numeral 40.
• the base portion of the shoot 58 which abuts the top surface 30 of the disc 28b has expanded radially and the shoot tissue has grown into the area of this phloem sponge 36 which surrounds the shoot 58.
• the cup 24 includes a porous and hydrophobic oxygen sparging probe 23, which admits oxygen to the solution 22 through an external oxygen fitting 25 so that the oxygen content of the solution 22 can be maintained for optimal plant growth.
• a graft interface 70 is seen to include a first portion 72 and a second portion 74, which may be brought into mating engagement and secured by a bracket 76 which rotates about a flange and groove combination 78 and, through the engagement of external and internal threads 80 and 82, respectively, causes the first and second portions 72 and 74 to abut and seal at the O-ring 84.
• an end cap 86 is releasably secured to the portion 72 by an O-ring 88 and a bracket 90 which bears on a flange and groove combination 92 and mating internal and external threads 94.
• the outer casing of the interface 70 is preferably formed from transparent or translucent polymeric material, although minor parts such as the brackets 76 and 90 or other components nearer the end cap 86 may be opaque or darker if desired. However, the shoot-containing second portion 74 and the areas of the portion 72 immediately adjacent thereto may be transparent or translucent to assist in the inhibition of the formation of roots in the developing explant tissue.
• the assembly 100 includes a plurality of hollow fibers 102 which are disposed longitudinally to the long axis of the assembly 100. As is shown in Fig. 4, each of the fibers has a shape similar to a haplostele in natural vascular plant tissue, that is, a hollow fiber 102 having a cylindrical wall 104 enclosing a lumen 106. In Fig. 3, it should be noted that both ends
• Fig. 5 shows a disc 28 which has been formed by sealing the plurality of fibers 102, such as are described with respect to Fig. 3, in the urethane casting material 120 throughout the length of the assembly 100, and thereafter, cutting the individual discs 28 from the assembly 100 in a manner which does not obscure the fiber lumens.
• the perforate surface formed as described with respect to Figs. 3 and 5 includes the urethane casting material 120 which fills the interstices between the hollow fibers 102, thus sealing the fibers and permitting fluid flow through the assembly 100 only through the lumens 106.
• the fibers are disposed to provide a perforate surface formed by a plurality of parallel channels having a diameter (the lumen diameter of the fiber) of from about 30 to 65 microns.
• the fibers are cast at the surface 116 with a density of about 8,000 fibers per square inch.
• the assembly 100 is disposed within the interface housing 72, and the cast sealing members 112 and 114 are seen to be cast in a manner so as to bond to the housing 72 at their outer perimeters.
• an aseptic seal is provided at either end of the housing with respect to the outer surfaces of the fibers 102.
• This aseptic seal is preferred due to the fact that as the nutrient solution proceeds through the lumens of the fibers 102, as further described, air or oxygen may be fed under adequate pressure through an inlet 130.
• the hollow fibers 102 are produced from a material having a wall porosity sufficient to allow oxygen to pass through the cylindrical walls 104 in amounts which are sufficient to provide a proper level of oxygen in the nutrient solution yet is hydrophobic in order to resist the flow of liquid throughout the pores.
• a wall porosity of ranging from about 0.005 to about 0.1 microns in a polypropylene fiber material is appropriate for this purpose.
• the oxygen. is seen to flow through the inlet 130 into the open spaces 132 within the housing portion 72 and between the seals 112 and 114. As is shown in Fig. 3, the hollow fibers between the seals 112 and 114 are unsealed and open to allow perfusion of the oxygen.
• the nutrient solution is applied to the housing 70 by a solution inlet fitting 138, thereafter filling the internal area 140 within the end cap 86 and adjacent the sealing member 114. From this area, the nutrient solution may flow through the lumens 106 of the hollow fibers 102 toward the end 108 of the fiber assembly 100. While the nutrient solution may be drawn a short distance through the hollow fibers, as in the hollow fiber discs 28 described with respect to Fig. 1,
• a hydraulic pressure of about 2 p.s.i. is sufficient to force the the solution through the lumens to the surface 116.
• various apparatus such as the fitting 142, here shown to include a temperature probe 144, may be included in the housing 70.
• this portion is disposed at the distal end of the interface housing 70.
• distal which is employed in the medical sciences to indicate a location remote from the point of attachment or origin, is used to describe a plant portion away from the center of the natural organism, e.g., the portion of a plant or shoot disposed in the direction of longitudinal growth.
• proximal is used to indicate a direction along the plant axis which is toward the origin of growth, for example, in natural plants the direction toward the roots.
• a phloem sponge 150 is seen to be disposed at the distal end of the housing portion 72 and abutting the porous surface 116 of the hollow fiber assembly 100.
• This phloem sponge 150 is formed from a biocompatible porous substance having an open cell size and pattern which is neointima with respect to plant tissue.
• the sponge 150 includes a plurality of projections 152, each having a central aperture 154 having an internal diameter of about 2 cm.
• callus or explant tissue is inoculated onto the perforate surface 116 through the apertures 154, under clean room conditions. This tissue is indicated by the reference numerals 156 in Fig. 2.
• the distal housing portion 74 is seen to be constructed from a rigid polymeric material forming a circumferential side 160, and a distal wall 162 including a plurality of apertures 164 which are disposed essentially coaxially with the projections 152 of the phloem sponge 150.
• the side 160 includes a circumferential external thread 165 which is disposed about the wall 162, and permits the attachment of a cover such as the cover 42 shown in Fig. 1.
• a major portion of the interior of the portion 74 is formed from a molded, transparent, resilient and biocompatible polymeric material such as a room temperature vulcanization silicone polymer, indicated by the reference numeral 166.
• a polymer which is compressible and extensible to the extent of radial limb growth is preferred so that support means for the growing limb or branch is provided,
• a room temperature vulcanization silicone product manufactured by Dow-Corning Medical Products and sold under the trademark Silastic Q 7-4840 medical grade liquid silicone rubber has been found effective for this purpose.
• This polymer may be molded into the portion 70, and includes recesses 168 which loosely mate with the projections 152, and a proximal surface 170 which abuts the phloem sponge 150.
• the portion 74 When the portion 74 is connected to the portion 72, sufficient space remains between the surface 170 and the perforate surface 116 to allow transfer of fluid by the sponge 150. Fluid which is exuded from the natural plant phloem is absorbed by the sponge 150, and may be removed through a port 172 which is sealed by a one-way valve 174 such as the duck-bill type valve shown in the drawing.
• the valve 174 permits draining of excess fluid while maintaining the aseptic environment for tissue growth.
• Plant growth channels 176 are seen to extend from each of the recesses 168 through the polymer 166 to an outer or distal edge 178 of the housing portion 74.
• the shoots thus formed will grow distally through the phloem sponge apertures 154 and further through the channels 176.
• the tissue 156 expands radially, it will grow into the phloem sponge 150.
• the tissue 156 differentiates to form aerial plant portions, i.e., shoots, from the growth conditions, these shoots will extend longitudinally through the channels 176.
• the periderm tissue will bear against the walls of the channels 172 of the polymer 166, thus providing an aseptic seal for the shoot as it grows through the channel 176.
• This seal preserves the callus at the perforate surface 116, and prevents any degeneration of the enclosed shoot which may result from the perfusion of the nutrient solution beyond the callus tissue.
