WO2009137135A2 - On-orbit procedures for adapting plants and animals to hostile environments - Google Patents

Abstract

The present invention provides methods for adapting plants and animals to survive outside their native environments. In particular, undifferentiated cells from plants or animals are replicated under weightless conditions in which cell replication or proliferation is accelerated and sustained. Under such conditions, the undifferentiated cells can be "forced" to express sets of genes useful for survival in particular environmental conditions. In this manner, cells surviving prolonged exposure to specific environmental conditions can be selected for and cultivated to produce an organism adapted to that particular environment in an accelerated manner. Methods of identifying specific genes associated with adaptation of a plant or animal to a specific environment are also disclosed.

Description

ON-ORBIT PROCEDURES FOR ADAPTING PLANTS AND ANIMALS TO HOSTILE ENVIRONMENTS

CROSS REFERENCE

This Application claims priority to U.S. Provisional Application No. 61/029,053, which was filed on February 15, 2008, which is incorporated in its entirety by reference.

BACKGROUND OF THE INVENTION

[0001] A weightless condition on space orbit has produced many effects on visitors to that region. Gravity still is present on orbit and through the course of interplanetary travel, but normal plant and animal functions as is known on Earth do not function in the same manner. If gravity acts as a dominant force in a weightless condition, then the reproduction of cells would follow the normal or close to normal pattern experienced on Earth. Evidence indicates that it is weightlessness and not gravity that drives the biology of both plants and animals on space orbit and during interplanetary transits and human travel.

[0002] Weight is a physical attribute and force parameter. Weight is a condition wherein the "escape force" [as a function of the angular velocity of a mass (angular momentum)] around a gravitational mass is less than the specific value of the gravitational force. That is, when a body is at rest on the Earth (stationary), it has an angular velocity due to the Earth's rotation. This angular velocity opposes, to some extent, the gravitational force that draws the mass to the center-of-gravity point of Earth. As the angular velocity of the mass increases, the inertial force that opposes the gravitational force increases, to the extent that a sufficient increase will result in the gravitational force equaling the inertial force.

[0003] This is the case for masses that have been launched from Earth and are in an orbit of equilibrium around the Earth, i.e. "on orbit". If the Earth had no angular velocity (i.e. - it did not rotate), the weight of a mass would be greater than for a mass on a rotating Earth. It should be noted that the Earth's rotation has only a small effect on "gravity" (actually on "weight"), about 0.5%.

[0004] Weight is a condition that results from either (a) the presence of a mass within a gravitational field and/or (b) a mass that is subjected to an external force that accelerates that mass (inertial gravity). Weight, as described in (a) or (b), results from a "mass-acceleration" force so universal and common on Earth that it is normally perceived as an ever-present attribute, state, or condition and is not included in many patent process and protocol parameter descriptions. "Weight" is a normal condition and physical parameter that affects "Living Systems" on Earth. Perceived weightlessness is experienced on orbit or in orbiting spacecraft that have reached a constant velocity on orbit or en-route to interplanetary destinations. [0005] Biological organisms, and specifically most higher-ordered biological organisms, including plants, animals, including humans, hereafter referred to as "High Order Living Biological Systems " (HOLBS) are adapted to Earth's gravity. The effects of weightlessness on plants and animals are expressed by physiological effects that alter the physiology and the morphology of the HOLBS, causing deleterious, irreversible, compromising, and transmuting effects from exposure to such conditions. For example, microgravity has been shown to have an impact on an astronaut's body in space. The effects of gravity in plants and animals and the biological mechanisms involved in adapting to weightlessness may be studied under real microgravity conditions. For example, research on astronauts has shown that body function is disturbed in microgravity. Space agencies are therefore continuing their research in hope of eventually reducing or eliminating some of these undesirable physical effects that appear during a stay in space.

[0006] Generally, exposure of living systems to weightlessness results in biological degradation. This degradation is a result of biological processes that have been fundamentally altered due to the absence of an essential force, gravity that is an essential component of those biological processes. Prolonged exposures to a weightless environment correlate to increased biological alterations and degradations. It is the attribute of "weightlessness" or "an apparent absence of gravity induced- force" and/or the "absence of inertially-produced force" that is the critical physical parameter which affects physiological process of living systems in a weightless environment.

[0007] On-orbit environments, e.g., as would be found on the International Space Station (ISS), are referred to as zero-g, zero-gravity, and gravity-free environments. These terms are misleading and incorrect. The term "weightless" is more correct and can be equated to "micro-gravity" for our purpose. [0008] The Earth's "gravitational field," whether in LEO, GEO or other orbits (600 km altitudes, etc), is still present, i.e. - 90+% of the gravitational field and gravitational force amplitude remains. More precisely - static, on-orbit environments exhibit "weight-free" conditions, wherein gravity forces remain substantial as a consequence of a continuing presence of Earth's gravitation field. For example: On Earth at Sea Level: Assumption: Gravity = 1.00, Weight = 1.0. On Orbit at 600 km: Then Gravity = 0.98, Weight = 0.0. Static environment: zero local acceleration.

[0009] All mass, including the space vehicle and pay load will be at zero weight on a earth bound scale that measures "weight". However, it should be noted that gravity is still present.

[0010] The force of gravity between a spacecraft and Earth is directly proportional to the product of their masses and indirectly proportional to the square of the distance between them. Acceleration of a mass into orbit overcomes the force of gravity and the mass will enter what is considered to be a "free-fall" effect. The mass in orbit may be a combination of many objects (masses) that appear to be weightless in relation to the other masses in an apparent weightless "free-fall" environment. Gravity forces are still present, but the HOLBS are not able to function properly without a force that mimics gravity.

[0011] As noted above, HOLBS exhibit marked physiological and biological changes when resident on-orbit, e.g., given that this environment is where Gravity = 0.95 - 1.00 and Weight = 0.00 - 0.10, it is concluded that it is the attribute of "weightlessness" that links these activities and processes, and that it may appear to be constant without regard to gravity.

[0012] More specifically and to further clarify, it may be stated that on Earth, all higher-order living systems, plants, animals, including humans, proceed with biological processes under the influence of a "constant acceleration of their mass." On Earth, this constant acceleration is a result of Earth's gravity, and the endless, largely constant, angular acceleration associated with Earth's gravitational force. It is recognized that there are slight (= < 0.5%) variations in gravitational forces and Earth rotation angular velocities that occur depending upon the location and region the mass is on the Earth. [0013] On orbit, in a static environment (no apparent inertial acceleration is present and angular velocity is relatively constant), near-weightlessness (commonly referred to as zero-gravity or micro-gravity) conditions are achieved. These terms are misleading and the terms should be: zero-weight or micro-weight or some equivalents. The term "weightlessness" is being used herein to collectively refer to these conditions. It is therefore evident that it is the attribute of "weightlessness", not gravity, that is critical to active biological processes and components of plants, animals, humans, and higher-order living systems.

[0014] Furthermore, it is evident that commonly referred to conditions such as "hyper-gravity," are misnomers, as the term has been associated with the forces resulting from the use of centrifuges that create "artificial gravity", when in actuality, they produce centripetal, angular acceleration forces that are more accurately "inertia produced angular accelerations," and may be viewed as or termed "inertial gravity" or more accurately "hyper- weight". Linear accelerations also apply here: in the form of artificial gravity, especially on long space flights in the acceleration and de- acceleration phases. In summary, an angular (or linearly) induced acceleration of a mass will result in a force upon that mass that causes that mass to possess "weight". Weight is an attribute of acceleration of mass (evidenced elementarily by its unit of measure being in - meters/per second/per second).

