Patent Application
20210337752
The present application is directed generally to plant micropropagation, and more particularly, to plant tissue culture devices and methods of culturing and harvesting plant shoot tips.
The labor input per plant is one of the biggest factors in overall system efficiency of plant micropropagation because the worker time under the laminar flow hood is about two-thirds of the cost of growing a plant tissue culture. Prior studies of work flow in the laminar flow hood quantified the labor components of planting, cutting, and removing the plant. See, e.g., Alper, et al., Mass handling of watermelon microcuttings. ASAE. 37(4):1337-1343 (1994). In these prior studies, planting and cutting times were shown to be equal in affecting the total efficiency of the plant micropropagation system. Id. However, current vessels used in plant tissue cultures, such as a standard GA7 Magenta vessel 10 (see, e.g.,
Embodiments of the present invention are directed to multiple-cut methods of culturing and harvesting plant shoot tips.
According to embodiments of the present invention, methods of culturing and harvesting plant shoot tips include providing a sterile vessel sized and configured to hold at least one plant comprising one or more root masses and a first set of shoot tips, cutting across a base of the first set of shoot tips with the one or more root masses held in the vessel to cut a first plurality of cut shoot tips at a first time, then growing a second set of shoot tips from the one or more root masses in the vessel, and then cutting across the base of the second set of shoot tips with the one or more root masses held in the vessel to cut a second plurality of cut shoot tips at a second time.
The second set of shoot tips may have an increased multiplication ratio providing more shoot tips than that the first set of shoot tips.
The cutting may be carried out using a single direction motion of a cutting tool above and across a base of the vessel.
The cutting may be carried out using a reciprocating motion of a cutting tool above and across a base of the vessel.
The cutting may be carried out using an electric knife.
The cutting may be carried out using a blade that is axially stationary and moved either automatically using a robotic arm or other electromechanical member or moved manually across and above the base.
The growing of the second set of shoot tips may include a growth period in a range of about 1 week to about 3 weeks, optionally comprising a growth period in a range of about 1.5 weeks to about 2.5 weeks.
The first plurality of cut shoot tips and the second plurality of cut shoot tips may be collected in a sterile receiver as microcuttings. The microcuttings may be placed in one or more different sterile vessels with nutrients in a greenhouse to grow into full grown plants.
The method of culturing and harvesting plant shoot tips may further include growing a third set of shoot tips from the one or more root masses held in the vessel after cutting the second set of shoot tips, and cutting across the base of the third set of shoot tips with the one or more root masses held in the vessel to cut a third plurality of cut shoot tips at a third time.
The method may further include growing a fourth set of shoot tips from the one or more root masses held in the vessel after cutting the third set of shoot tips, and then cutting across the base of the fourth set of shoot tips with the one or more root mass held in the vessel to cut a fourth plurality of cut shoot tips at a fourth time. At least the base of the vessel may remain sterile over each growing step allowing for several harvests of shoot tips from the same root masses, and optionally at least one of the second, third, and fourth set of shoot tips may have an increased number of shoot tips per plant relative to the first set of shoot tips.
The method may further include adding nutrients and/or water to the one or more root masses after a respective cutting. In some embodiments, the one or more root masses after the cutting of the first plurality of cut shoot tips may include a rooted matrix with one or more buds thereby allowing for rapid re-growth of one or more additional shoots to yield the second set of shoot tips.
The vessel can have a base releasably attached to a housing. The base may have a first height and the housing may have a second height. The second height of the housing can be about 2 times to about 10 times greater than the first height of the base. The base can hold the one or more root masses and can be sized and configured to allow the first and second set of shoot tips to grow a distance extending outwardly from the base and to be exposed when the housing is detached from the base.
The vessel may have a base with a height that is between about 0.5 inches and about 2 inches. The base may have a lateral width that is greater than the height. The base may hold the one or more root masses with the first and second set of shoot tips allowed to grow above the base.
The vessel can include a permeable substrate in the base configured to hold the root mass and comprising plant nutrients.
The base may hold a soilless substrate, and optionally a plant nutrient and moisture, along with the one or more root masses.
The base may be configured to hold the one or more root masses such that new shoot buds forming the second set of shoot tips after the cutting of the first plurality of cut shoot tips are allowed to grow above the height of the base during the growing step.
The housing may have a sidewall and/or a top with one or more permeable membranes.
The at least one plant may be a vascular plant.
The at least one plant may be a gymnosperm or an angiosperm.
The at least one plant may be a dicot or a monocot.
The at least one plant may be cannabis.
The at least one plant may be edible microgreens.
The at least one plant may be a potato.
The at least one plant may be a fruit tree or a timber tree.
The at least one plant may be an ornamental plant.
The vessel may include a base, and optionally an impermeable rigid or semi-rigid base, and a releasably attached housing. The base can be configured to hold the one or more root masses and the first and second sets of shoot tips can grow a distance extending outwardly from the base into the housing during the growing steps.
The vessel may further include a plant support member that is coupled to the base and that is configured to allow the first and second sets of shoot tips to grow through and above the plant support member. The support member may extend or reside at a height that defines a cut height for the cutting of the first plurality of cut shoot tips and second plurality of cut shoot tips. The base and/or the housing may optionally be visually transmissive.
