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Title:
METHODS FOR PLANT CLONING USING AEROPONICS
Document Type and Number:
WIPO Patent Application WO/2021/046508
Kind Code:
A1
Abstract:
Materials and methods for clonal propagation of plants from plant cuttings using aeroponics are provided. For example, methods of seed increase including harvesting a plurality of cuttings from a single mother plant, rooting the cuttings in an aeroponics enclosure, and transferring the rooted cuttings to soil for reproductive development are provided. The methods can be used for any plants of interest. Advantages of the methods include reduced time and costs associated with generation of a full-grown plant from a cutting.

Inventors:
UPPGAARD ANDERS (US)
CAMPBELL BENJAMIN (US)
Application Number:
PCT/US2020/049652
Publication Date:
March 11, 2021
Filing Date:
September 08, 2020
Export Citation:
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Assignee:
CALYXT INC (US)
International Classes:
A01G31/00
Domestic Patent References:
WO1985003195A11985-08-01
WO2016164652A12016-10-13
Foreign References:
US20070113472A12007-05-24
US20190216029A12019-07-18
Other References:
ANGEL R. DEL VALLE-ECHEVARRIA ET AL: "Aeroponic Cloning of Capsicum spp.", HORTICULTURAE, vol. 5, no. 2, 16 April 2019 (2019-04-16), pages 30, XP055743126, DOI: 10.3390/horticulturae5020030
Attorney, Agent or Firm:
ROBINSON, Lisbeth, C. (US)
Download PDF:
Claims:
CLAIMS

1. A method for producing a daughter plant from a plant cutting, comprising: securing a plant cutting having a rooting portion harvested from a mother plant in an aeroponic growth media in an aeroponic growth enclosure; providing a nutrient mist to the rooting portion at a misting cycle to promote root formation.

2. The method of Claim 1, wherein the nutrient mist does not comprise a plant hormone.

3. The method of Claim 1 or Claim 2, wherein the aeroponic enclosure is maintained at a temperature from about 16 °C to about 32 °C.

4. The method of any one of the preceding claims, wherein the aeroponic enclosure is maintained at a light cycle with a ratio of 16:8 to 12:12 hours of light to dark, 16:8 to 22: 2 hours of light to dark, 2:22 to 22:2 hours of light to dark, or 2:22 to 24:0 hours of light to dark.

5. The method of any one of the preceding claims, wherein the misting cycle comprises misting for 100 consecutive seconds every about 400 seconds.

6. The method of any one of the preceding claims, wherein the nutrient mist has electric conductivity equivalent to of about 600 ppm NaCl.

7. The method of any one of the preceding claims, wherein the nutrient mist is an aqueous solution comprising cations selected from a group consisting of ammonium, calcium, copper, iron, manganese, potassium, zinc, magnesium, and combinations thereof.

8. The method of any one of the preceding claims, wherein the nutrient mist is an aqueous solution comprising anions selected from a group consisting of borate, carbonate, phosphate, nitrate, sulfate, molybdate, and combinations thereof.

9. The method of any one of the preceding claims, wherein the nutrient mist is an aqueous solution comprising ammonium molybdate, ammonium nitrate, calcium carbonate, calcium nitrate, copper EDTA, iron DTPA, iron EDDHA, iron EDTA, manganese EDTA, potassium borate, potassium nitrate, zinc EDTA, magnesium phosphate, phosphoric acid, potassium phosphate, potassium sulfate, magnesium sulfate, ammonium phosphate, magnesium carbonate, potassium carbonate, monopotassium phosphate, calcium carbonate, and magnesium nitrate.

10. The method of any one of the preceding claims, wherein the nutrient mist has a pH from about 5.5 to about 6.0.

11. The method of any one of the preceding claims, wherein the temperature of the nutrient mist is from about 19 °C and about 25 °C.

12. The method of any one of the preceding claims, wherein the temperature of the nutrient mist is about 21 °C.

13. The method of any one of the preceding claims, wherein the plant is a monocotyledon or dicotyledon.

14. The method of any one of the preceding claims, wherein the plant is a soybean plant, a potato plant, a wheat plant, or a tomato plant.

15. The method of any one of the preceding claims, wherein the plant cutting is harvested from a mother plant having at least one branch with meristem.

16. The method of Claim 15, wherein the cutting is produced by cutting the mother plant stem at an angle between about 30 degrees and about 90 degrees.

17. The method of any one of Claims 1-16, wherein the mother plant has a cotyledon and harvesting includes cutting the mother plant below the cotyledon.

18. The method of any one of Claims 1-16, wherein the mother plant has a cotyledon and harvesting includes cutting the mother plant above the cotyledon.

19. The method of any one of the preceding claims, wherein the mother plant is a genetically modified plant or a genetically edited plant.

20. The method of Claim 19, wherein the mother plant is a To plant.

21. The method of any one of the preceding claims, wherein the daughter plant is grown to maturity without transferring the daughter plant into a new growth media.

22. The method of any one of the preceding claims, wherein the plant cutting is harvested from a dicotyledonous mother plant having been grown under conditions that promote shoot elongation, optionally wherein the mother plant has a branch.

23. The method of claim 22, further comprising harvesting an apical cutting from the main stem of the mother plant to promote formation of a plurality of axillary shoots, and optionally inserting the apical cutting in the aeroponic growth media in the aeroponic growth enclosure.

24. The method of claim 23, further comprising:

(a) harvesting an apical cutting and a plurality of axillary cuttings from the main stem or optional branch, after formation of axillary shoots, wherein each axillary cutting comprises an axillary shoot, an internode portion, and a leaf; and

(b) securing the apical cutting and each internode portion of the plurality of axillary cuttings in the aeroponic growth media in the aeroponic growth enclosure; whereby a plurality of daughter plants are produced.

25. The method of any one of claims 22-24, wherein the conditions that promote shoot elongation include low light conditions.

26. The method of any one of claims 24-25, wherein each axillary cutting includes at least two leaves, optionally at least three leaves.

Description:
METHODS FOR PLANT CLONING USING AEROPONICS

BACKGROUND

[0001] Modem plant breeding has been supercharged with new technologies such as gene editing. Gene editing technology enables humans to address agricultural challenges relatively quickly, with the potential to meet the needs of a rapidly growing population. However, gene editing is challenging, requiring significant resources to create a single product.

[0002] Cloning can help ensure important plants are not lost in production. Cloning enables generation of multiple plants from a single mother plant, with progeny (“clones”) having genetics identical (or substantially identical) to the mother and to each other. However, many plants, including soybean, cannot be cloned. This current inability to clone plants of interest requires a large expenditure of resources to ensure that plants of interest survive and create viable seed.