[0015] Thus, it is submitted that a linkage exists between mass-acceleration (as a mass-energy function) and mass-density (as a mass function), and that the linkage of these two attributes has specific direct and indirect effects upon certain biological processes that occur in plants and animals, including humans and higher-order living systems (HOLBS), where these effects impact functions of cellular replication, reproduction, regeneration, creation, differentiation, specialization, function, cell lifespan, suspended animation, and cell death.

[0016] It is evident that specific, fundamental biological processes possessed by plants, animals, humans and higher-order organisms and living systems and their growth, development, and life-cycles are affected by weightlessness, and, that these living processes differ critically and profoundly when those processes occur in a "weight" (mass-accelerating) environment versus a "weightless" (mass-zero- accelerating) environment. Therefore it is submitted that it is the attribute or characteristic of "weightlessness" that determines and assures and drives certain essential plant, animal (including humans) and higher-living system processes and not the presence of gravity, nor the presence of a gravitational field or its effects. [0017] Experiments and the data derived from visitors to weightlessness indicate that weightlessness plays a role in the development of human cells. See e.g., Longnecker et al, ed., "Review of NASA's Longitudinal Study of Astronaut Health", January 20, 2004, The Institute of Medicine, which is herein incorporated by reference in its entirety. The Earth's gravitational force influences the developing cells to differentiate into specialized cells and which in plants may be branches or roots and in animals the brain or the legs. In weightlessness, the differentiation of the primordial cells in both plants and animals cannot occur. The continuous reproduction of the primordial tissues will continue with the right nutrient system producing undifferentiated cells in both plants and animals until terminated. See International Application No. PCT/US07/85821, filed November 28, 2007, which is herein incorporated by reference in its entirety. The plant studies described therein and conducted on the space transportation system (STS)-118 mission demonstrated cell replication rather than cell differentiation and that the cells replicating in space demonstrated a greater mass than the control cells on Earth, thereby demonstrating replication of undifferentiated cell in weightlessness as noted in the above mentioned International patent application. These results then allow a further expansion of the basic logic to include the acceleration of gene expression in undifferentiated cells on orbit and using the protocol discussed herein to provide for a acceleration of the natural selection process for plants and animals.

[0018] The accelerated and sustained proliferation of undifferentiated cells on orbit provides an opportunity to "force" the cells to express genes that will enable them to adapt to specific environmental conditions. In other words, imposing environmental constraints on the undifferentiated cells while they are proliferating will result in a subset of cells expressing the necessary genes which enable the cells to survive in that specific environmental condition. One can then cultivate an organism from these selected cells that will survive in that particular environmental condition. For example, undifferentiated cells from a species of citrus plant can be propagated at cooler temperatures on orbit. Cells surviving the cool temperature conditions can be returned to earth and cultivated to produce a citrus plant than can thrive in cold temperature environments. [0019] These techniques are applicable to both plant and animal cells. "Hardy" organisms can be produced by selecting undifferentiated cells expressing genes for survival in particular environments, including extreme environments, such as the surface of Mars. The present invention recognizes the advantages of replicating primordial cells on orbit, and utilizes these advantages to expand the range of cell function thus accelerating the evolution of organisms. Therefore, the present invention provides methods for adapting plants and animals to survive outside their native environments.

SUMMARY OF THE INVENTION

[0020] The present invention provides methods of adapting a plant or animal to grow in a hostile environment or an environment outside of the plant's or animal's native environment. In one embodiment, the method comprises culturing undifferentiated cells from a plant or animal in a weightless condition that mimics at least one element of the hostile or non-native environment to which the plant or animal is to be adapted; selecting the cells that replicate in said condition; and cultivating said selected cells to produce plants or animals, wherein the plants or animals are adapted to grow in said hostile environment.

[0021] In another embodiment, the method further comprises evaluating the resultant plants or animals in the hostile environment. The organisms may be evaluated on various criteria including length of survival, growth rate, reproductive capability, cell structure, hardiness in hostile or non-native environments and other gene expressions including but not limited to those enumerated above.

[0022] Various environmental stimuli can be used in the methods of the invention to induce the adaptation of the plant or animal to the hostile environment. These stimuli may include excessive heat, excessive cold, low barometric pressure, excessive radiation, high carbon dioxide levels, low humidity, high humidity, drought conditions and duration of sunlight exposure and other environmental factors to mimic conditions in a climate other than the present native climate of the plant or animal.

[0023] The present invention also provides methods of identifying genes associated with adaptation of a plant or animal to a hostile environment. In one embodiment, the method comprises culturing undifferentiated cells from the plant or animal in a weightless condition that mimics at least one element of the hostile environment to which the plant or animal is to be adapted; selecting the cells that replicate or proliferate in said condition; examining the gene expression profile of the selected cells in comparison to the gene expression profile of control cells; and identifying genes that have a change in expression level, wherein the identified genes are associated with adaptation to the hostile environment. The selected genes that are differentially expressed in the various environmental conditions can be further used to produce transgenic plants and animals with the desired adaptive characteristics by introducing these genes into cells that mature into plants or animals. [0024] In another embodiment, the method can be used for the production of vaccines to be used in animals and humans. By stressing pathogenic microorganisms on orbit, modified strains of microorganisms can be produced. These can include less virulent and/or more virulent strains of bacteria and viruses, which can then be utilized for the production of improved vaccines.

BRIEF DESCRIPTION OF THE FIGURES

[0025] Figure 1. Gene expression profiles of Arabidopsis thaliana seedlings on orbit and on Earth. The gene expression pattern from orbit is plotted versus the gene expression pattern on Earth (panel A). Panel B depicts a subset of the gene expression profile to illustrate heat-shock and CAB (light) genes. The lines labeled with 4x and 10x illustrate four-fold and ten- fold threshold levels of expression, respectively.

DETAILED DESCRIPTION OF THE INVENTION

[0026] It has been recognized that the essential and critical constellation of processes associated with the reproduction of cells is uniquely and profoundly different in a weightless condition, than where those processes are conducted or performed or executed in an environment where (a) gravity that results in mass-acceleration exists, (b) on Earth, (c) in artificial gravity environments [i.e. - inertial gravity environments e.g. - centrifuges, so-called hyper-gravity, more correctly termed hyper-weight) systems, units, devices, or facilities.] The nature of all reproduction processes are influenced by gravity and cells continue to replicate under the influence of the gravitational force, following a genetic code prescribed in the genes that respond to the force of gravity. A fertilized egg replicates, and begins forming unions and congregate into a mass that expands in exponential fashion (1, 2, 4, 8, etc.) to eventually form an embryo, then differentiated cells and tissue. However, in weightless conditions, a fertilized egg can be replicated indefinitely to form cultures of undifferentiated cells that do not develop into an embryo. Methods of preparing such undifferentiated cell cultures in weightless environments and their use have been extensively described in International Application No. PCT/US07/85821, filed November 28, 2007, which is herein incorporated by reference in its entirety. Plant experiments on the STS-118 mission demonstrated that plants replicate in weightlessness and since both plants and animals follow the Kreb's Cycle, animal cells similarly replicate in weightlessness.