• the polymer 166 provides support for the developing shoot. Additionally, as the shoot extends beyond the outer edge 178, the polymer 166 serves as a means for retaining the vascular system of the limb or branch in fluid communication with the perforate xylem surface 116 and the phloem sponge 150.
• a wide variety of retaining means may be provided, depending upon the extent of plant growth required and the size of the generated limb.
• a radially expanding plant stem generates force.
• the silicone polymer is preferred due to the fact that the polymer expands while maintaining sufficient compressibility to provide a support and sealing function to the limb.
• the polymer 166 may be formed with air bubbles immediately adjacent the channels 176 to lessen the initial resistance of the polymer to the radial expansion of each shoot.
• a biocompatible stainless steel wire screen mesh may be incorporated within the polymer 166 near the surface 170 through which the tissue and shoots must grow, thereby captivating the branches which will extend beyond the channels 176.
• the channels may be formed from or include a microencapsulated epoxy, including both catalyst and resin cells which rupture and reseal as the limb extends radially and distally to form an epoxy bond between the living portion and the polymer.
• Microencapsulated epoxy anaerobic resins used in this manner remain dormant until the crushing action between the limb and the limb retention housing causes the beads to burst and mix forming a region of cured epoxy resin of superior strength.
• a microencapsulated epoxy resin such as that sold by the 3M Corporation under the trademark Scotch-Grip 2353, commonly used for securing threaded fasteners, is preferred.
• a preferred use of microencapsulated epoxy resin precursors in connection with the limb retaining means is described in more detail with respect to Fig. 7(B).
• Fig. 6 details another form of retaining means which may be employed in the housing portion 74.
• a disc member 180 is seen to be trifurcated into distinct sections 182, 184 and 186. Further, the member 180 includes an opening 188 which bears ridges and valleys 190 on the lateral surfaces thereof.
• the disc 180 may be disposed midway between the surfaces 162 and 170 with the channel 176 axially disposed through the opening 188 on the disc.
• the periderm contacts the ridges 190 to provide support and retention for the shoot, and as the limb expands further the individual sections of the disc member expand radially to accommodate limb growth.
• a plurality of disc members 180 may be provided around each of the channels 176.
• the adaptation of existing nutrient solutions to this end can be attained without undue experimentation.
• steps be taken to eliminate or neutralize endotoxins which are produced by the plant and which are retained within the closed artificial vascular system.
• the nutrient solutions described in the examples are seen to include about 0.18 weight percent polyvinyl-polypyrrolidone which has been shown to be effective in binding and neutralizing phenolic endotoxins produced by a variety of plants, thus allowing uninhibited growth.
• the normal concentration of macroelements in known growth solutions is unnecessary, and often deleterious, to growth in the closed-system plant graft interface of the invention.
• an interface housing 200 includes a casing 202 formed from a transparent polymeric material.
• the casing 202 is seen to include a plurality of hollow fiber bundles 204 which extend longitudinally through the casing.
• Each end of the bundles 204 is embedded in a cast urethane sealing member embedded, respectively, within a distal end 206 and a proximal end 208 of the casing 202 in a manner which is essentially similar to the construction of the sealing members 112 and 114 shown in Fig. 2.
• the fiber bundles provide advantages in that the total number of fibers required is substantially less than the number required for the hollow fiber assembly 100 in Fig. 2, where the nutrient solutions are fed directly to the plant tissue by the fibers 100.
• the construction of these fiber bundles 204 is as described in detail with respect to Figs. 3 and 5.
• the casing 202 is also seen to include an oxygen inlet 210 to enable membrane oxygenation of the nutrient solutions and a one-way valve similar to the valve 174 of Fig. 2, which is not specifically shown, is disposed toward the distal end 206 of the casing.
• the proximal end 208 includes threads 212 which are adapted to receive an end cap 214 which is attached to
• the end cap 214 includes a thermocouple probe 220, a nutrient solution inlet 222, and a port 224 which is closed by a fitting 226.
• the port 224 allows access to the nutrient solution within the casing 202 for testing if desired.
• the distal ends of the hollow fiber bundles 204 are seen, in combination with the cast sealing member which is similar to the member 112 in Fig. 2 but is not specifically shown by a reference numeral in Fig. 7, to form a perforate surface 228 which receives a lower retaining bracket 230.
• the bracket 230 is seen to include circumferential notches 232 which engage flanges 234 to prevent rotation of the lower retainer bracket within the casing 202 and to allow for peripheral drainage from the distal portion of the casing beyond the bracket 230.
• the bracket 230 also includes a plurality of apertures 236 and a central aperture 238 which mate, respectively, with each of the hollow fiber bundles 204.
• the hollow fibers 204 in Fig. 7A are employed to permit membrane oxygenation of the nutrient solution, as the xylem interface is provided by fiber discs as later described.
• the housing 202 may be modified to permit plant growth directly from the fibers 204 in the manner described with respect to Fig. 2.
• the fibers 204 may not be employed and oxygenation provided by a sparging tube inserted within the casing in the manner described with respect to the interface shown in Fig. 1.
• a sponge member 240 including apertures 242 and 243, and a culture carousel 250, which also contains a plurality of apertures 252 which are coaxial with the hollow fiber bundles 204.
• the sponge 240 draws nutrient solution by capillary action from the phloem sponges later described.
• the bracket 230, sponge 240 and culture carousel 250 are secured to the casing or housing 202 by the abutting lower edge 258 of a second housing portion 260.
• the assembly of the above-mentioned apparatus, including the positioning of an upper retaining bracket 265 within the housing portion 260, will be described in more detail with respect to Figs. 7(B) and 7(C).
• the second housing portion 260 is secured to the casing 202 by a collar 264 which bears on a flange 266.
• the collar 264 includes internal threads 268 which engage external threads 270 of the casing 202.
• Internal threads 272 of the housing 260 engage with external threads 274 at a lower or proximal edge 276 of a cover 278.
• An internal annular flange 277 serves to secure the upper retainer bracket 265, as is shown in more detail in Fig. 7(C).
• the cover 278 is seen to include ports 280 and 282, which are adapted to receive caps of various gas/water vapor permeability. These caps are serially engaged and removed as shown in
• the cap 284 may be nonpermeable to such vapor and thus useful in retaining high humidity conditions during the initial growth of the shoots as hereinafter described.
• a second cap 286 is semipermeable to gas/water vapor so that the developing shoots may be hardened, during growth, toward natural humidity.
• the cap 288 is totally permeable and is used in the final growth process prior to removal of the cover 278.
• Figs. 7(B) and 7(C) the construction and use of the interface housing 200 will be described in more detail.
• Fig. 7(B) the lower retainer bracket 230, drain sponge 240, culture carousel 250 and upper retaining bracket 265 are shown.
• the lower bracket 230 includes, in addition to the apertures 236, at least one aperture 238, here shown to be centrally located and larger in diameter, for purposes which will later be described.
• the sponge 240 includes a central aperture 244
• the culture carousel 250 includes a central aperture 254.
• the central apertures are adapted to receive a feed sponge 290 having a groove 292 which receives a bottomless retainer cup 294 which, in turn, rests within the aperture 254.
• the sponge 290 mates with the edge of the aperture 244 in the phloem sponge 240.
