CA2692722A1 - Micro-plot field trial system - Google Patents

Abstract

A micro-plot field trial system is provided to replace or augment the conventional small plot research trials. Seeds to be tested are arranged on a planting or seed tape at a predetermined spacing to eliminate the need to thin the plants once emerged from the seeds. Furthermore the quantity of the seeds needed for each trial is reduced which therefore reduces the trial area needed for each trial. The seed tape is created using a robotic apparatus to ensure precise placement and spacing of the seed within a water soluble tape. The seeds are enclosed in the tape which are then planted and harvested for analysis. The seed tape is created in a controlled environment providing accuracy and reliability and can be accurately reproduced so multiple locations can be planted. The reduced number of seeds required means that a micro-plot considerably smaller than traditional trial plots can be utilized.

Description

MICRO-PLOT FIELD TRIAL SYSTEM
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority from United States Patent Application No.
60/948,438 filed July 6, 2007, which is herein incorporated by reference.

TECHNICAL FIELD
The present disclosure relates to seed variety testing system in particular to small plot research field trials.

BACKGROUND
The seed industry relies heavily on annual small plot testing to determine the commercial merit of thousands of new plant cultivars. In the field, small research plots provide the only means to evaluate the agronomic and relative field performance of new cultivars in comparison to existing cultivars. Seed companies rely on small incremental improvements of new cultivars in comparison with existing products to maintain competitiveness and progression in the marketplace.
Screening new cultivars is the largest expenditure of energy and capital in the seed development process. The standard method of field-testing uses conventional two-row replicated yield trials, which require substantial capital and labor investment and are prone to largely variable results.

The standard method of field-testing uses conventional two-row replicated yield trials, which require substantial capital and labor investment. Trials commonly consist of 20 to 50 entries (cultivars of interest) arranged in a statistical design, which is replicated 2 to 4 times within a given location. Seed is packaged prior to planting in small envelopes (packets). In the case of corn hybrid testing, 40 to 75 seeds of each entry are counted and put into small packets with each packet representing one row. It is common to package 6 to 8 packets of the same entry (2 row plot = 2 packets per plot times 3 to 4 replications per location). The packets are then placed in planting order by arranging the packets in trays, which are placed on the planter when planting. Once the packets are in planting order they are ready for the field. Specialized research planters are used to plant small research plots.

Conventional small plots (experimental units) are often 2 rows wide at 76 cm (30 inch) spacing and are 5.2m (17 feet) to 7m (23 feet) in length. Research planters facilitate planting over a specific row length and upon finishing each plot, remaining seeds are removed and seed for the next plot is planted. The process of planting and removing seeds is repeated across the testing location. When plants are at the V5-V6 growth stage (5-7 leaves), each row is shortened to the specified length and plants within each row of each plot are thinned to the desired population.
Plots are often over planted due to the inaccuracy of the planting mechanism used.
Agronomic notes are taken throughout the season on each plot prior to harvest.
Specialized harvest equipment is used to harvest the plots in a conventional manner (collect seeds). This harvest equipment is designed to collect the seeds from each plot, separating them from the plant matter. The harvest equipment then measures the weight and moisture of the grain collected for each plot. This data is then used in statistical analysis to calculate the final yield of each entry within the trial.

There is thus a need to design a system that would reduce the area required for a trial and thus the number of seeds required for each trial and decrease variability.
SUMMARY

An improved seed cultivar testing system designed to replace or augment conventional small plot research trials is provided.

The present disclosure provides a method for performing micro-plot plant trials of a seed cultivar, the method comprising the steps of: determining a seed arrangement for the seed cultivar for a seed tape using a statistical design to be tested in a plant trial; creating the seed tape on a water soluble planting tape, wherein the seeds contained by the tape are placed using a predetermined quantity and spacing based upon the statistical design; planting the seed tape in a micro-plot environment;
collecting observations on the resulting plants; harvesting the plants; and analyze the collected plants; wherein the spacing of the seeds within the seed tape is determined to eliminate the need to thin the plants once emerged from the seeds, reducing the quantity of the seeds and the trial area required for the micro-plot.

There is provided a method of creating a seed tape using a robotic apparatus, the method comprising the steps of: receiving seed tape configuration information wherein the tape configuration defines seed type and position of the seed within the seed tape based on seed characteristics of the seed cultivar to the tested, the seed tape configuration designed to eliminate the need to thin the plants once emerged from the seeds, reducing the quantity of the seeds and the trial area required for the micro-plot; selecting a seed receptacle containing a particular seed cultivar from a plurality of seed receptacles; extracting a seed from the selected seed receptacle using a probe having a vacuum tip, wherein the seed is picked up by the probe by a vacuum created at the tip of the probe; placing the extracted seed into a water soluble tape, wherein the seed is dropped into the tape by removing the vacuum from the probe tip; advancing the seed tape a predetermined distance based upon the received tape configuration; and encapsulating the seed in the tape.

There is also provided an automated apparatus for making plant seed tape comprising: a digital controller programmed for the number of seeds and seed spacing; a plurality of seed containers, each container contain seeds of a particular seed cultivar; an arm with a vacuum probe to draw a seed from a selected container and deposit it on a water soluble tape; a air-brush sprayer to deliver fine water mist moisten the water soluble tape to allow it to adhere to itself, and encasing the seed;
a closure system to capture the seed in place the water soluble tape; a drive for advancing the tape in a pre-set amount to ensure the correct seed spacing on the tape; one or more guides for guiding the tape onto a spool; and a talc delivery for applying a small blast of talc powder with each rotation of the spool to ensure easy removal of the tape from the spool.

There is also provided a computer readable medium containing instructions for creating a seed tape using a robotic apparatus, the instructions which when executed by a processor perform the steps of: receiving seed tape configuration information wherein the tape configuration defines seed type and position of the seed within the seed tape and is based on seed characteristics to the tested, the seed tape configuration designed to eliminate the need to thin the plants once emerged from the seeds reducing the quantity of the seeds and the trial area required for the micro-plot; selecting a seed receptacle from a plurality of seed receptacles; extracting a seed from the selected seed receptacle using a probe having a vacuum tip, wherein the seed is picked up by the probe by a vacuum created at the tip of the probe; placing the extracted seed into a water soluble tape, wherein the seed is dropped into the tape by removing the vacuum from the probe tip; advancing the seed tape a predetermined distance based upon the received tape configuration; and encapsulate the seed in the tape.

BRIEF DESCRIPTION OF THE DRAWINGS
Further features and advantages will become apparent from the following detailed description, taken in combination with the appended drawings, in which:

FIGURE 1 shows a perspective view of a robot that mechanically loads a predetermined number of individual seeds from each entry into a water-soluble film;
FIGURES 2a-d shows placement of a seed into the water-soluble film;

FIGURE 3 shows top view of the robot;

FIGURE 4 shows a front view of the robot;
FIGURE 5 shows a side view of the robot;

FIGURE 6 shows a method of performing micro-plot field trials;

FIGURE 7 shows method of creating a seed tape for micro-plot field trials;

FIGURE 8 compares the yield (FIG. 8A) and moisture (FIG. 8B) of 17 corn hybrids from two locations of a conventional performance trial to two locations of a micro-plot trial;

FIGURE 9 compares the yield (FIG. 9A) and moisture (FIG. 9B) of 13 corn hybrids from two locations of conventional performance trial to two locations of the micro-plot trial system;

FIGURE 10 provides a summary of the harvest yield (dry bushel/acre) by treatment (two row = 1; one row = 2; micro = 3) of five corn hybrids over four locations in a micro-plot field trial;.

FIGURE 11 provides a summary of the harvest yield (dry bushel/acre) by treatment (two row = 1; one row = 2; micro = 3) of five corn hybrids over four locations in a micro-plot field trial;

FIGURE 12 provides a summary of the combined grain moisture by treatment (two row = 1; one row = 2; micro = 3) of five corn hybrids over four locations;

FIGURE 13 shows the adjusted yield (dry bushel/acre) of entries by soil type from 8 plants per micro-plot from 9 locations;

FIGURE 14 shows the adjusted yield (dry bushel/acre) of entries by soil type from 6 plants per micro-plot from 9 locations;

FIGURE 15 shows a processor for controlling the robot.

It will be noted that throughout the appended drawings, like features are identified by like reference numerals.

DETAILED DESCRIPTION
Embodiments are described below, by way of example only, with reference to Figs.
1-15. The present disclosure provides a system and method for performing micro-plot field trials for seed cultivar testing designed to replace or augment conventional small plot research trials.

There are several limitations to selecting cultivars from limited data sources.
Selection of testing locations often does not reflect the majority of the environments within the targeted region. Trials are often located in high yield environments which create difficulties evaluating hybrid adaptation. Soil texture, soil temperature, crop rotation and fertility are examples of parameters varying across growing regions.
Disease and insect pressure also vary greatly across growing regions due to crop rotation and weather patterns. Trial locations are often not exposed to insect and disease pressures due to the limited locations planted. In addition, companies are often limited to the number of testing locations due to the narrow optimum planting window. Planting on either side of this optimum date results in reduced yield and therefore does not accurately predict how cultivars will perform under commercial scale.