The plant support member may be formed of thermoformed copolymer.
An edge of the plant support member may form an interference fit within the vessel.
The plant support member may be concave in shape and may apply a spring-like pressure on the shoot tips growing within the vessel such that the shoot tips cannot force the plant support member upward during growth.
The plant support member may have an open mesh or a grid configuration.
The mesh or grid configuration may include laterally spaced apart apertures that have an inverted funnel shape with a larger end residing further away from a bottom of the base to allow increased space for lateral expansion during plant growth.
The methods may further include removing the housing before the cutting steps and optionally rotating the base from a first orientation to a second orientation between each cutting step.
The cutting steps may be carried out to provide dozens of shoot tips as the first and second set of cut shoot tips.
The methods may further include reattaching the housing to the base for the growing of the second set of shoot tips.
The growing steps may be carried out in vitro in a sterile environment with the base of the vessel maintaining sterility during the growing of each of the first, second, third, and fourth sets of shoot tips.
The first plurality of cut shoot tips and the second plurality of cut shoot tips may have an increased rooting percentage and/or an increased percentage of survival in a greenhouse environment compared to at least one standard set of shoot tips collected individually using a scalpel from a corresponding root mass.
Further embodiments of the present invention are directed to a plant tissue culture device.
The plant tissue culture device may include a sterile base, optionally an impermeable rigid or semi-rigid base, and a sterile housing releasably attached to the sterile base. The base may have a first height and the housing may have a second height. The second height of the housing may be about 2 times to about 10 times greater than the first height of the base.
The base may be sized and configured to hold one or more plant root masses therein and may allow the one or more plant root masses to grow and produce a set of shoot tips with the set of shoot tips residing in the housing above a top edge of the base. The one or more root masses may optionally be held in a matrix of soilless nutrient material in the base.
The housing may include one or more permeable membranes.
The plant tissue culture device may further include a plant support member that is coupled to the base and configured to allow the set of shoot tips to grow through and above the plant support member. The plant support member may reside at a height that defines a cut height for cutting of the set of shoot tips.
The plant support member may be formed from a thermoformed copolymer.
An edge of the plant support member may form an interference fit with the base of the device.
The plant support member may be concave in shape and may apply a spring-like pressure on the shoot tips growing within the device such that the shoot tips cannot force the plant support member upward during growth.
The plant support member may have an open mesh or a grid configuration.
The mesh or grid configuration may include a plurality of laterally spaced apart apertures that have an inverted funnel shape with a larger end residing further away from a bottom of the base to allow increased space for lateral expansion during plant growth.
It is noted that aspects of the invention described with respect to one embodiment, may be incorporated in a different embodiment although not specifically described relative thereto. That is, all embodiments and/or features of any embodiment can be combined in any way and/or combination. Applicant reserves the right to change any originally filed claim or file any new claim accordingly, including the right to be able to amend any originally filed claim to depend from and/or incorporate any feature of any other claim although not originally claimed in that manner. These and other objects and/or aspects of the present invention are explained in detail in the specification set forth below.
The present invention now is described more fully hereinafter with reference to the accompanying drawings, in which embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein. Well-known functions or constructions may not be described in detail for brevity and/or clarity.
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises” and/or “comprising”, when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items.
As used herein, phrases such as “between X and Y” and “between about X and Y” should be interpreted to include X and Y. As used herein, phrases such as “between about X and Y” mean “between about X and about Y.” As used herein, phrases such as “from about X to Y” mean “from about X to about Y.”
In general, embodiments of the present invention provide a plant tissue culture device that may be used to provide a vessel and culturing and harvesting tool to allow mechanized cutting of elongated plant shoot cuttings for plant micropropagation. Related methods of culturing and harvesting plant shoot tips are also provided. Embodiments of the present invention will now be discussed in greater detail with reference to
Referring to
As shown in
In some embodiments, both the base 102 and the housing 104 of the plant tissue culture device 100 are sterile. As used herein, “sterile” means free from contamination by other organisms, such as, for example, bacteria, insects, fungi, viruses, and weeds. Optionally, in some embodiments, the base 102 may be an impermeable rigid or semi-rigid, self-supporting base 102. As used herein, the term “rigid” means that the base 102 is unable to bend or be forced out of shape, i.e., not flexible. As used herein, the term “semi-rigid” means that the base 102 has a self-supporting shape, but may flex when sufficient force is applied. As used herein, the term “self-supporting” means that the base 102 will retain a pre-formed shape when apart from the housing 104 and/or when holding a plant root mass or masses 140 therein (see, e.g.,
In some embodiments, the housing 104 may comprise at least one permeable membrane 108. In some embodiments, the at least one permeable membrane 108 may be used for ventilation. The at least one permeable membrane 108 may allow for gases, such as, oxygen, nitrogen, and carbon dioxide to enter and exit the device 100 and/or to allow water evaporation. For example, as shown in
In some embodiments, the membrane(s) 108 may be covered by a removable cover 108c to reduce water evaporation from the device 100. For example, as shown in
As shown in
As shown in
In some embodiments, each set of shoot tips 1501, 1502 may be cut using a cutting tool 160. As shown in
Referring to
As shown in
An alternative plant support member 112′ according to embodiments of the present invention in illustrated in
Referring to
Methods of culturing and harvesting plant shoot tips 150 are also provided by embodiments of the invention.