• a lower edge 296 of the sponge 290 when fully inserted in the assembly, abuts the central hollow fiber bundle 204 and receives solution from the perforate surface thereof.
• the sponge 290 is seen to include a top surface 298 which receives a hollow fiber disc 299 which is similar to the disc 28 shown in Fig. 5.
• the bottomless retainer cup 294 abuts the top of the disc 299, and rests within and is restrained from lateral movement by the edge 256 of the carousel central aperture 254.
• a neointimal phloem sponge 300 having an aperture 301, rests within the retainer cup 294 atop the disc 299.
• the apparatus indicated by reference numerals 294-301 is collectively referred to as a tissue culture apparatus 302.
• the small apertures 236 and 242 are shown to be adapted to receive a shoot growth apparatus 306 which comprises a silicone rubber cap 308 having upper and lower circumferential flanges 310 and a plurality of openings 312.
• a neointimal phloem sponge 314 having a central aperture 315 is inserted into the cap 308 so that the sponge 314 protrudes, in part, through the openings 312.
• a hollow fiber disc 316 Directly below the sponge 314 is a hollow fiber disc 316, and thereafter a feed sponge 318.
• a cap 308 is seen to be disposed with the flanges 310 above and below the lower retaining bracket 230.
• the feed sponge 318 is seen to protrude below the bracket 230 and positioned so as to contact the fiber bundle 204 which, although not shown in Fig. 7(C), lies directly below each of the shoot growth appratus 306.
• the feed sponge 318 is seen to be positioned to feed nutrient solution to the hollow fiber disc 316, where it is transported by capillary action to a shoot 320 which is seen to be growing from the fiber disc 316 in the absence of root formation.
• a proximal portion 322 of the shoot 320 is seen to have grown and expanded radially on the disc 316 within the aperture 315 of the phloem sponge 314, and the natural phloem tissue has expanded radially and grown into the artificial phloem sponge 314.
• Natural phloem fluid exuded by the shoot 320 is thus conducted by the porous sponge 314, and thereafter to the sponge 240 and drained through the notch 232 into a cavity 233 and thereafter from the casing 202 as described with respect to Fig. 2.
• Fig. 7(C) the lower retaining bracket 230 is seen to be supported by a bracket 330 extending internally from the distal end 206 of the casing 202, and is sealed against the bracket 330 by an O-ring 323 therebetween.
• a shoot-retaining means 330 which includes a silicone cap 332 which is similar to the cap 308 described above in that it includes upper and lower circumferential ridges 334, but the cap 332 does not include lateral openings 308.
• the cap 332 accepts the upper edges of mating support fittings 336 and 338 as is shown more clearly in Fig. 7(C).
• the fittings 336 and 338 are seen to include a plurality of shoot-support plates 340 which, when the fittings 336 and 338 are combined, form a series of longitudinal apertures which support the length of the shoot.
• the fittings also include upwardly extending arms 342a and b which, when the fittings are combined, form an upwardly extending hollow arm 342 which is inserted through one of the plurality of apertures 344 in the upper retaining bracket 265.
• the fittings 336 and 338 also include twistlock flanges 346 which enable the fastening of the shoot support means 330 to the culture carousel by engagement with the
• twistlock receiving brackets 348 on the carousel 250 are
• the systemic plant interface of this embodiment is initially employed to grow a number of small shoots from tissue culture, followed by the dissection of individual shoots which are then inserted into the separate shoot growth apparatus and shoot retaining means to permit the further growth of the initial shoots to form branches.
• the casing 202 with the end cap 214 attached is placed in a vertical position, and the lower retaining bracket 230, sponge 240 and culture carousel 250 are sequentially inserted atop the perforate surface 228 as described, and are secured to the casing 202 by the pressure of the housing portion 260 on the flange 251 of the carousel 250.
• the sponge 290, disc 299 retainer cup 294 and phloem sponge 300 are then inserted through the central apertures 238, 244 and 254, and appropriate callus or explant tissue is then seated within the aperture 301 atop the disc 299. Thereafter, the cover 278 is attached to the top of the housing 260. Appropriate growth solution is then introduced through the inlet 222 through an exterior fluid level control to maintain the solution at the level of the surface 228, until a plurality of shoots form in the plant tissue. It should be noted that the growth of plant t issue culture requires that the tissue be initially loaded, and grown, under clean room conditions to prohibit bacterial growth and contamination of the nutrient solutions and tissue.
• the apparatus 306 has preferably been previously inserted into an aperture 236 of the lower retaining bracket 230, and the sponge 240 has been placed as is shown in Fig. 7(C) so that the walls of one of the apertures 242 abut the phloem sponge 314 within the apparatus 306.
• the apertures in the sponge 240 may assume a variety of shapes as long as communication with the phloem sponge 314 and drainage from the natural phloem of the shoot within the apparatus 306 is obtained to enable drainage of highly viscous photosynthates, e.g., isoprene compounds.
• the silicone cap 308 may have openings 312 which allow insertion of the inwardly extending edges of an X-shaped aperture such as is indicated by the reference numerals 243, which may extend to the natural phloem tissue. Following insertion of the apparatus 306 and placement of sponge 240, the culture carousel is then positioned as shown in Fig. 7(C).
• the apparatus is equally adaptable to having each of the shoot growth apparatus installed prior to the growth of the explant tissue to form shoots, and the excised shoots may then be placed on the xylem discs 316 by widening the aperture 313 of the resilient cap 308 to insert the excised shoots therein.
• tissue may be placed directly to the hollow fiber discs 316.
• tissue may be similarly applied to the fiber disc 299, and after shoots of acceptable dimensions are formed they may be excised and transferred to the individual discs 316.
• selected shoots from field plants may be obtained and disinfected, and then sealed at the point of incision with dental impression compounds or other appropriate material to inhibit air locking of the natural vascular system of the shoot. Under laminar flow/clean room conditions, shoots obtained in this manner have been further disinfected with a mercuric chloride solution (0.01M) for several minutes. Thereafter, these shoots were cut to acceptable dimensions and introduced into the individual shoot growth sites on the hollow fiber disc.
• the shoot support fittings are carefully placed around the shoot stem extending through the coaxial apertures formed by the plates 340, and the assembled fittings 336-338 are then engaged with the culture carousel 250 by the twist lock fittings 346 and 348.
• the abutting portions of the fittings 336-338 are coated with microencapsulated epoxy resin prior to this assembly to bond the fittings into a rigid unit.
• the silicone cap 332 is positioned atop the assembled fitting 336-338 with the shoot extending through the aperture 333.
• the upper retaining bracket 265 is placed over the caps 332 between the flanges 334, with the arms 342 extending
• the entire apparatus 330 may be assembled and attached to the carousel 250, and the shoot may be inserted through the respective apertures of the apparatus until the proximal end 322 is seated within the aperture 315 atop the disc 316.
• RTV silicone rubber or other biocompatible polymers are introduced through the hollow arms 342 to fill the interior of the fitting 336-338. Further, the space surrounding the fittings 336-338 may be filled with such polymers to produce a limb support and retention means similar to that described with respect to Fig. 2. Thereafter, the cover 278 is again secured to the housing 200, with caps providing the appropriate permeability attached, and the interface housing 200 is charged with nutrient solution. After the contamination-free growth of all shoots has been confirmed, the charged housing 200 may be transferred to a natural environment for further growth.
