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Flame retardants include but are not limited to aluminium trihydroxide ATH , magnesium hydroxide MDH , phosphonate esters, triphenyl phosphate, phosphate esters, ammonium pyrophosphate and melamine polyphosphate. When the container is constructed of cellulosic material, it can optionally contain clay, alum, waxes, binders, glues, surfactants and barriers such as plastic or metallized layers.
The cellulosic material may be porous and may possess multiple layers comprising, or alternatively consisting of, a variety of papers including but not limited to craft paper, bond paper, recycled paper, recycled newsprint, construction paper, chip board, cup stock, copier paper, wax paper, and coated papers.
In the presently disclosed invention, artificial seeds can be produced using a paper or a plastic container. The paper or plastic, to be used for container construction, has the following properties to be suitable for such application: The paper containers can be porous in nature, and can be degradable over the course of at least 5 years in soil. The plastic containers can be porous or non-porous, and may or may not be degradable in soil. The plastic material is either thermoplastic or thermoset materials.
In an embodiment, wax paper can be used to prepare the paper containers. In this case the size of the wax paper container can be around 1. The cylindrical containers can have flat ends at the top and the bottom.
In one embodiment, the bottom end of the container is crenellated see Figure 2. As used herein, "crenellation" means the creation of an irregular edge via the use of tabs of material extending from the edge and indentations into the edge. The size of crenellation can be from 0. In another embodiment, crenellation can be from 0. Artificial seeds can also comprise one or more of a nutrient source Figure 1 , 5 , solid objects such as pieces of cotton Figure 1, 6 , insecticides, fungicides, nematicides, antimicrobial compounds, antibiotics, biocides, herbicides, plant growth regulators or stimulators, microbes, molluscicides, miticides, acaricides, bird repellant s , insect repellant s , plant hormones, rodent repellant s , fertilizers, hydrogels,.
Molluscicides include, but are not limited to, metaldehyde or methiocarb. Acaricides include, but are not limited to, ivermectin or permethrin. A bird repellent is defined as a substance that repels birds. Bird repellants include, but are not limited to, methyl anthranilate, methiocarb, chlorpyrifos and propiconazole. A rodent repellent is defined as a substance that repels rodents.
Rodent repellents include, but are not limited to, thiram and methiocarb. Insect repellents include, but are not limited to, N,N-diethyl-m-toluamide, essential oils and citronella oil. Miticides include, but are not limited to, abamectin and chlorfenapyr. Plant hormones include, but are not limited to, abscisic acid, auxins, cytokinins, ethylene and gibberellins.
Plant growth regulators include, but are not limited to, paclobutyrazol, ethephon, and ancymidol. As used herein, "superabsorbents" means absorbents which absorb water or aqueous solutions resulting in a hydrated gel such that the weight of the gel is 30 times or greater the weight of the dry superabsorbent.
Superabsorbents include, but are not limited to, superabsorbent polymers, crosslinked poly sodium acrylate , crosslinked poly acrylic acid , crosslinked poly acrylic acid salts, acrylic acid modified starch, crosslinked copolymers of acrylic acid with poly ethylene glycol acrylate, poly ethylene glycol methacrylate, poly ethylene glycol diacrylate, acrylamide, vinyl acetate, acrylic acid salts, bisacrylamide, N-vinyl pyrrolidone, acrylate esters, methacryrlate esters, styrenic monomers, diene monomers and crosslinkers.
The superabsorbent may be present in the artificial seed in a dry or swollen state. It may be swollen with water or aqueous solutions, including but not limited to nutrient solutions, fertilizer solutions and antimicrobial solutions. The superabsorbent may also be mixed with soil or other components of the nutrient media. In one embodiment, the. The compartment may be connected or not with the compartment containing the regenerable plant tissue.
The compartment may be separated by a screen or mesh from the compartment containing the tissue. Microbes include but are not limited to beneficial microbes, nitrogen fixing bacteria, rhizobium, fungi, azotobacter, microrhyza, microbes that release cellulases, and microbes that participate in degradation of the artificial seed container. The soil suitable for application inside the container where the regenerable plant tissue is to be inserted to grow should be able to provide aeration, water, nutrition, and anchorage to the growing regenerable plant tissue.
It can also include natural soils such as sand, silt, loam, peat, and mixtures of these soils. The artificial seed of the disclosed invention comprises airspace 2 within the container. The artificial seed can also contain closures Figure 1, 1.
Closures are defined as lids, caps or objects that cover openings. In one embodiment the closure may be separable from the container. The regenerable plant tissue may be capable of lifting off or shedding the separable closure during its growth. Separable closures include but are not limited to caps, inserts, flat films, dome shaped caps and conical caps.