Increasing the environments/locations a cultivar is tested in, within its adapted geographic region, can greatly increase the knowledge gained on a cultivar.
The more information collected on a cultivar increases the confidence and accuracy of the data collected. Using this data, breeders can make more informed decisions on cultivar selections advanced to commercial products.

Spatial competition between corn plants has been shown to have negative effects on grain yield per plant, kernel number per plant and kernel weight per plant as the plant population is increased. Dominate plants quickly develop within a population which affects the plants ability to partition resources. Industry widely accepts that uneven emergence is the primary reason for lower expected yields.

Border rows are often used when evaluating advanced corn hybrid yield to account for deviations in plant height of hybrids. The treatment of these border rows can influence the final outcome of the trial. A study comparing the treatment of border rows (i.e. the population of border rows) found significant differences in mean grain yield of hybrids and a significant interaction between hybrids and treatments.
Although surrounded by the identical hybrid, differences in the treatment of border plants have been shown to influence the outcome of harvested plots.

The disclosed micro-plot field trial system uses very small plot size and, in the example of corn consist of a one row plot containing 1 to 12, preferably 6 to 8 seeds per plot spaced uniformly at 0.2m (7 inch) to simulate commercial field scenario but may be smaller based upon the particular seed type. In this example, entries can be arranged one after the other and are spaced 25cm (10 inch) from the last seed of the previous entry but can be varied based upon the particular seed growth requirements. The potential exists to evaluate as few as one plant depending on the testing or observation required. By the term "entry" or "entries" it is meant to refer to an individual cultivar or seed type. Any plant seed can be used such as for example for field trials of corn, beans, peas, tobacco, cereal grains or other agricultural crops.
The number of seeds and the spacing between each seed can be adjusted according to the plant species to be tested. In the micro-plot system the plot size is approx. 10% the size of conventional two row plots. Depending on the testing required , with molecular screening techniques plot size could be reduced to as small as one plant and less than 5 feet (1.52m) in length.

One of the significant difference between the conventional and the micro-plot system is the ability to use conventional farm planting equipment to plant replicated research trials. This system eliminates the need for specialized and expensive equipment used for planting conventional research trials. Within a few minutes, the conventional commercial scale planter can be equipped with a modified seed tube and spool holder , the trial planted and the standard equipment replaced resulting in minimal interruption to the cooperators' planting progress. The ability to easily ship or courier the equipment needed for the system allow trials to be planted efficiently and eliminates much of the liability of transporting conventional research equipment.
The efficiency of the system facilitates a large number of locations to be planted within the limited optimum planting window (approximately 9 days). The ease of the micro-plot system allows individual producers to cooperate in the testing cycle with little or no supervision and with little interruption to their own progress.
Allowing cooperators to plant research trials allows for grower observation and input in the selection process. The micro-plot system potentially allows for hundreds of cooperator planted trials consisting of the same entries to be planted within the limited planting window. Using standard two row replicated trials it is common for companies to plant only two or three locations in any given maturity zone. The ability to evaluate cultivars over multiple locations greatly increases the efficiency and accuracy of cultivar selection and segregation of the genetic by environment interaction (i.e. the ability of a variety to perform within a specific growing region or environment). Testing over more locations sooner results in less time to get the test seed to market and a higher commercial success rate.

The disclosed micro-plot system increases the efficiency of the testing program and allows for increased testing locations utilizing a similar labor force as a commercial testing program. It is often difficult to acquire labor for seed preparation prior to spring planting. The micro-plot system eliminates the requirement of counting and packaging seed prior to planting commercial trials. The process is automated with a unique pick and place robot in a seed laboratory environment to generate highly accurate seed tapes. The automation of this process allows for one person to assemble large numbers of trials in minimal time. The micro-plot system also eiiminates the need for shortening and thinning rows within plots which is required using the conventional system. Reallocating labor in the testing program allows for increased efficiency within testing programs.

The ability to easily adjust trial design and the opportunity to basically pre-plant the trial in a lab environment allows for sophisticated and elaborate research trial layouts. For example seed spacing/plant population studies are easily accomplished on a cultivar or bio-tech event.

The micro-plot system allows the trials to be prepared in a controlled laboratory environment up to the soil insertion under tight supervision. This is an important consideration when testing unregistered genetically engineered crops. The area available for this type of testing is regulated and limited. By using micro-plot testing many more new products can be tested and advanced, allowing more rapid commercial introduction of the crop. The micro-plot system can also be controlled to induce environmental stress for the purpose of selection, such as covering large areas to mimic drought conditions. Micro-plots would allow for much smaller enclosures and more varieties tested.

Accurate seed spacing and the ability to have multiple sites of a specific trial allow for the reduction of plant number per plot to produce statistically valid data (see the Examples). Plant entries are arranged using statistical trial designs. Uniform seed spacing and small plot size allow for collection of data under ideal conditions reducing the variation within the trial. A reduction in plot size reduces the overall trial size and limits the amount of field variability, thus reducing the need for replicating entries at any given location. Replication of data is done by planting multiple locations of the same entry list and combining the data for statistical analysis. A systematic check entry is included at the beginning of the trial arrangement, within the trial and again at the end of the trial. This check allows for a measure of the amount of variability across the plot.

The ease and expense of the micro-plot system also allows breeding/selection programs to evaluate many new cultivars without requiring large quantities of seed.
Currently, trials are restricted in the number of entries that can be tested due to the limitations of enough area for trials or the quantity of seed available for testing. An entry list for a specific trial is determined first on the theoretical merit of the hybrid and secondly by the amount of seed available. It is not uncommon to require 400 to 3000 kernels of an entry to participate in multiple location trials. Breeders must make the same parental cross (this creates a new cultivar) many times to collect enough seed for conventional testing. With the micro-plot system, plots are much smaller than conventional systems and require very small quantities of seed.
For example, the system requires only a few seeds per plot per location. Smaller requirements of seed for a particular entry to be included within a trial allows breeders to create more experimental cultivars for screening, thereby increasing the probability of identifying a superior variety in the testing/screening program. Seed that would otherwise go into the testing cycle could be used to increase the seed available for seed production or commercialization. Reallocating resources and energy to create crosses and develop lines from bulking up seed quantities for testing could increase the efficiency and success of breeding and testing programs.
Enclosing the seeds in a tape and wrapping the tape on a spool greatly reduces the risk of un-intentional environmental exposure to seed of non-registered bio-tech events. The enclosed tape also reduces the risk of seed mixing or theft of the seed and ensures that the seed is positioned accurately when planted.

Prior to planting, seeds of each entry are placed in a predetermined order based upon the trial design requirements. The seeds are then robotically placed in a water-soluble film and rolled on a spool as shown in Figures 1 to 5. The tape/film provides a means of ensuring placement of the seeds in a precise manner ensuring results of the micro-plot trial. The robot 100 comprises an air actuated computer controlled device which is engineered to place seeds from specially designed containers 104 one seed 140 at a time in a specific order at specific spacing onto a water-soluble tape 112. The computer controlled robot 104 can be programmed to place the seed 140 onto a tape 112 to reflect the research trial design and allows for precise placement and complicated trial designs without the opportunity of human error or fatigue. This allows for the manufacturing of many multiples of each trial design facilitating wide scale testing at multiple locations.

A supply spool 110 of water-soluble tape 112 is attached to the spool holder and the tape 112 is threaded through the machine 100. A digital controller 170 is programmed for the number of seeds per plot, seed spacing on tape and the number of plots per tape based upon the micro-plot trial design, a secondary controller 172 may be used to control the robot movements. Seed of different cultivars are stored in containers 104 placed on a rotating table 102 at identified locations 103. Each container contains a specific seed or cultivar for selection by the robot via one or more probes 105a and 105b for placement in the tape 112.
Container locations on the table 102 are identified for ensuring correct placement of the seed container 104. Not all spaces 103 on the table 102 may be utilized for holding seeds if not required. The containers 104 for the seeds may be of various shapes such as oval, rectangular or square based upon the configuration and operation of the robot 100. The containers 104 or receptacles may form part of the table or may be removable containers that are place on the table. The containers may also have a concave shape to aid in seed extraction. The rotating table requires the probes to only be moved in one direction horizontally in addition to vertically. However, the table 102 may be of varying shapes and the selection probes may move in 3 coordinate space (x, y, z) to accommodate the table shape thus not requiring movement of the table itself.

As shown in Figure 1, the circular table 102 is rotated so that the appropriate seed container 104 is placed under probes 105a or 105b mounted on frame 109 to extract seeds from the appropriate container 104. The probes 105a and 105b are actuated by a respective air piston 106a and 106b in the vertical direction and by 107a and 107b in the horizontal direction. The movement and actuation of the probes can be controlled by a secondary processor 172. The air piston 106a and 106b drives the associated vacuum probe 105a and 105b into the seed container 104. Vacuum suction draws a seed onto the probe end, a digital negative vacuum pressure sensor detects the presence of a seed and will either trigger the continuation of the process or will cause the plunger to be deployed back into the seed container until full negative pressure (a seed) is achieved. The vacuum holding the seed is turned off dropping the seed 140 through the funnel 120 and onto the tape 112 which rolls into a trough as a result of the tape closure system. The movement of the probes 105a and 105b may alternatively be controlled by motors rather than pistons.