[0027] When in a weightless and/or near-weightless environment, typically in space and on-orbit, but also in any and all such equivalent non- weight and weightless circumstances, there exists cellular materials and wherein a process or processes are performed in those environments, specifically in the absence of a "weight-forcing condition" (a circumstance where weightlessness prevails), specific genetic attributes will be altered, i.e., either switched on-from-off or switched off- from-on, and that certain of these gene characteristics may be identified, as to their function and nature, and may consequently and purposefully selected and altered in order to achieve specific, valuable, and useful outcomes, to derive targeted cellular characteristics and accordingly, behavior, performance, and function, and/or that result in the securing, development, and/or enhancement of certain preferable and valuable attributes of said cells, whether as part of a cell, a cellular mass, a cellular volume, or a living organism, in whole or in part.

[0028] Wherein these cellular components and sub-components including cellular elements, genes, DNA, RNA, enzymes, hormones, etc., may be tied individually or collectively in structure, process, and function, such that they act to produce characteristics, attributes and capabilities both cellular and when in-complex and mass as may be observed with a living organism or organisms, such that they express living behaviors which include, but are not limited to increased or decreased or modulated metabolic rate, i.e. respiration, transpiration, anabolic and catabolic activity levels or both, up and/or down regulated, increased, decreased, altered or modulated nutrient uptake, increased, decreased, altered, or modulated waste and byproduct output, modification or alteration of water and fluid uptake or release, alteration of structure, i.e., modification, adaptation, or variation of cell wall thickness, structure, or composition, alternation of character as relates to external environmental pH (acid / base) conditions, salinity and mineral presence, ionics and salts, water, temperature variations, both high and low extremes as well as cellular character, performance and viability in the presence of rapid temperature excursions, or combinations of external chemical, thermal, physical, energy, electromagnetic, radiation, solar, vibrational, acoustic, magnetic, paramagnetic, gravitational, weight- forcing conditions. In other words, all external conditions which may affect or interact with the cells, cellular mass or volumes, components thereof, or organisms in whole or in part may be modulated. [0029] Also contemplated by this invention is the alteration of those biological and cellular processes, activities, reactions, motions, behaviors, process induced reactions and behaviors and the like which are deliberately conducted or affected or driven or occur in a "zero-weight-force" (so-called microgravity) environment. This includes, but is not limited to, the alteration of genes, DNA, RNA, enzymes, hormones, and all biologies known to those competent in the art of biology and cellular sciences, which result in the creation of and/or development of cellular components, cells, cellular masses and volumes such that they or their complexed resulting cellular organisms, may be altered, modified, and enhanced to yield valuable and useful new cellular types and variant organisms including, but not limited to, cows resistant to mastoid staphylococcus disease, cattle resistant to mad cow disease, wheat resistant to wheat rust, soybean crops resistant to soybean rust, agriculture crops resistant to ralstonia, citrus capable of growing in Northern latitudes, Jatropha, Camelina, and other third generation bio fuel crops capable of being grown in the Continental US latitudes, citrus resistant to canker and greening, interbreeding of Tamarix to overgrown existing invasives in the Western US to eliminate water losses, tailored halophyte production and growth, minimization of waterway invasives through retailoring of hydrilla, water hyacinth and egeria species, modification of grapevines to eliminate grape blight, modification of corn, soy, wheat, rye, rice and multiple other types of food crops enabling them to be grown and viable in drought conditions, in harsh wind and climate environments, or in adverse soils and a wide variety of other biological and cellular and organism modifications, alterations, and restructurings of similar usefulness and value and nature.

[0030] The present invention recognizes the value of the accelerated and sustained proliferation or replication of undifferentiated cells from plants and animals in weightless conditions as an opportunity to select for organisms adapted to specific, even harsh, environments. By exposing the proliferating or replicating undifferentiated cells to one or more "non-natural" environmental conditions while in orbit (or weightlessness), one can "force" the cells to express genes that enable the cells to adapt and survive in these abnormal environments. The selected cells can be cultivated to develop into an organism that would be adapted to the specific environmental conditions to which its primordial cells were exposed. [0031] The present invention comprises "replication processes" that occur in a weightless environment as contrasted to the normally occurring reproductive processes that result in progress or development through normal maturation stages and cycles resulting in the differentiation of cells which are known as "reproductive processes" on Earth. However, on orbit in weightless or microgravity conditions, cellular activities occur and are referred to as "replication" which can also be referred to as "duplication" or "proliferation" of copies of cells that are identical in structure and function to their originating predecessors. This process is known as replication and results in an undifferentiated cell replicating or duplicating itself without differentiating into a more specialized cell with a predetermined function. Thus, the process allows for the production of large amounts of undifferentiated plant and animal cells in the weightless or microgravity environment.

[0032] As used herein, the term "undifferentiated" means a primordial state of a cell or cells capable of differentiation and proliferation to produce progeny cells that can be physiologically, biochemically, morphologically, anatomically, immunologically, physiologically, or genetically distinct from the primordial state. [0033] As described above, the present invention provides methods of adapting plants and animals to survive in a hostile environment, wherein the method comprises culturing undifferentiated cells from the plant or animal in a weightless condition that mimics at least one element of the hostile environment to which the animal or plant is to be adapted; selecting the cells that proliferate in said condition; and cultivating said selected cells to produce plants and animals that are adapted to grow in that particular hostile environment.

[0034] Any suitable means for achieving reduced gravity or microgravity conditions can be used for performing the method. In one embodiment, the method is performed under reduced gravity or microgravity conditions in space, e.g., aboard the Space Shuttle, the Space Station, a sounding rocket, or a satellite. In another embodiment, the method is performed under reduced gravity or microgravity conditions simulated on Earth using a machine or other device suitable for this purpose. [0035] As used herein, the term "hostile environment" is used interchangeably with "non-natural environment" and means an environment in which the plant or animal does not normally exist or survive. By way of example, a hostile environment for a banana plant would be the Arctic Circle. Another example of a hostile or non-natural environment for almost any plant or animal would be the surface of Mars. [0036] Many elements of the particular hostile environment of interest can be chosen as the selection pressure to "force" the undifferentiated cells from the plant or animal to express a subset of genes that will enable them to adapt to the hostile environment. As used herein, the term "force" means to apply a selection pressure to the population of proliferating undifferentiated cells to obtain cells that survive in the condition of interest. Some environmental elements suitable for use include, but are not limited to, temperature, such as excessive heat or excessive cold, high or low concentrations of carbon dioxide, barometric pressure, radiation levels, humidity levels, oxygen concentration, low sunlight exposure, extreme drought, extreme salinity, and the presence of environmental toxins.

[0037] In one embodiment, the present invention provides a method of adapting a plant to grow in a hostile environment, wherein the method comprises culturing undifferentiated cells from the plant in a weightless condition that mimics at least one element of the hostile environment to which the plant is to be adapted; selecting the cells that proliferate in said condition; and cultivating said selected cells to produce mature plants, wherein the mature plants are adapted to grow in said hostile environment.

[0038] In one embodiment, the method further comprises evaluating the mature plants in the hostile environment. The plants may be evaluated on several criteria including, but not limited to, length of survival, growth rate, reproductive capability, cell structure, and gene expression.

[0039] In one embodiment, plants suitable for use in the methods of present invention include dicotyledons. In certain exemplary embodiments, the dicotyledons may include leguminous plants and other large seed dicots, e.g., peanuts, soybeans, common beans, squash, zucchini, peppers, melons, cucumbers and others. Other dicots for use in the invention include potatoes, tomatoes, alfalfa, canola, apples, and pairs. In certain other embodiments, a plant suitable for use in the invention can be a woody dicot, including pome fruits, citrus crops, and vegetable crops. [0040] In other embodiments, plants suitable for use in the methods of present invention include be monocotyledons. In certain exemplary embodiments, the monocotyledons include may include corn ("maize"), rice, wheat, barley, sorghum, rye, banana, plantains, and other grasses.