[0003] One example of a plant of interest is a To plant. Some agricultural products are created in a lab and start as a single plant containing the desired edits; this plant is referred to as a To plant. This To plant creates seed, and the seed is bulked until there is sufficient seed to plant desired acreage (potentially thousands). A key problem faced by scientists is high risk of loss with unique To gene edited plants. Once a To plant is created, the young plant requires time and resources to mature into a seed-producing plant. The process of making To plants that produce viable seed is long, difficult, and expensive. Thus, every plant that is created is extremely valuable, and a need exists for methods for cloning of plants of interest such as To plants.

SUMMARY

[0004] Disclosed herein are methods for aeroponic cloning of plants. In one aspect, provided herein is a method for producing a daughter plant from a plant cutting obtained from a mother plant, comprising: securing a plant cutting having a rooting portion in an aeroponic growth media in an aeroponic growth enclosure; providing a nutrient mist to the rooting portion at a misting cycle to promote root formation. [0005] In some embodiments, the nutrient mist does not comprise a plant hormone. In some embodiments, the aeroponic enclosure is maintained at a temperature from 16 °C to 32 °C. In some embodiments, the aeroponic enclosure is maintained at a light cycle with a ratio of about 16:8 to 12:12 hours of light to dark. In some embodiments, the light cycle is about 12:12 hours of light to dark. In some embodiments, the light cycle is about 2:22 to 22:2 hours of light to dark. In some embodiments, the light cycle is about 2:22 to 22:2 hours of light to dark.

[0006] In some embodiments, the misting cycle comprises misting for about 100 consecutive seconds every about 400 seconds.

[0007] In some embodiments, the nutrient mist has an electrical conductivity of about 1200 mS/m (i.e., corresponding to a NaCl value of about 600 ppm). In some embodiments, the nutrient mist is an aqueous solution comprising ammonium molybdate, ammonium nitrate, calcium carbonate, calcium nitrate, copper EDTA, iron DTPA, iron EDDHA, iron EDTA, manganese EDTA, potassium borate, potassium nitrate, zinc EDTA, magnesium phosphate, phosphoric acid, potassium phosphate, potassium carbonate, potassium sulfate, magnesium carbonate, magnesium sulfate, ammonium phosphate, potassium carbonate, monopotassium phosphate, calcium carbonate, and magnesium nitrate. In some embodiments, the nutrient mist has a pH from about 5.5 to about 6.0. In some embodiments, the nutrient mist has a pH of about 5.6 to about 5.8.

[0008] In some embodiments, the temperature of the nutrient mist is from about 19 °C to about 25 °C. In some embodiments, the temperature of the nutrient mist is about 21 °C.

[0009] In some embodiments, the plant is a monocotyledon or dicotyledon. In some embodiments, the plant is a soybean plant, a potato plant, a wheat plant or a tomato plant. [0010] In some embodiments, the plant cutting is produced from a mother plant having at least one branch with meristem. In some embodiments, the plant cutting is produced by cutting the plant stem of the mother plant at an angle ranging from about 30 degrees to about 90 degrees. The mother plant can be a plant with a cotyledon. In some embodiments, the cutting is made by cutting the mother plant stem below the plant’s cotyledon and above root tissue. In some embodiments, cutting is made by cutting the plant above the plant’s cotyledon at a region of epicotyl tissue or cutting along a branch. [0011] In some embodiments, the mother plant is a genetically modified plant or a genetically edited plant. In some embodiments, the mother plant is a To plant. In some embodiments, the daughter plant is grown to maturity without transferring the daughter plant into a new growth media.

[0012] In some embodiments, the secured plant cutting generates roots in about 7 days or less, about 6 days or less, about 5 days or less, or about 4 days or less.

[0013] In some embodiments, the daughter plant reaches maturity in about 30 days or less, about 35 days or less, about 40 days or less, about 45 days or less, about 50 days or less, or about 55 days or less after the plant cutting is secured.

[0014] In some embodiments, the plant cutting is harvested from a dicotyledonous mother plant that has been grown under conditions that promote shoot elongation. Optionally, the mother plant has a branch. The method can further include harvesting an apical cutting from the main stem of the mother plant to promote formation of a plurality of axillary shoots, and optionally inserting the apical cutting in the aeroponic growth media in the aeroponic growth enclosure.

[0015] In some embodiments the method further includes (a) harvesting an apical cutting and a plurality of axillary cuttings from the main stem or optional branch, after formation of axillary shoots, wherein each axillary cutting comprises an axillary shoot, an internode portion, and a leaf; and (b) securing the apical cutting and each intemode portion of the plurality of axillary cuttings in the aeroponic growth media in the aeroponic growth enclosure; whereby a plurality of daughter plants are produced. The conditions that promote shoot elongation can include low light conditions. Each axillary cutting can includes at least two leaves, optionally at least three leaves.

[0016] In another aspect, provided herein is a plant, e.g., a plant clone, obtained using a method disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The foregoing aspects and many of the attendant advantages of the claimed subject matter will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings. [0018] FIGURES 1A-F show an exemplary soybean growth cycle entirely in aeroponics (i.e., “rapid cycling”)(A) Eleven day old seedlings inserted in foam cups and secured in the aeroponics enclosure; (B) Vegetative growth at day 18; (C) Vegetative growth at day 25; (D) Reproductive growth (first flower) at day 28, (E) First pod at day 36; and (F) Pods ready for harvest at day 51. Gm (Bert) plants were grown in 12/12-hour light/dark conditions. Air temperatures during the day were about 27 °C and dropped to about 25.5 °C during the night. Two experiments were run in parallel, and viable seeds were ready to plant a second generation after about 60 days.

[0019] FIGURE 2 is a photograph of a soybean seed pod harvested at day 51. The seeds were dried for 10 days, and planted in rockwool at day 61 (not shown).

[0020] FIGURE 3 is a table of the experimental setup and results of the soybean rapid cycling experiments.

[0021] FIGURE 4A-B demonstrate a soybean cloning experiment, according to one or more embodiments of the present disclosure. (A) plant cutting (branch with an apical meristem) was harvested from a 27-day old mother plant grown in aeroponics; (B) The plant cutting was rooted within 8 days of securing in the aeroponic growth medium; and (C) produced 1 mature pod within 37 days.