According to embodiments of the present invention, the growing step(s) may be carried out in vitro in a sterile environment with at least the base 102 of the vessel 100 maintaining sterility during the growing of the first and second set of shoot tips 1501, 1502.
According to some embodiments of the present invention, the cutting step(s) may provide a multitude (dozens) of new shoot tips 150 from the first plurality and the second plurality of cut shoot tips 150c1, 150c2. As shown in
In some embodiments, the methods may further comprise removing the housing 104 of the vessel 100 before performing the cutting step(s) (see, e.g.,
In some embodiments, methods of the present invention may further comprise adding nutrients and/or water to the one or more root masses 140 after a respective cutting (block 300). In some embodiments, the one or more root masses 140 after the cutting of the first set of shoot tips 1501 may comprise a rooted matrix 140m with one or more buds within the interior chamber 102i of the base 102 (
In some embodiments, as each of the first and second sets of shoot tips 1501, 1502 are cut, the respective first plurality and second plurality of cut shoot tips 150c1, 150c2 may be collected in a sterile receiver 120 as microcuttings 150m (block 290) (see, e.g.,
In some embodiments, the second set of shoot tips 1502 has an increased multiplication ratio providing more shoot tips 150 than that of the first set of shoot tips 1501. According to embodiments of the present invention, the first plurality of cut shoot tips 150c1 and the second plurality of cut shoot tips 150c2 may have an increased rooting percentage and/or an increased percentage of survival in a greenhouse environment compared to at least one standard set of shoot tips 150 collected individually using a scalpel from a corresponding root mass 140 (i.e., single cut systems, for example, shown in
In some embodiments, the growing step for the second set of shoot tips 1502 may comprise a growth period (e.g., T2) in a range of about 1 week to about 3 weeks. Optionally, the growing step for the second set of shoot tips 1502 may comprise a growth period (e.g., T2) in a range of about 1.5 weeks to about 2.5 weeks.
In some embodiments, methods of the present invention may further comprise growing a third set of shoot tips 1503 from the one or more root masses 140 held in the vessel 100 after cutting the second set of shoot tips 1502 (block 240) (see also, e.g.,
In some embodiments, methods of the present invention may further comprise growing a fourth set of shoot tips 1504 from the one or more root masses 140 held in the vessel 100 after cutting the third set of shoot tips 1503 (block 260) (see also, e.g.,
According to embodiments of the present invention, the base 102 of the vessel 100 can remain sterile over each growing step and may allow for several harvests of shoot tips (1501 to 150n) from the same root mass 140. In some embodiments, at least one of the second, third, or fourth set of shoot tips 1502, 1503, 1504 may have an increased number of shoot tips 150 per plant relative to the first set of shoot tips 1501. The same housing 104 of a different housing 104 can be used for growing each subsequent set of shoot tips 1502, 1503, 1504. The housing 104 can remain sterile over each growing step.
Devices and methods of the present invention may be suitable for use with a variety of different plants. Exemplary types of plants that may be suitable for use include, but are not limited to, a vascular plant, a gymnosperm or an angiosperm, a dicot or a monocot, cannabis, edible microgreens, a potato, a fruit tree or a timber tree, or an ornamental plant.
The present subject matter will be now be described more fully hereinafter with reference to the accompanying EXAMPLES, in which representative embodiments of the presently disclosed subject matter are shown. The presently disclosed subject matter can, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the presently disclosed subject matter to those skilled in the art.
The following EXAMPLES provide illustrative embodiments. Certain aspects of the following EXAMPLES are disclosed in terms of techniques and procedures found or contemplated by the present inventor to work well in the practice of the embodiments. In light of the present disclosure and the general level of skill in the art, those of skill will appreciate that the following EXAMPLES are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently claimed subject matter.
Several experiments were performed to investigate the effects of processes on worker efficiency under the laminar flow hood, and the quality of Petunia shoot tips in lab and the greenhouse. The multiple-cut system utilized the plant tissue culture device 100 described above (
The first experiment (Example 1) tested the effect of a scalpel and forceps cutting system and using agar media with a GA7 Magenta vessel (Magenta Corp., Chicago, Ill., USA) in a single batch cycle standard control (e.g.,
The first experiment aimed to screen the effects of Petunia genotypes Ragtime and Suncatcher, Oasis® media (Grower Solutions, Kent, Ohio, USA) in the “smart” vessel, cutting cycles, and fed-batch technique on Petunia microcutting and the microcutting quality in the greenhouse.
Plant material: Petunia x hybrida (3832 Ragtime and 3859 Suncatcher) stage II tissue cultures were obtained from (Ball Horticultural Co., West Chicago, Ill., USA). Stock plantlets were grown on Basal MS medium (Murashige and Skoog, 1962) supplemented with 0.01% w/v thiamine HCl, 0.05% w/v nicotinic acid, 0.05% w/v pyridoxine HCl, 10.0% w/v myo-inositol, 0.2% w/v glycine, 0.65% w/v agargel (agar and phytagel, Sigma A3301), 0.5% v/v iron sulfate EDTA, and 3% w/v sucrose. Medium pH was adjusted to 5.8. Media are autoclaved in vessels at 121° C. for 25 minutes.