[0041] In another embodiment, the plant may be from the genus Jatropha. Jatropha is a genus of approximately 175 succulent plants, shrubs and trees (some are deciduous, like Jatropha curcas L.), from the family Euphorbiaceae. The hardy Jatropha is resistant to drought and pests, and produces seeds containing up to 40% oil. When the seeds are crushed and processed, the resulting oil can be used in a standard diesel engine, while the residue can also be processed into biomass to power electricity plants and jet engines. Thus, Jatropha curcas is a promising candidate for future bio fuel and energy production. Therefore, expanding the range of habitats in which it can survive is of great interest and importance.

[0042] In another embodiment, the organism may be a lichen. Lichens are composite organisms consisting of a symbiotic association of a fungus (the mycobiont) with a photosynthetic partner (the photobiont or phycobiont), usually either a green algae or cyanobacterium. The morphology, physiology and biochemistry of lichens are very different to that of the isolated fungus and alga in culture. Lichens occur in some of the most extreme environments on Earth — arctic tundra, hot deserts, rain forests, rocky coasts and toxic slag heaps.

[0043] In another embodiment, the organism may be an algae or fungus by themselves. In other embodiments, the algae or fungus may be associated with other primitive organisms, such as lower plants, including, but not limited to, Thallophytes, Chlorophyceae (for example, green algae, spirogyra, or vaucheria) and Phycomycetes (for example, algae fungi, bread mold, or water mold).

[0044] In another embodiment, the undifferentiated cells may be from a plant that has been genetically modified to result in a specific phenotype. There are numerous examples of plants that have been transformed with specific genes so that the resulting transgenic plants exhibit a particular characteristic, such as resistance to a particular pathogen or increased size of fruit. For instance, herbicide resistant plants as disclosed in U.S. Patent No. 7,169,970, plants that have enhanced nitrogen assimilation as disclosed in U.S. Patent No. 6,107,547, and tomatoes with a delayed ripening phenotype as disclosed in U.S. Patent No. 5,952,546 are just a few of the various examples of genetically-modified plants that have been created. Undifferentiated cells may be obtained from any of the many varieties of transgenic plants for use in the methods of the present invention.

[0045] In some embodiments, certain plant species known to be suitable for use as biofuels may be modified, tailored, altered, enhanced, by on-orbit, weightless processing of the cells and/or cellular components, such that higher-energy by weight or by volume biofuel product may be produced from said plant seeds and cultivars, and that these species may include the known, 1st, 2nd, 3rd, and 4th generation biofuels, including but not limited to corn, soy, sugar cane, sugar beet, sweet sorghum, maize, palm, pinnata, switchgrass, rapeseed, miscanthus, hemp and other known suitable biofuels of the 1st generation, as well as Jatropha (as described above), Camelina, Manihot, and algae, including in particular marine algae, as third generation biofuels, including halophytes, particularly Salicornia. This embodiment further contemplates modifications, alterations, and optimizations of these biofuel species as a result of weightless (microgravity) cellular processes executed in the zero-force weightless environment on-orbit, as claimed herein, that enable and result in the production of useful and valuable genetically altered and tailored biofuel and crop outputs and end-products.

[0046] Methods of obtaining undifferentiated plant cells are well known in the art. A mass of undifferentiated plant cells may be obtained by aseptically removing a small piece of plant tissue from a selected organ, such as from the root, stem, etc., and placing it in a sterile medium containing appropriate nutrients. Such a tissue explant will grow and proliferate into a large number of the same type of plant cells or of related plant cells, without specialization of these cells to form specific plant organs such as roots or leaves, etc. These cells may be referred to as a heterogeneous population or colony of undifferentiated plant cells comprised of single cells as well as aggregates of cells. This type of uninterrupted cell growth and multiplication without the formation of specific plant organs is known as undifferentiated cell growth.

[0047] In one embodiment, the undifferentiated plant cells to be used in methods of the invention may be obtained from the undifferentiated parenchyma from the apical meristems of the plant. Reproduction and use of apical cell reproduction has greatly increased the numbers of plants in a vegetative reproduction process. The process depends on the isolation of the reproducing cells at the tip of a plant or plant part (root, branch, etc.) known as the meristem and successful cloning of the limited number of cells at the undifferentiated stage of development at the tip of the plant or other actively growing portions of the plant (root, cambium, etc.). Suspension cultures of undifferentiated cells may be prepared from meristem isolates. [0048] Alternatively, the undifferentiated plant cells suitable for use in the methods of the invention may be obtained by proliferation or replication of diploid cells formed by the union of pollen (sperm) and ovule (egg) from the particular plant species of interest under weightless conditions as described in International Application No. PCT/US07/85821, filed November 28, 2007, which is herein incorporated by reference in its entirety. It is possible to replicate and produce undifferentiated parenchyma resulting from the unification of pollen (sperm) and egg (ovary) in plants that are unified on Earth, preserved prior to any division of the united single cell, and transported immediately to orbit for the purpose of producing undifferentiated cells capable of replicating identical cells for production of tissues used for parts of plants, and the plant itself, including, but not limited to stems, roots, flowers, seeds, fruits, and other tissues. The union of pollen and ovary (egg) in zero gravity will produce a cell that will go to mitosis and then reproduce that cell continually en masse or until a genetic break down in the cell(s) may occur that would disrupt the exponential reproduction of the same mitosis. The newly formed cells can subsequently be used in the methods of the present invention.

[0049] The following example outlines an experiment for adapting a species of citrus plant to grow in colder climates. This example is for illustration purposes only and in no way limits the scope of the invention. As described above, various types of plants may be used in the methods of the invention. Similarly, many different elements of a hostile environment may be used as selective pressures to adapt the plants.

Example - Method of adapting a citrus plant to thrive in cold climates [0050] Suspension cultures have been widely used for tissue culture and mass clonal propagation of a diverse array of higher plants, and also as models for studies of cell development and differentiation. Analysis of these suspension cultures determine structural and genetic changes in undifferentiated plant cells submitted to the effects of environmental elements, such as abnormal temperatures. In addition, cell growth and replication are assessed visually. Structural changes are performed through histological analyses, including light microscopy, transmission electron microscopy (TEM), and if feasible, scanning electron microscopy (SEM). Genetic analyses is performed to evaluate differential gene expression under the specific environmental condition.

[0051] Cell suspension cultures are initiated for a variety of citrus tree (e.g. Citrus sinensis) that has superior fruit, but is not cold tolerant below 28°F. Cultures are prepared by excising the undifferentiated parenchyma cells from the apical meristems of the plant about one day before space shuttle launch. The cell suspensions are cultured on MS medium modified with 1 mg/L 2,4-D. Once a significant amount of cells are produced, they are transferred to 10-ml opticells. Also WPM (woody plant culture medium) medium (Lloyd and McCown, 1986) modified as the MS medium above may be used for woody species of plants.