• each individual housing 200 in this embodiment, will enable the growth of eighteen separate plant product-bearing limbs.
• Fig. 7 is particularly advantageous due to the fact that when limb growth and support is completed or no longer desired, the portion distal to the surface 228 of the casing 202 may be removed and discarded and the components comprising the casing 202 may be reused for the growth of new limbs.
• the fitting 336-338 may be injected with RTV silicone or microencapsulated epoxy adhesive, and the space surrounding the fitting 336-338 may also be injected with similar material, producing a limb support and retention means similar to that described with respect to Fig. 2.
• Fig. 7(A) may be altered to provide for bubble oxygenation if desired.
• the hollow fiber bundles 204 may be omitted and a thin disc of longitudinal hollow fibers may be inserted at the distal end of the casing 202 solely to control the flow of solution to the artificial xylem discs.
• the nutrient solution flows through the inlet 222, with appropriate solution drainage means at the distal end, and the permeable oxygen sparging probe is inserted in the oxygen inlet 210 to oxygenate the solution without relying on the permeability of the hollow fiber bundle or bundles.
• systemic plant interface of the invention in the growth of rootless, vascular plants will be described in the following examples.
• Example One A variety of shoots of various plant species have been grown in a systemic plant interface such as that shown in Fig. 2. In each such trial, a variation of the known Linsmaier-Shoog growth medium was employed. However, the traditional formulation of this medium was modified for use in the closed-loop system by reducing a proportion of macroelements to reduce vitrification, and including an endotoxin-inhibiting formulation. While a wide variety of endotoxin-binding agents may be employed, polyvinyl-polypyrrolidone in concentrations of from about 0.05 to about 0.2 weight percent are effective for this purpose.
• a field shoot obtained from a Mission fig tree (Ficus carica) was excised and the proximal end of the shoot was applied to the perforate surface 116 of the housing 70 through one of the channels 176 and apertures 154. When inserted into the housing, this shoot had a diameter of about 3mm and a length of about 85mm.
• the apparatus 70 was then mounted in a horizontal position in a supporting yoke, and the nutrient solution shown in Table 1 was recirculated into the housing 70 through the solution inlet fitting 138. The solution migrated through the hollow fibers 100 under a pressure of 2 p.s.i., which was produced by a positive pressure of sterile nitrogen in the vessel containing the nutrient solution.
• the solution was absorbed by the phloem sponge 150, delivered to the xylem interface and transferred to the natural xylem of the plant. Excess solution, photosynthates and endotoxins from the plants were, exited from the housing 70 through the valve 174. The flow of the nutrient solution through the housing occurred, under these conditions, at less than 6.5 milliliters per minute.
• the shoot had grown from 85mm to 100mm in length and exhibited new leaf growth.
• the protective cover was removed and within five weeks the branch had reached a length of 135mm, and additional leaf growth had occurred.
• Example Two In addition to the subgrowth of plants in the subtropical category (Mission fig), plants of the citrus variety were also grown. Tissue culture from the Carrizo and Swingle orange were obtained, and pressed onto a hollow fiber disc such as that indicated by the reference numeral 298 of the tissue culture
• Fig. 7(B) After a period of about four weeks, shoots having a length of about 80mm were excised, and inserted into the housing 70 as described. The nutrient solution was then circulated with the protective cover in place, and after eleven weeks the shoots had reached a length of 90mm. After removal of the protective cover, the shoots continued to grow for a period of four weeks to a length of 95mm. New leaf growth was exhibited during this period. Further with respect to the citrus family, a
• Swingle orange shoot was grown from callus tissue, inserted into the housing 70, with results identical to that described for the shoots initially grown from tissue culture. Also, field shoots from the Pink Grapefruit variety were excised, disinfected and grown with similar results in the absence of roots.
• Example Three In addition to plants of the citrus family, plants of the maleus family have also been grown. Macintosh apple shoots, obtained from the growth of tissue culture, were inserted into the housing 70 and grown over a period of twenty-seven weeks. In the initial stages, the shoots grew from an initial length of 90mm to a length of 95mm with the protective covering in place over a period of twenty weeks. Following the removal of the protective cover, the shoots extended to a length of 110mm over a period of seven weeks. In a similar manner, Jonathan apple shoots were isolated from tissue culture and grown in the housing 70, in the absence of roots, for a period of twenty-nine weeks.
• Example Four Example Four
• plants from the prunis family were also grown from tissue culture in the manner described in Example Three.
• Shoots from the Colt cherry, Suncrest peach and Myrobalan plum were grown from tissue culture on the perforate surface provided by the artificial xylem discs described above, inserted into the housing 70, and grown over a period of twentyfive weeks.
• the Cherry Colt shoots initially were inserted into the housing 70 at a length of 90mm, and grew to a length of 100mm within this time period.
• the Suncrest peach shoots grew from 105mm to 125mm during this time. In each instance, the protective cover was left in place until the last eight weeks of growth.
• apparatus which is useful in the propagation and growth of tissue culture.
• artificial plant interface is employed in a method for preparing rooted plantlets for transfer to a natural environment.
• a growth chamber for a vascular plant having xylem and phloem tissue which comprises a container supporting an artificial xylem surface formed from a plurality of hollow fibers, each having a lumen and an outer surface. The proximal ends of the fibers are bonded to each other at the other surfaces, and having open lumens to form a perforate artificial xylem surface. This surface is supported within the container such that a second or distal end of the fibers extends into a chamber adapted to contain a nutrient medium.
• the container also includes an absorptive neointimal phloem interface disposed adjacent the xylem surface, and means for providing a flow of rinse solution through the phloem interface.
• Means for the inlet and outlet of both the nutrient medium and the rinse solution may also be provided, and preferably both of said outlets include means to inhibit the passage of pathogenic substances such as bacteria, fungus and vi ruses from the container to isolate contamination.
• the container may be used in a method for propagating plant tissue and growing vascular plants having xylem and phloem tissue. The method comprises placing a totipotent plant cell or explant tissue on the artificial xylem surface described above, the plant cell or tissue being placed on the surface in fluid communication with the absorptive phloem interface, and caused to grow by applying nutrient media into the nutrient media chamber, through the lumens to the plant tissue.
• the method includes rinsing accumulated phytotoxins from the phloem interface with a rinse solution, which preferably is isotonic with respect to natural fluids within the growing tissue.
• a propagation chamber constructed in accordance with the invention is shown in Figs. 9(A)-9(C) as designated by the reference numeral 410.
• the chamber 410 may be constructed from a variety of resinous or ceramic materials, but preferably is molded from a high impact polystyrene or polycarbonate material which will withstand the repeated autoclaving required by tissue culture techniques.
• the chamber 410 is seen to include a lower tray 412 which forms a reservoir or chamber 414 for the containment of a nutrient medium as later described.
• the lower tray 412 is seen to include tapering sides 416 supporting a rinse solution receiving tray 418, which rests on supporting flanges 420 of the tray 412.
• an upper support tray 422 Disposed above the tray 418 is an upper support tray 422, which is seen to enable the lower tray 412, the rinse solution receiving tray 418 and the upper support tray 422 to be joined as a unitary member by rolling the lower edge of the tray 422 beneath the flange 420 after softening the lower edge with a heat-sealing means.