The separable closure may be attached to the container using an adhesive or degradable material. The degradable material may be hydrolytically degradable, oxidatively degradable, biodegradable, compostable or photodegradable. The caps or lids may also be attached by simple physical means including but not limited to insertion or crimping.
As used herein, "nutrient source" means nutrients which can help sustain and provide for the growth of the plant from the regenerable tissue. Suitable nutrients include, but are not limited to, one or more of water, soil, coconut coir, vermiculite, an artificial growth medium, agar, a plant growth regulator, a plant hormone, a.
Micronutrients include but are not limited to cobalt chloride, boric acid, ferrous sulfate and manganese sulfate. The nutrient source can also contain extracellular.
Journal of Arid Environments, , 75, 2, The nutrient source can also contain hormones and plant growth regulators including but not limited to, gibberellic acid, indole acetic acid, naphthalene acetic acid NAA , ethephon, 6-benzylamino purine 6-ABP , 2,4-dichlorophenoxyacetic acid 2,4- D , paclobutrazole, ancymidol and abcissic acid. The nutrients can be present in an aqueous solution or aqueous gel solution, such as those well known in the art of plant propagation, including but not limited to natural and synthetic gels including: In one embodiment, the nutrients can be present in a silicate gel.
Such a silicate gel can be formed by neutralizing a solution of sodium or potassium silicate with acid. In one embodiment, subsequent washing or soaking steps may be used to remove the excess salts. Optionally, the gel can then be infused with nutrients through soaking or other processes. Alternatively, the silicate gel can be formed from silicic acid, or from other precursors, including but not limited to alkoxysilanes, silyl halides, or silazanes.
When the container comprises a nutrient source, the regenerable plant tissue within the container is partially embedded or in contact with the nutrient source and can be partially exposed to the airspace within the container.
The term "partially exposed to an airspace", as used herein, refers to a regenerable plant tissue that is either in contact with or has been partially embedded i. The regenerable plant tissue can be partially or fully surrounded by the nutrient source. The regenerable plant tissue can also be placed on top of the nutrient source. As used herein, "airspace" means a void in the container that is empty of any solid or liquid material, and filled by atmospheric gasses such as air, for example.
An airspace, as defined herein does not include the collective voids in a porous or particulate material. It is advantageous for the function of the artificial seed that the airspace be free of obstructions that limit the growth of the regenerable plant tissue with exception of the limits of the container wall.
As used herein, "an unobstructed airspace" means an airspace that is continuous and uninterrupted between any part of the regenerable plant tissue and any region of the container. As used herein, "tapered" means narrowing or becoming progressively narrower along a dimension.
Other possible methods include using plant cuttings, embryos from natural seeds or somatic embryos obtained through somatic embryogenesis. In one embodiment meristems can be excised to form explants and cultured to increase the tissue mass. The term "explant" as used herein, refers to tissues which have been excised from a plant to be used in plant tissue culture. The regenerable plant tissue of the invention may also be genetically modified.
This genetic modification includes, but is not limited to, herbicide resistance, disease resistance, drought tolerance, and insect resistance.
Genetically modified also known as transgenic plants may comprise a single transgenic trait or a stack of one or more transgene polynucleotides with one or more additional polynucleotides resulting in the production or suppression of multiple polypeptide sequences. Transgenic plants comprising stacks of polynucleotide sequences can be obtained by either or both of traditional breeding methods or through genetic engineering methods.
These methods include, but are not limited to, breeding individual lines each comprising a polynucleotide of interest, transforming a transgenic plant comprising a gene with a subsequent gene and co- transformation of genes into a single plant cell.
As used herein, the term "stacked" includes having the multiple traits present in the same plant i. In one non-limiting example, "stacked traits" comprise a molecular stack where the sequences are physically adjacent to each other. A trait, as used herein, refers to the phenotype derived from a particular sequence or groups of sequences.
Co-transformation of genes can be carried out using single transformation vectors comprising multiple genes or genes carried separately on multiple vectors. If the sequences are stacked by genetically transforming the plants, the polynucleotide sequences of interest can be combined at any time and in any order.
The traits can be introduced simultaneously in a co-transformation protocol with the polynucleotides of interest provided by any combination of transformation cassettes. For example, if two sequences will be introduced, the two sequences can be contained in separate transformation cassettes trans or contained on the same transformation cassette cis.
Expression of the sequences can be driven by the same promoter or by different promoters. In certain cases, it may be desirable to introduce a transformation cassette that will suppress the expression of the polynucleotide of interest. This may be combined with any combination of other suppression cassettes or overexpression cassettes to generate the desired combination of traits in the plant. It is further recognized that polynucleotide sequences can be stacked at a desired genomic location using a site-specific recombination system.