In vitro micropropagation: Sixteen shoot tips grown on Oasis® media in a “smart” vessel (
Cutting cycles and fed-batch techniques: The shoot tips in the “smart” vessel were cut every two weeks with electric knife (
Greenhouse acclimatization: Non-rooted shoot tips T1 and T2 were transferred directly to the soilless mix (Fafard 3B) where six shoot tips from each vessel transferred to six cells in the 1206 pack. Plants grew for 2 weeks under mist frequency of 8 sec every 10 min during the daylight hours (latitude=34.67350, and longitude=−82.8326) in Fafard 3B soilless mix (Canadian sphagnum peat moss, 7.62/20.32 cm processed pine bark, perlite, vermiculite, wetting agent, starter fertilizer and dolomitic limestone; Sun Gro Horticulture, Agawam, Mass., USA).
Measurements: Multiplication ratio of T1 and T2 and plant cutting rate per min were counted in the laboratory. Survival %, % rooted, shoots length (cm), and numbers of leaves were counted in the greenhouse.
Multiplication ratio=the number microcuttings/initial shoot tips in the vessel
Plant cutting rate per min=the multiplication ratio/the cutting time (min)
The system efficiency was calculated by using the equation below:
System efficiency=the sum of multiplication ratio for each cutting cycle×the average of plant cutting rate of the cutting cycles.
Experimental design and data analysis: A complete random design (2×2×2×3 full factorial) was conducted with replicates of the treatments. The factorial model terms were considered significant at P<0.05 (Table 3). The experiment design, analysis, and graphs were created using JMP version 12.0 (SAS Inst., Cary, N.C., USA).
Labor components in the laminar flow hood: Plant multiplication, with the scalpel and forceps in agar and GA7 Magenta vessel system, Petunia T1 was 0.86±0.5 fold and T2 was 1.76±0.5 fold (
System Efficiency: After two cutting cycles, the total efficiency of scalpel and forceps in agar and GA7 Magenta vessel system and electric knife multiple-cut in Oasis® media and the “smart” vessel system were calculated by using the numbers from the previous paragraphs and the equation below:
System Efficiency of scalpel and forceps in agar and GA7 Magenta vessel system: (0.86+1.76)×{(0.96+1.0)/2}=2.6×
System Efficiency of electric knife in Oasis® media and the “smart” vessel system: (0.7+0.76)×{(0.96+1.1)/2}=1.4×
The systems efficiency was compared with a single cut by the scalpel and forceps from the agar and GA7 Magenta vessel system 1x. The stool cutting of two cycles increased the systems efficiency comparing with the single cut per culture. The electric knife was faster than the scalpel and forceps cutting tools but the regrowth of new T2 shoot tips was lower in Oasis® media than agar media which reduced the efficiency of the electric knife multiple-cut system.
Plant quality in the greenhouse: The quality of the microcutting was measured by the growth in the greenhouse. Petunia cuts had 97±7% survival percentage after 2 weeks in the greenhouse. Media fed-batch techniques increased % rooted. LSMean differences student's t test analysis showed that the nutrient media fed-batch and batch culture were significantly difference from water fed-batch at 95% confidence. The % rooted from media fed-batch treatment was 90±11%, % rooted from batch culture was 87±11%, and % rooted from water fed-batch was 62±11%. Ragtime had longer shoots than Suncatcher (
The experiment was performed to investigate the effect of long duration (three months), cutting cycles (six cycles), and fed-batch techniques on Petunia Ragtime shoot culture in the lab and the quality in the greenhouse.
Plant material: Petunia x hybrida (3832 Ragtime) as described above.
In vitro micropropagation: Sixteen shoot tips were cultured in Oasis® media in the “smart” vessel (
Cutting cycles and fed-batch techniques: The shoot tips were cut every two weeks with two cutting system: the electric knife in Oasis® media and the “smart” vessel (
Greenhouse acclimatization: Non-rooted shoot tips T1, T2, T3, T4, T5, and T6 were transferred directly to the soilless mix (Fafard 3B) for 2 weeks as described above.
Measurements: Multiplication ratio of T1, T2, T3, T4, T5, and T6 and plant cutting rate per min were counted in the laboratory. Survival %, % rooted, shoots length (cm), and numbers of leaves were counted after two weeks of growth in the greenhouse. The system efficiency was calculated. The calculations were as described above. The vessel initial mass and the vessel mass before and after the cutting were measured for each cycle and the water loss from the vessel was measured by:
Shoot tips fresh mass (g)=Vessel mass before cutting (g)−Vessel mass after cutting (g)
Water loss (mL)=Vessel initial mass (g)−Vessel mass before cutting (g)−Shoot tips fresh mass (g)
Experimental design and data analysis: A complete random design (2×3×6 full factorial) was conducted with two replicates of treatments and six replicates of the control. The factorial model terms were considered significant at P<0.05 (Table 4). The experiment design, analysis, and graphs were created using JMP version 12.0 (SAS Inst., Cary, N.C., USA).