Referring to Figure 5, the operation of the tape closure system encapsulates the seeds within the water soluble film/tape. Water container 122 holds water applied to the film to encapsulate seeds. An air-brush 124 provides a fine water mist using controlled pulses of air pressure to deliver water from the water container to moisten the film sufficiently to adhere to itself. A pulse drive motor (not shown) rotates the attached seed spool 160, sufficiently to advance the tape 112 the appropriate pre-determined amount to ensure the correct seed 140 spacing on the tape 112. The digital sensor 200 or encoder above a roller 202 measures the linear motion of the tape and controls the pulse drive motor.

As shown in Figure 2, the seed 140 is dropped in to the tape 112 at a predefined order and location along the tape length. The tape closure system, forms a trough or "V" shape of the tape 112 is formed by two pairs of guides 114 to receive the seeds as shown in Figure 2a. A fine mist of water from reservoir 122 can then be provided by an air brush 124 to aid in sealing the tape 112. The tape, being slightly moist from the steam adheres to itself ensuring security. The tape is then pressed by primary rollers 130a to encapsulate the seeds as shown in Figure 2b.
Secondary rollers 130b and tertiary rollers 130c add further pressure to the film to adhere the two sides of the "V" together. As shown in Figure 2c and 2d seeds are then individually encapsulated at defined intervals. The tape 112 is guided on the spool by a pulse controlled piston with pulleys 180. A heat source 190 ensure that excess moisture is removed to mitigate any possible degradation or adherence of the tape to itself prior to planting. The controlled motion of the tape through the robot aids in the even distribution of the tape on the spool and ensures a snag free unwinding of the spool 160 when planting the tape 112. A small blast of talc powder is applied to the tape 112 and spool 160, by the talc powder applicator 162 from reservoir 164, with each rotation of the spool 160. The spool may be enclosed 166 to ensure containment of the talc. This is done to ensure easy removal of the tape from the spool by absorbing any free moisture that may be present from the steam delivery system. Once the robot 100 has completed its cycle the spool 160 is removed, an empty spool is put into position and the cycle repeats itself.

After each seed 140 placement in the tape 112, it is advanced through a predefined distance for placement of the next seed. If the next seed is to be extracted from a different container, the table 102 is rotated by a motor 168 to move the desired seed container 104 to a location for the probe 105a or 105b to be moved in to position for extraction of the desired seed.

The water soluble tape used dissolves within minutes of being planted in the soil ensure precise seed placement with in the plot. A systematic check entry (control plot) seed can be placed at the beginning of the trial, within the trial and again at the end of the trial. This check measures the amount of variability across the length of the trial based upon know characteristics of the check entry. Identification can also be added to the beginning or end of the tape such as an bar code or radio frequency identification device (RFID) to aid in identifying the particular tape at the planting stage.

Figure 6 shows a method for performing the micro-plot field trial system. A
location for the field trial is selected (step 602). The characteristics of the seeds can be used to define placement within the seed tape or the trial locations (step 604).
The characteristics may be based upon desired aspects to be analyzed such growth characteristics related to moisture requirements, spacing, light requirements, soil conditions, etc. The configuration of the seed tape is determined (step 606) based upon trial design conditions. For example, position and spacing of seeds may be randomized or position of different types of seeds may be randomized. One or more seed tapes are generated by the robot (step 608). Based upon the trial requirements, multiple tapes may be generated either with the same seed layout or with different configurations if randomization is required. The seed tape is planted (step 610) in selected location. Tapes of the same configuration may be planted all multiple locations improving the number of environments that data can be collected for the trial seeds.

Many seed tape planting devices are known in the art, however to utilize the seed tape, a modified seed tube is used to facilitate planting. This modified seed tube can be used with commercial planters such as for example John DeereT"", KinzeTM
and WhiteTM planters, and therefore provides an easy adaptation to existing equipment, eliminating the need to use specialized equipment. The planter is lowered into the soil and the tape is covered by soil behind the planting unit to hold it in place. The forward motion of the planter feeds the tape off the spool, down the tube and into the seed trench. The tape follows the identical path of a conventionally planted seed. Plots are identified by the larger plant spacing between entries compared to between plants. During the growing stages of the plans agronomic notes or observation data are collected taken on each plot prior to harvest (step 612).

Individual plots are then harvested (step 614) by a micro-plot harvester at the desired growth stage. The micro-plot harvester machine is compact enough to be transported in the back of a small truck and narrow enough to fit between the bordering rows of the micro-plot trial (approx 60 inches). The actual harvesting unit is attached to a commercial scale lawn tractor or equivalent with parallel linkage and a hydraulic cylinder tool provide adjustable harvest height. Once the plants from the plot are harvested. The plant is weighed and removed, after which the machine moves forward to repeat the process for each consecutive plot. In the case of corn an adjustable stripper plates strip the ear off the corn plant as the stalk is pulled down through. Gathering chains with rubber covered projections every 4 inches drag the husk covered ears up to the husking rollers. Counter rotating rubber covered rollers pinch off and remove the husk cover from the ears and drop the husks to the ground. A double chain drag conveyor elevate the ears up to the sheller. A rubber covered rotating shelling drum pull the ears down and through an ever decreasing concave space, removing the kernels from the ear. The shelled kernels fall through a grated screen and the cobs roll out onto the ground.
The shelled kernels are collected in a weighing pan, which is suspended from load cells. A moisture probe is present in the bottom of the weighing pan. Grain weight and grain moisture data are measured and recorded on the data logger and the weighing pan rotates and dumps the corn on the ground. The harvester advances down the row and harvests the next plot. Although corn has been described any tape of plant or grain may be harvested. The harvested plants (step 616) analyzed such as by weight or moisture measurements. This data is statistically analyzed to calculate the final yield of each entry within the trial.

Figure 7 shows a method of generating a seed tape for the micro-plot trial system.
The tape configuration is determined either by an automated process or by a pilot designer (step 702). The tape configuration is based upon seed characteristics and trial requirements. The configuration may be received in computer readable code (step 704) or programmed at an input device at the robot for controlling computer processors 170 and 172 for operation of the robot. The particular seed and the position within the tape is defined in the input process. In commencing creation of the seed tape, the defined seed is selected from the respective container by a seed probe (step 706) as identified in the programming code. The table is turned so that the container that is to be accessed by the probe is in position under the travel path of the probe. Position sensor 204 on the table 102 enable accurate monitoring on table position and can rotate the table to the appropriate container position by measuring rotation distance to a predefined position. The probe 106a or 106b extracts the seed 140 from the appropriate container 104 using vacuum suction at the tip of the probe. The extracted seed is then placed in the tape 112 (step 708).
The tape is advanced and is sealed (step 710) and spooled onto a spool 160. If the seed selection of the particular seed is complete (YES at step 712) and the tape is complete (YES at step 714) identification can be added or inserted to the tape (step 716) if required. The tape spool is then completed (step 718) and ready for planting.
If additional seeds of the same type are to be placed on the tape (NO at step 712) then the next seed is extracted from the container (step 706). If different seed is to be placed in the tape (NO at step 714) the appropriate seed container is selected at step 704 by rotation of the table or movement of the probes. At the completion of the method the tape spool can be removed from the robot and provided for planting.
The micro-plot system is an alternative to conventional replicated yield testing. The system provides the opportunity to plant replicated trials in multiple locations with commercial equipment. The system has the opportunity to increase the efficiency and accuracy of cultivar development and screening programs without the capital investment currently required. The ease and expense of the system allow seed companies to screen and evaluate large numbers of new hybrids over many locations, which can increase the productivity of their program.

The micro-plot system has unlimited applications. The micro-plot system has been evaluated as a replacement for standard conventional corn hybrid yield trials.
This testing system can be applied to a wide range of crops and can be used for plant population, herbicide resistance, tillage and nutrient studies to name a few.

The following examples illustrate the improvement of the micro-plot trial system or conventional trial system.

EXAMPLE 1: Corn Hybrid Performance in a Micro-plot Compared to a Traditional Hybrid Corn Performance Trial Plot This example shows whether corn hybrid performance is relative for yield and moisture in a micro-plot compared to the traditional hybrid corn performance trial plot.

Materials and Methods Seventeen corn hybrids that were entered in table 5 of the Ontario Corn Committee Performance trials and 13 corn hybrids that were entered in table 6 of the Ontario Corn Committee Performance trials. These performance trials use commercial research methods to evaluate parameters of corn hybrids and chosen entries (corn hybrids) from each table were compared to the micro-plot system.