[0052] OptiCell™ is a sterile, sealed cell culture environment between two optically clear gas-permeable growth surfaces in a standard microtiter plate-sized plastic frame with specially designed ports for access to the contents. OptiCell™ allows an ideal environment for cell growth, microscopy, treatment, selection, separation, harvest, storage, and shipping. Optically clear gas-permeable growth surfaces allow diffusion of oxygen and carbon dioxide for optimal cell growth and permit microscopic examination at any stage of any cell process. OptiCell™ is compatible for use with standard, phase contrast, confocal, and high-resolution time-lapse video microscopes and takes up a fraction of the space of conventional cell culture devices. Access ports allow aseptic access to the interior and its contents. [0053] Each OptiCell™ ("opticell") contains about 10-12 ml of cell suspension. Opticells are maintained in quiescent conditions for both ground and space environments and are evaluated periodically through visual observations for cell growth and development. Under microgravity (space) conditions, opticells are arranged in a C-Hab hardware developed by Bioserve, University of Colorado, and comprised of 6 individual Opticell cell culture systems, peristaltic pumps and a control circuit board. The C-Hab hardware allows the transfer of 1 ml of suspension from one opticell to the next during transfer of cells to fresh medium. An aluminum base and an extruded aluminum outer box with a clear optical window provide the second level of containment. Visual evaluations in space are performed with the aid of video cameras. The C-Hab is associated with CSI camera modules. Each of the camera modules contains up to three analog color video cameras, fitted with either microscope adaptors or standard lenses for macroscopic view. This allows the observation of cell growth and replication throughout the period of experimentation in space. Still images Op^eg^s) ^are fed to the ground periodically during the entire period of the experiment, thus generating a time lapse for cell growth and replication. The hardware and related control software are tested and evaluated previous to launch. [0054] On orbit, in the opticells the experimental cell suspension cultures are subjected to temperatures of 25°F for a predetermined period of time, such as for several weeks or about three months or more on the International Space Station (ISS). Also on orbit, corresponding control cell suspension cultures would be exposed to the optimal growing temperature for that species of citrus. Cells exposed to each growing temperature would be returned to Earth and a portion used for further analyses (see below). The other portion would immediately be separated into individual cell containers with agar and cultured to determine which cells survived. The cells that survived can be nurtured to mature trees and then subjected to temperatures of 25 ⁰F to determine the level of cold tolerance achieved. Other parameters of the mature trees would also be measured, including yield of the trees, length of survival, and growth.

[0055] Samples from suspension cells maintained in opticells, under both experimental (25°F) and control (greater than 28°F) temperature conditions are collected and compared for histological and genetic analysis. For histological analyses, cell suspensions are prepared for light and electron microscopy. Opticells are compatible for use with standard, phase contrast, confocal, and high-resolution time-lapse video microscopes. Cells are examined microscopically on either opticell growth surface or in between. Oil immersion lenses (up to 100X) are used on the membrane without disruption or contamination. The membrane is sectioned for small scale staining and microscopy. Additional samples are removed and fixed in glutaraldehyde for subsequent evaluation of cell ultrastructure through TEM and SEM.

[0056] By the term control or control cells or microorganisms within the meaning of the present invention, is meant cells or microorganisms grown on earth or in a gravity environment as compared to the cells or microorganisms grown in a weightless or microgravity environment as described herein or on the Space Station. Additionally, control or control cells or microorganisms can also mean those cells or microorganisms grown under a normal non-stressed environment, as compared to the stress environment factors and stimuli, as set forth herein, such as in [0022] and in other portions of this application.

[0057] Gene expression analyses are performed to evaluate possible genes that are either up-regulated or down-regulated in response to the colder temperature. Suspension cultures maintained in space are fixed in RNAlater (Ambion) liquid preservative through the Kennedy Space Center fixation tube (KFT), hardware designed to provide proper containment of fixatives for biological samples in space placed inside the C-hab environment. RNA is isolated and compared for suspension cultures in both temperature conditions to evaluate gene expression. Molecular biology techniques for reverse transcriptase polymerase chain reaction (RT-PCR) and/or copy-DNA amplified fragment length polymorphism (cDNA- AFLP) and gel electrophoresis are performed according to well-known techniques to those skilled in the art and are used for gene expression analyses. Microarray analysis of gene expression is performed. Results of the microarray data identify the genes involved in the tolerance factor for cold. Genes involved in cold-tolerance adaptation will typically show at least a four- fold change in expression compared to the control cells exposed to the normal growing temperature.

[0058] Additional evaluations of the cell suspensions may also be conducted including, but not limited to, cell growth rates, cell densities, subculture frequency, and size and condition of cells. [0059] The above-described techniques are also applicable to adapting animals to hostile environments. In certain embodiments, the animal is a mammal. As used herein, the term "mammal" refers to any mammal. Nonexclusive examples of such mammals include, but are not limited to, animals such as dogs, cats, horses, cattle, sheep, and goats. In other embodiments, the animal may be a bird. In yet other embodiments, the animal may be an aquatic species.

[0060] In one embodiment, the invention provides a method of adapting an animal to grow in a hostile environment, wherein an element of the hostile environment is selected from the group consisting of heat, cold, excessive radiation, high carbon dioxide levels, low humidity, high humidity, chemical pollutants, disease, and drought conditions.

[0061] In some embodiments, the undifferentiated cells from animals suitable for use in the methods of the invention can be embryonic stem cells. Methods for isolating embryonic stem cells are well known to those of skill in the art, including, but not limited to, somatic nuclear transfer, cell fusion, and genetic manipulation techniques that create totipotent cells that are capable of generating all the tissues of the entire animal.

[0062] Alternatively, the undifferentiated animal cells can be obtained by methods comprising forming a diploid cell by uniting two haploid cells and proliferating the diploid cell in a weightless condition, wherein the diploid cell replicates itself but does not differentiate into specialized cells and tissues. Such methods are described extensively in International Application No. PCT/US07/85821, which is herein incorporated by reference in its entirety.

[0063] More specifically, the egg and sperm are united using standard in vitro fertilization (IVF) techniques for harvesting human or animal eggs, collecting sperm and inseminating the egg with the sperm in a laboratory dish in IVF culture medium. The dish is then placed in an incubator at a controlled temperature which should be the same temperature as the female species' body. It generally takes 18 hours for fertilization of the egg to be complete.

[0064] Culture conditions and media for culturing the undifferentiated cells according to the methods of the invention are well known to the skilled artisan. For example, a medium useful for the isolation of embryonic stem cells is "ES medium." ES medium consists of 80% Dulbecco's modified Eagle's medium (DMEM; no pyruvate, high glucose formulation, (Invitrogen or Sigma), with 20% fetal bovine serum (FBS; Hyclone), 0.1 mM β-mercaptoethanol (Sigma), 1% non-essential amino acid stock (Sigma or other known sources). Preferably, fetal bovine serum batches are compared by testing clonal plating efficiency of a low passage mouse ES cell line. FBS batches must be compared because it has been found that batches vary dramatically in their ability to support embryonic cell growth, but any other method of assaying the competence of FBS batches for support of embryonic cells will work as an alternative. But any known media for culturing the replicating stem cells can be used and tested by the scientists performing these experiments to select the appropriate medium to obtain optimum results. Appropriate plant cell culture media known to skilled persons can be selected to culture undifferentiated plant cells according to the present invention.