[0022] FIGURES 5A-G show soybean rooting experiments. These experiments were performed to evaluate the aeroponic capacity for rooting plant cuttings without using hormones. (A) A plant cutting was harvested from a juvenile mother plant with scissors by cutting the main stem below the cotyledon (i.e., within the hypocotyl region); (B) the hypocotyl stem cutting was secured in the aeroponic growth medium at day 0; and (C) the stem cutting formed several extended roots within 4 days. (D) A cutting was harvested from a juvenile mother plant with scissors by cutting the main stem above the cotyledon within the epicotyl region; (E) the epicotyl stem cutting was secured in the aeroponic growth medium at day 0; (F) the stem cutting showed signs of stress at day 4; (G) the cutting formed several roots by day 5. Stress from having no roots is demonstrated by wilting leaves; plants recovered once the root system established. Stem cuttings comprising the cotyledon grew faster and regenerated roots earlier than epicotyl stem cuttings.

[0023] FIGURES 6(A)-(B) show an exemplary soybean rooting experiment. The depicted rooted stem cutting, harvested by cutting the stem above the cotyledons but below the first true leaves, grows roots directly from epicotyl stem tissue after growth in aeroponic conditions for 6 days, with no hormones. (A) side view and (B) perspective view.

[0024] FIGURE 7 shows a mother wheat plant with a main stem and two side tillers. [0025] FIGURE 8 shows three individual daughter wheat plants divided from the mother wheat plant (main stem and tillers)of FIGURE 7.

[0026] FIGURE 9 shows a secured wheat plant cutting from a mother plant. The mother tissue (i.e., plant cutting) has no roots at the time of insertion in the growth medium. [0027] FIGURE 10 shows the rooted wheat cutting grown from the cutting of FIGURE 9, after two days in the aeroponic growth enclosure. New roots have formed, enabling the clone to uptake nutrients and grow into a mature plant. The nutrient mist did not include plant hormones.

[0028] FIGURE 11 shows the rooted wheat cutting grown from FIGURE 9, after four days in the aeroponic growth enclosure. The new roots have increased in size, and the daughter plant demonstrates new vegetative growth.

[0029] FIGURE 12 shows the daughter wheat plant grown from the wheat cutting of FIGURE 9 after thirty days in the aeroponic growth enclosure. The daughter plant has produced a full head of seed and is considered a fully mature plant. This mature wheat plant is a genetic clone of the mother plant (i.e., genetically identical or virtually identical to the mother plant).

[0030] FIGURE 13 shows the three daughter plants derived from the mother plant depicted in FIGURE 7. All daughter plants have reached maturity and produced viable seed.

[0031] FIGURE 14 shows a mother potato plant.

[0032] FIGURE 15 shows a potato plant cutting cut from the mother plant in FIGURE 14.

[0033] FIGURE 16 shows the potato plant cutting of FIGURE 15 after the cutting harvested from the mother plant is affixed in a foam insert, ready for securing in the aeroponics growth enclosure.

[0034] FIGURE 17 shows a rooted potato plant cutting 7 days after the cutting harvested from the mother plant was secured in an aeroponic growth enclosure. The cutting developed strong roots without the use of added hormones. [0035] FIGURE 18 shows a daughter potato plant 21 days after the cutting harvested from the mother plant was secured in an aeroponic growth enclosure.

[0036] FIGURE 19 shows a daughter potato plant that has produced an asexual tuber 54 days after the cutting harvested from the mother plant was secured in an aeroponic growth enclosure.

[0037] FIGURE 20 shows an example potato clone that has produced sexual flowers 54 days after the cutting harvested from the mother plant was secured in an aeroponic growth enclosure.

[0038] FIGURE 21 shows rooted tomato plant cuttings harvested from a mother plant after 7 days in an aeroponic enclosure and exposed to a hormone- free nutrient mist. [0039] FIGURE 22 illustrates a strategy for clonal propagation of dicots, according to one or more embodiments of the present disclosure.

DETAILED DESCRIPTION

[0040] Disclosed herein are methods for clonal propagation of plants, for example, from plant cuttings, using aeroponics. The methods can be advantageously used for any plants of interest, such as plants previously shown to be unable to be cloned, as well as plants that are known to be propagated by cloning (e.g., potato). Thus, a mother plant can be any seed plant, gymnosperm or angiosperm, discovered in uncultivated soil, cultivated from seed, or cultivated from an asexually propagated plantlet (e.g., via micro propagation, conventional clonal propagation, or aeroponic clonal propagation according to a method of the present disclosure). Advantageously, the method can be applied to the propagation of herbaceous plants such as annual crop plants. In some cases, the mother plant is a chimera or the mother plant includes a bud sport. Exemplary advantages of the methods include reduced time and costs associated with generation of a full-grown plant from a cutting, as compared with the resources required for cultivation from seed because the germination and seedling emergence stages are by-passed. Suitable plant varieties for cloning using the methods include, for example, soybean, potato, wheat, canola, com, and others.

[0041] The term “plant cutting” refers to any aerial plant material, other than a mature seed, that can be removed from a mother (donor) plant and grown separately to produce a daughter plant. Generally, the plant cutting includes an aerial portion of the mother plant such as a leaf, an unrooted shoot, or unrooted stem portion. In some cases, the plant cutting includes a rooted stem portion (e.g., tiller).

[0042] The methods of the disclosure allow generation of multiple copies from a mother plant without using seed, and generally do not require the use of any added growth hormones to do so. The ability to eliminate use of hormones using the methods of the present disclosure offers several advantages, such as reduced cost, increased efficiency (e.g. elimination of a reagent and a method step), market desirability, and consumer preference. The methods also allow growth of daughter plants year-round, in different rooms and under different growth conditions to optimize growth conditions and observe phenotype stability; yet minimize loss of plants of interest due to death or lack of viable seed. A single plant of interest, such as a To gene-edited plant, can generate multiple plants for subsequent use, such as stock plant maintenance, seed bulking, faster time to field trials, speed breeding, etc.

[0043] In contrast to currently available methods, where a single plant may or may not produce viable seed, embodiments of the present disclosure enable production of multiple genetically identical (or virtually identical) “daughter” plants from a single mother plant in parallel; for example, one daughter plant can be grown in soil, and the others can be grown in aeroponics. A “virtually identical” daughter plant includes plants with some degree of somaclonal variation, but which exhibit substantially similar genotype, phenotype, and vigor as the mother plant. The germplasm of a virtually identical daughter plant can be at least 90%, at least 95%, at least 97%, or at least 99% identical to the germplasm of the mother plant. The percentage of “identity” for a daughter plant genetic sequence is determined by comparing two optimally aligned sequences over the comparison window, which may include gaps (e.g., deletions or additions) as compared to the mother plant genetic sequence. After alignment, the number of matched positions (i.e., positions where the identical nucleic acid base residue occurs in both sequences) is determined and then divided by the total number of positions in the comparison window. This result is then multiplied by 100 to calculate the percentage of identity. The daughter plant in soil typically takes about 90 to about 120 days to produce mature seed, if the plant survives to maturity, and the seed may have low viability (e.g., less than 75% germination rate). The daughter plants grown in aeroponics can serve as a backup: if the daughter plant in soil dies without producing a sufficient number of viable seed, the grower still has access to valuable genetics. Additionally, using the methods disclosed herein, the daughter plants can reach maturity in less than about 60 days, enabling production of up to six generations of breeding in one year.