• a transparent top cover 424 is attached to the upper support tray 422 by lower flange portions 426 which engage latch members 428 that are formed on the external surface of the upper support tray 422.
• the sides 430 and flanges 426 of the top cover 424 form a recess (not bearing a specific reference numeral) which receives an 0-ring 432 to form an air-tight and aseptic seal between the top cover 424 and the upper tray 422.
• the top cover 424 is also seen to include a carbon dioxide inlet 434, and microbial shield-water vapor diffusion membrane means 436, which may be adapted to and employed for the purposes described with respect to the ports and caps 280-288 as shown in Fig. 7(A).
• the membrane 436 provides significant advantages in that it permits gas transfer without the introduction of microbial contamination into the propagation or growth chamber.
• the membrane reduces stress on the cover and cover seal during autoclaving.
• This vapor diffusion membrane means provides significant advantages in any chamber which is employed for the growth of plant tissue in a sterile environment in the presence of a nutrient medium. Such chambers unavoidably require the inlet and egress of gases and water vapor due to temperature changes in the surrounding environment.
• a vapor diffusion membrane means having a porosity which permits the passage of gases and water vapor and inhibits the passage of pathogenic substances into the chamber.
• a porosity of about 0.2 microns is sufficient to inhibit the passage of virus, bacteria and fungus through the membrane, but as the thickness of the membrane increases the porosity may increase up to about 60 microns if a sufficiently convoluted pathway through the membrane is provided.
• the carbon dioxide inlet 434 is seen to have a longitudinal bore 438 in addition to a transverse bore leading to the growth chamber 440 within the chamber 410, to enable the feed of carbon dioxide to a plurality of the propagation chambers in parallel, with a carbon dioxide atmosphere being maintained within the growth chamber 440 under positive pressure.
• An artificial xylem member 450 is supported within the lower tray 412 by a supporting collar 452 which engages the internal edges of the receiving tray 418 by flanges 454.
• the xylem member 450 includes a plurality of hollow fibers 456, which are sealed at a proximal end in a cast sealing member 458 in a manner similar to that described with respect to Fig. 4.
• the distal ends of the fibers are not restrained by a cast medium.
• the fibers 456 will be described in more detail in the description of Fig. 10. From a top view as shown in Fig. 9(C), the cast sealing member 458 is seen to comprise a rectangular perforate or artificial xylem surface.
• the artificial xylem surface 458 and one of the fibers 456 is shown in a figurative cross-sectional view.
• the proximal end 460 of the fiber is seen to have an open lumen 462, while the distal end 464 is seen to have an end which is heat sealed.
• the fibers 456 are made from materials described with respect to the fibers 102, but in preparing the xylem member 450 the fibers are first heat sealed at both ends, and then the proximal end is sealed in an appropriate casting resin. Subsequently, a transverse cut is made across the cast member to form a planar surface having a plurality of open lumens from the fibers.
• This method of forming an artificial xylem surface and fiber assembly is also adaptable to form the hollow fiber assembly 100 described in Figs. 2 and 3.
• the hollow fiber 456 has a lumen diameter of up to about 120 microns, and is shown to have transverse channels 466 which indicate a porosity of from about 0.01 to 0.45 microns.
• the fibers are preferably made from a hydrophilic resinous material, and the hydrophilic porosity provided by the channels 466 permits the influx of fluid, i.e., nutrient solution, which is then transported by capillary action to the proximal end 460, that is, the surface of the artificial xylem means 458.
• an absorptive phloem interface 470 such as a neointimal sponge, is disposed adjacent a proximal surface 472 of the artificial xylem member 450.
• a plurality of totipotent plant cells or explant tissue is inserted through incisions 474 in the phloem sponge 470 and placed on the artificial xylem surface 472.
• the plant tissue is thus capable of seating on the surface 472 and forming xylem tissue in fluid communication with the lumens 462, and is also capable of fluid communication with the phloem interface sponge 470.
• the plant tissue then begins to propagate as described above, eventually forming a plurality of shoots which extend through the incisions 474 above the sponge 470 into the growth chamber 440.
• nutrient solution is applied to the chamber or reservoir 414 through a medium inlet 480.
• the medium fills the reservoir 414 to the level of a medium outlet 482, which is seen to be of a height which is sufficient to allow the distal ends of the fibers 456 to extend into the nutrient medium.
• the flow of medium through the outlet 482 is partially blocked with a porous plastic filter which inhibits the flow of contaminating particulate or pathogenic material such as bacteria or viruses. This is done to isolate any contamination which would otherwise be introduced into other chambers during use.
• oxygen may be introduced through a sparging inlet 484 into a sparging tube 485 if desired.
• an isotonic, sterile rinse solution may be introduced into the chamber 410 through a rinse solution inlet 486 which is seen to include threads or flanges 488 for the attachment of the fluid delivery tubing, as are all the fluid inlets and outlets described herein.
• the rinse media is a sterile, deionized aqueous solution adjusted to the physiological pH of the plant, but any solution which is isotonic and capable of removing endotoxins or waste material exuded by the plant tissue into the phloem sponge is acceptable.
• the rinse solution flows through the inlet 486 to a valley 490 which is formed between internal side walls 492 and 494 of the upper support tray 422.
• the valley 490 is seen to be generally rectangular in Fig. 9(C), and the rinse solution thus courses through detents 496 in the internal side wall 494 onto the sponge 470, where it flows by gravity through a porous plastic collar 498 into the rinse solution receiving tray 418.
• the rinse solution exits the chamber 410 through a rinse solution outlet 499 which is equipped with a porous plastic filter to isolate the chamber 410 in a manner similar to that described with respect to the medium outlet 482.
• the plastic collar 498 has a sufficient porosity to regulate the flow of fluid through the neointimal sponge 470, and preferably has a porosity of about 10 microns.
• explant tissue having a diameter of about 7mm is placed on the xylem surface 472 through the incisons 474, and a nutrient solution allowed to flow through the chamber 410 is described.
• a nutrient solution allowed to flow through the chamber 410 is described.
• the growth conditions may be maintained and the tissue will repeatedly generate shoots until so much callus is formed on the tissue that further shoot growth ceases. As many as 20-25 repeated shoot growths may be sustained from a single tissue explant when propagated with the apparatus and method described.
• the growth chamber 510 is not dissimilar to the propagation chamber 410, in that it is constructed of resinous material, preferably polystyrene or polycarbonate resin, which will withstand repeated autoclaving.
• the chamber 510 includes a lower tray 512, forming a nutrient solution containing reservoir or chamber 514 contained in part by sides 516, and a rinse solution receiving tray 518 which also serves a support function as hereinafter described.
• the tray 518 is attached to the sides 516 by resting on a flange 520 and a heat sealed edge 521 of an upper support tray 522.
• the tray 522 supports a top cover 524 which is attached by flanges 526 which engage latch members 528 which depend from a side 530 of the top cover.
• An O-ring 532 is seen to seal the top cover 524 to the upper support tray 522.
• the top cover 524 also includes a CO 2 inlet 534 and vapor diffusion membrane means 536.
• the CO 2 inlet 534 is also seen to include a longitudinal bore 538 for purposes described in the description of Fig. 9.
• the chamber 510 includes individual receptacles 540. In this embodiment, fifteen such receptacles are provided, each formed by molded peaks 542 in the upper support tray 522.