In some embodiments the polynucleotides encoding the polypeptides, alone or stacked with one or more additional insect resistance traits can be stacked with one or more additional input traits e. Thus, the polynucleotide embodiments can be used to provide a complete agronomic package of improved crop quality with the ability to flexibly and cost effectively control any number of agronomic pests.
Transgenes useful for preparing transgenic plants include, but are not limited to, the following:. A Plant disease resistance genes. Plant defenses are often activated by specific interaction between the product of a disease resistance gene R in the plant and the product of a corresponding avirulence Avr gene in the pathogen.
A plant variety can be transformed with cloned resistance gene to engineer plants that are resistant to specific pathogen strains. See, for example, Jones, et al, Science A plant resistant to a disease is one that is more resistant to a pathogen as compared to the wild type plant.
B Genes encoding a Bacillus thuringiensis protein, a derivative thereof or a synthetic polypeptide modeled thereon. See, for example, Geiser, et al, Gene Accession Numbers , , and Other non-limiting examples of Bacillus thuringiensis transgenes being genetically engineered are given in the following patents and patent applications and hereby are incorporated by reference for this purpose: C A polynucleotide encoding an insect-specific hormone or pheromone such as an ecdysteroid and juvenile hormone, a variant thereof, a mimetic based thereon or an antagonist or agonist thereof.
See, for example, the disclosure by Hammock, et al, Nature D A polynucleotide encoding an insect-specific peptide which, upon expression, disrupts the physiology of the affected pest. For example, see the disclosures of, Regan, J. See also, US Patent Number 5,, to Tomalski, et al, who disclose genes encoding insect-specific toxins. E A polynucleotide encoding an enzyme responsible for a hyperaccumulation of a monoterpene, a sesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivative or another non-protein molecule with insecticidal activity.
F A polynucleotide encoding an enzyme involved in the modification, including the post-translational modification, of a biologically active molecule; for example, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme, a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, a phosphatase, a kinase, a phosphorylase, a polymerase, an elastase, a chitinase and a glucanase, whether natural or synthetic.
See also, Kramer, et al, Insect Biochem. G A polynucleotide encoding a molecule that stimulates signal transduction. For example, see the disclosure by Botella, et al. H A polynucleotide encoding a hydrophobic moment peptide. I A polynucleotide encoding a membrane permease, a channel former or a channel blocker. For example, see the disclosure by Jaynes, et al. J A gene encoding a viral-invasive protein or a complex toxin derived therefrom.
See, Beachy, et al. Coat protein-mediated resistance has been conferred upon transformed plants against alfalfa mosaic virus, cucumber mosaic virus, tobacco streak virus, potato virus X, potato virus Y, tobacco etch virus, tobacco rattle virus and tobacco mosaic virus.
K A gene encoding an insect-specific antibody or an immunotoxin derived therefrom. Thus, an antibody targeted to a critical metabolic function in the insect gut would inactivate an affected enzyme, killing the insect. L A gene encoding a virus-specific antibody. See, for example, Tavladoraki, et al, Nature M A polynucleotide encoding a developmental-arrestive protein produced in nature by a pathogen or a parasite. Thus, fungal endo alpha- 1 ,4-D-polygalacturonases facilitate fungal colonization and plant nutrient release by solubilizing plant cell wall homo-alpha- 1,4-D-galacturonase.
See, Lamb, et al. The cloning and characterization of a gene which encodes a bean endopolygalacturonase- inhibiting protein is described by Toubart, et al. N A polynucleotide encoding a developmental-arrestive protein produced in nature by a plant. For example, Logemann, et al. Q Detoxification genes, such as for fumonisin, beauvericin, moniliformin and zearalenone and their structurally related derivatives.
For example, see, US Patent Numbers 5,,; 5,,; 5,,; 5,,; 6,,; 6,,; 6,, and 6,, R A polynucleotide encoding a Cystatin and cysteine proteinase inhibitors. See, US Patent Number 7,, T Genes conferring resistance to nematodes. This includes the Reg locus that may be utilized as a single locus conversion. A A polynucleotide encoding resistance to a herbicide that inhibits the growing point or meristem, such as an imidazolinone or a sulfonylurea. B A polynucleotide encoding a protein for resistance to Glyphosate resistance imparted by mutant 5-enolpyruvlphosphikimate synthase EPSP and aroA genes, respectively and other phosphono compounds such as glufosinate phosphinothricin acetyl transferase PAT and Streptomyces hygroscopicus phosphinothricin acetyl transferase bar genes , and pyridinoxy or phenoxy proprionic acids and cyclohexones ACCase inhibitor-encoding genes.