[0044] Volume production has been a limiting factor in rapid seed bulking of a new variety. Multiple plant cuttings can be harvested from a mother plant over the course of its life or at a single time. As illustrated in FIG. 22, a dicotyledonous mother plant can be grown under conditions that promote stem or shoot elongation, to increase the internode length, and thereby permit harvest multiple apical and axillary plant cuttings from the mother plant for insertion into the aeroponics enclosure at one time. For example, soybean plants exposed to low light conditions and 16-hour day length develop branches with greater intemodal distance than plants grown under maximum light. When the internodal distance is at least about 7.5 cm, and at least one branch has grown to have several nodes below the apical meristem, the terminal bud of the branch can be removed (e.g., pinched or cut for securing in a aeroponic growth medium) to initiate axillary shoot formation. After axillary shoots have developed, plant cuttings can be harvested along the branch of the mother plant from the apical tip and at one or more nodal segments. Each axillary cutting can include a node with the growing axillary shoot, an attached leaf, and a length of the internode sufficient for securing in the aeroponics growth medium. Axillary cuttings can include at least two leaves/node (e.g., 3 or more leaves). Within about 7 days in the aeroponics growth enclosure, the plant cuttings will form roots. In some cases, the axillary cuttings initiate root formation more quickly than an apical cutting from the same mother plant (e.g., 12-24 hours more quickly), and the root structure includes a greater number of roots than the apical cutting from the same mother plant (e.g., about 2-5 times the number of roots). Without being bound by theory, the larger leaf size, the number of leaves, and the thicker stem of the axillary cutting compared with apical cuttings provide better post severance photosynthesis for root formation and overall health of the cutting. Rooted cuttings can be transferred to soil when the root structure is sufficiently developed for nutrient and water uptake (e.g., when the roots have a bristle brush appearance).

[0045] The methods disclosed herein can shorten the time in which a plant typically reaches maturity, compared with seed-grown plants, field-grown plants, or plants produced using conventional clonal propagation methods. In some embodiments, the plant reaches maturity in about 30 days or less, about 35 days or less, about 40 days or less, about 45 days or less, about 50 days or less, or about 55 days or less.

[0046] The methods disclosed herein use aeroponic techniques. Aeroponics is the process of growing plants in an air or mist environment without the use of soil or an aggregate medium. The plants can be suspended plants above spray heads. The spray heads dispense nutrient solution as a fine mist on the plant roots, or on the wound of a plant cutting. Aeroponics expedites plant growth through optimization for nutrient dosage, spray time, light synchronization, growth phases, and pH levels for a given plant species or variety.

[0047] The present invention allows for variance in temperature and photoperiod, based on the plant (e.g., species or cultivar) and desired plant stages (e.g. vegetative growth vs. reproductive). In some embodiments, the methods disclosed herein include growing plants at temperature ranges from about 5 °C to about 40 °C, from about 16 °C to about 32 °C, from about 18 °C to about 25 °C, or from about 23 °C to about 25 °C. Typical photoperiods can range from 18 h light/6 h dark to 20 h light/4 h dark for vegetative state, and from 14 h light/10 h dark and 12 h light/12 h dark for reproductive stages. A person of skill in the art can determine the most optimal range of temperatures and light cycle for the specific plant/need.

[0048] Aeroponic systems have been described in the art. Aeroponic roots hang in open air, with no mechanical resistance from soil. This is in contrast to hydroponic systems where plant roots are typically submerged in water. Aeroponic systems allow roots to grow freely, more rapidly and larger than in soil, which in turn provides the nutritional support for larger foliage, bloom, and fruit growth in the canopy. Dangling roots absorb essential minerals from the nutrient spray solution and enable increased oxygen intake for respiration. Aeroponics accelerates growth while reducing nutrient and water usage (see, for example, www.aessensegrows.com/en/why-aeroponics).

[0049] In one aspect, provided herein is a method for producing a daughter plant from a plant cutting harvested from a mother plant, comprising: securing a plant cutting having a rooting portion in an aeroponic growth media in an aeroponic growth enclosure; and providing a nutrient mist to the rooting portion at a misting cycle, to promote root formation. [0050] In some embodiments, the aeroponic enclosure is maintained at a temperature from 16 °C to 32 °C. In some embodiments, the aeroponic enclosure is maintained at a light cycle that comprises 16:8 to 12:12 hours of light to dark. In some embodiments, the aeroponic enclosure is maintained at a light cycle that comprises 24:0 to 22:2 hours of light to dark. In some embodiments, the nutrient mist does not comprise a hormone, e.g., a plant rooting or growth hormone.

[0051] As used herein, “aeroponic growth media” is a material, such as synthetic foam, which does not comprise soil and can hold the plant’s stem and root mass in place as the plant grows. The material is exposed to light on one top side and nutrient mist on the bottom side. As used herein, a “rooting portion” is the portion of a plant cutting which is inserted below the top side of the aeroponic growth media. The “rooting portion” includes the wounded portion of the plant cutting, and does not necessarily include root tissue at the time of insertion. Plant cuttings produced by dividing a plant with tillers, suckers, stolons, bulbs, tubers or rhizomes can include a rooting portion with some root tissue.

[0052] The methods disclosed herein can be used to clone or propagate plants, e.g., monocotyledonous and dicotyledonous plants, from mother plant parts, e.g., plant cuttings. In some embodiments, the mother plant is from the family Solanaceae, Poaceae, or Fabaceae. In some embodiments, the mother plant is soybean, tomato, or potato plant. In some embodiments, the mother plant is from a Triticum genus, e.g., a wheat plant. In some embodiments, the methods can be used to propagate or clone genetically edited or genetically modified plants, such as mother To plants. In some embodiments, the methods can be used to clone any mother plant of interest or value, or part thereof (e.g., chimera or bud sport).