Labor components in the laminar flow hood: The multiplication ratio and plant cutting rate per min for the stool shoot cutting of six cycles were listed in Table 1. The scalpel and forceps in agar media and GA7 Magenta vessel had higher multiplication ratio in the first four cycles (T1 to T4) than the electric knife multiple-cut in Oasis® media and the “smart” vessel system (
System Efficiency: After six cutting cycles, the total efficiency of the scalpel and forceps in agar and GA7 Magenta vessel system and electric knife in Oasis® and the “smart” vessel system were calculated using the numbers from Table 1 by the equations below:
System Efficiency of scalpel and forceps in agar and GA7 Magenta vessel system: (1.13+1.87+1.0+1.33+0.75+0.63)×{(1.05+1.0+0.53+0.96+0.77+0.68)/6}=8.39×
System Efficiency of electric knife multiple-cut in Oasis® media and the “smart” vessel system: (0.65+0.5+0.54+0.53+0.78+0.68)×{(1.6+1.6+0.96+0.69+1.85+1.93)/6}=7.95×
The stool cutting of six cycles improved the total efficiency of the scalpel and forceps from the agar and GA7 Magenta vessel and the electric knife multiple-cut in Oasis® media and the “smart” vessel systems comparing with a single cut by scalpel and forceps from the agar and GA7 Magenta vessel system 1x. Both systems had similar system efficiency with the long stool cutting.
Plants quality in the greenhouse: The electric knife multiple-cut in Oasis® media and the “smart” vessel system had 100±10% survival in the greenhouse through the cutting cycles but the scalpel and forceps in agar and GA7 Magenta vessel system was affected by the cutting cycles and reduced from 100±10% in T1 to 53±10% in T6. All the microcuttings had rooted in the greenhouse. The shoot length and the number of leaves of Ragtime, after 2 weeks in the greenhouse were affected by the cutting cycle. The quality of the microcutting (shoot length and number of leaves) was reduced through the long-term culture.
The electric knife multiple-cut in Oasis® media and the “smart” vessel system was faster than the scalpel and forceps in agar and GA7 Magenta vessel system but the damage of the small new shoot tips during the cutting might reduce the number of the shoot tips and that reduced the multiplication ratio. System efficiency of both cutting systems was the same but the plant from the electric knife in Oasis® media and the “smart” vessel system had greater survival in the greenhouse. Adding media or water to the Oasis® system may not provide an advantage on the system efficiency or the quality in the greenhouse. But the reduction of total volume from the evaporation and removing plants was higher in the batch treatment that lost 54±1.2 mL during the six cycles (12 weeks). The fed-batch techniques, media and water lost 2.0±2.2 mL and 0.54±1.6 mL respectively after 12 weeks. The reduction in the media volume was higher in the batch culture than fed-batch culture in the “smart” vessel. The results indicated that the water evaporation from the “smart” vessel not removing plant tissues (cutting) caused the loss in the volume of the batch culture although the ventilation membranes were closed with Aluminum foil to reduce the evaporation (
Two experiments were performed, one to compare the scalpel and forceps in agar and GA7 Magenta vessel system (“C”) with the electric knife multiple-cut in Oasis® and the “smart” vessel system (“0”) and in agar media and the “smart” vessel (“A”). The other experiment was performed to optimize the media volume in Oasis® media.
Plant material: Petunia x hybrida (3832 Ragtime) as described above.
In vitro micropropagation: Eight Petunia Ragtime shoot tips were grown on Oasis® media (160 mL, 200 mL, 240 mL, and 280 mL) in liquid MS medium as described above. Eight shoot tips grown on agargel media of 300 mL MS medium (300 mL medium volume was added to reach the height of Oasis® foam in the “smart” vessel so the cutting would be in the same level) supplemented with 0.65% w/v agargel (Sigma A3301) as described above. Six shoot tips grown on agargel MS medium in GA7 Magenta vessel as control system.
Cutting cycle systems: The shoot tips in the “smart” vessels were cut every three weeks with electric knife (
Measurements: Multiplication ratio of T1, T2, T3, and T4, plant cutting rate per min, and the length of the first three nodes in Petunia shoot (cm) were measured in a lab. The system efficiency and volume loss (mL) were calculated as described above.
Experimental design and data analysis: Two complete random designs (3×4 full factorial) and (Oasis® media volume I, 4×4) full factorial were conducted with four replicates of treatments and the control. The factorial model terms were considered significant at P<0.05 (Table 5 and Table 6, respectively). The experiment designs, analysis, and graphs were created using JMP version 12.0 (SAS Inst., Cary, N.C., USA).
