WO2015162625A2 - Three way matrix sampling device for seeds and a method thereof - Google Patents

Abstract

The present invention relates to three way matrix sampler that facilitate in carring out DNA based genetic purity assessment of seed lot from crop plants using three way sampling method The sampler comprise of multiple long rectangular boxes which are arranged horizontally and vertically one over the other, providing space between the horizontal and vertical boxes called retainer. The horizontally arranged rectangular boxes used for row bulking and vertically arranged boxes for column bulking, whereas the retainer boxes are used for retaining seed parts for further analysis to trace out accurate contaminant. The arrangement of rectangular boxes give rise to grid like structure to the sampler that has a potential use low- cost, rapid and accurate identification of contaminants from the seeds to be tested.

Description

THREE WAY MATRIX SAMPLING DEVICE FOR SEEDS AND A METHOD THEREOF

FIELD OF THE INVENTION

The present invention relates to a sampl ing device, more particularly to a portable device that facilitates sorting of seeds based on three way sampling technique for DNA based genetic purity testing of seed lots of various varieties / hybrids at seed testing laboratories and identify the contaminants if any.

BACKG ROUND ART

Agriculture is the back bone of Indian economy, about 50% of the total workforce is employed in Agriculture. India ranks second worldwide in farm output. Agriculture and all ied sectors accounted for 14% of the GDP in 20 1 1 - 1 2 . Among all the inputs seeds are the most basic, critical and vital input for the sustained growth of,agricultural production. To a large extent, response of all other inputs depends on quality of seeds. It is estimated that the direct contribution of quality seed alone to the total production is about 1 5 - 20% depending upon the type of crop. The old scripture, Manu Smriti quotes "Subeejam Sukshetre Jayale Sampadyathe" i.e., Good seed in good soil yields abundantly. Seed quality has been treated as sacred, being an important factor in the improvement of agriculture and agrarian societies. The igveda, 2000 BC indicate the importance accorded to seed and the mother earth.5th century autilya Artha Shasthra, surapalas vrikshayurveda mentioned importance of seed and mentioned about seed treatments to ensure good germination.

Quality seeds are ones that are highly viable, genetically uniform and free from seed borne pathogen. Among various qualities of seeds like, germination, longevity etc., Genetic Purity (GP) of the seed lot is of top priority in order to have the fullest potential yield of the crop plants. Farm yield can be enhanced up to 10-15 % by using genetically pure good quality seeds. The genuineness of the variety/hybrid is one of the most important characteristics of good quality seed. Maintenance of high level of genetic purity in hybrid seed is a major challenge to exploit the moderate level of heterosis. For instance, it is estimated that for every I percent impurity in rice hybrid seed, the yield reduction is 100 kg per hectare. Moreover, with ever increasing number of seed varieties, the chances of admixture of seeds are high due to post harvest mishandling. Many a time seed distributors voluntarily contaminate the quality seed with poor quality seed for profit. The use of seeds with low genetic purity results in segregation of the traits, lower yields and genetic deterioration of varieties. Thus, it is important to maintain genetic purity to. provide best quality seed. Genetic purity (GP) is tested to verify any deviation from genuineness of the variety/hybrid during its multiplications. Genetic purity test is compulsory for seed certification of all different classes of seeds (breeder, foundation and certified) in India. As per the Indian Minimum Seed Certification Standard (IMSCS), 98 % genetic purity is an essential prerequisite for commercialization of certified seeds depending upon crops. However, 100 % genetic purity is mandatory for breeder seeds of all varieties and hybrids. The genetic purity during multiplication stage is prone to contamination due to the presence of pollen shedders and out crossing with foreign pollens etc., besides contamination by physical inter mixing. Seed certification officials visit every seed production field and certify the standing crop plants fit for commercial seed production after visually examining for isolation distances, presence of pollen shedders and off types plants etc., In order to ensure the quality of seeds, the seeds harvested from certified seed production field are processed and submitted for seed quality testing at seed certification centres in India.

Traditionally, apart from conducting germination and physical purity tests, seed testing officials carry out GROW OUT TEST (GOT) for assessing the genetic purity of the seed lot during subsequent season. In this method, 100 individual seeds are sown in the field along with four replications i.e, a total of 400 seeds and scored for their morphological description of respective CLiltivars all through the life stages. The estimated number of true to type cultivar is calculated out of 400 seeds and expressed as genetic purity percentage.