[0053] The methods of the disclosure can expedite daughter plant maturation and seed generation. In some embodiments, a daughter plant grown by the methods disclosed herein can produce viable seeds about 50% faster, about 40% faster, about 30% faster, about 25% faster, about 20% faster, or about 10% faster than a genetically identical (or virtually identical) plant of substantially identical size and substantially identical age grown in soil or hydroponically in optimized growth conditions, such as temperature and/or light conditions. As used herein, “optimized growth conditions” are growth conditions resulting in the fastest time to maturity and/or seed production by a plant. [0054] In the methods disclosed herein, a nutrient mix is delivered to the rooting portion of a plant cutting by misting the rooting portion with a misting composition or a nutrient mist comprising a mixture of plant nutrients. The nutrient mix is mixed with water and delivered to the plant.

[0055] Concentration of the nutrient components in the nutrient mist can be controlled by measuring and adjusting the electrical conductivity (EC) of the mix. For example, a commonly used method of determining the total dissolved solids in an aqueous solution (TDS) is measuring specific conductivity to detect the presence of ions and converting the EC to a concentration of a specific salt or a mixture of salts, for example, by ppm (TDS or NaCl). The conductivity of sodium chloride solutions is close to that of mineral nutrients of the nutrient mist composition. Thus, ppm NaCl (or NaCl value) can be used as an indicator of the total mineral nutrient concentration, even if the composition does not contain sodium or chloride ions. For NaCl, EC (mS/m) = 2*(ppm NaCl). The aeroponics system can be configured to add nutrient components based on the NaCl ppm or EC being below a specific threshold. The ppm (NaCl) value can be set to a specified number, such as 500 ppm. Nutrient components are then added to a water tank at indicated ratios, until the detected EC reaches about 1000 mS/m. The plants then absorb nutrients from the water which is returned to the tank; and the nutrients are replenished at specified ratios to keep the ppm (NaCl) at 500, based on the detected EC. In one embodiment, the following nutrient components are added at the following ratio: about 30% micronutrient component, about 30% bloom-promoting component, about 30% root growth-promoting component, and about 10% mineral supplement component by volume until the conductivity of the total mixture reaches 1000 mS/m, which corresponds to 500 ppm NaCl. [0056] In some embodiments, the misting is done on a cycle. Cycle times can be adjusted based upon desired result. For example, water restriction facilitates root growth, and high moisture facilitates plant growth. In some embodiments, the misting cycle comprises misting for 100 consecutive seconds every 400 seconds. In some embodiments, the misting cycle includes 200 seconds on, 200 seconds off or 300 seconds on and 100 seconds off.

[0057] In some embodiments, the nutrient mist comprises a source of potassium, a source of nitrogen, a source of phosphorus, and plant microelements. In some embodiments, the nutrient mist is an aqueous solution comprising cations selected from a group consisting of ammonium, calcium, copper, iron, manganese, potassium, zinc, magnesium, and combinations thereof. In some embodiments, the nutrient mist is an aqueous solution comprising anions selected from a group consisting of borate, carbonate, phosphate, nitrate, sulfate, molybdate, and combinations thereof.

[0058] The nutrient mist comprises an aqueous composition comprising a plant nutrient mix. In some embodiments, the nutrient mist is an aqueous solution comprising ammonium molybdate, ammonium nitrate, calcium carbonate, calcium nitrate, copper EDTA, iron DTPA, iron EDDHA, iron EDTA, manganese EDTA, potassium borate, potassium nitrate, zinc EDTA, magnesium phosphate, phosphoric acid, potassium phosphate, potassium carbonate, potassium sulfate, magnesium carbonate, magnesium sulfate, ammonium phosphate, potassium carbonate, monopotassium phosphate, calcium carbonate, magnesium nitrate, or a combination thereof. In some embodiments, the nutrient mist is an aqueous solution comprising ammonium molybdate, ammonium nitrate, calcium carbonate, calcium nitrate, copper EDTA, iron DTPA, iron EDDHA, iron EDTA, manganese EDTA, potassium borate, potassium nitrate, zinc EDTA, magnesium phosphate, phosphoric acid, potassium phosphate, potassium carbonate, potassium sulfate, magnesium carbonate, magnesium sulfate, ammonium phosphate, potassium carbonate, monopotassium phosphate, calcium carbonate, and magnesium nitrate.

[0059] In some embodiments, the nutrient mist comprises the following components: a micronutrient component, a root growth-promoting component, a bloom-promoting component, and a mineral supplement component.

[0060] In some embodiments, the micronutrient component comprises ammonium molybdate, ammonium nitrate, calcium carbonate, calcium nitrate, copper EDTA, iron DTPA, iron EDDHA, iron EDTA, manganese EDTA, potassium borate, potassium nitrate, and zinc EDTA. In some embodiments, the micronutrient component has a guaranteed analysis of about 5% total nitrogen, about 1% soluble potash (K2O), about 5% calcium, about 0.01% boron, about 0.0005% cobalt, about 0.01% chelated copper, about 0.1% chelated iron, about 0.05% chelated manganese, about 0.0008% molybdenum, and about 0.015% chelated zinc. Commercial micronutrient components can be used in the nutrient mists disclosed herein, for example, FloraMicro (General Hydroponics, Santa Rosa, USA). [0061] In some embodiments, the bloom-promoting component comprises magnesium phosphate, phosphoric acid, potassium phosphate, potassium carbonate, potassium sulfate, magnesium carbonate, and magnesium sulfate. In some embodiments, the bloom-promoting component has a guaranteed analysis of about 5% available phosphate (P2O5), about 4% soluble potash (K2O), about 1.5% water-soluble magnesium, and about 1.0% combined sulfur. Commercial bloom-promoting components can be used in the nutrient mists disclosed herein, for example, FloraBloom (General Hydroponics, Santa Rosa, USA).

[0062] In some embodiments, the root growth-promoting component comprises ammonium phosphate, magnesium carbonate, magnesium nitrate, magnesium sulfate, potassium carbonate, potassium nitrate, monopotassium phosphate, and potassium sulfate. In some embodiments, the root growth-promoting component has a guaranteed analysis of about 2.0% total nitrogen, about 1.0% available phosphate (P2O5), about 6.0% soluble potash (K2O), and about 0.5% water-soluble magnesium. Commercial root growth- promoting components can be used in the nutrient mists disclosed herein, for example, FloraGro (General Hydroponics, Santa Rosa, USA).

[0063] In some embodiments, the mineral supplement component comprises calcium carbonate, magnesium nitrate, and iron DTPA. In some embodiments, the root growth- promoting component has a guaranteed analysis of about 1.0% total nitrogen, about 5.0% calcium, about 0.1% iron, and about 1.5% water-soluble magnesium. Commercial root growth-promoting components can be used in the nutrient mists disclosed herein, for example, CaliMagic (General Hydroponics, Santa Rosa, USA).