Trials were planted using a 4 row John DeereTM 7000 Maxi-Merge planter. The performance trial plots were planted in 76cm (30 inch) rows. Each plot was 2 rows wide and 5.8m long (19 feet long). 55 kernels per row were planted using a traditional cone type seeder and thinned to 33 kernels per row resulting in a desired plant population of 74,000 plants per hectare (pph) (30,000 plants per acre (ppa)).
The performance trials were analyzed as a randomized complete block design.
The micro-plots were planted using a seed tape planting technique. A micro-plot consisted of 9 kernels of the hybrid (entry) and one purple marker to indicate the end of a plot. All plots were equally spaced in a continuous row on fiberglass drywall tape to represent the final population of the performance trials (74,000 pph).
Modified seed tubes were installed on the planter to plant the tape. Three consecutive plants with uniform emergence within each plot were staked and marked for harvest. Seeds that did not emerge were planted with a purple marker.
The micro-plot was a one replication trial with the first, last and every fifth plot a check hybrid. The check hybrid was MZ540, but any suitable check may have been used. A moving means analysis was used to analyze the data. The performance trials were harvested by machine using electronic weighing and moisture equipment.
The tape trial was harvested by hand, shelled, weighed and moisture determined using a Dickey John GACTM II moisture meter. Grain yields were converted to 15.5% moisture. Plots were fertilized and maintained according to provincial recommendations.

Results Figure 8A compares the yield of 17 hybrids from 2 locations in table 5 of the OCC
performance trial to 2 locations from micro-plot trial. Figure 8B compares the moistures of the same 17 hybrids. Figure 9A compares the yield of 13 hybrids from 2 locations in table 6 of the OCC performance trial to 2 locations from micro-plot trial. Figure 9B compares the moistures of the same 13 hybrids.

The data from the OCC and micro-plot trials were combined and a split plot analysis was used to determine if there were significant differences between the two methods of performance testing. The reliability of such an analysis is questionable, however, there was no significant difference for yield and moisture in the table 5 or table 6 comparisons. The data suggests that the hybrid performance for yield and moisture in a micro-plot is relative to that in the OCC performance trials.

EXAMPLE 2: Corn Hybrid Performance in a Micro-plot Compared to a Traditional Hybrid Corn Performance Trial Plot This example determined whether corn hybrid performance is relative for yield and harvest moisture when comparing the technique of seed tape planting and cone planting in small plot hybrid performance trials.

Materials and Methods Four commercial corn hybrids MaizexTM MZ533, NK BrandT " N65-M7, MaizexTM
MZ540 and PrideTM K542 were used in this trial.

Corn was planted using a 4 row John DeereTM 7000 Maxi-Merge planter. All plots were planted in 76cm (30 inch) rows. In a first trial the plots were 2 rows wide and 19 ft. long and in second trial each plot was 4 rows wide and 5.8m (19 ft.) long (two center rows used for harvest). Plots were managed according to provincial recommendations. A 3 replication, split plot design was used with seeding technique the main plot and hybrid the split plot. To construct the seed tapes, 33 kernels/row (30,000 ppa) were equally spaced and wrapped in fiberglass drywall tape. Modified seed tubes were installed on the planter to plant the tape. The cone seeded plots were planted using a traditional 32 cell cone type seeder that is typically used to plant hybrid performance trials. The cone seeded plots were planted at 55 kernels/row and thinned to 33 kernels/row. The plots were machine harvested and yields converted to 15.5% moisture.

Results In both first and second trials there was no significant difference in yield between the technique of seed tape and cone seeded plots(conventional plots) (Table I).
While there was a significant difference for moisture between the two techniques in the first trial the combined data over the two trials shows no significant difference (Table 1). Likewise, there was no significant difference between seeding technique (main plot) and hybrid (sub plot) for both yield and moisture (Table 2).

Table 1: Yield and moisture for main plot (tape seeded and cone seeded).

Yield (bu/ac) %H20 CV % 7.2 2.6 LSD (.05) 8 0.5 Pr>F 0.4086 0.5424 Tape 230 21.9 Cone 228 22.0 Table 2: Two- way table for yield and moisture data Yield %H20 CV % 7.2 2.6 LSD (.05) 16 0.6 Pr> F 0.4315 0.1339 Tape 228 22.2 Cone 237 22.4 Tape 232 21.8 Cone 227 21.7 Tape 232 22.1 Cone 220 21.9 Tape 229 21.4 Cone 227 22.0 Based on data over 2 years seeding technique has no effect on the performance of a corn hybrid for the parameters of yield and moisture.

Example 3: Corn Hybrid Performance in a Micro-plot Compared to a Traditional Hybrid Corn Performance Trial Plot The objective of this example was to determine if the standardized plot size can be reduced in corn hybrid testing when evaluating advanced corn hybrids in performance trials.

Materials and Methods Five locations were planted in a split plot design randomized twice within each location. Plots were bordered on either side by one row of a common cultivar, MZ
540, to alleviate any border affects on the plots. Each main plot consisted of treatments varying in experimental unit size of two rows (5.2m long, 76cm apart), a one row plot (5.2m long) and a micro-plot (1.4m long). All planting populations were at 74 400 plants/ha (30 000 plants/ac). Within each main plot, sub plots consisted of five commercial corn hybrids (MZ 540, MZ 4422Bt, MZ 4433Bt, MZ 4655Bt, MZ
535). Recommended fertilizer rates were applied according to provincial recommended rates.

Two and one row plots were planted with a John Deere 7000 corn planter equipped with an AlmacoTM cone system. The micro-plot was planted using a modified seed tube which mounted on the John Deere 7000 planter unit.

Prior to planting the micro-plot, eight seeds of each entry were placed within Pval TM
film (CWS-10) at 0.2m spacing (74 400 plants/ha planting population) in the predetermined randomized order and rolled on a wooden spool for transport to the field for planting. The two and one row plots were over planted with 40 seeds per row and later thinned to 32 plants per row at the V6 growth stage. A 0.5m to 1 m gap was also left between plots of the two and one row plot to separate plots.

Prior to harvest, the number of plants per plot was recorded to determine the final population. From each plant within the micro-plot, individual ears were shelled and the grain weight measured. The grain samples were then combined from the eight plants and pooled grain moisture was measured. The remaining plots were harvested and shelled using a modified single ear corn sheller and the combined grain weight and moisture was measured. Harvest yield was determined using the following equation:

Grain Yield (bu/ac) = (((harvested grain (grams) x 1000g/kg x 2.2lbs/kg) /(47.32 /
(100 - grain moisture)*100)) / plot area (acres) Micro-plot harvest yield was calculated from the pooled weight of the eight plants and the pooled weight of the middle six plants. Harvest moisture was measured using a LabtronicsTM moisture meter.

The trial was analyzed as two separate experiments to determine if the outside plants within each micro-plot had any influence on the analysis. Therefore, separate analysis were performed using harvest data from eight plants and the middle six plants within each micro-plot.

Analysis of variance (ANOVA) was performed to determine significant differences among main plots and sub plots. Adjusted means were calculated for grain weight and moisture for the main plots and sub plots. Residuals were plotted against predicted values and blocks to visually inspect for deviations from homogeneity and independence. Outliers were determined by visually inspected plot residual values.
The Shapiro-Wilk statistic was calculated to test if the residuals confirmed normality.
Using the adjusted means of the hybrids by treatment, a Spearman Rank Correlation was performed to determine significant differences among the hybrid rank within each main plot. Means were also evaluated visually to determine differences in hybrid ranking.

All statistical procedures were performed using GLM, UNIVARIATE and PLOT
procedures of SASTM v.8.2 (SAS Institute, Cary, NC). The Type One error rate (a) was 0.05 for all analysis unless specified.

Results Yield analysis using 8 plants/micro-plot ANOVO suggests significant differences among locations, treatments and hybrids in the eight plants per micro-plot test. Blocks, treatment* block and hybrid*treatment were not significant, as shown below.

Analysis of variance results for grain yield using 8 plants.
Source df MS F-value P>F
Location 3 18868.66 65.64 0.0150 Block 1 240.72 0.84 0.4567 Treat 2 14679.21 51.07 0.0192 Treat*Block 2 287.46 0.33 0.7206 Hybrid 4 2289.72 2.62 0.0402 Hybrid*Treat 8 380.35 0.44 0.8969 R2 = 0.5620 CV = 12.09 The model was able to explain 56.2% of the total variation and had a CV of 12.09.
The Shipiro-Wilk statistic indicates the residual distribution was normal.
Visual observation of the residuals showed a slight tendency for the residuals to get larger from the two-row plot to the micro-plot but otherwise appeared independent and homogeneous.

Adjusted yield from the micro-plot was significantly different from the two-(P=0.0103) and one-row plot (P=0.0227) (Table 3, 4). The two-row and one-row plots were not significantly different (P=0.1644). The one-row and micro-plot was estimated to have 9.43bu/ac and 37.57bu/ac more yield than the two-row plot, respectively (Table 3, 4). Visual analysis of the adjusted hybrid harvest means by treatment also suggests that the micro-plot yields were significantly larger than the two row and one row plots (Figure 8).

Table 3: Adjusted mean yield (bu/ac) of the two row plot, one row plot and micro-plot of five hybrids over four locations.

Adjusted Mean Treatment (bu/ac) SE
Two row 227.25 4.809 One row 236.68 5.892 Micro 264.82 4.674 Table 4: Contrast and estimate of the adjusted treatment means over the four locations.