GOT has many disadvantages, for e.g., it is time consuming (takes one full growing season for completion), space demanding and often does not allow the unequivocal identification of genotypes. Moreover, it is subjective as several aspects of plant phenotype (morphology, yield, etc.) get influenced by environmental conditions. Further, there is a possibility that adverse climatic conditions (like heavy rain or wind) that can damage or destroy the crop and make it difficult for data collection.

Apart from field based GOT method, that uses morphological marker; laboratory based biochemical markers, molecular markers are available for genetic purity analysis. Among these, molecular markers (RAPD, SSR, RFLP, AFLP etc.,) are mostly employed with DNA fingerprinting technology and particularly SSR because of its co-dominant character, high polymorphism, and comparatively less technical complexity. Despite of all advantages, molecular Marker Assisted seed testing (MAT) is not popular in major seed production agencies or testing laboratories, because of non-availability of cultivar specific marker (primer) information, high cost, technical complexity and requirement of trained staff. The cost involved in estimating the GP using GOT is less expensive than molecular marker involved DNA fingerprinting method.

In order to reduce the cost involved in DNA based genetic purity analysis using molecular tools, two different DNA bulking strategies were attempted and have been disclosed in scientific reports.

Indian patent application 2270/CHE/20I 1 discloses DNA based genetic purity testing technique for commercial seed lot. The technique involves bulking single leaf bits from 50 seedlings which were analyzed through PCR involving SSR marker, RM 3630 primer, in 8 replications. Presence of extra alleles other than the allele 'specific' and 'unique' to the test variety is notified as offtype in one of the bulked sample of 50 seedlings. The extra allele confirm that at least one of the 50 seedlings tested is an off type. If one replication reveals different allele, it can be interpreted that the lot contains at least 2 per cent genetic contamination. The inference will be that "If two replication reveals a different allele the lot can be rejected" since the genetic contamination level

exceeds the permissible limit of 2 per cent. Though this technique reduced the cost of analysis through minimizing sample number (400 to only 8 samples), this application is silent on identifying the contaminant seed and is merely focussed on selecting and rejecting the seed lot. Moreover, the chance of misinterpretation of result is inevitable when the seed lot contain more than two contaminants per DNA bulk and it is merely focussed on selecting and rejecting the seed lot.

Nas et al. (2000) proposed a two dimensional DNA sampling strategy involving a 10 x 10 grow out matrix for assessment of genetic purity of rice hybrid using AFLP molecular marker. This strategy was originally developed for hybrid rice seed production and it can be used for pure line varieties of rice and other crops. In this strategy, 100 seeds were germinated in 10 row ^χ 10 column grow out matrix and two leaf bits from each seedling is collected for DNA based fingerprinting. Each leaflet from respective row and column was separately bulked and used for identification of contamination with any other variety. Rows and columns involving the off-type showed a fingerprint pattern different (with extra bands) from the control and were considered as impure rows and columns. Plants located on hills where the impure rows and columns intersect were considered as suspected off-types. Sundaram et al. (2008) used the same two dimensional bulking of DNA samples strategy involving 20 x 20 grow out matrix for detection of contaminants in a commercial seed lot of CMS line (IR 58025 A) using multiplex SSR markers. It was reported that by adopting this DNA bulking strategy, analysis of all four hundred seedlings is not required and total number of PCRs can be restricted to just twenty reactions. Further, it was identified that the results were comparable with conventional grow out test (GOT).

US patent US 8561346 B2 discloses an automated seed sampler including, a sampling station, a sampler for removing material from a seed in the sampling station, a seed conveyer for conveying the seed from the sampling station to a compartment in a seed tray and a conveyor for conveying the material removed from the seed to a corresponding compartment in a sample tray. The method of this invention comprises feeding seeds individually to a sampling station, removing a sample from the seed in the sampling station; conveying the sample to a compartment in a sample tray, and conveying the seed to a corresponding compartment in a seed tray. The samples can be tested, and the seeds can be sorted according to the results of the testing of their corresponding samples. Here each & every seed is sliced in to two halves and endosperm portion of seed used for DNA fingerprinting. Other embryo portion sorted and used for sowing, only if DNA based GP testing qualified or rejected. Though this sampler collect and sort the .seed sample, this sampler is not suitable for bulking of seed sample that reduces the cost involved in DNA based GP testing. Thus, there is a need of an user-friendly device, which could be used routine to carry out bulk sampling of seeds for accurate, DNA based GP testing of seed lots at seed_^ testing laboratories in India. Further, there is a need of practical tool that utilises DNA based sampling strategy for assisting seed certification officials to arrive decision to certify or reject the commercial seed lots.