US Patent Number 5,, to Barry, et al. See also, US Patent Numbers 6,,; 6,,; 6,, Bl; 6,,; 5,,; 5,,; 5,,; 4,,;. Glyphosate resistance is also imparted to plants that express a gene encoding a glyphosate oxido- reductase enzyme as described more fully in US Patent Numbers 5,, and. In addition glyphosate resistance can be imparted to plants by the over expression of genes encoding glyphosate N-acetyltransferase. See, for example, US Patent Numbers 7,,;.
EP Application Number 0 to Kumada, et al. The nucleotide sequence of a phosphinothricin-acetyl-transferase gene is provided in EP Application Numbers 0 and 0 to Leemans, et al. See also, US Patent Numbers 5,,; 5,,; 5,,; 5,,; 5,,; 5,,;. Exemplary genes conferring resistance to phenoxy proprionic acids and cyclohexones, such as sethoxydim and haloxyfop, are the Accl-Sl, Accl-S2 and Accl-S3 genes described by Marshall, et al. D A polynucleotide encoding a protein for resistance to Acetohydroxy acid synthase, which has been found to make plants that express this enzyme resistant to multiple types of herbicides, has been introduced into a variety of plants see, e.
Other genes that confer resistance to herbicides include: Protoporphyrinogen oxidase protox which is necessary for the production of chlorophyll. The protox enzyme serves as the target for a variety of herbicidal compounds. These herbicides also inhibit growth of all the different species of plants present, causing their total destruction.
F The aad-1 gene originally from Sphingobium herbicidovorans encodes the aryloxyalkanoate dioxygenase AAD-1 protein. The trait confers tolerance to 2,4- dichlorophenoxyacetic acid and aryloxyphenoxypropionate commonly referred to as "fop" herbicides such as quizalofop herbicides. The aad gene, derived from Delftia acidovorans, which encodes the aryloxyalkanoate dioxygenase AAD protein that confers tolerance to 2,4-dichlorophenoxyacetic acid and pyridyloxyacetate herbicides by deactivating several herbicides with an aryloxyalkanoate moiety, including phenoxy auxin e.
H A polynucleotide molecule encoding bromoxynil nitrilase Bxn disclosed in US Patent Number 4,, for imparting bromoxynil tolerance;. I A polynucleotide molecule encoding phytoene crtl described in Misawa, et al, Plant J. See, Knultzon, et al, Proc. For example, see, Van Hartingsveldt, et ah, Gene See, Shiroza, et ah, J. D Altered antioxidant content or composition, such as alteration of tocopherol or tocotrienols.
E Altered essential seed amino acids. For example, see, Lyznik, et al, Plant Cell Rep Other systems that may be used include the Gin recombinase of phage Mu Maeser, et al. Including but not limited to flowering, ear and seed development, enhancement of nitrogen utilization efficiency, altered nitrogen responsiveness, drought resistance or tolerance, cold resistance or tolerance and salt resistance or tolerance and increased yield under stress.
A For example, see: B WO describing genes, including CBF genes and transcription factors effective in mitigating the negative effects of freezing, high salinity and drought on plants, as well as conferring other positive effects on plant phenotype. F For plant transcription factors or transcriptional regulators of abiotic stress, see, e. B Over-expression of maize zinc finger protein gene Zm-ZFPl using a seed preferred promoter has been shown to enhance plant growth, increase kernel number and total kernel weight per plant US Patent Application Publication Number.
E Modulating expression in a plant of a nucleic acid encoding a Stelike polypeptide or a homologue thereof gives plants having increased yield relative to control plants EP In some embodiments the stacked trait may be in the form of silencing of one or more polynucleotides of interest resulting in suppression of one or more target pest polypeptides.
In some embodiments the silencing is achieved through the use of a suppression DNA construct. In some embodiments one or more polynucleotides encoding the polypeptides or fragments or variants thereof may be stacked with one or more polynucleotides encoding one or more polypeptides having insecticidal activity or agronomic traits as set forth supra and optionally may further include one or more polynucleotides providing for gene silencing of one or more target polynucleotides as discussed infra.
The target gene may be endogenous or transgenic to the plant. The term "suppression" includes lower, reduce, decline, decrease, inhibit, eliminate and prevent. A suppression DNA construct may comprise a region derived from a target gene of interest and may comprise all or part of the nucleic acid sequence of the sense strand or antisense strand of the target gene of interest.
Suppression DNA constructs are well-known in the art, are readily constructed once the target gene of interest is selected, and include, without limitation, cosuppression constructs, antisense constructs, viral-suppression constructs, hairpin suppression constructs, stem-loop suppression constructs, double-stranded RNA-producing constructs, and more generally, RNAi RNA interference constructs and small RNA constructs such as siRNA short interfering RNA constructs and miRNA microRNA constructs.
The complementarity of an antisense RNA may be with any part of the specific gene transcript, i. Cosuppression constructs in plants have been previously designed by focusing on overexpression of a nucleic acid sequence having homology to a native mRNA, in the sense orientation, which results in the reduction of all RNA having homology to the overexpressed sequence see, Vaucheret, et ah, Plant J.