Parameter Estimate SE T Value Pr > Itl F Value Pr > F
two vs one -9.43 7.640 -1.23 0.2206 4.63 0.1644 two vs micro -37.57 6.706 -5.6 <.0001 95.4 0.0103 one vs micro -28.14 7.521 -3.74 0.0003 42.56 0.0227 The adjusted hybrid yield was ranked by treatment. The ranking was consistent between the 3 treatments. MZ 540 and MZ 4433Bt were not significantly different and had the largest yields within all treatments. All hybrids within the two-row treatment were not significantly different. Significant differences did occur between hybrids in the one row and micro-plots. There were no significant Spearman rank correlation between the ranking of the two-row plot and both the one row plot (P =
0.1041) and the micro-plot (P = 0.1881).

Table 5: Adjusted yield (bu/ac) and standard error ranked in ascending order of the five hybrids by treatment.

Two Row Plot One Row Plot Micro-plot Mean Mean Mean Yield Yield Yield Hybrid (bu/ac) SE Hybrid (bu/ac) SE Hybrid (bu/ac) SE
MZ535 219.47a 11.217 MZ4655Bt 221.66c 12.304 MZ4655Bt 248.39b 10.451 MZ4655Bt 224.48a 10.451 MZ535 228.09bc 11.601 MZ4422Bt 261.19b 10.451 MZ4422Bt 226.67a 11.218 MZ4422Bt 228.92bc 12.304 MZ535 263.88ab 10.451 MZ540 229.82a 10.451 MZ4433Bt 249.62ab 12.304 MZ540 265.92ab 10.451 MZ4433Bt 235.82a 10.451 MZ540 255.12a 12.304 MZ4433Bt 284.73a 10.451 Yield analysis using 6 plants/micro-plot When analyzing the data from 6 plants within the micro-plot there were significant differences among locations (P=0.0152) and treatments (P=0.0213). There were no significant differences among blocks, treatment*block, hybrids and hybrid*treatments, as shown below.

Analysis of variance results for grain yield using 6 plants.

Source df MS F-value P>F
Location 3 19409.70 64.84 0.0152 Block 1 779.59 2.60 0.2479 Treat 2 13770.37 46.00 0.0213 Treat*Block 2 299.36 15.17 <0.0001 Hybrid 4 2010.76 2.22 0.0737 Hybrid*Treat 8 382.75 0.42 0.9052 R2 = 0.5511 CV = 12.34 The model was able to explain 55.1% of the total variation and had a CV of 12.34.
The Shipiro-Wilk statistic indicates the residual distribution was normal.
Visual observation of the residuals showed a slight tendency for the residuals to get larger from two-row plot to the micro-plot but otherwise they appeared independent and homogeneous.

Adjusted yield from the micro-plot was significantly different from the two (P=0.01 14) and one row plot (P=0.0253) (Table 6, 7). The two row and one row plot were not significantly different (P=0.1737). The one row and micro-plot was estimated to have 9.28bu/ac and 36.42bu/ac more yield than the two row plot, respectively (Table 6, 7). Visual analysis of the adjusted hybrid harvest means by treatment also suggests that the micro-plot yields were significantly larger than the two row and one row plots (Figure 9).

Table 6: Adjusted mean yield (bu/ac) of the two row plot, one row plot and micro-plot of five hybrids over four locations.

Treatment Adjusted Mean SE
(bu/ac) Two row 227.27 4.901 One row 236.55 6.005 Micro 263.69 4.763 Table 7: Contrast and estimate of the adjusted treatment means over the four locations.

Parameter Estimate SE T Value Pr > itl F Value Pr > F
two vs one -9.28 7.786 -1.19 0.2366 4.31 0.1737 two vs micro -36.42 6.834 -5.33 <.0001 86.10 0.0114 one vs micro -27.14 7.665 -3.54 0.0006 38.02 0.0253 The adjusted hybrid yield was ranked by treatment. The ranking was consistent between the 3 treatments. MZ 540 and MZ 4433Bt were not significantly different in all treatments. All hybrids within the two row treatment were not significantly different. Significant differences did occur between hybrids in the one row and micro-plots. There was more similarity between significant hybrids within the two row plot and the micro-plot than the two row plot and the one row plot. There were no significant Spearman rank correlation between the ranking of the two row plot and both the one row plot (P = 0.1041) and the micro-plot (P = 0.5046).

Table 8: Adjusted yield (bu/ac) and standard error ranked in ascending order of the five hybrids by treatment.

Two Row Plot One Row Plot Micro-plot Mean Mean Mean Yield Yield Yield Hybrid (bu/ac) SE Hybrid (bu/ac) SE Hybrid (bu/ac) SE
MZ535 219.54a 11.432 MZ4655Bt 221.54c 12.540 MZ4655Bt 246.87b 10.651 MZ4655Bt 224.48a 10.651 MZ535 227.90bc 11.823 MZ4422Bt 263.19ab 10.651 MZ4422Bt 226.67a 11.433 MZ4422Bt 228.80bc 12.540 MZ540 264.09ab 10.651 MZ540 229.82a 10.651 MZ4433Bt 249.50ab 12.540 MZ535 264.88ab 10.651 MZ4433Bt 235.82a 10.651 MZ540a 255.00a 12.540 MZ4433Bt 279.41a 10.651 Moisture analysis using 8 plants/micro-plot When analyzing the moisture data, there were significant differences among locations (P=0.0374) and hybrids (P=<0.0001). There were no significant differences among blocks, treatments, treatment*block and hybrid*treatment , as shown below:

Analysis of variance results for moisture.

Source df MS F- P>F
value Location 3 135.95 25.90 0.0374 Block 1 0.62 0.12 0.7635 Treatment 2 6.44 1.23 0.4490 Treatment*Block 2 5.25 1.31 0.2741 Hybrid 4 30.95 7.75 <0.0001 Hybrid*Treat 8 2.54 0.64 0.7456 R2 = 0.6272 CV= 9.20 The model was able to explain 62.7% of the total variation and had a CV of 9.2. The Shipiro-Wilk statistic indicates the residual distribution was normal (P=0.4741).
Visual observation of the residuals showed them to be independent and homogeneous. The micro-plot did have higher moistures than the one and two row treatments but this was not significant (Table 9, 10 and Figure 12).

Table 9: Mean adjusted grain moisture (%) by treatment of five hybrids planted in a split plot design over four locations.

Treatment Adjusted SE
Moisture N

Two Row 21.66 0.325 One Row 21.32 0.398 Micro 22.20 0.316 Table 10: Estimate and contrast of the treatment means of five hybrids planted in a split plot design over four locations.

Parameter Estimate SE T Value Pr > itl two vs one 0.34 0.517 0.65 0.5168 two vs micro -0.54 0.453 -1.19 0.2366 one vs micro -0.88 0.509 -1.72 0.0883 The increased yield from the micro-plot in comparison to two and one row plots occurred in both the 8 and 6 plant micro-plot. Higher yields were not unexpected since corn plants determine yield at a very early stage. Corn plants have been shown to differentiate into hierarchies (dominate plants) at very early stages (V4) which is maximized until flowering and remains constant until senescence. Corn yield is also determined at the 4-6 leaf stage of growth. Both the two and one row plots were over-planted and were thinned at the V6 stage once the growing point had emerged from the ground. Up to this point in growth, seedlings have determined the competition around them and have altered their growth habits accordingly. The increased competition may have influenced the plants to allocate more resources to vegetative growth at a time when reproductive growth is determined. The micro-plot treatment was not thinned and the competition between plants was theoretically identical. Within commercial fields, the intra specific competition pressure from neighboring plants may be larger than in our micro-plots due to the added variation exposed to the corn on larger scales. Seeding depth, plant spacing, nutrient availability and soil type are a few factors which can create variability influencing plant growth.

Comparison between the 8 and 6 plant micro-plot determined that the 8 plant plot accounted for 1% more variation and had a slightly lower CV. These results may be explained by the decrease in the plant number sampled. Data from missing plants from the plot it would have a larger effect on the micro-plots of the smaller sample set per experimental unit causing increased variability in the trial. In a similar experiment, plant height and plot population were significant covariates and they were able to account for more variation within the experiment.

There is little difference when comparing the ranking of yield results by hybrid from the 8 and 6 plants sampled from the micro-plot results compared to the one and two row plots. Over all the treatments, MZ 540 and MZ 4433Bt were at the top of the ranking and MZ 4655Bt was close to the bottom. This reflects the performance of these hybrids in commercial production. MZ 540 was not ranked in the top two positions in both the micro-plot analyzed as 8 and 6 plants however it was not significantly different from the top ranked hybrid. This may be due to this hybrids plant short plant height compared to the other hybrids. Measuring plant height may account for differences in height and may more accurately reflect the hybrids full potential. By eliminating the plants on either side of the micro-plot a more consistent ranking was obtained in comparison to the two row plot.

Harvest moisture was not significant among the treatments however there were significant differences among hybrids. Differences among hybrids were expected due to the range in maturities used in the experiment. The rate of grain drying is more a function of plant phenotype and the weather at harvest.