[0065] The cells are cultured in 3 -dimensions by simply suspending the cells in a closed culture vessel in the weightless environment which will keep the cells suspended without the need for any agitation as the cell will not settle to the bottom of the vessel that they would in a gravity environment. Any known methods of 3- dimensional cell culture can be used to culture the replicating undifferentiated stem cells, which could include culturing methods from Mina Bissell's laboratory, such as for example disclosed in J. Cell. ScL, 2003 June 15; 116(Pt 12):2377-88. [0066] Various methods for culturing stem cells, e.g., embryonic stem cells (ESCs), may be used with the present invention. Typically, ESCs are grown in adherent culture systems such as on tissue culture plates. In certain aspects, culture plates for use in the invention may comprise a gel matrix such as a collagen or hydrogel matrix (e.g., a MATRIGEL™). In various embodiments, culture plates may be coated with, e.g., collagen IV, fibronectin, laminin, and vitronectin in combination may be used to provide a solid support for embryonic cell culturing and maintenance, as described in Ludwig et al. (2006). Matrix components which may be used with the present invention to coat tissue culture plates includes a collagen such as collagen IV, laminin, vitronectin, Matrigel™, gelatin, polylysine, thrombospondin (e.g., TSP-I, -2, -3, -4 and/or -5), and/or ProNectin-F™. Three dimensional support matrices for use in tissue culture have been previously described for example in U.S. Publication Nos. 20060198827 and 20060210596, each incorporated herein by reference. The skilled artisan will recognize that in certain aspects adherent tissue culture cells may be defined by the cell density or confluency. Thus, in some cases, methods of the invention involve expansion of proliferating cells from a high density to a lower density to facilitate further cell proliferation. For example, methods for expanding cells according to the invention may involve a first population of embryonic stem (ES) cells that is between about 50% and 99% confluent. For example, in certain aspects the first population of ES cells may be about or less than about 60%, 70%, 80%, 90% or 95% confluent. Furthermore, in certain aspects expansion or passage of adherent ES cells may involve seeding separated cells in fresh growth media. As used herein the term "seeding" cells means dispersing cells in growth media such that the resultant cell culture(s) are of approximately uniform density. Thus, seeding of cells may involve mixing separated cells with fresh growth media and/or spatially dispersing separated cells over the surface of a tissue culture plate. [0067] Undifferentiated propagation of adherent colonies of ESCs may be accomplished with a Knockout (KO) serum- free culture system without the use of feeders by plating and growing the colonies on extracellular matrices (ECM) within a feeder-conditioned KO-DMEM medium supplemented with KOSR and fibroblast growth factor 2 (FGF2). Media available from commercial sources, such as Gibco Invitrogen Corporation, Grand Island, NY. Furthermore, it has been suggested that feeder conditioning may be replaced by substituting the medium with high concentrations of FGF2 and noggin. Alternatively, feeder conditioning was replaced by transforming growth factor- 1 and human leukemia inhibitory factor (LIF) (in addition to FGF2) and growing the cells on human fibronectin, or by serum- free media supplemented with soluble factors including FGF2, activin A, transforming growth factor-βl (TGF-βl), pipecolic acid, GABA, LiCL and culturing the cells on ECM components. In general, a key limitation of ESC culture systems is that they do not allow the propagation of pure populations of undifferentiated stem cells and their use typically involves some level of background differentiation. The stem cells most commonly follow a default pathway of differentiation into an epithelial cell type that grows either as a monolayer of flat squamous cells or form cystic structures. Most probably, this form of differentiation represents differentiation of human ESC (hESC) into extraembryonic endoderm.

[0068] In these adherent culture systems of colonies, the ESCs are most commonly propagated (mechanically and/or by using enzymatic digestion) as clusters, on a small scale. These culture systems are labor-intensive, highly variable, may contain undefined factors, and do not provide steady-state operating conditions. Most importantly, they do not typically allow for large scale production of standardized homogenous undifferentiated ESCs needed for the aforementioned uses. [0069] Suspension culture bioreactors offer several advantages over the conventional use of static monolayer cultures. These systems facilitate the large-scale expansion of the cells in a homogeneous culture environment, thus decreasing the risk of culture variability. They are also less labor-intensive to operate and offer the possibility of computer control and monitoring of the culture conditions. Although bioreactors have been used to expand neural stem cells, mouse ES cells and differentiating hESCs within embryoid bodies (EBs), only recently some progress has been made towards the development of protocols for the feeder- free expansion of undifferentiated hESCs in suspension systems (see US20070212777, or J. Biotechnology, Vol. 132 (2), 227- 236 (2007), which are herein incorporated by reference in its entirety). [0070] The present invention also provides methods of identifying genes associated with adaptation of a plant or animal to a hostile environment. Previous experiments demonstrated that seedlings from Arabidopsis thaliana grown under microgravity (weightless condition) exhibited a change in expression of select genes compared to their counterpart controls grown on Earth. The microarray data (Figure 1) from these experiments showed that 182 genes were differentially expressed with at least a fourfold change in expression. Some of the differentially expressed genes were identified as heat-shock and/or CAB (light) genes. Using the same principles on which these experiments are based, the present invention provides methods for identifying specific genes differentially expressed between a control condition and a particular environmental condition. These identified genes may play a role in the adaptation of the plant or animal to that particular environment.

[0071] In one embodiment of the invention, the method comprises culturing undifferentiated cells from a plant or animal in a weightless condition that mimics at least one element of the hostile environment to which the plant or animal is to be adapted; selecting the cells that proliferate in said condition; and examining the gene expression profile of the selected cells in comparison to the gene expression profile of control cells; and identifying genes that have a change in expression level, wherein the identified genes are associated with adaptation to the hostile environment. [0072] The change in expression level is at least 2-fold, at least 4-fold, at least 6-fold, at least 10-fold, at least 15-fold, or at least 20-fold. The change in expression level could be an increase in expression or a decrease in expression. Thus, particular environmental stimuli may produce both up-regulation and down-regulation of specific genes.

[0073] In one embodiment, the selected genes that are differentially expressed in the various environmental conditions can be further used to produce transgenic plants and animals with the desired adaptive characteristics by introducing these genes into cells that mature into plants or animals.

[0074] Therefore, the present invention further encompasses a plant or animal or undifferentiated cell thereof produced by the methods described herein, wherein said plant, animal or undifferentiated cell thereof comprises at least one identified gene that has a change in expression level as compared to the gene expression profile of control cell, wherein the identified genes are associated with adaptation to the hostile environment.

[0075] In one embodiment, a transgenic plant tolerant to environmental stresses, such as low temperature, freezing, and dehydration stresses, can be produced by introducing DNA encoding the protein of the interest into a host plant using genetic engineering techniques. Methods for introducing the gene into a host plant include indirect introduction such as the Agrobacterium infection method and direct introduction such as the particle gun method, polyethylene glycol method, liposome method, and microinjection method.

[0076] In the present invention, while the host for the transformant is not particularly limited, it is preferably a plant. The plant may be any cultured plant cells, the entire plant body of a cultured plant, plant organs (such as leaves, petals, stems, roots, rhizomes, or seeds), or plant tissues (such as epidermis, phloem, parenchyma, xylem, or vascular bundle). Plants may be monocotyledonous plants such as rice, maize, and wheat. When a cultured plant cell, plant body, plant organ or plant tissue is used as the host, the Agrobacterium infection method, particle gun method, or polyethylene glycol method can be employed to introduce the DNA encoding the protein of the present invention to transform this host plant by introducing a vector into plant sections. Alternatively, a vector can be introduced into a protoplast by electroporation to produce a transformed plant. [0077] For example, when a gene is introduced into Arabidopsis thaliana by the Agrobacterium infection method, the step of infecting the plant with an Agrobacterium containing a plasmid comprising the gene of interest is essential. This step can be performed by the vacuum infiltration method [Ci? Acad. Sci. Paris, Life Science, 316:1194 (1993)]. Specifically, Arabidopsis thaliana is grown in a soil composed of equivalent portions of vermiculite and perlite. The Arabidopsis thaliana is immersed directly in a culture fluid of an Agrobacterium, containing a plasmid comprising the gene of interest, placed in a desiccator, and then sucked with a vacuum pump to 65-70 mmHg. Then, the plant is allowed to stand at room temperature for 5-10 min. The plant pot is transferred to a tray, which is covered with a wrap to maintain humidity. On the next day, the wrap is removed. The plant is grown in that state to harvest seeds.