[0064] In some embodiments, the components of the nutrient mist are aqueous solutions of the plant nutrients as described above. In some embodiments, the nutrient mist is prepared from a mixture comprising about 30% micronutrient component, about 30% bloom-promoting component, about 30% root growth-promoting component, and about 10% mineral supplement component by volume. In some embodiments, the components are mixed together and further diluted to a desired concentration and/or conductivity. [0065] In some embodiments, the nutrient mist is an aqueous plant nutrient composition that has an electrical conductivity equivalent to about 50 ppm to about 3000 ppm NaCl (about 100 to 6000 mS/m) . In some embodiments, the nutrient mist is an aqueous plant nutrient composition that has an electrical conductivity equivalent to about 600 ppm to about 1200 ppm NaCl (about 1200 to about 2400 mS/m). In some embodiments, the electrical conductivity can be adjusted up or down depending on the growth stage of the daughter plant; i.e., as the daughter plant grows, larger amounts of dissolved nutrients can be provided. In some embodiments, the aqueous plant nutrient composition has an electrical conductivity equivalent to about 600 ppm (1200 mS/m), about 800 ppm (1600 mS/m), or about 1000 ppm NaCl (2000 mS/m). In some embodiments, the composition has an electrical conductivity equivalent to about 600 ppm NaCl (1200 mS/m).

[0066] The nutrient mist is kept and delivered to the rooting portion of the plant cutting (or roots of a rooted cutting/daughter plant) at a specified temperature, for example, in a range of about 5 °C to about 33 °C. In one embodiment, the temperature is kept in a range from about 19 °C to about 21 °C. In another embodiment, the temperature is about 21 °C. In some embodiments, it is advantageous to control the temperature within 2 degrees of the specified temperature. Any suitable means of keeping the temperature of the misting composition within the required range can be used.

[0067] The pH of the misting composition can range from about 5 to about 7. In some embodiments, the pH is from about 5.5 to about 6.5. Any suitable means of adjusting pH can be used to control pH of the composition within the required range, for example, phosphoric acid can be used to decrease the pH, and potassium carbonate or potassium hydroxide can be used to increase the pH.

[0068] The methods disclosed herein advantageously do not require the use of plant growth hormones. In some embodiments, the misting composition does not contain a plant hormone. Eliminating hormone use reduces cost of production by reducing the materials needed, and increasing efficiency by process simplification. As some individuals desire to minimize their exposure to hormones, including plant growth hormones, the present invention offers a method and product that meets this need.

[0069] Any suitable plant cutting can be used in the methods disclosed herein. In some embodiments, the plant cutting is produced from a plant having at least one branch with meristem. In some embodiments, the cutting is produced by cutting the plant stem at a 45-degree angle. For dicot plants, the cutting can made by cutting the plant below the plant’s cotyledon (e.g., hypocotyl region, above any root tissue, or the cutting is made by cutting the plant above the plant’s cotyledon (e.g., epicotyl region, or above). In some cases, the plant cutting comprises one or more of a nodal segment, a terminal bud, an apical dome, an intemodal segment, a true leaf, a seed leaf, an axillary shoot, an adventitious bud, an adventitious shoot, a bud sport, and a periclinal chimera. The plant cutting can be harvested from a juvenile or mature plant, including from new growth on a mature plant. The cutting can be procured using a plant propagation tool such as secateurs, scissors, a knife (e.g., bud knife, grafting knife), razor blade, scalpel, and the like. The plant cutting can be treated to reduce the risk of pest (insect) vectoring by dipping or spraying the plant material with a mild detergent and/or dilute alcohol. In some embodiments, the plant cutting is not treated with any plant hormone to induce rooting.

[0070] In some embodiments of the methods disclosed herein, the plant cutting generates roots in about 7 days or less, about 6 days or less, about 5 days or less, about 4 days or less, about 3 days or less, or about 2 days or less.

[0071] As used herein, the term "about" refers to +10% of the stated value.

[0072] While each of the elements of the present invention is described herein as containing multiple embodiments, it should be understood that, unless indicated otherwise, each of the embodiments of a given element of the present invention is capable of being used with each of the embodiments of the other elements of the present invention and each such use is intended to form a distinct embodiment of the present invention.

[0073] The referenced patents, patent applications, and scientific literature referred to herein are hereby incorporated by reference in their entirety as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference. Any conflict between any reference cited herein and the specific teachings of this specification shall be resolved in favor of the latter. Likewise, any conflict between an art-understood definition of a word or phrase and a definition of the word or phrase as specifically taught in this specification shall be resolved in favor of the latter. [0074] As can be appreciated from the disclosure above, the present invention has a wide variety of applications. The invention is further illustrated by the following examples, which are only illustrative and are not intended to limit the definition and scope of the invention in any way.

EXAMPLES:

[0075] The vast majority of agricultural products are derived from flowering plants.

All flowering plants can be divided into either monocotyledons (monocots) or dicotyledons

(dicots). Monocot and dicot plants differ from each other in flowers, stems, roots, and leaves, and thus grow and develop very differently from one another. The ability of the methods of the present disclosure to be used with success across monocots and dicots is significant.

[0076] The examples described below demonstrate that both monocots and dicots can be easily cloned using the methods of the disclosure, thus overcoming many of the challenges of creating new plant products. The present disclosure is applicable to plants beyond monocots and dicots.

A. Cloning of an exemplary dicot (soybean)

[0077] A pilot study was conducted using soybean, a dicot, as an exemplary species. Mother soybean plants were grown from seed in aeroponic conditions. Seeds were germinated in a water-soaked rockwool substrate in close-bottomed flats with a clear domed lid at 25 °C using a 12/12 light/dark cycle. After 7 days, the germinated seedlings were removed from the rockwool and affixed in foam cups situated in the growth tray of the aeroponic unit (FIGURE 1A). The mother plants were grown for 25 days, at which point they had multiple branches (1-3 branches) with meri stems (FIGURE 1C). These meristem branches were cut from the mother plant using a sharp razor at a 45-degree angle. Each branch, which is genetically identical (or virtually identical) to the mother plant (i.e., not a bud sport), was affixed in a new foam cup with one inch (about 2.5 cm) of stem tissue protruding from the base of the foam collar (i.e., “rooting portion”) (FIGURES 4A-C). The foam cups containing the plant cuttings were then inserted into the aeroponic growth tray under the conditions as disclosed below. After 8 days, the plant cuttings had produced roots, and after 37 days the daughter plants were fully mature and had produced pods containing viable, mature seeds.