Though all the prior known DNA bulking strategies minimize the cost of laboratory based genetic purity analysis by reducing sampling size, misinterpretation of result occurs where the seed lot contains more than two contaminants per DNA bulk (Row/column). This may be the case always where seed testing laboratory receives seed sample that may contains~more than two contaminations per seed lot.

Yet more cumbersome is to collect, sort and bulk the leaf samples from GOT field containing 400 plants, to form two-way sampling matrix that reduces the cost of DNA based genetic purity testing. Moreover, seed testing laboratory receives "submitted seed sample" truly representing the larger seed lot for genetic purity testing. As all the above strategy requires leaf sample from a minimum of 5-6 days old seedling for DNA isolation, the time taken for seeds to germinate and misinterpretation of results due to biased selection of only germinating seeds (ignoring non-viable/dorinant/poor quality contaminant seeds) are the major drawbacks. Yet, random sampling and bulking of seed sample is humanly impossible at seed testing laboratory. Thus, there is a need of an user friendly device to sort the seed samples and assess the genetic purity of seed lots of variety/hybrids so that the seeds produced in dry season can be released for commercial cultivation in the ensuing wet season.

It is also required to develop rapid test methodology for quick genetic purity assessment within the shortest possible time to ensure client satisfaction and over comes the requirement of live germinating seeds for genetic purity assessment. This holds good for assessing genetic purity of spurious seed lot which is adulterated/intentionally contaminated with poor quality (dormant/ non viable) seeds. Further, there is a need to devise method for identi fication of genetic purity of legal samples that are dead (Non viable guard sample). Apart from this, there should be an ergonomical ly simple and user friendly protocol for daily routine testing and handling of thousands of sample.

OBJ ECTS OF THE I VENTION

The object of the invention is to provide a portable device for sorting seed samples for assessing the genetic purity of seed lots and identify the contaminants, if any at seed testing laboratories.

Yet another objective of the invention is to provide a portable device that facil itate seed officials to use direct seed based three dimensional DNA sampling strategy for testing parental line purity of hybrid seed. Yet another object of the invention is to provide a portable device that facilitates seed officials to arrive at wel l informed decision to certify or reject the commercial seed production field. Yet another object of the invention is to provide a method of sorting seed samples from the desired seed lots as per the designed three-way sampling method using the developed matrix sampler.

SUMMARY OF INVENTION The afore-mentioned objectives are served by the present invention by way of providing a portable device having multiple rectangu lar boxes arranged horizontal ly and vertical ly for seed bulking.

In the present invention, horizontally arranged rectangular boxes enable row bulking and vertically arranged rectangu lar boxes enable column bulking. The spaces between the rows aand columns give rise to retainer boxes among them. The rectangular boxes of the said device are provided with seed drawers to col lect the seeds. The seeds spread over the top surface of the said device are sliced at least twise in three substantial ly equal parts and get arranged in such a way that the first part part is loaded in rows for row bulking, the second part is loaded in columns for column bulking and the third part of the seed is stored in retainer boxes for second time analysis, if required.

The present invention also provides a Method for sorting seeds for assesing the genetic purity of desired seed lot, the method comprising: placing the seeds on the top surface of the matrix sampler; slicing seeds randomly placed on the corss-intersection of rows and columns; sorting sliced seed parts in rows, columns and retainer boxes of the matrix sampler; wherein, one part of the sliced seed gets arranged in rows; one part gets arranged in columns; and the remaining third part gets retained in the retainer box;

The present invention would be clear from the ensuing description.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

Figure 1: illustrates diagrammatic representation of 5X5 sampling device.

Figure 2: illustrates right side, perspective view of sampling device.

Figure 3: illustrates 2X2 sampling device.

Figure 4: illustrates photographic image of portable sampling device to be used at seed testing laboratories.