Labor components in the laminar flow hood: In the scalpel and forceps in agar and GA7 Magenta vessel system (“C”), multiplication ratio for the four cycles was 1.110.5 fold T1, 1.3±0.5 fold T2, 1.2±0.5 fold T3, and 0.95±0.5 fold T4 (
The scalpel and forceps used 1.0±0.1 min/vessel to cut 6 shoot tips in GA7 Magenta vessel but the electric knife used 0.41±0.1 min/vessel to cut 8 shoot tips from the “smart” vessel in both media “O” and “A”. Plant cutting rate per min for the scalpel and forceps in agar and GA7 Magenta vessel system was 1.4±1.2 min−1 for T1, 1.21±1.2 min−1 for T2, 0.9±1.2 min−1 for T3, and 1.3±1.2 min−1 for T4 (
System Efficiency: After four cutting cycles, the total efficiency of the scalpel and forceps in agar and GA7 Magenta vessel system and electric knife in Oasis® media and in agar with the “smart” vessel systems were calculated using the numbers from the previous paragraphs by the equations below:
System Efficiency of scalpel and forceps in agar and GA7 Magenta vessel system: (1.1+1.3+1.2+0.95)×{(1.4+1.2+0.9+1.3)/4}=5.46×
System Efficiency of electric knife multiple-cut in Oasis® media and the “smart” vessel system: (0.78+1.2+0.65+1.5)×{(1.6+2.8+2.6+4.9)/4}=14.54×
System Efficiency of electric knife multiple-cut in agar media and the “smart” vessel system: (1.3+1.8+2.4+1.0)×{(1.6+4.0+6.1+7.5)/4}=31.2×
Agar media in the “smart” vessel had the highest system efficiency comparing with a single cut by the scalpel and forceps from the agar and GA7 Magenta vessel system 1x and the other systems.
Plant quality in laboratory: Shoot node length was measured after the cut directly to test the quality of the stool shoots in different cutting systems. The scalpel and forceps in agar and GA7 Magenta vessel system (“C”) had shoot node 1.1±0.2 cm from T1 cycle and reduced to 0.5±0.2 cm at T4 cycle (
Two experiments were performed, one to compare the scalpel and forceps in agar and GA7 Magenta vessel system (“C”) with the scalpel and forceps in Oasis® media and the “smart” vessel system (“KO”) and in agar media and the “smart” vessel (“KA”). Scissors were tested as a cutting tool in Oasis® media and the “smart” vessel (“SO”) and in agar media and the “smart” vessel (“SA”). The other experiment was performed to optimize the media volume in Oasis® media.
Plant material: Petunia x hybrida (3832 Ragtime) as described above.
In vitro micropropagation: Eight Petunia Ragtime shoot tips were grown on Oasis® media (160 mL, 200 mL, 240 mL, and 280 mL) in MS medium as described above. Eight shoot tips grown on agargel media of 280 mL MS medium supplemented with 0.65% w/v agargel (Sigma A3301) as described above. Six shoot tips grown on agargel MS medium in GA7 Magenta vessel as control system.
Cutting cycle systems: The shoot tips in the “smart” vessels were cut every two weeks with scissors (“SA” and “SO”) (see, e.g.,
Measurements: Multiplication ratio of T1, T2, T3, and T4, plant cutting rate per min and the length of the first three nodes in Petunia shoot (cm), shoot tips fresh mass (g) were counted in a lab. System efficiency was calculated for each cutting system as described above.
Experimental design and data analysis: Two complete random designs (5×4 full factorial) and (Oasis® media volume II, 4×4) full factorial were conducted with four replicates of treatments and the control. The factorial model terms were considered significant at P<0.05 (Table 7 and Table 8, respectively). The experiment designs, analysis, and graphs were created using JMP version 12.0 (SAS Inst., Cary, N.C., USA).
Labor components in the laminar flow hood: The multiplication ratio and cutting rate per min for the four cycles in all cutting systems are list in Table 2. All systems had 1.0±0.2 fold multiplication ratio at T1 cycle (
However, scissors in agar media and the “smart” vessel increased the cutting rate twice (2.1±0.1 min−1) as the scalpel and forceps in agar media and GA7 Magenta vessel (1.0±0.1 min−1) Scissors had higher plant cutting rate (1.9±0.1 min−1) than scalpel and forceps (0.9±0.1 min4) in Oasis® media and the “smart” vessel. Scissors in Oasis® and agar media and the “smart” vessel (“SA” and “SO”) had its highest plant cutting rate per min than other systems (“C”, “KA”, and “KO”) (
System Efficiency: The systems efficiency was compared with a single cut by scalpel and forceps from the agar and GA7 Magenta vessel system 1x. After four cutting cycles, the total efficiency of the scalpel and forceps in agar and GA7 Magenta vessel system (“C”) and scalpel and forceps in Oasis® media and agar with the “smart” vessel systems (“KO” and “KA”), and scissors in Oasis® media and agar with the “smart” vessel systems (“SO” and “SA”) were calculated using the numbers from Table 2 by the equations below:
System Efficiency of scalpel and forceps in agar and GA7 Magenta vessel system (“C”): (1.0+1.5+0.46+1.0)×{(0.92+0.80+0.48+0.73)/4}=2.9×
System Efficiency of scalpel and forceps in Oasis® media and the “smart” vessel system (“KO”): (1.0+0.68+0.82+1.0)×{(0.88+0.79+0.92+1.0)/4}=3.14×
System Efficiency of scalpel and forceps in agar media and the “smart” vessel system (“KA”): (1.0+1.3+1.4+1.75)×{(0.91+1.1+1.5+1.0)/4}=8.7×
System Efficiency of scissor in Oasis® media and the “smart” vessel system (“SO”): (1.0+0.76+0.86+1.0)×{(2.2+1.5+1.7+2.2)/4}=10.3×
System Efficiency of scissor in agar media and the “smart” vessel system (“SA”): (1.0+1.0+1.5+1.6)×{(1.8+1.3+2.3+2.9)/4}=10.6×
The system efficiency of scissor in agar or Oasis® media and the “smart” vessel was higher than the scalpel and forceps in GA7 Magenta and the “smart” vessels. The efficiency of scalpel and forceps in agar media was higher than Oasis® media in the “smart” vessel.