[0078] Further studies were conducted to determine rooting efficiency of different plant cuttings and to evaluate the aeroponic capacity for rooting plant cuttings without using hormones. Plant tissue below the cotyledon is expected to be easier to root than plant tissue above the cotyledon. As shown in FIG. 5A-G, plant cuttings that include the cotyledon grew faster and regenerated roots a day earlier than plant cuttings from the epicotyl region. Even without hormone, however, epicotyl stem cuttings developed roots

(FIG. 6A-B). Normally, such cuttings do not root unless exposed to hormones. These results demonstrate that the aeroponics system is a powerful rooting technology. B. Cloning of an exemplary dicot (potato)

[0079] A study was performed using potato, a dicot, as an exemplary species. Mother potato plants were grown in sterile tissue culture vessels for 30 days. These mother potato plants were then removed from the tissue culture vessels and affixed into foam collars, with 0.5 inches (about 1.25 cm) of the stem protruding from the base of the foam collar (i.e., rooting portion). The foam inserts containing the mother potatoes were then placed into the aeroponic growth tray, where they began growing in aeroponic conditions. After 14 days, the mother potatoes were removed from the aeroponics, cuttings were harvested, and then the mother plant plus the cuttings were placed back into the aeroponic unit. The cloning processes included the following. Using a sharp razor, cuttings were taken from the mother plant (FIGURES 14-15). The cuttings were then placed into their own, individual foam inserts, with 0.1-0.5 inches (about 0.25-1.25 cm) of stem protruding from the base of the foam collar (FIGURE 16). The foam inserts with the plant cuttings were then placed back into the aeroponic units for continued growth. 7 days after placement, the cuttings had produced new roots (FIGURE 17). 21 days after placement, the cuttings produced a full set of roots, along with increased vegetative growth (FIGURE 18). 54 days after placement, the daughter plants were fully mature and produced asexual reproductive tubers (FIGURE 19) and sexual reproductive flowers (FIGURE 20).

C. Cloning of an exemplary monocot (wheat)

[0080] A study was performed using wheat, a monocot, as an exemplary species. Mother wheat plants were grown from seed in aeroponic conditions. Seeds were germinated in individual rockwool plugs that were inserted into plastic grow cups in the aeroponics. The wheat plants grew for 21 days in aeroponic conditions. At this point, each plant had multiple tillers (2-5). The mother plants were removed from the aeroponics and placed on a workstation (FIGURE 7). Each mother plant consists of a main shoot and secondary tillers, all which meet at the base of the plant. The base of the plant also contains the root structure,

[0081] For the cloning process, a sharp razor was used to cut the wheat plant into multiple pieces. Each individual tiller was cut off from the main shoot (FIGURE 8). The roots, which are attached to the base of the main shoot, were kept on each tiller when possible. Often, the main stem had strong root systems, but the smaller, outer tillers would not have any roots after the cutting process (FIGURE 9). Once the cutting process was complete, the mother plant was divided into three daughter plants having identical (or virtually identical) genetics. Each daughter plant was affixed into a new foam cup with 0.5 inches (about 1.25 cm) of stem tissue (and any associated root tissue) protruding from the base of the foam collar. The foam cups containing the daughter plants were then inserted into the aeroponic growth tray and grown under the conditions as disclosed below.

[0082] Daughter plants that were originally rootless, produced new root structures within 4 days of being secured in the aeroponic system (FIGURES 10-11). Each daughter plant was able to reach reproductive stages and produce heads with viable seed (FIGURES 12-13).

D. Cloning of an exemplary dicot (tomato)

[0083] A study was performed using tomato, a dicot, as an exemplary species. Mother tomato plants were grown in sterile tissue culture vessels for 30 days. The mother tomato plants were then removed from the tissue culture vessels and affixed into foam collars, with 0.5 inches (about 1.25 cm) of the stem protruding from the base of the foam collar. The foam inserts containing the mother tomato plants were then placed into the aeroponic growth tray, where they began growing in aeroponic conditions. After 14 days, the mother tomato plants were removed from the aeroponics, cuttings harvested, and then the mother plant plus the cuttings were placed back into the aeroponic unit. The cloning processes involved the following steps: Using a sharp razor, cuttings were taken from the mother plant. The cuttings were then placed into their own, individual foam inserts, with 0.1-0.5 inches (about 0.25-1.25 cm) of stem protruding from the base of the foam collar. The foam inserts with the cuttings were then placed back into the aeroponic units for continued growth without added hormone. After 7 days in the aeroponic unit the cuttings had produced new roots (FIGURE 21).

E. General aeroponic conditions

[0084] The aeroponic system was set to a set of conditions that, as discovered by the inventors, promote aeroponic plant cloning, e.g., conditions that promote growth and/or maturation of both dicot and monocot plants, such as wheat, potato, and soybean plants, from plant cuttings. A nutrient tank was used to feed the aeroponic system, with appropriate parameters for best results. The parameters in this embodiment were as follows: plants were grown on a set misting cycle of 100 seconds on, 300 seconds off. The electric conductivity was set equivalent to 600 ppm NaCl using a nutrient mixture with a ratio of 30% GH Flora Micro, 30% GH Flora Bloom, 30 % GH Flora Gro, 10% GH CaliMagic mixed with deionized, reverse osmosis water. The pH was set to 5.85 and the pH stabilized using Botanicare pH Up (potassium hydroxide or potassium carbonate) and Botanicare pH Down (phosphoric acid-based) pH stabilizers. Water temperature was controlled using Pentair water chillers that cool the nutrient tank to a setpoint of 21 °C. The air temperature was set to 28 °C during the day, and 25 °C during the night. The photoperiods were controlled using an automatic light timer, with the day length set to 12 hours of light, and 12 hours of darkness, at an intensity of 300 pmol m V 1 .

[0085] Several plant species, including soybean, are considered recalcitrant species for plant transformation: these species are difficult to transform and to regenerate into mature, seed-bearing plants. This difficulty means that each gene edited plant created in the laboratory is extremely valuable, and often one of a kind. These valuable plants are susceptible to death at every stage of transformation, in tissue culture, and during transfer from tissue culture to soil. Additionally, growth in greenhouse conditions can vary significantly, requiring long time periods for production of mature seed. By contrast, aeroponic systems of the present invention are fully controlled and can consistently produce plants that produce mature seed in less than 60 days for some crops.