• the receptacles 540 are each adapted to retain an artificial xylem member 550 which is retained by a supporting collar 552 which engages a flange 554 on the receiving tray 518.
• the xylem member 550 includes fibers 556 and a cast sealing member 558 which provides a proximal surface 560 which includes open fiber lumens similar to those described with reference numeral 462 in Fig. 9.
• the fibers 556 may be identical, except as to the overall dimension which will be apparent in the drawings, to those described in Fig. 10.
• Each receptacle 540 is also adapted to receive, disposed above the xylem member 550, a supporting absorptive phloem interface collar 570 which is adjacent a perforate xylem surface 572 of the xylem means.
• the neointimal sponge 570 is laterally enclosed by a porous plastic cincture 573 which abuts side walls 574 of the receptacles 540.
• the phloem sponge 570 is enclosed at an upper edge by a retention seal 576, and includes a slot 578 which facilitates insertion of shoots as hereinafter described.
• the growth chamber 510 is adapted to employ nutrient and rinse solutions in a manner similar to that referred to above with respect to the propagation chamber 410, and thus includes a growth medium inlet 580, a nutrient medium outlet 582 which is detailed in Fig. 1KB) but not specifically shown in Fig. 11(A), and an oxygen-sparging inlet 584 which communicates with a sparging tube 585.
• the growth chamber 510 includes a rinse solution inlet 586 which is seen to include, as do all fluid inlets and outlets in this Figure, flanges or threads 588 for the connection of tubing or the like.
• the rinse solution inlet 586 provides for the flow of solution to a plurality of valleys 590 formed by side walls 592 and 594 of the upper support tray 522.
• the construction of the growth chamber 510 is best understood in terms of the method for growth of shoots therein.
• Shoots from the propagation chamber 410 are excised from the growing tissue when the shoots have attained approximately the size of toothpicks.
• the shoots After being excised with a scalpel under sterile conditions, the shoots are inserted into the phloem sponge 570 through the slot 570, extending about 2mm beynd the bottom of the sponge, and the phloem sponge is then inserted into the porous cincture 573 and secured into one of the receptacles 540 with the lower end of the shoot abutting the artificial xylem surface 572.
• nutrient solution is applied to the chamber or reservoir 514 through a medium inlet 580.
• the medium fills the reservoir 514 to the level of a medium level 582, which is seen to be of a height which is sufficient to allow the distal ends of the fibers 556 to extend into the nutrient medium.
• the hydrophilic porosity provided by the channels 446 of the fibers 556 permits the influx of fluid, i.e., the nutrient solution, which is then transported by capillary action to the proximal end or top 572 of the artificial xylem means 558.
• the non-porous fibers described above may be employed in the chambers 410 and 510.
• the flow of medium through the outlet 582 is partially blocked with a porous plastic filter which inhibits the flow of contaminating particulate or pathogenic material.
• Plant hormones known as "pulsing" agents may be added to both the nutrient and rinse solutions to induce the preferred aspects of plant growth.
• the isotonic rinse solution is caused to flow into the valleys 590.
• the support tray 522 is essentially rectangular, and includes communicating longitudinal and transverse valleys.
• the rinse solution flows through the valleys to a level which causes the solution to flow through the detents 596 which are disposed on either side of the individual receptacles 540.
• This rinse solution then flows through the phloem sponge 570, by the force of gravity, and exits through the porous cup 572. Thereafter, the rinse solution collects at the bottom of the rinse solution receiving tray 518 and exits the growth chamber 510 through the rinse outlet 599.
• the use of root-growth hormones in the nutrient solution in the growth chamber 510 provides significant advantages in a method for plant propagation.
• the shoot and collar may be planted in a soil-like medium for further growth.
• a variety of solid or soil-like media may be employed, such as vermiculite or a wide variety of other potting mixes.
• the shoot grows vigorously on the artificial xylem surface 572, due to the fact that the xylem tissue is in communication with the artificial xylem fibers.
• root-growth hormones induce the lateral formation of roots, which grow into the phloem sponge 570.
• the shoot and the root formation is of sufficient size and is physiologically prepared for existence in a natural environment, the shoot may be easily removed from the growth chamber 510 and the xylem surface 572, as the roots by this time have begun to sustain the vascular flow within the plant. While the plant tissue has been intimate with the hollow fibers on the artificial xylem surface, the removal of the plant from that surface does not severely injure the shoot or affect its further growth.
• the established shoots may be planted, along with the sponge 570, in potting mix and will develop rapidly into mature plants.
• This method of propagating tissue culture into mature plants provides substantial advantages.
• survival is substantially improved.
• the artificial root system and the hydrophilic hollow fiber allow for a microbial shield while permitting nutrients to flow to the tissue as needed.
• the rinse system may be automated and conducted in parallel for a large number of individual chambers, thus eluting plant endotoxins and greatly increasing the survival of the shoots.
• This type of rinse system has been impossible with previous propagation methods, which rely on the formation of roots in agar and inhibit nutrient flow and plant growth prior to root formation. Further, it has been found that superior shoot growth is obtained through the maintenance of lower humidity in the propagation and growth chambers.
• a portable systemic xylem interface which enables the unitary transfer of the growing shoot and the supporting interface to a new environment.
• the term "portable" refers to a plant-supporting xylem interface which is removable, along with the plant, from the propagation vessel. As is shown in Fig.
• a systemic interface including a body portion; an artificial surface, including perforations, which is capable of mating with natural xylem tissue and transferring nutrient solution thereto; and wicking means in contact with the perforate surface.
• the interface which is in some respects a mechanical fastener, is designated by the reference numeral 610.
• the fastener 610 is preferably formed from a resinous material, and most preferably is formed from an autoclavable material such as a polycarbonate resin if repeated usage is desired.
• the fastener 610 is seen to include a lower body portion 612 and an upper body portion 614.
• the portion 614 is seen to comprise a basin-like member 616 which is essentially circular from an overhead view.
• the basin is seen to contain a wicking sponge member 618 which is preferably formed from an autoclavable material (i.e., an open-cell material wherein the open-cell or sponge-like nature is not destroyed by the heat required to sterilize the fastener). While a neointimal sponge material such as that employed in the artificial phloem is not required, the Merocel sponge has been found advantageous for this use. However, any material which provides the capillary dispersion of fluid, as described, may be employed.
• the sponge 618 is seen to contact a perforate surface 620 which provides the artificial xylem surface of the interface 610.
• the surface 620 may be formed from discs of hollow fibers such as that shown in Fig. 5 , or may be formed from capillary-pore membrane laminates such as those sold by the Nuclepore Corporation. These are polyester or polypropylene films, or laminates of both such polymers, which are exposed to sufficient X- or other radiation, or other means, to form a perforate surface which is capable of mating with natural xylem tissue.
• a Nuclepore or similar film having perforations of about 0.2 micron and a perforation density of about 3 x 10 8 perforations/cm 2 ⁇ 15% has been found to be advantageous in that it mates with natural xylem tissue and also serves as a barrier to pathogens.
• any of the perforate surfaces which are employed to form the surface 620 may be sealed inside the top of the side walls 622 of the basin 616, as shown in Fig. 12, with silicone or other appropriate adhesives, or a more pliable perforate surface such as the Nuclepore material may be wrapped or formed around the outside edges of the side wall 622.