DETAILED DESCRIPTION OF THE EMBODIMENTS

This patent describes the subject matter for patenting with specificity to meet statutory requirements. However, the description itself is not intended to limit the scope of this patent. The principles described herein may be embodied in many different forms. Illustrative embodiments of the invention now will be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the invention are shown. Indeed, the invention may 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 satisfy applicable legal requirements. Like numbers refer to like elements throughout. In the broadest aspect, the present invention contemplates a portable device that assist in carrying out three way sampling method for assessing the genetic purity of seed lot at seed tesing laboratories. The developed device essentially simplifies the three way method by direct row and column bulking and helps in Marker Assisted seed Testing (MAT). In the present invention, seeds are randomly selected from the seed lot received at the seed laboratories for testing and spread on the top of the portable device or the three way matrix sampler as being termed here. The seeds settled on to the cross intersection of rows and columns are retained and the remaining seeds are cleared from the surface. Such seeds getting placed on the cross- intersection of rows and columns are sliced at least twice in three substantially equal parts to be sorted in the said matrix sampler. For sorting, the two parts of each seeds are considered for row and column bulking, while the third part is retained in the retainer boxes for further assessment, if required (figure I ).

I I The first part of every seed gets loaded in the horizontally aligned rows for row bulking, whereas the second part of the seed gets loaded in the vertically aligned column for column bulking. Thereafter, DNA is isolated from bulked rows and columns seeds. The isolated DNA of respective rows and columns is then al lowed to undergo PCR assay with SSR markers. The amplified DNA is then subjected to DNA electrophoresis, and the probable contaminant seed are traced out by studying and ^' analyzing the gel picture of the electrophoresed DNA samples. In case of noted contamination from the tested samples, the third part retained is now tested for further analysis and accurate results.

Such a designed device that enables sorting of seed lots in a three way matrix facilitates seed testing officials and researchers to use direct seed based three dimensional DNA sampling strategy for testing parental line purity of hybrid seed at laboratory level.

In one of the embodiments, the device as in figure 4, primari ly comprises of multiple long rectangular boxes which are arranged horizontal ly and vertical ly one over the other i.e. horizontal boxes are arranged over the vertical boxes. The space that arise between the horizontal and vertical boxes is called retainer. The rectangular boxes are also provided with drawers to fit in the rows and columns, where the seed samples are loaded. The shutters in the device protect the seeds from falling and inter- mixing. The arrangement of rectangular boxes give rise to grid like structure to the said device in the form a matrix sampler (Figure 2). This grid like structure can be used as three way matrix. Overall arrangement give rise to three way matrix wherein each column and each row assist in column and row DNA bulking.

The matrix arrangement can be used for performing three way sampling method for which each seed gets sliced in three substantially equal parts either manually using the cutting means such as knife / scissors/ blades etc. or automatic slicing by use of laser rays which are made to penetrate through the seeds retained at the corss-intersection of the rows and columns at the top surface of the device. The resulting each piece is placed in the sampler in such a way that one portion of the sliced seed is placed in a retainer box and rest of the two portion are added in corresponding row and column. Similarly, all the retainer boxes are filled and other sliced parts are added in corresponding rows and columns.

In an exemplary embodiment, Figure 3 illustrates 2X2 three way sampler which is created using four rectangular boxes, two horizontal and two vertical. There are four retainer boxes among the horizontal ad vertical boxes in which third part of each four seeds is retained i.e. 1 *, 2*, 3* and 4*. From rest of the two parts, first part is placed in rows, i.e. , 2', 3' and 4'and second part is placed in column i.e. 1, 2, 3 and 4. The sliced seeds collected in rows can be directly used in row bulking and the sliced seed collected in columns can be used for column bulking. Example I : Now foe instance, suppose 25 seeds are to be tested in the laboratory, then 5X5 matix (Figure 1 , 2) wi l l be required having 10 long rectangular boxes of which .5 are arranged vertically and rest 5 are arranged horizontal ly over the vertical boxes. The arrangement wi ll give rise to 25 boxes among them wh ich are to be used as retainer boxes.

In the simi lar way. for sampl ing and sorting of 400 seeds which is the minimum number of seeds required for test samples, a -20X20 matix of horizontal and vertical ly aligned boxes is developed. The arrangement would be capable of sorting 400 seeds at a given time to perform Marker Assisted seed Testing (MAT). These sorted samples can be used for parental purity analysis of hybrid seed using molecular techniques including m icrosatell ite SSR markers at laboratory level.