This example shows that the micro-plots with uniform spacing significantly differed from the two and one row plots however, the ranking and trend of hybrid performance was consistent over all treatments. Sampling 8 or 6 plants within the micro-plot did not differ considerably. The 8 plant plot did account for more variation and a slightly lower CV. However, by eliminating the border rows on either end of the plot did result in more consistent hybrid ranking when compared to the two row plot. The moisture between hybrids did not differ between treatments.

Example 4: Micro-plot Analysis over Multiple Locations The objective of this example was to determine if the standardized plot size can be reduced in corn hybrid testing when evaluating advanced corn hybrids in performance trials on a multiple location testing regime.

Materials and Methods In this example, twelve locations were planted in a modified randomized complete design replicated once per location. Plots were bordered on either side by a random hybrid chosen by the producer (often the hybrid bordering the plot was the hybrid planted in the entire field). A border of MZ 4422Bt was included on either end of the plot and as the control hybrid (check hybrid). Each plot consisted of 20 hybrids randomized across the location. Within each location, a systematic control hybrid was included after every 4 plots to account for any variation that may exist across the trial area. Experimental unit size was one row consisting of 8 plants equally spaced at 0.2m spacing resulting in a planting population of 74 400 plants/ha (30 000 plants/ac). Hybrids were included in the trial based on commercial hybrids marketed in the geographic region and various precommercial hybrids deemed adapted to the area.

Agronomic information was collected on all the locations and included parameters such as planting date, harvest date, soil type, previous crop, fertility, herbicide used.
Prior to planting the micro-plots, eight seeds of each entry were placed within Pval film (CWS-10) at 0.2m spacing (74 400 plants/ha planting population) in the predetermined randomized order and rolled on a wooden spool for transport to the field for planting. Planting was executed using an adaptor specifically designed to plant such trails.

The parameter of plant height was measured at the V6, V10 and R2 from the ground to the emerged collar. Plant height at the R2 stage was taken to the flag leaf collar.
Prior to harvest, the number of plants per plot was recorded to determine the final population. Three locations were discarded at harvest due to poor plant stands.
Each micro-plot was harvested on a by plant basis. Grain was collected from all the ears from individual plants, grain shelled and the grain weight measured. The grain samples were then combined from the eight plants and pooled grain moisture was measured. Harvest yield was determined using the following equation:

Grain Yield (bu/ac) = (((harvested grain (grams) x 1000g/kg x 2.21bs/kg) /
(47.32 / (100 - grain moisture)*100)) / plot area (acres) Micro-plot harvest yield was calculated from the pooled weight of the eight plants and the pooled weight of the middle six plants. Harvest moisture was measured using a Labtronics moisture meter.

The trial was analyzed as two separate experiments to determine if the outside plants within each micro-plot had any influence on the analysis. Therefore, separate analysis were performed using harvest data from eight plants and the middle six plants within each micro-plot.

Analysis of variance (ANOVA) was performed to determine significant differences among entries with regard to grain yield and plant height. Adjusted means were calculated for grain weight, moisture and plant height. Residuals were plotted against predicted values and blocks to visually inspect for deviations from homogeneity and independence. Outliers were determined by visually inspected plot residual values. The Shapiro-Wilk statistic was calculated to test if the residuals confirmed normality. Using the adjusted means of the hybrids, a Spearman Rank Correlation was performed to determine significant differences among the micro-plot trial and the data set of the Ontario Corn Committee trials which use a standard two row experimental unit.

Agronomic data was visually evaluated to determine if the locations had commonalities among them. If commonalities existed, the data was separated into those groups and the common factor was analyzed.

All statistical procedures were performed using GLM, UNIVARIATE and PLOT
procedures of SAS v.8.2 (SAS Institute, Cary, NC). The Type One error rate (a) was 0.05 for all analysis unless specified.

Results By plant analysis of grain yield ANOVA suggests significant differences among entries, blocks and the covariate parameters of plant height and plot population. There were no significant differences among plant position or entry x plant position. The Shipiro-Wilk statistic indicates the residual distribution was not normal (P=0.0001). Visual observation of the residuals showed them to be independent and homogeneous. The adjusted means of plant position (Table 11) suggest that there are no significant differences in plant position. Significant differences among plant height of the entries shown by the difference of 45cm between the tallest hybrid and the shortest hybrid (Table 12).Table 11: ANOVA analysis of the micro-plots by plant position within the plot measured in grams per plant.

Std.
Pltnum Estimate Error 1 220.2 13.20 2 213.7 13.20 3 213.6 13.20 4 216.5 13.22 5 214.7 13.21 6 215.9 13.20 7 218.8 13.20 8 214.1 13.22 Table 12: ANOVA of the micro-plot entries by yield per plant on 9 locations.

Entry Adjusted Yield by Std. Error Plant (grams) EX 4550Bt 241.2 14.16 MZ 4422Bt 238.9 14.13 MZ 4433Bt 232.4 14.13 MZ 4422Bt 228.8 14.04 MZ 4422Bt 225.7 14.05 MZ 4422Bt 224.6 14.02 MZ 4422Bt 224.3 14.07 MZ 4422Bt 222.9 14.00 MZ 540 222.7 14.06 EX
5469Bt/RR 219.5 14.06 EX 5462Bt 219.2 14.04 EX 45-66RR 219.0 14.06 EX 4161Bt 217.4 14.01 EX 54-55RR 215.3 14.20 EX 5466HX 214.4 14.08 EX 4565 210.8 14.21 MZ 4655Bt 210.7 14.06 MZ 4422Bt 209.9 14.19 EX 54-71 RR 209.3 14.09 MZ 535 209.1 14.16 MZ 535HX 207.8 14.11 EX 4561 HX 200.5 14.13 MZ 366 199.9 14.12 DKC 50-18 198.9 14.03 MZ 36-66RR 195.3 14.06 EX 4562HX 195.2 14.25 Since plant position was not a significant factor, further analysis was performed using a pooled value for per plot yield at both 6 and 8 plants per plot.

Yield analysis using 8 plants/plot ANOVA suggests significant differences among entries, blocks and plant population.
There were no significant differences among the covariate entry plant height.
The model was able to explain 79.0% of the total variation and had a CV of 9.84.
The Shipiro-Wilk statistic indicates the residual distribution was normal (P=0.9815).
Visual observation of the residuals showed them to be independent and homogeneous. The adjusted means by entry are shown in Table 13.

Table 13: ANOVA adjusted means of grain yield pooled from 8 plants of the micro-plot over 9 locations.

Adjusted Std. Yield Entry Grain Yield Error (bu/ac) (g/plot) EX 4550Bt 1657.9 51.71 264.7 MZ 4422Bt 1637.7 51.03 261.5 EX 5469Bt/RR 1621.7 51.39 255.7 EX 54-71 RR 1611.7 51.19 249.5 MZ 540 1608.3 51.03 244.9 EX 5466HX 1599.0 51.19 251.2 MZ 443313t 1595.3 51.61 254.1 EX 4161 Bt 1581.8 51.55 255.8 MZ 535 1569.7 51.37 251.0 EX 546213t 1553.9 51.16 243.6 MZ 4655Bt 1544.6 51.41 245.3 EX 4565 1544.4 54.34 251.2 MZ 535HX 1530.3 51.25 242.3 EX 54-55RR 1508.5 52.65 238.4 EX 45-66RR 1501.6 51.43 238.8 EX 4562HX 1468.7 51.53 233.7 EX 4561 HX 1444.6 51.34 232.5 MZ 366 1359.9 51.43 217.2 MZ 36-66RR 1350.9 51.41 218.0 DKC 50-18 1331.0 51.93 216.3 Visual analysis of the hybrid ranking using the adjusted means of the pooled results do not significantly differ from the hybrid ranking results from the by plant analysis.
Yield analysis using 6 plants/plot ANOVA suggests significant differences among entries, blocks and plant population.
There were no significant differences among plant height. The model was able to explain 75.7.0% of the total variation and had a CV of 10.79. The Shipiro-Wilk statistic indicates the residual distribution was normal (P=0.5386). Visual observation of the residuals showed them to be independent and homogeneous.
The adjusted means by entry are shown in table 14.

Table 14: ANOVA adjusted means of grain yield pooled from 6 plants of the micro-plot over 9 locations within southwestern Ontario.