F. Axillary Cloning of Exemplary Dicot (Soybean)

A study on growing conditions to harvest multiple axillary cuttings from a single mother plant was conducted using soybean, a dicot, as an exemplary species. Mother soybean plants were grown from seed in aeroponic conditions. Seeds were germinated in a water- soaked rockwool substrate in close-bottomed flats with a clear domed lid at 25 °C using a 12/12 light/dark cycle. After 7 days, the germinated seedlings were removed from the rockwool and affixed in foam cups situated in the growth tray of the aeroponic unit. The mother plants were grown for 25 days with the lights far from the plants in 16h of day length to encourage greater stem elongation. At day 25 soybean plants have 1-3 branches, and under low light, the branches have sufficient length of their intemodes for cuttings to be secured in the aeroponic system (at least about 7.5 cm long), and to have several nodes below the apical meristem. To initiate growth of axillary meristems, the apical meristem was taken from the main stem or one of the branches and the plant cutting was inserted into a new foam cup as described above. Removal of the apical meristem encouraged axillary shoot growth. After the topmost axillary shoot showed any amount of new growth, each node with the growing axillary shoot, the attached leaf, and a length of the stem (about 7.5 cm for the aeroponic interface) was harvested along the length of the stem or branch of the mother plant from the apical cutting (FIGURE 22). Each individual cutting was placed into the aeroponics unit as described above. These axillary cuttings showed comparatively improved root development including faster rooting, which resulted in the cutting having more roots in less time relative to apical meristem cuttings. Within 7 days, the plant cuttings had sufficient root development to be transferred to soil. This method provides soil-grown daughter plants at an improved rate for seed bulking or speed breeding program than methods using apical meristem.

[0086] Thus, the methods disclosed herein overcome multiple challenges faced by skilled artisans working towards creating new plant products, such as gene-edited plant products.

[0087] One current challenge is producing seed from a desired plant that yield progeny having the desired characteristics. The present disclosure addresses this need by enabling creation of multiple daughter plants from a single mother plant. The daughter plants can be grown in different areas to ensure that if one area is affected by pests, drought, etc., the daughter plants growing in other areas will still be alive and reach maturity. [0088] The maturation and floral induction of photosensitive plants, such as the normally short day plant soybean, can be manipulated to increase seed yield. For example, by adjusting the length of day with supplemental lighting and/or the use of winter nurseries (e.g., in Florida, Texas, Arizona, California, Hawaii, Puerto Rico and New Zealand) can correct the balance of opposing vegetative and reproductive pathways to increase yield while reducing cycle time. Methods of the present disclosure can further reduce the cycle time and/or increase yield. For example, a typical winter nursery seed increase program for soybean using supplemental lighting to suppress floral induction can take about 120 days, and provides a seed increase of about 80 (i.e., 80 lb seed harvested for each lb planted). Using the axially cloning method described above, a plurality of axial cuttings from a single mother plant can be rooted in an aeroponic growth enclosure and transferred to soil for maturation and floral induction, resulting in more than 2,000 seed within 124 days day after planting (DAP) mother plant (TABLE 1) — a 25X increase over the conventional seed increase methods in about the same time. Moreover, the axial cuttings can be serially propagated by taking cuttings from the daughter plants to substantially increase seed yield from a single mother plant (e.g., >10,000 total seeds from all series). Seed increase usually requires several rounds of planting and harvesting to offer seed for sale to growers. The seeds produced by serial cuttings can be used to reduce the number of seed increase rounds using conventional large-scale methods that are necessary to generate sufficient quantities of seeds to meet commercial demands by providing greater seed number in the first round. Additionally, the daughter plants can be grown to maturity at different speeds. This means that material from a single mother plant can be used in seed increase and breeding efforts in parallel, and products can reach the market faster and more efficiently.

TABLE 1: Exemplary seed increase experiment for serial cuttings harvested at 34 and 89 DAP (mother plant).

[0089] The second challenge overcome by the methods disclosed herein is speed to market. Normally, a single gene edited plant is taken from tissue culture and grown to maturity in soil, which can take upwards of 100 days to harvest mature seed. The methods disclosed herein allow rapid cycling to occur, which can bring daughter plants from that same mother plant to maturity in about 50 days, for example, rather than 100 days. This reduction in time can Theses axillary cuttings showed comparatively improved root development (i.e., faster rooting resulting in more roots in less time relative to apical meristem cuttings) a full year from the product development timeline, and is extremely valuable to gene editing companies desiring to market products faster and more cheaply than their competitors. [0090] Using conventional cloning methods, tissue culture rooting, soil acclimation, and soil maturation occur in three transfer steps. As used herein, “rapid cycling” means that the three transfer steps of conventional cloning methods are combined into one step. In method disclosed herein, tissue culture plants are transferred to aeroponics growth media where they root and are grown to maturity - a one step process, which saves times and resources. FIGURES 1-3 depict exemplary “rapid cycling.”

[0091 ] Additionally, the cloning methods of the present invention can be used on any plant with desirable characteristics, including conventionally bred, wild- type plant, or a plant that has been modified using any techniques available in the art. For example, soybean take about two to three weeks to root in tissue culture. In aeroponics, a soybean shoot or branch can root in less than seven days (FIGURE 5). The present invention can efficiently root many types of plant tissue, shortening the timeline for bringing a gene edited product to the market.

[0092] The technology of the present disclosure is valuable for multiple reasons. Gene editing and breeding companies can benefit from effectively having an insurance policy on each plant of interest. For example, To plants are extremely expensive and time consuming to create. A company could spend two years to develop a To plant with desired edits; if the plant dies before producing seed, the time and money invested are lost. Plants derived from To are also highly valuable and include, without limitation, Ti, Fi, F2, etc. Cloning techniques of the present invention provide the opportunity to keep valuable genetics alive and to expedite time to market. The present disclosure enables propagation of generations of daughter plants, providing clonal lineages that do not exhibit a loss of vigor or degeneracy due to rooting -related stress. For example, a mother plant can be cloned into several daughter plants in a first cycle and subsequent cloning cycles can be completed using any number of mother plants, daughter plants, or a combination.

[0093] The present disclosure furthers offers a method to prevent/minimize plant death from prolonged plant tissue culture. Companies that use tissue culture to produce gene edited plants often suffer plant loss due to extended time in tissue culture. Tissue culture is an artificial environment for a plant, and carries increased death rates compared to growing a plant from seed in soil. Loss can also occur when plants are transferred to soil. This is due to challenges faced by plants in acclimating to soil conditions from laboratory conditions: laboratory conditions are optimized for plant growth (e.g., perfect lighting, nutrients available, absence of pests).

[0094] The present disclosure minimizes loss by enabling transfer of plants from tissue culture to aeroponic culture instead of soil. The plant can also be removed from the tissue culture system at an earlier stage. Additionally, the transition from laboratory conditions to aeroponics is gentle: aeroponics provides high humidity to induce root growth and has minimal or no pests.