• the lower portion 612 is seen to comprise a sheathlike cylindrical casing 624 which surrounds a plurality of hydrophilic , e . g. , surfactant-treated, hollow fibers 626 similar to the fibers 456 in Figs. 9 and 10, which are seen to extend upwardly to contact the sponge 618.
• the casing 624 includes openings 628 to permit the flow of medium around the fibers 626, as described.
• the fibers are seen to be engaged, at the lowest portion of the casing 624, in a sealing member 630, i.e., a plug formed from appropriate material which captivates the fibers at the lower end of the casing 624.
• a sealing member 630 i.e., a plug formed from appropriate material which captivates the fibers at the lower end of the casing 624.
• a similarplug 632 captivates the fibers at an upper end of the casing 624, that is, where the casing 624 joins with the basic 616.
• These plugs which may be formed from a urethane resin or by a heat sealing means, captivate the fibers so that fiber shrinking does not occur during autoclaving. Since the shrinkage will reduce both the pore and lumen size of the fibers, such sealing at both ends of the casing 624 is preferred.
• the upper plug 632 also serves to limit the flow of nutrient solution to that which wicks through the fibers 626, as more fully described in the description of the use of the interface 610.
• any number of fibers may be disposed within the interface 610, with the limits being established by the need for sufficient capillary profusion to the sponge 618, and the cost of the fibers.
• about four to eight fiber strands each having a sufficient lumen diameter and wall porosity are preferred.
• the sufficiency of the lumen diameter is established by the ability of the fibers to wick the fluid to the sponge 618.
• the sufficiency of the wall porosity is determined by the ability of the fibers to serve as a barrier to the transport of pathogens during use.
• a wall porosity of 0.2 micron will serve as a barrier to the flow of most pathogenic agents.
• the fibers are seen to be looped from the sponge 618 to the plug 630, and then returned to the sponge.
• individual lengths of fibers may be employed.
• a wide variety of automated methods may be employed to manufacture the interface 610, and the fibers may be inserted in a sewing technique whereby the fiber is fed from a continuous spool through a needle-pointed mandrel guide which pierces a thin, disposable retention material at the top and bottom of the fastener to suspend the fiber in a relatively taut condition until the sealing at the ends of the casing 624 is completed.
• the polyurethane or silicone sealing material is then inserted into the respective ends of the casing 624, e.g., through the recesses 628, and curing then completed.
• centrifuging may be employed to force the sealing material toward the ends of the casing 624.
• the interface 610 is also seen to include a sealing flange 634, for purposes hereinafter described.
• the flange 634 may be a threaded member to facilitate the fastening of the interface 610 into other apparatus.
• twist-lock fasteners, tapered slip-fit points or other retaining means and gaskets to facilitate the retention and release of the interface 610 in other apparatus, and the sealing of the upper growing portion 614 from the nutrient flow, may be employed as later described.
• the interface 610 may be shown in combination with the growth chamber 510, which is shown in Figures 11.
• the receptacles 540 of the growth chamber 510 can readily be adapted to securely retain the fastener 610, by appropriate retention or fastener means therebetween.
• the dimensions of the fastener 610 may be altered, or engaging means such as turning lugs included, so that the interface 610 can be inserted, fastened and the flanges 634 can be adapted to provide sealing means between the growth surface 620 of the interface and the nutrient solution chamber 514 of the growth chamber.
• the interface 610 is disposed in a manner so that the hollow fibers 626, within the casing 624, are in contact with the nutrient solution so that nutrients are wicked to the sponge 516, through the perforate surface 620 and to the growing plants.
• the sealing of the growth surface from the nutrient solution is of significant importance in the growth of shoots, due to the fact that the influx of pathogens to the plants is blocked by the minimal porosity of the hollow fibers. Accordingly, external contaminants cannot reach the growing plants, and any internal contaminants (produced within the plant itself) cannot pass from the individual interface unit 610 and cross-contaminate the medium which is shared with other growing shoots.
• an adjustable vapor diffusion means 536 may be constructed whereby the adaptation and hardening of a growing plant to natural humidity are greatly facilitated.
• a series of holes or perforations of increasing diameter or surface area can be formed or otherwise provided in the cover 524, each of which is sealed with a diffusion membrane means.
• the diffusion membrane may be the same for each of the holes.
• a sliding cover enables the covering of all holes, or the exposure of one or more, depending on the humidity required within the growth chamber. It has been found that the permeability of the diffusion membranes is significantly dependent on the surface area of the membrane, and thus the progressive uncovering of larger holes allows more diffusion of vapor.
• the shoot and root formation When used in connection with the chamber 510, when the shoot and root formation is of sufficient size and is physiologically prepared for existence in a natural environment, the shoot need not be removed from the surface 620, but the entire combination of the plant and the interface 610 may be easily removed and planted to minimize temporary damage to the plant.
• the interface 610 could be employed as the initial interface for the growth of explant tissue, and once the shoot has grown to an appropriate size the shoots and the interface can be transferred to a growth chamber as a unit.
• shoots can be formed, grown to significant size, and transferred to environments for further grow-out without having to separate the plant xylem from the artificial xylem interface.
• FIG. 12 An alternative use is shown by the use of the cap 636 in Fig. 12.
• This cap may be constructed from a variety of resilient or other resinous material,, and is seen to include an upper sealing surface 638 and a basin 640 formed by a side wall 642. Following the initial growth of the plant in a growth chamber, the basin 640 may be partially filled with growth medium and the casing 624 is then inserted into the cap 636, as shown by the arrow on Fig. 12. The solution is then sealed around the wicking fibers by the sealing means 634 and 638, allowing transport of the rootless plant to the next growth cycle, or shipment to final use, without disruption of nutrient feed to the living portion.
• FIG. 13 additional apparatus which is used to accomplish this objective is shown.
• a rooting cartridge 650 is seen to comprise an essentially bell-shaped core of open-cell sponge material 652. While a wide variety of pre-formed or molded cellular sponges may be employed, use of a polyvinyl acetyl sponge such as the Merocel sponge has again been found to be advantageous.
• the sponge 652 is seen to be covered by a non-toxic, essentially non-permeable covering 654. This covering has been formed from a food-grade vinyl coating known as "plastisol", and preferably is a non- leaching plastisol. Alternatively, a non-leaching coating can be applied to the sponge prior to the vinyl coating to function as a barrier against the perfusion of the coating into the sponge and to facilitate removal of the coating at time of planting in soil.
• the coating of the sponge 52 may be employed for the coating of the sponge 52, if the coating is non-permeable so as not to inhibit plant growth.
• the liquid-applied vinyl i.e., plastisol
• the sponge 52 is coated with a coating that is non-permeable so as not to inhibit plant growth.
• the liquid-applied vinyl i.e., plastisol
• the rooting cartridge 650 is seen to include a xylem interface 656 mounted atop a cylindrical collar 658.
• the interface 656 may be any perforate surface which will mate with the xylem tissue of the plant, although in this instance the use of the track-etch, capillary-pore membrane manufactured by Nuclepore Corporation having a thickness of 2 to 4 mils, and perforations of about 0.2 microns on 10r-30 micron centers is advantageous.
• any factitious surface having perforations which will mate with some or all of the natural plant xylem tissue is within the purview of this invention.