Adjusted Grain Yield Std.
Entry (g/plot) Error Yield (bu/ac) EX 4550Bt 1449.8 49.68 308.6 EX 5469Bt/RR 1441.5 49.40 303.0 MZ 442213t 1440.4 49.54 306.7 MZ 540 1437.0 49.26 291.8 EX 54-71 RR 1436.2 49.53 296.4 EX 5466HX 1435.1 49.33 300.7 MZ 443313t 1418.2 49.96 301.2 EX 4161 Bt 1403.0 49.50 302.6 MZ 465513t 1393.5 49.37 295.1 MZ 535 1359.1 49.30 289.8 EX 4565 1342.0 52.61 291.1 EX 546213t 1339.2 49.30 279.9 EX 45-66RR 1333.1 49.36 282.7 MZ 535HX 1325.4 49.97 279.8 EX 54-55RR 1309.7 50.20 276.0 EX 4562HX 1294.4 50.00 274.6 EX 4561 HX 1275.0 49.30 273.6 MZ 366 1201.8 49.51 256.0 MZ 36-66RR 1190.7 49.77 256.1 DKC 50-18 1165.8 50.01 252.6 Hybrid ranking is similar to the results obtained from the analysis using 8 plants/plot.
Grain moisture analysis using 8 seeds/plot ANOVA suggests significant differences among entries, blocks and plant population.
There were no significant differences among plant height. The model was able to explain 81.7.0% of the total variation and had a CV of 5.79. The Shipiro-Wilk statistic indicates the residual distribution was not normal (P=0.0001).
Visual observation of the residuals showed them to be independent and homogeneous.

Table 15: ANOVA adjusted means of grain moisture from a pooled sample of 8 plants per plot over 9 locations.

Hybrid Maturity Adjusted Entry (CHU) Moisture (%) Std. Error MZ 540 3300 24.9 0.42 EX 54-71 RR 3250 23.7 0.42 EX 5462Bt 3250 22.7 0.42 EX 5466HX 3250 22.5 0.42 EX 5469Bt/RR 3250 22.2 0.42 EX 54-55RR 3250 22.1 0.43 MZ 535HX 3250 21.9 0.42 MZ 4655Bt 3150 21.7 0.42 EX 45-66RR 3150 21.6 0.42 EX 4562HX 3100 21.5 0.42 MZ 4433Bt 3100 21.4 0.43 EX 4550Bt 3100 21.3 0.42 MZ 4422Bt 3100 21.2 0.42 MZ 366 3050 21.2 0.42 MZ 535 3250 21.1 0.42 EX 4561 HX 3100 20.6 0.42 MZ 36-66RR 3050 20.4 0.42 EX 4161 Bt 3050 20.2 0.42 DKC 50-18 3050 19.8 0.43 EX 4565 3100 19.8 0.45 Visual analysis of the hybrid ranking by moisture indicate that the moisture of the hybrids expectedly correspond to hybrid maturity. From Table 15, the higher the grain moisture the longer the hybrid maturity. This relationship is common and generally accepted by industry.

Comparison between the Micro-plot to OCC Trials Using the adjusted means from 8 plants per micro-plot there appears to be a strong relationship between the results from both testing procedures. Visual analysis of the hybrid ranking suggests hybrids that have high yield results in the OCC trials also have high yield results in the micro-plot trials. Those hybrids with low yield results appear also to be consistent among both testing procedures. The same correlation holds true for grain moisture. This is apparent in tables 16, 17, 18 and 19.

Table 16: Micro-plot data from 9 locations compared to the yield and moisture results from the Ontario Corn Committee trials of 2 locations planted in a replicated complete block design with 3 replications per location.

OCC Trials (Table 5) Micro-plot - 8 plants Hybrid bu/ac moist Hybrid bu/ac moist MZ 4433Bt 239.8 21.9 MZ 4433Bt 290.7 21.4 EX 4550Bt 235.3 22.0 EX 4161 Bt 290.0 20.2 MZ 4422Bt 230.9 22.1 EX 4550Bt 288.8 21.3 EX 4561 HX 228.7 20.6 MZ 442213t 288.0 21.2 MZ 465513t 226.4 22.3 MZ 465513t 272.5 21.7 EX 4562HX 222.0 21.4 EX 4562HX 264.8 21.5 EX 45-66RR 219.8 22.0 EX 45-66RR 263.5 21.6 EX 4161 Bt 217.6 21.2 EX 4561 HX 262.6 20.6 MZ 366 208.7 21.4 DKC 50-18 248.2 19.9 MZ 36-66RR 206.5 21.6 MZ 366 244.4 21.2 DKC 50-18 204.2 20.1 MZ 36-66RR 238.0 20.4 Table 17: Micro-plot data from 9 locations compared to the yield and moisture results from the Ontario Corn Committee trials of 2 locations planted in a replicated complete block design with 3 replications per location.

OCC Trials (Table 5) Micro-plot - 6 plants Hybrid bu/ac moist Hybrid bu/ac moist MZ 4433Bt 239.8 21.9 EX 4161 Bt 293.3 20.2287 EX 4550Bt 235.3 22.0 MZ 4433Bt 292.5 21.4169 MZ 4422Bt 230.9 22.1 EX 4550Bt 289.5 21.2905 EX 4561 HX 228.7 20.6 MZ 4422Bt 287.4 21.2461 MZ 4655Bt 226.4 22.3 EX 4561 HX 273.3 20.6412 EX 4562HX 222.0 21.4 EX 4562HX 272.4 21.512 EX 45-66RR 219.8 22.0 MZ 4655Bt 272.3 21.6773 EX 4161 Bt 217.6 21.2 EX 45-66RR 261.8 21.5773 MZ 366 208.7 21.4 DKC 50-18 247.9 19.869 MZ 36-66RR 206.5 21.6 MZ 366 240.9 21.235 DKC 50-18 204.2 20.1 MZ 36-66RR 238.5 20.444 Table 18: Micro-plot data from 9 location compared to the yield and moisture results from the Ontario Corn Committee trials of 6 locations planted in a replicated complete block design with 3 replications per location.

OCC Table 6 Micro-plot - 8 plants Hybrid bu/ac moist Hybrid bu/ac moist EX 5466HX 236.9 19.9 EX 5469Bt/RR 283.6 22.2 MZ 535 234.6 19.2 MZ 535 282.0 21.1 MZ 540 234.6 21.3 EX 5466HX 280.0 22.5 EX 4562HX 234.6 19.2 MZ 540 277.4 24.9 EX 546213t 234.6 19.5 MZ 535HX 273.1 21.9 MZ 535HX 232.3 19.7 EX 546213t 270.8 22.7 EX 54-55RR 223.1 20.7 EX 54-55RR 266.9 22.1 EX 5469Bt/RR 223.1 20.7 EX 4562HX 264.8 21.5 EX 45-66RR 202.4 19.9 EX 45-66RR 263.5 21.6 Table 19: Micro-plot data from 9 locations compared to the yield and moisture results from the Ontario Corn Committee trials of 3 locations planted in a replicated complete block design with 3 replications per location.

OCC Table 6 Micro-plot - 6 plants Hybrid bu/ac moist Hybrid bu/ac moist EX 5466HX 236.9 19.9 EX 5469Bt/RR 289.1 22.235 MZ 535 234.6 19.2 MZ 535 285.5 21.121 MZ 540 234.6 21.3 EX 5466HX 283.6 22.5016 EX 4562HX 234.6 19.2 MZ 540 277.7 24.8905 EX 5462Bt 234.6 19.5 EX 4562HX 272.4 21.512 MZ 535HX 232.3 19.7 EX 54-55RR 268.1 22.0585 EX 54-55RR 223.1 20.7 MZ 535HX 267.8 21.9169 EX 5469Bt/RR 223.1 20.7 EX 5462Bt 266.1 22.6995 EX 45-66RR 202.4 19.9 EX 45-66RR 261.8 21.5773 Data Segregation Visual analysis of the agronomic data collected about specific management characteristics indicate that soil type has two factors which may be partitioned to yield more information on individual hybrids (Table 20).

Table 20: Adjusted moisture, mean yield (bu/ac) from 8 plants/plot, mean yield (bu/ac) from 6 plants/plot, soil type, planting date, previous crop and tillage practice of the microplot locations.

Adjusted Mean Mean Yield Yield Planting Previous Tillage Location Moisture (8 Plants) (6 Plants) Soil Type Date Crop Used (%) (bu/ac) (bu/ac) Tilbury 19.4 238.8 236.2 clay loam 28-Apr corn conventional Melbourne - A 19.9 243.8 249.5 sandy loam 3-May corn minimum Blenhiem 19.6 259.5 258.5 clay loam 8-May soybeans no till Dresden 20.0 270.0 271.8 sandy loam 10-May wheat conventional PainCourt 23.5 284.9 288.4 clay loam 3-May wheat conventional Melbourne - B 21.9 289.4 292.1 clay loam 5-May alfalfa conventional Chatham 24.0 291.3 288.2 sandy loam 2-May soybeans conventional Thamesville 20.9 292.3 291.9 sandy loam 4-May corn conventional Tilbury 26.1 313.0 314.9 clay loam 31-May wheat conventional ANOVA using data from 8 plants / micro-plot suggests significant differences among soil type, soil*entry, plant population and the covariate plant height. The Shipiro-Wilk statistic indicates the residual distribution was normal (P=0.1691).
Visual observation of the residuals showed them to be independent and homogeneous.
ANOVA using data from 6 plants / micro-plot suggests significant differences among soil type, soil*entry, plant population. The covariate plant height was not significant.
The Shipiro-Wilk statistic indicates the residual distribution was normal (P=0.1396).
Visual observation of the residuals showed them to be independent and homogeneous.