Recent work has described the use of "hairpin" structures that incorporate all or part, of an mRNA encoding sequence in a complementary orientation that results in a potential "stem-loop" structure for the expressed RNA PCT Publication WO. In this case the stem is formed by polynucleotides corresponding to the gene of interest inserted in either sense or anti-sense orientation with respect to the promoter and the loop is formed by some polynucleotides of the gene of interest, which do not have a complement in the construct.
This increases the frequency of cosuppression or silencing in the recovered transgenic plants. Methods and Protocols Yet another variation includes using synthetic repeats to promote formation of a stem in the stem-loop structure. R A interference refers to the process of sequence-specific post-transcriptional gene silencing in animals mediated by short interfering RNAs siRNAs Fire, et al , Nature The corresponding process in plants is commonly referred to as post-transcriptional gene silencing PTGS or RNA silencing and is also referred to as quelling in fungi.
The process of post-transcriptional gene silencing is thought to be an evolutionarily-conserved cellular defense mechanism used to prevent the expression of foreign genes and is commonly shared by diverse flora and phyla Fire, et al, Trends Genet. Such protection from foreign gene expression may have evolved in response to the production of double-stranded RNAs dsRNAs derived from viral infection or from the random integration of transposon elements into a host genome via a cellular response that specifically destroys homologous single-stranded RNA of viral genomic RNA.
Short interfering RNAs derived from dicer activity are typically about 21 to about 23 nucleotides in length and comprise about 19 base pair duplexes Elbashir, et al, Genes Dev. Dicer has also been implicated in the excision of and nucleotide small temporal R As stR As from precursor RNA of conserved structure that are implicated in translational control Hutvagner, et al, Science As such, miRNA molecules of the invention can be used to mediate gene silencing via interaction with RNA transcripts or alternately by interaction with particular gene sequences, wherein such interaction results in gene silencing either at the transcriptional or post-transcriptional level.
Methods and compositions are further provided which allow for an increase in RNAi produced from the silencing element. In such embodiments, the methods and compositions employ a first polynucleotide comprising a silencing element for a target pest sequence operably linked to a promoter active in the plant cell; and, a second polynucleotide comprising a suppressor enhancer element comprising the target pest sequence or an active variant or fragment thereof operably linked to a promoter active in the plant cell.
The combined expression of the silencing element with suppressor enhancer element leads to an increased amplification of the inhibitory RNA produced from the silencing element over that achievable with only the expression of the silencing element alone. In addition to the increased amplification of the specific RNAi species itself, the methods and compositions further allow for the production of a diverse population of RNAi species that can enhance the effectiveness of disrupting target gene expression.
As such, when the suppressor enhancer element is expressed in a plant cell in combination with the silencing element, the methods and composition can allow for the systemic production of RNAi throughout the plant; the production of greater amounts of RNAi than would be observed with just the silencing element construct alone; and, the improved loading of RNAi into the phloem of the plant, thus providing better control of phloem feeding insects by an RNAi approach.
Thus, the various methods and. As used herein, a "suppressor enhancer element" comprises a polynucleotide comprising the target sequence to be suppressed or an active fragment or variant thereof. It is recognize that the suppressor enhancer element need not be identical to the target sequence, but rather, the suppressor enhancer element can comprise a variant of the target sequence, so long as the suppressor enhancer element has sufficient sequence identity to the target sequence to allow for an increased level of the RNAi produced by the silencing element over that achievable with only the expression of the silencing element.
Similarly, the suppressor enhancer element can comprise a fragment of the target sequence, wherein the fragment is of sufficient length to allow for an increased level of the RNAi produced by the silencing element over that achievable with only the expression of the silencing element.
It is recognized that multiple suppressor enhancer elements from the same target sequence or from different target sequences or from different regions of the same target sequence can be employed. For example, the suppressor enhancer elements employed can comprise fragments of the target sequence derived from different region of the target sequence i. Further, the suppressor enhancer element can be contained in an expression cassette, as described elsewhere herein, and in specific embodiments, the suppressor enhancer element is on the same or on a different DNA vector or construct as the silencing element.
The suppressor enhancer element can be operably linked to a promoter. It is recognized that the suppressor enhancer element can be expressed constitutively or alternatively, it may be produced in a stage-specific manner employing the various inducible or tissue -preferred or developmentally regulated promoters that are discussed elsewhere herein.
In specific embodiments, employing both a silencing element and the suppressor enhancer element the systemic production of RNAi occurs throughout the entire plant.
In further embodiments, the plant or plant parts of the invention have an improved loading of RNAi into the phloem of the plant than would be observed with the expression of the silencing element construct alone and, thus provide better control of phloem feeding insects by an RNAi approach.
