WO2015162625A2 - Dispositif d'échantillonnage à matrice trois voies pour semences et procédé associé - Google Patents

Dispositif d'échantillonnage à matrice trois voies pour semences et procédé associé Download PDF

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Publication number
WO2015162625A2
WO2015162625A2 PCT/IN2015/000176 IN2015000176W WO2015162625A2 WO 2015162625 A2 WO2015162625 A2 WO 2015162625A2 IN 2015000176 W IN2015000176 W IN 2015000176W WO 2015162625 A2 WO2015162625 A2 WO 2015162625A2
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WIPO (PCT)
Prior art keywords
seed
boxes
seeds
rows
horizontally
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PCT/IN2015/000176
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English (en)
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WO2015162625A3 (fr
Inventor
R. Dhandapani
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Indian Council Of Agricultural Research (Icar)
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Publication of WO2015162625A2 publication Critical patent/WO2015162625A2/fr
Publication of WO2015162625A3 publication Critical patent/WO2015162625A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5097—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving plant cells

Definitions

  • 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.
  • 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.
  • GP Genetic Purity
  • 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.
  • GROW OUT TEST GROW OUT TEST
  • 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.
  • molecular markers are available for genetic purity analysis.
  • 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.
  • RAPD molecular Marker Assisted seed testing
  • MAT molecular Marker Assisted seed testing
  • the cost involved in estimating the GP using GOT is less expensive than molecular marker involved DNA fingerprinting method.
  • 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
  • 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.
  • 100 seeds were germinated in 10 row ⁇ 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.
  • 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.
  • 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.
  • 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.
  • 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;
  • 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.
  • the developed device essentially simplifies the three way method by direct row and column bulking and helps in Marker Assisted seed Testing (MAT).
  • MAT Marker Assisted seed Testing
  • 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 ).
  • 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.
  • the device as in figure 4 represents 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.
  • all the retainer boxes are filled and other sliced parts are added in corresponding rows and columns.
  • 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.
  • shutters 1 ,2,3 in Figure 4
  • the presence of shutters 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 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.
  • 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.
  • 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.

Abstract

La présente invention concerne un dispositif d'échantillonnage trois voies qui facilite la mise en œuvre d'une évaluation de la pureté génétique basée sur l'ADN d'un lot de semences issu de plantes cultivées au moyen du procédé d'échantillonnage trois voies. Le dispositif d'échantillonnage comprend de multiples boîtes rectangulaires et longues qui sont agencées horizontalement et verticalement les unes au-dessus des autres, formant un espace entre les boîtes horizontales et verticales appelé retenue. Les boîtes rectangulaires disposées horizontalement sont utilisées pour le regroupement en rangées et les boîtes disposées verticalement pour le regroupement en colonnes, alors que les boîtes de retenue sont utilisées pour conserver des parties de semence pour analyse ultérieure en vue de tracer un contaminant précis. L'agencement de boîtes rectangulaires forme une structure de type grille pour le dispositif d'échantillonnage avec une utilisation potentielle pour l'identification rapide, précise et à faible coût de contaminants dans les semences à tester.
PCT/IN2015/000176 2014-04-21 2015-04-21 Dispositif d'échantillonnage à matrice trois voies pour semences et procédé associé WO2015162625A2 (fr)

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IN179DE2014 2014-04-21
IN179/DEL/2014 2014-04-21

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WO2015162625A3 WO2015162625A3 (fr) 2016-01-14

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108871852A (zh) * 2018-08-08 2018-11-23 新疆农业科学院经济作物研究所 一种棉花考种铃防错辅助采收取样装置

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998030721A1 (fr) * 1997-01-10 1998-07-16 Pioneer Hi-Bred International, Inc. Amplification et analyse genetiques sur la base d'une hybridation
JP2009506174A (ja) * 2005-08-26 2009-02-12 モンサント テクノロジー エルエルシー 脂肪酸組成物のハイスループット・スクリーニング

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108871852A (zh) * 2018-08-08 2018-11-23 新疆农业科学院经济作物研究所 一种棉花考种铃防错辅助采收取样装置

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