• the membrane 656 and collar 658 may be assembled previously, such as by securing the flexible Nuclepore membrane to the collar with appropriate adhesive, and inserted into a cylindrical opening 660 in the body of the rooting cartridge 650. Preferably, this insertion is performed prior to the coating of the sponge 652 with plastisol, and the perforate surface of the membrane 656 is protected by a protective covering, not specifically shown, which is retained by lugs 662 on the interior surface of the cylindrical collar 658.
• the collar 658 is made of a resilient resinous material, i.e., the collar and the lugs 662 have sufficient memory to permit expansion to permit the insertion and removal of appropriate objects, and the sealing of the inserted object against the transfer of liquid to the xylem interface 656, as hereinafter described.
• This insertion and sealing will be described in more detail with respect to a wicking cartridge 664 which is seen to be disposed within the cylindrical opening 660.
• This cartridge is seen to include a casing 666 which comprises a lower sheath-like portion 668 and an upper basin portion 670.
• the portions 668 and 670 may be identical to the portions 612 and 614 in Fig. 12, aside from the fact that the xylem interface 656 in Fig.
• the lower portion 668 includes, although not specifically shown, hollow fibers embedded in a sealing resin at either end of the portion 668, with the ends of the fibers extending into a sponge 672 which extends above the basin portion 670.
• the lower portion 668 also includes recesses 674 which permit liquid to contact the hollow fibers within the sheath portion 668 when that portion is immersed in liquid.
• Fig. 12 which describes a fiber, sheath and sponge arrangement which is identical to that of the cartridge 664, liquid flows by capillary action through the porous walls of the fibers, through the lumens, to the sponge 672.
• the wicking cartridge 664 is inserted into the cylindrical opening 660 and elevated until the circumferential tang 675 on the basin portion 670 is inserted between the lugs 662 of the collar 658.
• the sponge 672 abuts the xylem interface 656 so that any nutrient medium wicked to the sponge is transferred through the perforate surface to be absorbed by the natural plant xylem tissue, and the abutting of the tang 675 with the collar 658 seals the wicking cartridge 664 within the rooting cartridge 650.
• the construction of the rooting cartridge 650 facilitates the insertion of the shoot or explant tissue which is to be grown on the xylem surface 656.
• the circular tang 673 is seen to include the resilient collar 658 and the lugs 662 in a manner which minimizes or eliminates the flow of pathogens therebetween.
• the construction of the rooting cartridge 650 facilitates the insertion of the shoot or explant tissue onto the artificial xylem surface 656.
• the vinyl coating 654 is seen to cover a channel 676 which communicates with the perforate xylem surface.
• the vinyl is pierced directly above the channel, and the vinyl above the channel 676, and the channel itself, is expanded with a mandrel to permit the insertion of the material onto the xylem surface.
• a number of manufacturing techniques may be employed in this regard.
• the shoot may be inserted with a mandrel into the channel 676 and a vacuum applied through the cylindrical opening 660 to pull the shoot tight against the xylem surface.
• the cartridge 664 is inserted to permit the profusion of the nutrient medium to the plant.
• Channel 676 and the size of the opening formed in the vinyl covering may be appropriately sized to retain the shoot firmly against the perforate surface during this procedure. It should be noted that both the vinyl covering and the sponge 652 offer sufficient resilience to permit significant expansion of the shoot during growth.
• Cell culture can be injected through the vinyl covering onto the perforate surface with a hypodermic needle.
• a tubular member may be cast within the channel 676 and extend above the top of the rooting cartridge 650. Cell culture can be injected through the top of this tubular member onto the perforate surface, and the top may be clipped off after the shoot has formed to permit further growth.
• the sterility of the growing tissue is not compromised until the shoot has formed and natural plant callus has covered the surface 656 and sealed the vascular system against the influx of pathogens.
• the rooting cartridge 650 may be equipped with one or more aeration port 680 which provides a proper air-medium mixture to the sponge 652.
• Multiple aeration ports may be provided depending upon the sponge size and density, and the particular varietal being grown therein.
• the port 680 is formed by coating a protrusion on the sponge with the plastisol and, after coating, shearing the end of the protrusion with a knife to expose the sponge.
• a cap 682 which includes a hydrophilic membrane 684 is bonded to the port 680 to provide the proper gas exchange with the sponge for proper air-medium ratio.
• the hydrophilic membrane 684 is similar to the membrane 436 described with respect to Figure 9.
• the lower portion of Fig. 13 shows a medium delivery and support device 710 which provides mechanical support to the rooting cartridge 650.
• the device 710 is molded of high-impact resistant, autoclavable polymeric material such as polycarbonate.
• the device is a sterile reservoir designed to accept plant growth liquid medium for the rooting cartridge 650.
• the device comprises a rigid polymeric casing 712 which forms a cylindrical side wall 714, a central retaining stanchion 716 and an inner protrusion 718.
• the stanchion 716 includes a removable insert 720 comprising a cylindrical, threaded collar 722 bearing a first and second silicone diaphragm 724 and 726, respectively. Each of these diaphragms is perforated at the center to form a passageway 728.
• the side wall 714 is seen to include a fluid inlet 730 and a fluid outlet 732. Fluid enters a reservoir 734 which is formed by the walls of the device 710, and fills the interior reservoir 734 until the liquid rises to the level of the lower diaphragm 726.
• the outlet port 732 is positioned at a height which controls this liquid level.
• the entire rooting cartridge 650 is then placed in the device 710.
• the sponge 652 is wedged tightly between the side wall 714 and the stanchion 716, and the sheath portion 668 enters the passageway 728.
• the diaphragms 724 and 726 enter a retaining groove 686 on the wicking cartridge 664, to close the reservoir 734 to the outside environment.
• the basin portion 670 of the wicking cartridge 664 is seen to engage tightly with the collar 722 to provide additional sealing to the transmission of pathogens to the nutrient medium.
• the rooting cartridge 650 will be apparent in the context of the previous description of the induction of lateral root formation into the sponge 570 in Fig. llA. In this instance, however, the rooting cartridge 650 may be planted in toto after removal or partial removal of the coating 654 and hardening of the roots within the sponge 652.
• the overall shape of the cartridge 650, and the device 710, facilitates the development of a root ball and the rooting cartridge 650 may be easily transferred to soil or soil-like medium for ultimate plant growth.
• this coating may be partially degradable over time to allow the eventual growth of the roots into the soil when planted.

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• Life Sciences & Earth Sciences (AREA)
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• 1987
  • 1987-05-27 EP EP19870903947 patent/EP0308422A4/en not_active Withdrawn
  • 1987-05-27 AU AU75491/87A patent/AU7549187A/en not_active Abandoned
  • 1987-05-27 JP JP87503653A patent/JPH02500003A/ja active Pending
  • 1987-05-27 WO PCT/US1987/001232 patent/WO1987007113A1/en not_active Application Discontinuation
  • 1987-05-29 IL IL82718A patent/IL82718A0/xx unknown
• 1988
  • 1988-01-29 DK DK046588A patent/DK46588A/da not_active Application Discontinuation
  • 1988-05-12 KR KR1019890700096A patent/KR910003642B1/ko not_active Application Discontinuation
  • 1988-11-29 FI FI885538A patent/FI885538A/fi not_active IP Right Cessation

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