In specific embodiments, the plants, plant parts and plant cells of the invention can further be characterized as allowing for the production of a diversity of RNAi species that can enhance the effectiveness of disrupting target gene expression.
In specific embodiments, the combined expression of the silencing element and the suppressor enhancer element increases the concentration of the inhibitory RNA in the plant cell, plant, plant part, plant tissue or phloem over the level that is achieved when the silencing element is expressed alone. As used herein, an "increased level of inhibitory RNA" comprises any statistically significant increase in the level of RNAi produced in a plant having the combined expression when compared to an appropriate control plant.
Some embodiments relate to down-regulation of expression of target genes in insect pest species by interfering ribonucleic acid RNA molecules. Typically, when a transgenic plant comprising a recombinant DNA construct or suppression DNA construct in its genome exhibits increased drought tolerance relative to a reference or control plant, the reference or control plant does not comprise in its genome the recombinant DNA construct or suppression DNA construct.
One of ordinary skill in the art is familiar with protocols for simulating drought conditions and for evaluating drought tolerance of plants that have been subjected to simulated or naturally-occurring drought conditions. Other techniques for evaluating drought tolerance include measuring chlorophyll fluorescence, photosynthetic rates and gas exchange rates. A drought stress experiment may involve a chronic stress i.
The regenerable plant tissue can be obtained from any plant species, including crops such as, but not limited to: In one embodiment, the regenerable plant tissue used in the artificial seed can be from sugarcane. The regenerable plant tissue can be prepared using several methods including excision of meristems from the top of the sugarcane stalks, followed by tissue culture on solid or liquid media, or temporarily immersed in liquid nutrients and combinations thereof.
In one embodiment, the regenerable sugarcane tissue can be prepared using tissue culture on a solid medium, followed by temporary immersion in liquid nutrient media. The meristem tissue can be allowed to grow and proliferate using a proliferation medium. The proliferation medium can include, but is not limited to, culturing in various liquid nutrient media, culturing on solid media, temporary immersion in liquid nutrient media, and any variations thereof.
In one embodiment, the proliferation medium used in the current method comprises MS nutrients and can additionally comprise ingredients not limited to: However, other nutrient formulations such as the well known in the art Gamborg's B-5 medium, other carbon sources such as glucose and mannitol, other cytokinins, such as kinetin and zeatin can also be used. The meristem tissues can be allowed to proliferate from about 3 weeks to about 52 weeks.
Temperature control for growth of the regenerable plant tissues can be achieved using constant temperature incubators as is well known in the relevant art. Following growth of the meristem tissue, proliferated buds are formed which contain independent meristem structures capable of differentiating into shoots, and subsequently into well-formed plantlets at later stages.
As used herein, "proliferated bud tissue" means a meristematic tissue with the capacity to multiply and self-regenerate into similar meristem structures. Over time, the base of this tissue, which was the original plant tissue, can blacken due to polyphenol production and can be removed by mechanical trimming methods well known in the relevant art. During the steps described above, the meristem tissue can be subjected to light to allow for growth.
The light can be produced by various devices suitable for this purpose such as fluorescent bulbs, incandescent bulbs, the sun, plant growth bulbs and light emitting diodes LEDs. The amount of light required for growth of the meristem tissue can vary from 1 hour photoperiod to 24 hours photoperiod.
After the meristem tissue forms the proliferated bud tissue, it can then be cut into small pieces fragmented to form tissue fragments. These tissue fragments can be 0. Alternatively, they can be mm in size. These tissue fragments can then be cultured for weeks further to form plantlets, which are suitable for encapsulation in the artificial seeds.
The culturing processes to form the plantlets can include, but is not limited to, culturing in various liquid nutrient media, culturing on solid media, temporary immersion in liquid culture, and any variations thereof. The plantlets that are formed in these processes possess shoots, with or without roots.
Artificial seeds of the type described in the present invention comprise a container assembly. The container assembly may be prepared using any variety of materials disclosed above.
In the present method, the regenerable plant tissue, which has been further cultivated to produce a plantlet may be used. The plantlet may be partially embedded into a nutrient-containing agar plug at the bottom of the container of the artificial seed such that part of the tissue e. The plantlet can be oriented or not, and can be trimmed to fit inside the container. Alternately, the plantlet can be placed in a soil layer in the container, such that airspace is present above it.
In the present method it is desirable to create an airspace within the container. The purpose of the airspace is to allow rapid gas exchange with the plantlet, helping to sustain the tissue and allow it to grow. The container can possess porosity which can allow a rate of gas transport such that equilibrium can be maintained between the airspace and the outside environment.