Plant quality in the laboratory: Shoot node length and fresh mass were measured after the cut directly to test the quality of the stool shoots in different cutting systems. The scalpel and forceps in agar and GA7 Magenta vessel system had 0.6±0.1 cm lengths of the three first nodes (tips cut) through cutting cycles (
All cutting systems had shoot tips with 0.2±0.05 g fresh mass at T1 cycle then the fresh mass reduced after the first cut T1 (
The quality of Ragtime microcutting was higher in agar media than Oasis® media and the “smart” vessel system. Shoot and root systems in agar media and the “smart” vessel was larger than plants from Oasis® media and the “smart” vessel (
This experiment was performed to compare the scalpel and forceps in agar and GA7 Magenta vessel system (“C”) with the scalpel and forceps in Oasis® media and the “smart” vessel system (“KO”) and in agar media and the “smart” vessel (“KA”). A blade and grid were tested as cutting tools in Oasis® media and the “smart” vessel (“BO”) and in agar media and the “smart” vessel (“BA”). Fed-batch technique was tested only in Oasis® media and the “smart” vessel system.
Plant material: Petunia x hybrida (3832 Ragtime) as described above.
Six shoot tips were grown in a GA7 Magenta vessel (Magenta Corp., Chicago, Ill., USA) in a cooler room 12° C. for 42 weeks under 20 μmol m−2s−1 light intensity (GreenPower LED production module deep red/blue 120 II0V, Philips, USA). Following the storage period, shoot tips were cut and six shoot tips (T0) were cultured on 50 mL MS medium described above in the Magenta vessel at 23±2° C. and under 30 μmol m−2s−1 cool white fluorescent lights for 16 hours a day. After two weeks, T1 microcuttings from the six T0 plants were cut using scalpel and forceps. Sixteen shoot tips T1 were cultured in the “smart” vessel on 280 mL MS medium described above. After two weeks T2 shoot tips were used in the experiment. Each subsequent cycle of tipping is designated Tn, where n=number of times tip removal was performed.
In vitro micropropagation: In the “smart” vessel (
Cutting cycle systems: The scalpel and forceps cut T1 microcuttings after two weeks. A sterile steel grid was put on the top of the plants (
The media to add mL=Initial vessel mass with the plant tissue and media+grid mass (if it is there)−the vessel mass after the cutting
Cutting schedule: Experiment started on August 30th
Measurements: Multiplication ratio of T1, T2, T3, T4, T5, and T6, cutting time (min), plant cutting rate per min, the length of the average of first three nodes in Petunia shoot (cm), the average of fresh mass of each microcutting (g), flowering tips %, hyperhydricity %, and water lost per vessel were measured in the laboratory. Shoot node length of the first three leaves was measured directly after the cut.
Multiplication ratio=(the number microcuttings−hyperhydricity tips)/initial shoot tips in the vessel
Plant cutting rate per min=the multiplication ratio/the cutting time (min)
The system efficiency was calculated by using the equation below:
System efficiency=the sum of multiplication ratio for each cutting cycle×the average of plant cutting rate of the cutting cycles
Water lost=the vessel initial mass (g)−the vessel mass before the cutting (g)
Experimental design and data analysis: The complete random design of cutting type, media type, cutting cycles, and fed-batch technique (2×2×2×5 full factorial) were conducted with two replicates of each treatment and four replicates of the control. The full factorial models with quadratic term of cutting cycle were selected by stepwise forward method and model terms were considered significant at P<0.05. The effect of factors on the responses, cutting time and cutting rate per min was tested by t-student test at 95% confidence interval. Design, data analysis, and graphs were created using JMP version 12.0 (SAS Inst., Cary, N.C., USA).
Labor components in the laminar flow hood: The multiplication ratio was affected significantly by the quadratic term of cutting cycles, the main terms of cutting type, media type and their interactions with cutting cycle. In the scalpel and forceps cutting system, the multiplication ratio reached the maximum (2.2±0.3 fold) in T4 in agar medium and in T5 in Oasis® medium (
The cutting type, the cutting cycles and their interaction could significantly affect the cutting time and plants cutting rate. In the smart vessel, the scalpel and forceps cutting system had longer cutting time (5.3±0.3 min/vessel) than the blade and grid cutting system (0.46±0.3 min/vessel). Thus, the blade and grid cutting system was 11 times faster than the scalpel and forceps cutting system. The plant cutting rate using the blade and grid was 2.7±0.6 min−1 in T2 and reduced to 4.13±0.6 min−1 in T6 in the agar media and 2.0±0.6 min−1 in T2 and reduced to 4.77±0.6 min−1 in T6 in Oasis® media. In the scalpel and forceps cutting system, plant cutting rate was 0.6±0.6 min−1 in T2 and reduced to 0.29±0.6 min−1 in T6 in the agar media and 0.4±0.6 min−1 in T2 and reduced to 0.36±0.6 min−1 in T6 in Oasis® media. The media type and fed-batch had no effects on the cutting time. The blade and grid system was faster than the scalpel and forceps cutting system and the blade and grid system could produce more microcuttings per min. In the GA7 magenta vessel with the scalpel and forceps cutting system, the cutting rate had 0.98±0.3 min−1 in T2 then reduced to 0.83±0.3 min−1 in T6.