Table 21: Adjusted yield (grams/plot) and moisture by soil type. Yield has also been expressed as bu/ac.

Plot Std. Yield Size Parameter Effect Type Estimate Error (bu/ac) 8 plants Yield/plant soil Clayloam 1684.1 b 32.96 267.0 8 plants Yield/plant soil Sandyloam 1411.4a 45.24 229.4 6 plants Yield/plant soil Clayloam 1455.5b 30.91 307.7 6 plants Yield/plant soil Sandyloam 1241.3a 40.95 269.0 8 plants Moisture soil clayloam 21.8b 0.37 8 plants Moisture soil sandyloam 19.8a 0.47 Adjusted yield and moisture are shown in Table 21 segregated by micro-plot size.
Clay soils tended to yield higher than sandy soil in both analysis using 6 and plants per micro-plot. The clay soil also had higher harvest moisture than sandy soils.

The significant interaction between soil types and entries suggests a significant genotype by environment interaction exists. While the trend for the entries was to have lower yield on the sandy loam soil some varieties actually performed better in these environments in comparison to the trial average (Figures 11 and 12).
Identifying genotype by environment interaction is important to position hybrids in environments were it has the highest yield potential.

FIG. 15 shows a digital controller 1500 computing environment for executing seed tape creation for controlling the robot apparatus in the form of computer readable code for execution as implemented by processors 170 and/or 172. The computer 102 comprises central processing unit (CPU) 1502 and associated memory 1510.
The CPU(s) may be a single processor or multiprocessor system or may be implemented by an application specific integrated circuit. In various computing environments, memory 1510 and storage 1540 can reside wholly on computer environment 1500, or they may be distributed between multiple computers.

Input devices 1530 such as a keyboard and mouse may be coupled to a bi-directional system bus. The keyboard and mouse are for introducing user input to a computer and communicating that user input to processor 1502 if required.
Computer 1502 may also include a communication interface 1508. Communication interface 1508 provides a two-way data communication coupiing via a network link to a network 1550 by wired or wireless connection or may provide an interface to other host devices by a direct radio frequency connection to enable retrieval of data or providing commands to remote robots 100. In any such implementation, communication interface 1508 sends and receives electrical, electromagnetic or optical signals which carry digital data streams representing various types of information. Display device 1520 is provided to facilitate programming and monitoring if required.

The CPU 1502 or similar device may be programmed in the manner of method steps, or may be executed by an electronic system which is provided with means for executing for operation of the classification and search engine. The storage device 1540 can be accessed through an input/output (I/O) interface and may include both fixed and removable media, such as magnetic, optical or magnetic optical storage systems, Random Access Memory (RAM), Read Only Memory (ROM) or any other available mass storage technology. The storage device or media may be programmed to provide such method steps for operation and control of robot 100 by CPU 1502. The storage device 1540 may also store operational information regarding the robot, such as error messages and operational and performance data.
The storage device 1540 may also contain data regarding seed cultivars to enable automated generation of tape configuration if required.

Memory 420 can provide code for operation and programming of the robot 100.
The tape configuration 1512, comprising a data file or executable code, is either entered by a user by an input device 1530 or retrieved from storage 1540. From the tape configuration 1512, commands for probe control 1514 and for controlling the tape 1516 are generated. The commands are the sent by a data interface to the appropriate devices for controlling the robot operation 100 to create the desired see tape.

It will be apparent to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the present disclosure as defined in the claims.

The embodiments described above are intended to be illustrative only. The scope of the invention is therefore intended to be limited solely by the scope of the appended claims.

Claims (27)

1. A method for performing micro-plot plant trials of a seed cultivar, the method comprising the steps of:

determining a seed arrangement for the seed cultivar for a seed tape using a statistical design to be tested in a plant trial;

creating the seed tape on a water soluble planting tape, wherein the seeds contained by the tape are placed using a predetermined quantity and spacing based upon the statistical design;

planting the seed tape in a micro-plot environment;
collecting observations on the resulting plants;
harvesting the plants; and analyze the collected plants;

wherein the spacing of the seeds within the seed tape is determined to eliminate the need to thin the plants once emerged from the seeds, reducing the quantity of the seeds and the trial area required for the micro-plot.

2. The method of claim 1 wherein the step of creating the seed tape is performed by an automated seed selection and encapsulation robot wherein the robot ensures exact placement and isolation of the seeds within the water soluble tape.

3. The method of claim 1 wherein the seed tape is less than 5 feet (1.52m) in length.

4. The method of claim 1 wherein the seed tape comprises 1 to 12 seeds of the seed cultivar.

5. A method of creating a seed tape using a robotic apparatus, the method comprising the steps of:

receiving seed tape configuration information wherein the tape configuration defines seed type and position of the seed within the seed tape based on seed characteristics of the seed cultivar to the tested, the seed tape configuration designed to eliminate the need to thin the plants once emerged from the seeds, reducing the quantity of the seeds and the trial area required for the micro-plot;

selecting a seed receptacle containing a particular seed cultivar from a plurality of seed receptacles;

extracting a seed from the selected seed receptacle using a probe having a vacuum tip, wherein the seed is picked up by the probe by a vacuum created at the tip of the probe;

placing the extracted seed into a water soluble tape, wherein the seed is dropped into the tape by removing the vacuum from the probe tip;
advancing the seed tape a predetermined distance based upon the received tape configuration; and encapsulating the seed in the tape.

6. The method of claim 5 wherein the step of encapsulating the seed in the tape further comprises:

forming the tape into a "V" shape configuration for receiving the seed;
applying a mist of water when the seed is placed in the tape;

folding the tape around the seed;
applying pressure to the seed tape;

7. The method of claim 6 wherein the step of encapsulating the seed in the tape further comprises:

applying heat to the seed tape to remove excess moisture; and applying talc to the tape prior to collection of the tape on a spool.

8. The method of claim 5 further comprising the step of adding identification to the tape.

9. The method of claim 8 wherein the identification is a bar code.

10. The method of claim 9 wherein the identification is an radio frequency identifier (RFID).

11. The method of claim 5 wherein the seed is a corn hybrid.

12. The method of claim 5 wherein the seed tape comprises 1 to 12 seeds of a cultivar.

13. The method of claim 5 wherein the seed tape is less than 5 feet (1.52m) in length.

14. The method of claim 5 further comprising the step of placing a marker seed at the beginning of the tape, within the tape, or at the end of the tape to provide a systematic check entry.

15. An automated apparatus for making plant seed tape comprising:

a digital controller programmed for the number of seeds and seed spacing;

a plurality of seed containers, each container contain seeds of a particular seed cultivar;

an arm with a vacuum probe to draw a seed from a selected container and deposit it on a water soluble tape;

a air-brush sprayer to deliver fine water mist moisten the water soluble tape to allow it to adhere to itself, and encasing the seed;

a closure system to capture the seed in place the water soluble tape;

a drive for advancing the tape in a pre-set amount to ensure the correct seed spacing on the tape;

one or more guides for guiding the tape onto a spool; and a talc delivery for applying a small blast of talc powder with each rotation of the spool to ensure easy removal of the tape from the spool.

16. The apparatus of claim 15 wherein a seed extraction mechanism is provided by one or more probes, each having a vacuum mechanism for individually extracting seeds from the plurality of containers.

17. The apparatus of claim 16 wherein the seed probes move by the digital controller actuating associated air pistons.

18. The apparatus of claim 17 wherein the plurality of containers are placed on a circular table, wherein the table is rotated to position a desired container underneath one or more probes by the digital controller.

19. The apparatus of claim 18 wherein the table position is determined by an encoder to determine the position of the table.

20. The apparatus of claim 15 wherein the closure system further comprises a pair of guides forming a trough or "V" shape for receiving the seeds.

21. The apparatus of claim 20 wherein the closure system further comprises one or more rollers for folding the tape together and enclosing the seeds.

22. The apparatus of claim 21 wherein the closure system further comprises a dryer for removing excess moisture.

23. The apparatus of claim 15 wherein the arm moves in two dimensions.

24. The apparatus of claim 15 wherein the arm moves in three dimensions and the seed containers are stationary.

25. The apparatus of claim 15 wherein the seed tape comprises 1 to 12 seeds of a cultivar.

26. The apparatus of claim 15 wherein the seed tape is less than 5 feet (1.52m) in length.

27. A computer readable medium containing instructions for creating a seed tape using a robotic apparatus, the instructions which when executed by a processor perform the steps of:

receiving seed tape configuration information wherein the tape configuration defines seed type and position of the seed within the seed tape and is based on seed characteristics to the tested, the seed tape configuration designed to eliminate the need to thin the plants once emerged from the seeds reducing the quantity of the seeds and the trial area required for the micro-plot;

selecting a seed receptacle from a plurality of seed receptacles;

extracting a seed from the selected seed receptacle using a probe having a vacuum tip, wherein the seed is picked up by the probe by a vacuum created at the tip of the probe;

placing the extracted seed into a water soluble tape, wherein the seed is dropped into the tape by removing the vacuum from the probe tip;
advancing the seed tape a predetermined distance based upon the received tape configuration; and encapsulate the seed in the tape.