[0078] Subsequently, the seeds are sown on MS agar medium supplemented with appropriate antibiotics to select those individuals which have the gene of interest. Arabidopsis thaliana grown on this medium are transferred to pots and grown there. As a result, seeds of a transgenic plant into which the gene of the interest has been introduced can be obtained. Generally, the genes are introduced into the genome of the host plant in a similar manner. However, due to differences in the specific locations on the genome into which the genes have been introduced, the expression of the introduced genes varies. This phenomenon is called "position effect." By assaying transformants with DNA fragments from the introduced gene as a probe by Northern blotting, it is possible to select those transformants in which the introduced gene is expressed more highly.

[0079] The confirmation that the gene of interest is integrated in the transgenic plant into which the gene of the present invention has been introduced and in the subsequent generation thereof can be made by extracting DNA from cells and tissues of those plants and detecting the introduced gene by PCR or Southern analysis, which are conventional methods in the art.

[0080] The expression level and expression site of a gene in a transgenic plant into which the gene of the present invention has been introduced can be analyzed by extracting RNA from cells and tissues of the plant and detecting the mRNA of the introduced gene by RT-PCR or Northern analysis, which are conventional methods in the art. Alternatively, the expression level and expression site can be analyzed directly by Western blotting or the like of the gene product of the present invention using an antibody against the above product.

[0081] In one embodiment, the gene of interest encodes a transcription factor. Because stress responses such as drought tolerance involve coordinated changes in many genes, the ability to affect many changes with one gene is an attractive proposition. Transcription factors can activate cascades of genes that function together to enhance stress tolerance. Transcription factors refer to a class of genes that control the degree to which other genes in a cell are activated. Transcription factors are able to recognize and bind to regions of DNA that have a specific sequence in the promoters of the genes they regulate. Thus, if a dozen genes all have that region of DNA somewhere in their promoters, they will all be regulated by the same transcription factor. Because transcription factors are key controlling elements of biological pathways, altering the expression levels of one or more transcription factors can control a variety of genes involved in the stress response. [0082] In another embodiment, the gene of interest encodes a stress-induced protein. In an exemplary embodiment, the gene of interest encodes a heat-shock protein. [0083] The control cells of the present invention to which the experimental cells are compared would include undifferentiated cells proliferating in a weightless condition exposed to the normal or native environmental element of that which is being varied in the experimental condition. For example, in a method to adapt a plant to grow in arid conditions, the undifferentiated cells from the plant would be exposed to a low humidity environment in the experimental condition and an environment having normal humidity for that particular plant species in the control condition. [0084] Gravity is a condition which may contribute to "weightlessness" or "weight" depending upon the presence of other forces, i.e. - other conditions, energies, or forces which result in the acceleration of mass present, and which may add to or oppose the effects of mass-acceleration that result from the presence of mass within a gravitational field. In a weightless condition (where the acceleration of mass is generally equal to and opposite the force that exists from gravitational conditions), gravity is a null-component of the system and does not have a unique, essential, critical, or driving effect upon the biological processes described and claimed herein. [0085] To clarify the present invention with respect to gravity, gravity forces and gravitational fields, it is recognized that gravitational fields and forces exist to varying degrees and at varying amplitudes on Earth, in the Earth, at altitudes above the Earth, on-orbit around the Earth, between Earth and Moon, and inter-planetary in the Solar System and beyond. The gravitational field forces in these regions vary from approximately IG on Earth, to 0.15G on the Moon, and 0.4G on Mars, etc., and at other varying amplitudes throughout the Solar System. It is recognized that these gravitational force scalars (values) remain, whether or not other inertial forces are present. It is recognized that gravitational forces at any particular point within the Solar System, vary temporally given the motions of the planets and heavenly bodies present within the System. It is recognized that gravitational forces present on local systems, e.g., Earth, Moon, Mars, are relatively constant and that local gravitational field amplitudes in these point regions are relatively constant in amplitude over time. [0086] The present invention encompasses the proliferation of undifferentiated cells from plants and animals in a weightless condition, including those systems used in current stem cell research and development and use of undifferentiated parenchyma in plants. Gravity is not the direct force or condition that drives biological processes in a weightless environment. Gravity on Earth, for example, is the condition that results in forces that drive the biological universe on Earth. Gravity is not absent on-orbit. Substantial gravitational fields exist on-orbit and in space. The gravity on orbit is approximately 95% the gravity on Earth and approximately 0% weight. The rules regarding reproduction of cells change in a weightless condition. Both plant and animal cells do not replicate in a weightless condition according to the rules on Earth. On Earth, gravity is a condition that ties to specific forces that govern the reproduction of cells.

[0087] All of the manufacturing processes, biological process, and related actions and activities conducted on Earth, are subject to and include a "mass-acceleration" component, a forcing component that results in a condition or characteristic known as "weightlessness" are encompassed by the present invention. [0088] The present invention further comprises the biological and living-system processes, manufacturing, cellular, replication, reproduction, creation, duplication, harvesting, development, maturation, and growth, processing storage (including suspended animation and shipping) that are conducted or performed, whether through human or autonomous-control, in (process) environments that have zero-acceleration of mass in orbit or in interplanetary travel and/or where the acceleration of mass is equal and opposite the gravity induced force present.

[0089] To further clarify those process environments where there is no mass- acceleration force present and/or where the acceleration of mass is equal and opposite the gravity induced force present, no mass-acceleration force is included as part of the biological, physiological, replication, reproduction, growth, harvesting, manufacturing, or production operation on orbit or in interplanetary travel unless artificially produced.

[0090] The present invention also comprises all processes and systems used in current stem cell research, development, processing, and manufacture, but with the additional step or condition of performing or conducting these processes in a "weightless" (zero- acceleration of mass) environment as defined in the present invention and as understood by persons skilled in the art of gravitational theories and sciences, and/or where the acceleration of mass is equal and opposite the gravity induced force present.

[0091] In another embodiment, the method can be used for the production of vaccines to be used in animals and humans. Strains of Salmonella on orbit have previously been shown to demonstrate increases in virulence. See Wilson et ah, PLoS One 12(3): e3923: 1-10.

[0092] The technology described in this invention can be used to "force" the expression of genes on orbit to produced microorganisms, such as a bacteria or virus, with desired properties for vaccine production. By stressing, for example, a pathogenic microorganisms on orbit, modified strains of microorganisms can be produced. These modified microorganisms can include less virulent and/or more virulent strains of bacteria and viruses, which can then be utilized for the production of improved vaccines. The vaccines produced by this method can be applied to treatment and/or prevention of many animal and human diseases, including, but not limited to, hoof and mouth disease and brucellosis. [0093] The foregoing detailed description has been given for clearness of understanding only and no unnecessary limitations should be understood therefrom as modifications will be obvious to those skilled in the art.