The presence of shutters ( 1 ,2,3 in Figure 4) is an added advantage of the sampler that assists in protecting the seed samples from free fall and intermixing once received inside row and column boxes during post sampl ing handling in DNA fingerprinting laboratory. The presence of seed drawers (4 in figure 4) in rectangular boxes helps store the seeds received inside the respective row and column boxes and assist in easy removal of bulked seeds for DNA isolation. The retainer boxes among the rows and columns of the matrix sampler helps store the seed part for further assessment to trace the accurate contaminant seed present in the seed lot. For verifying the probable contaminant, the third part of only that seed is considered that is expected to be contaminant as per the DNA fingerprinting result. The said third part is then processed for₌D A isolation, and undergo PCR assay with specific SSR markers. The sorting of seeds for testing genetic purity is not limited to only 400 seeds, it can be done on "n" number of seed samples depending upon the rectangular boxes in the matrix sampler. The arrangement of said matrix sampler in the above example is only exemplary. The said sampler enables NXN matrix depending upon the number of seeds to be tested. The present invention makes it relatively convenient to carry out the three sampling method for testing the genetic purity of seed lot of crop plants. Row bulking and column bulking becomes easier as the part of the seed collected in row or column can be directly used for DNA isolation. In addition, it also reduces cost and time. The said device reduces the complexity of the method for assessing the genetic purity in a large number of seed samples.

Further, the present device find its application in conjunction with seed based three way sampling method in various central referral seed testing laboratory, state seed testing laboratory, DNA finger printing lab of public and private seed organisations for assessing genetic purity of seed lots of any crop plants using molecular markers and identify the contaminants if any. Along with three way sampling method the present device can also be used effectively at laboratory level to identify the parental line purity of hybrid seeds at various public seed testing laboratories (STL's)/ seed companies/ research institutes in India and worldwide. Although the invention has been explained in relation to its preferred embodiment, it is to be understood that many other possible modifications and variations can be made without departing from the spirit and scope of the invention as herein described.

Since other moditications and changes varied to fit particular operating requirements and environments are apparent to those skilled in the art, the invention is not considered limited to the example chosen for purposes of disclosure, and covers all changes and modifications which do not constitute departures from the true spirit and scope of this invention.

This written description uses examples to disclose the invention, including the best mode, and also to enable any person skilled in the art to practice the invention, including making and using any devices or systems and performing any incorporated methods. The patentable scope of the invention is defined in the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal languages of the claims.

Claims

We claim:

1. A device for sorting seed samples for assessing genetic purity of desired seed lot, comprising:

horizontally and vertically arranged multiple rectangular boxes;

spaces between said horizontally and vertically arranged rectangular boxes giving rise to retainer boxes among them;

drawers provided to fit in said horizontally and vertically arranged rectangular boxes; and shutters configured with said horizontally and vertically arranged rectangular boxes to assist in protecting the seed samples from free- falling and inter- mixing during post sampling handling in DNA fingerprinting laboratory.

2. The device as claimed in claim 1, wherein the arrangement of horizontally and vertically aligned rectangular boxes give rise to grid like structure to the matrix, and wherein the horizontally arranged boxes rest over the vertically arranged boxes, the horizontal boxes represent, rows and vertical boxes represent columns.

3. The device as claimed in claim 1, wherein the drawers placed within the horizontally and vertically arranged rows and columns meant to load collected seed sample, wherein said seeds are sliced at least twice producing three seed parts;

wherein the first part of the seed is considered to be bulked in horizontally arranged rows; second part of the seed is considered to be bulked in vertically arranged columns; and the third part of the seed is retained in said retainer box for further assessment to locate accurate contam inant.

4. The device as claimed in claim 4, wherein the DNA is isolated from bulked rows and columns and said isolated DNA further undergo PCR assay with specific SSR markers and gel electrophoresis, where gel picture derived from electrophoresis suggests probable contaminant if any in the seed lot.

5. The device as claimed in claim 4, wherein the retained seed part of the seeds in the retainer box used to veri fy probable contaminant in the seed lot, wherein the retained part of on ly that seed is considered which is suggested as contaminant in the gel picture, .

6. The device as claimed in claim I , wherein the device is three way matrix sampler and designed to perform three way sampling/ sorting of collected seed selected from seed lot of crop plants (varieties / cu ltivars / parental lines / hybrids)

7. Method for sorting seeds for assesing the genetic purity of desired seed lot, the method comprising:

placing the seeds on the top surface of the matrix sampler;

slicing seeds randomly placed on the cross-intersection of rows and columns; sorting sliced seed parts in rows, columns and retainer boxes of the matrix sampler; wherein, one part of the sliced seed gets arranged in rows; and

one part gets arranged in columns; and the remaining third part gets retained in the retainer box.