Thus, as the plantlet consumes or releases oxygen or carbon dioxide, due to either respiration or photosynthesis, these gases are rapidly equilibrated with the outside atmosphere. In addition, the exposure of the plantlet to the airspace fosters the development of tissue that is better adapted to the harsher conditions the plantlet can be exposed to once it emerges from the seed for example reduced humidity, wind, higher light. In the artificial seed, the plantlet is exposed to less harsh conditions due to the protection of the container.
In the present invention, the airspace is also transparent to visible light, which allows the plantlet to perform photosynthesis. The airspace can also provide some thermal insulation for the plantlet.
The airspace may consist of multiple compartments. These compartments may be connected or adjoined and may be in communication with each other. To prevent fungal contamination of the artificial seed, the container can be treated with a solution of a fungicide prior to its assembly. Many fungicides can be used for this purpose.
Examples include, but are not limited to: Additionally, the container may comprise one or more antimicrobials, including but not limited to: Additionally, the container may comprise one or more antibiotics, including but not limited to: In order to prevent insect damage, the artificial seed may also comprise one or more insecticides.
Examples of suitable pesticidal compounds include, but are not limited to, abamectin, cyanoimine, acetamiprid, nitromethylene, nitenpyram, clothianidin, dimethoate, dinotefuran, fipronil, lufenuron, flubendamide, pyripfoxyfen, thiacloprid, f uxofenime, imidacloprid, thiamethoxam, beta cyfluthrin, fenoxycarb, lamda.
The artificial seed may comprise other crop protection chemicals, including but not limited to nematicides, termiticides, molluscicides, miticides and acaricides. In the process of artificial seed preparation and following addition of the plantlet, and in some cases, the nutrients, the opening in the container can be secured.
A container can have more than one opening. Alternatively, a container can have a top opening and a bottom opening. Depending on the design and method of planting, optionally one or both openings can be secured. Taking first place in both the Michigan and Los Angeles Cannabis Cups as well as the High Times Jamaican World Cup, this multiple award-winning hybrid's supremacy is no longer a secret, and consumers will search far and wide to get their hands sticky with Gorilla Glue 4.
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This sativa dominant Close. Mix and match any of the Sierra Essential cartridges! The message accompanying the picture was this: Shame on all of you. Lacking a mature and healthy Self that would normally be completely satisfied with the Love that it, itself, is, Burton has no choice but to seek love and acceptance externally.
And in the end, everyone is happy, or so they imagine. Again, Insider, thanks for the update [above]. Seekers trying to evaluate whether the Fellowship of Friends group is right for them should pay close attention.
Burton once again center stage, surrounded with gaudy trinkets, gold or marble statuettes, chandeliers, LED light strings. Those around him wearing the same lavender, purple and cream outfits, often his hand—me—downs. I find it hard to summon up any pity for the individuals, if you can call them that, embroiled in this mess.
Everyone there chose to give up all independence of thought and action to him, not understanding at first that this would become permanent. And now they are no longer interested or care. I feel pity for the situation, it is such a tragic waste of energy, resources and lives.
Every one, those on stage, members of the harem, and every member of the audience had sufficient warning before it was too late. Maybe not before being ensnared, but certainly after they joined. Everyone is complicit, and responsible for their own plight and those affected by their complicity. Looking at the photographs and videos posted here and at the REB blog, does not the lack of animation stand out?
This is an exhibition of the hive mind at work. The audience is completely passive, absorbing received knowledge, no questioning allowed, a one—way flow of energy from the superior to the inferior. Ordered to keep a self—hypnotizing, unblinking gaze on the source of, well, everything. Even the blob having to be openly prodded by minders does not break the trance, as he mutters the same memes over and over, constantly affirming the groupthink.
With the resistance to change institutionalized, death came unnoticed. Now we can see rigor mortis setting in, leading to the inevitable and final process of fossilization. Ames, reflecting for a moment upon the reason I expressed a degree of pity for those who continue to sleepwalk through life being hypnotized by a blathering Blobert and further self-hypnotized by group-think inside the hive-mind cocoon, somehow finding comfort in continued complicity in the charade….
But you make a good point regarding complicity of those who stay even though they must see the horror-of-the-situation — the damage done, the wasted human potential.
Some are simply not able to stand on their own as sovereign individuals and that seems to me a condition to be pitied — especially in cases where the person lacks the insight or intelligence to realize they unwittingly contribute to creating their own trap.
Yes, Ton, I may have come over as being judgmental, that was not intended, I was actually trying to describe the situation as dispassionately as possible. The leader has always been a dead man walking, and of course his disciples are following in his footsteps, their ambition and his wish perfectly matching. So much for the dynamics of the group as a whole. Nevertheless, each human being within the group always had and still has a choice—to individuate or not.