[0094] It is understood that the present invention is not limited to the particular methods and components, etc, described herein, as these may vary. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. It must be noted that as used herein, the singular forms "a," "an," and "the" include the plural reference unless the context clearly dictates otherwise.

[0095] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.

Claims

1. A method of adapting a plant to grow in a hostile environment comprising:

(a) culturing undifferentiated cells from the plant in a weightless condition that mimics at least one element of the hostile environment to which the plant is to be adapted;

(b) selecting the cells that replicate in said condition; and

(c) cultivating said selected cells to produce mature plants, wherein the mature plants are adapted to grow in said hostile environment.

2. The method of claim 1, further comprising:

(d) evaluating the mature plants in the hostile environment.

3. The method of claim 1 or 2, wherein undifferentiated cells are obtained from uniting a sperm and egg of a plant prior to culturing in a weightless condition or environment.

4. The method of claim 1 or 2, wherein the mature plants are evaluated for length of survival, growth rate, reproductive capability, cell structure, and gene expression, or combinations thereof.

5. The method of claim 1 or 2, wherein the plant is a dicotyledonous plant.

6. The method of claim 1 or 2, wherein the plant is a monocotyledonous plant.

7. The method of claim 1 or 2, wherein the plant comprises an algae contained in a lichen comprised further of a fungi.

8. The method of claim 1 or 2, wherein the plant is a lower plant selected from the group consisting of Thallopytes, Chlorophyceae, and Phycomycetes.

9. The method of claim 1 or 2, wherein said at least one element of the hostile environment is selected from the group consisting of heat, cold, low barometric pressure, excessive radiation, high carbon dioxide levels, low humidity, high humidity, extreme salinity, reduced or increased exposure to sunlight, and low water or drought conditions.

10. The method of claim 1 or 2, wherein the undifferentiated cells are obtained from the undifferentiated parenchyma from the apical meristems of the plant.

11. The method of claim 1 or 2, wherein the undifferentiated cells are obtained by uniting a pollen and an ovule from the plant to form a diploid single cell under conditions to support fertilization in a weightless condition or environment, wherein said single cell can replicate itself forming a diploid cell that will not develop into an embryo of differentiated cells and tissue.

12. The method of claim 1 or 2, wherein the plant is genetically modified to express a specific phenotype.

13. A method of identifying genes associated with adaptation of a plant to a hostile environment comprising:

(a) culturing undifferentiated cells from the plant in a weightless condition that mimics at least one element of the hostile environment to which the plant is to be adapted;

(b) selecting the cells that replicate in said condition;

(c) examining the gene expression profile of the selected cells in comparison to the gene expression profile of control cells; and

(d) identifying genes that have a change in expression level as compared to the gene expression profile of the control cell, wherein the identified genes are associated with adaptation to the hostile environment.

14. The method of claim 13, wherein undifferentiated cells are obtained from uniting a sperm and egg of a plant prior to culturing in a weightless condition or environment.

15. The method of claim 13 or 14, wherein the change in expression level is at least four- fold in comparison to the expression profile of control cells.

16. The method of claim 15, wherein the plant is a dicotyledonous plant.

17. The method of claim 15, wherein the plant is a monocotyledonous plant.

18. The method of claim 15, wherein the plant comprises an algae contained in a lichen comprised further of a fungi.

19. The method of claim 15, wherein the plant is a lower plant selected from the group consisting of Thallopytes, Chlorophyceae, and Phycomycetes.

20. The method of claim 15, wherein said at least one element of the hostile environment is selected from the group consisting of heat, cold, low barometric pressure, excessive radiation, high carbon dioxide levels, low humidity, high humidity, extreme salinity, reduced or increased exposure to sunlight and low water or drought conditions.

21. The method of claim 15, wherein the plant is genetically modified to express a specific phenotype.

22. A method of adapting an animal to grow in a hostile environment comprising:

(a) culturing undifferentiated cells from the animal in a weightless condition that mimics at least one element of the hostile environment to which the animal is to be adapted;

(b) selecting the cells that replicate in said condition; and

(c) cultivating said selected cells to produce mature animals, wherein the mature plants are adapted to grow in said hostile environment.

23. The method of claim 22, further comprising:

(d) evaluating the mature animals in the hostile environment.

24. The method of claim 22 or 23, wherein undifferentiated cells are obtained from uniting a sperm and egg of an animal prior to culturing in a weightless condition or environment.

25. The method of claim 24, wherein the mature plants are evaluated for length of survival, growth rate, reproductive capability, cell structure, and gene expression, or combinations thereof.

26. The method of claim 24, wherein the animal is genetically modified to express a specific phenotype.

27. A method producing at least one undifferentiated cell comprising uniting a sperm and egg of an animal prior to culturing in a weightless condition or environment to produce undifferentiated cells that replicate at a higher rate than in a weight or gravity condition or environment.

28. A method of identifying genes associated with adaptation of an animal to a hostile environment comprising:

(a) culturing undifferentiated cells from the animal in a weightless condition that mimics at least one element of the hostile environment to which the animal is to be adapted;

(b) selecting the cells that replicate in said condition;

(c) examining the gene expression profile of the selected cells in comparison to the gene expression profile of control cells; and

(d) identifying genes that have a change in expression level as compared to the gene expression profile of control cell, wherein the identified genes are associated with adaptation to the hostile environment.

29. The method of claim 28, wherein undifferentiated cells are obtained from uniting a sperm and egg of an animal prior to culturing in a weightless condition or environment.

30. The method of claim 28 or 29, wherein the change in expression level is at least four- fold in comparison to the expression profile of control cells.

31. The method of claim 30, wherein said at least one element of the hostile environment is selected from the group consisting of heat, cold, excessive radiation, high carbon dioxide levels, low humidity, high humidity, chemical pollutants, disease, and drought conditions.

32. The method of claim 30, wherein the animal is genetically modified to express a specific phenotype.

33. A method of producing stem cells comprising obtaining purified stem cells from an embryo of new-born animal and culturing said stem cells in a weightless condition or environment resulting in the cell replication of identical stem cells.

34. A method of producing undifferentiated cells from an animal, such as cells from liver, kidney, heart, skin, and other cells from the animal body including organs for the purpose of replication of undifferentiated cells including obtaining the undifferentiated cell from the animal, and culturing said undifferentiated cell in a weightless condition or environment resulting in the cell replication of identical undifferentiated cells.

35. The method of claim 34, wherein the undifferentiated cells is a subcutaneous skin cell obtained by harvested from the animal, wherein the culturing results in near exponential replication the cells overtime.

36. A plant or animal or undifferentiated cell thereof produced by the method of any one of claims 1, 2, 13, 14, 22, 23, 27-29 and 33-35, wherein said plant, animal or undifferentiated cell thereof comprises at least one identified gene that has a change in expression level as compared to the gene expression profile of control cell, wherein the identified genes are associated with adaptation to the hostile environment.

37. A method of adapting a pathogenic microorganism to grow in a hostile, non- native environment comprising:

(a) culturing the microorganism in a weightless condition that mimics at least one element of the hostile environment to which the microorganism is to be adapted;

(b) selecting the microorganisms that replicate in said condition; and

(c) cultivating said selected microorganism to produce a modified strain of microorganism, wherein the microorganism is are adapted to grow in said hostile environment.

38. The method of claim 37, further comprising:

(d) evaluating the microorganism to determine the properties of said modified strain.

39. The method of claim 38, wherein the virulence of the modified strain is different than the virulence of the original non-adapted microorganism.