System Efficiency: After six cutting cycles, the total efficiency of the different cutting systems were calculated by using the equation:
System efficiency=the sum of multiplication ratio for each cutting cycle×the average of plant cutting rate of the cutting cycles
The first cutting cycles was not considered because the blade and grid were applied after the first cut. The efficiency was calculated for the last five cycles.
System efficiency of the scalpel and forceps in the agar and the “smart” vessel system (“KA”): (1.9+2.2+2.2+2.1+1.7)×{(0.60+0.33+0.54+0.45+0.29)/5}=4.5×
System efficiency of blade and grid in agar and the “smart” vessel system (“BA”): (1.3+1.6+1.7+1.5+1.2)×{(2.7+8.78+11.05+5.0+4.13)/5}=46.2×
System efficiency of the scalpel and forceps in Oasis© and the “smart” vessel system (“KO”): (1.2+1.8+2.1+2.2+2.1)×{(0.42+0.29+0.39+0.27+0.36)/5}=3.3×
System efficiency of the blade and grid in Oasis® and the “smart” vessel system (“BO”): (0.5+0.96+1.2+1.2+0.97)×{(2.02+7.34+8.02+4.82+4.77)/5}=26.0×
System efficiency of the scalpel and forceps in GA7 Magenta vessel system (“C”): (1.7+1.8+1.7+1.9+1.3+0.72)×{(0.98+1.02+0.95+0.87+0.83)/5}=8.5×
The systems efficiency was compared with a single cut by the scalpel and forceps from the agar and GA7 Magenta vessel system 1x. The stool cutting of six cycles improved the total efficiency of the scalpel and forceps from the agar and Oasis® media in the “smart” vessel system and in agar GA7 Magenta vessel and the blade and grid in agar and Oasis® media and the “smart” vessel systems compering with a single cut by scalpel and forceps from the agar and GA7 Magenta vessel system 1x. In the “smart” vessel, both multiple cutting systems in agar medium had higher system efficiency than the multiple cutting systems in Oasis® medium. The blade and grid cutting system had the highest efficiency than the scalpel and forceps systems. In the scalpel and forceps cutting system, the efficiency was higher in GA7 Magenta vessel system than in the efficiency of the “smart” vessel systems.
Plant quality in laboratory: The cutting cycle and its interaction with media types had significant effects on the fresh mass of Petunia shoot tips (
The media types, cutting cycle and their interaction significantly affected the shoot nodes length (
Flowering %: The flowering % was significantly affected by media types, cutting cycle and their interaction (
Hyperhydricity %: The media types, cutting cycle, cutting types and their interactions, and fed-batch significantly affected the hyperhydricity percentage per vessel (
Water lost: Losing water from the “smart” vessel was affected by fed-batch treatments, cutting type, their interaction with cutting cycle, and cutting cycle. The vessels with the scalpel and forceps cutting system lost 7.0±2.5 mL of water after 12 weeks with fed-batch treatment and lost 58.1±2.5 mL of water after 12 weeks without fed-batch treatment (
The “smart” vessel systems with the two different types of cuttings with agar produced more plants and higher quality than Oasis® media, but with the continuous cutting, the multiplication ratio, fresh mass and the length of the microcuttings were reduced on agar medium (
In agar media, the “smart” vessel had more and larger shoot tips through the 12 weeks than GA7 Magenta vessel. The plant growth might be influenced by the large space of the “smart” vessel. The large vessels increased the fresh mass yield. That large space and the big number of plant reduce the system efficiency of scalpel and forceps cutting system comparing with GA7 Magenta vessel.
The grid had a negative effect on Petunia multiplication ratio and increased the hyperhydricity % and the water lost from the vessel. The grid might compress the Oasis® media. This pressure might affect the plant growth and increase buildup the water around the root in the Oasis® medium which increases the hyperhydricity % and water evaporation from the vessel. The volume of the medium may be reduced to control these problems the aluminum cover from the pores.
The foregoing is illustrative of the present invention and is not to be construed as limiting thereof. Although a few exemplary embodiments of this invention have been described, those skilled in the art will readily appreciate that many modifications are possible in the exemplary embodiments without materially departing from the novel teachings and advantages of this invention. Accordingly, all such modifications are intended to be included within the scope of this invention as defined in the claims. The invention is defined by the following claims, with equivalents of the claims to be included therein.
Petunia
Petunia*Cutting type
Petunia*Cutting cycle*Cutting type
Petunia*Cutting cycle
Petunia*Fed-batch techniques
Petunia*Cutting cycle*Fed-batch
The present application claims the benefit of and priority to U.S. Provisional Application Ser. No. 62/724,410, filed Aug. 29, 2018, the contents of which are hereby incorporated by reference as if recited in full herein.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2019/048741 | 8/29/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62724410 | Aug 2018 | US |