The psychological bonds are incredibly powerful. And the strongest fetters are the more effective because the wearers think they have escaped, that they are free, and the very opposite of captives or slaves. There are those who believe that Burton has the answers, generally and in the particular. And there are those who have experienced the cult and have broken away, who know both sides, who know the damage caused to themselves and others, and have taken responsibility for their lives, disappointments and joys and all.
Any of the latter are going to have empathy and sympathy for the plight of a captive. From what I can observe, there are many sub—categories in the group. There are those who have abdicated all responsibility for themselves, content and glad to leave their fate in the hands of Burton and his demons.
There are those who are happy to have a position of authority, spiritual or temporal, within the organization, and this is their main satisfaction. There are those who knowingly prostituted themselves, accepted the bargain, getting a Green Card and some luxury in exchange for labor and sexual favors. There are those who had not been able to make their way previously and stumbled upon the organization and found respite and a home.
There are those who were wounded, or needed a father figure. There are those who sought answers to the Big Questions, and are in the process of discovering that they are being fed pablum. There are those who are just plain tired, or are afraid of losing their circle of friends, or who are financially dependent on the organization. I can and do pity any one of these for the position they find themselves in. Yet no—one can take on the responsibility which is theirs and theirs alone.
If there is anyone from within the organization that is reading this, think about pride. Here in the western culture, pride is regarded as a sin—case closed. But I invite you to think of that aspect connected to self—respect and even self—love, the part of the psyche that takes care of the welfare of the Self. If you can observe and connect to that, then you can break the fetters and escape. You can hope—and act on that hope.
You may be used to putting your faith in Burton and his claims, how about putting faith in yourself and taking the plunge into the unknown? You are guaranteed interesting times, you will become alive in a totally different way, and every day will bring fresh adventures and surprises. Your days will no longer be scripted, and although you will probably miss some of those artificially induced highlights like concerts and ballets, your experiences will be genuine and yours alone to savor and learn from.
So, I encourage you to start respecting yourself and your potential and capacity for independence, and make the decision to break free. By January 1, , as the Fellowship entered its eleventh year, Burton had drawn an impressive 1, followers.
With Armageddon approaching, Burton predicted, "Our ark will be composed of 10, people in the year , and then the doors will close," Yet, in , membership peaked at around 2, Since that time, despite establishing Fellowship "teaching houses" in 60 cities around the globe and its elaborate array of recruiting websites , the group's ranks have dramatically ebbed. Today, the membership stands at roughly 1, many of whom were in that census.
All signs and omens suggest we are watching an institution a "church"? As "Insider" writes above, of the nearly 8 billion souls on the planet including 15, who at one time passed through the Fellowship only Burton and his 1, current followers now carry the flame of consciousness, and shall therefore claim their rightful place in Paradise. The obvious conclusion is that the Universe exists solely for the benefit of Robert Earl Burton and his Faithful As remaining members await Burton's next fever dream, it is time to truly ask yourselves, "Shall I still sleep or, finally awaken?
A true magnetic center must find the Fellowship; and there are true magnetic centers abounding in this world. It matters little how many groups exist on the earth that bear the name Gurdjieff or Ouspensky , as these groups remain Influence B. A properly developed magnetic center will not accept these organizations because it requires the nourishment of Influence C.
We are a young teaching and we have been afforded the great luxury of occupying the position of Higher School on earth.
The Fellowship of Friends is the greatest mystery of the Twentieth Century, and a series of shocks is necessary in order for this statement to be verified. We carry the sacred light of consciousness, as an esoteric school is the oldest civilized heritage on the earth.
Our School is a distinct descendant of the most eminent teachings in both recorded and unrecorded history. Tuesday, October 23, Pathologies on display. At a Fellowship of Friends meeting, Robert Burton mocks a follower's inability to control their "lower self.
Sunday, October 21, "Uh San Francisco and Alcatraz view, from wideasleep1's no joke! Alcatraz no longer serves as a prison, however San Quentin is quite close by.
Saturday, October 20, Rambling Man. Less than 19 hours to go. Some Burton followers are now in Oregon House waiting for the big event. Or the big non-event. But which version is being planned? Post-apocalyptic, or coping with cognitive dissonance following another failed prophesy? Somehow, meetings are not all that bad, even if Burton continues to ramble mindlessly from one associative topic to another.
Every member is a believer, a supporter, and a defender of Burton. Only the degree changes from one person to another. Now is not the time to express any doubts. People have really tightened the ranks.
Well, off to the mountain top with my day supply of cannabis and Oreo Cookies to enjoy the show. Insider's "T minus 12 hours" update: Oct 20, exactly 4: Twelve hours to go to nothing. People have been having a great time. In fact, if Burton keeps throwing bashes like this, followers will happily believe anything he says, do whatever he asks, and immediately forgive him for missing yet another prediction.
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