Patent Application: US-58560909-A

Abstract:
the present invention relates to haploid oil palm plants and homozygous doubled haploid oil palm plants . the invention also relates to methods for producing and selecting haploid and doubled haploid plants . more particularly , but not exclusively , the method may be used for selecting haploid and doubled haploid oil palm plants . haploid and doubled haploid plants are selected by a large - scale screening based on a combination of the phenotype with the use of molecular methods combined with flow cytometry techniques to identify haploid and doubled haploid plants . more particularly , a method for selecting haploid and doubled haploid plants is described comprising : germinating seeds ; selecting seedlings with atypical phenotype ; assessing heterozygosity using markers ; isolating cells from the seedlings and determining the dna content of the cells ; and isolating and purifying the dna and using defined molecular markers to characterise the genotype of the plant . the haploid oil palm plants may be used for producing homozygous doubled haploid oil palms : doubled haploids may be intercrossed to produce uniform f 1 hybrids of superior properties .

Description:
the following example is presented to further illustrate and explain the present invention and should not be taken as limiting in any regard . it shows the application of the invention to oil palm : but is similarly applicable to date palm . 1 the mesocarp was mechanically removed from oil palm seeds and the seeds were air dried for 24 hours at ambient temperature and then for 24 hours in an air - conditioned room at 25 ° c . to a seed moisture content of 15 - 18 %. the seeds were then stored , usually for one to three months in an air - conditioned room ( 25 ° c .) in plastic bags or trays ( although it is possible to store seeds for up to one year in this way ). 2 the seeds were soaked for three days to increase their moisture content to 18 - 20 % and then heat treated in plastic bags or trays for 40 to 60 days at 38 - 40 ° c . 3 after heating , the seeds were soaked for five days to raise their moisture content to & gt ; 22 % and then dried at ambient temperature for approximately four hours 4 the seed were transferred to a germination room where under ambient temperatures germination usually starts after 7 to 10 days and continues for two to three months . there were two large - scale morphological screens of oil palm seedlings for morphological off - types . the first consisted of 10 , 900 , 000 germinated seeds , of which 3 , 854 were identified as being morphologically deviant ( 3 , 801 ) or twin - seeded ( 53 ), with the remaining individuals all being deemed ‘ normal ’ ( see fig1 and 2 for examples of both types ). thus , in this instance 99 . 96 % of seeds evaluated were classified as exhibiting , a normal phenotype and 0 . 035 % being aberrant . in the second screen , approximately 10 , 000 , 000 commercial seedlings were screened , together with approximately 1 , 000 , 000 seedlings taken from breeding experiments . this trial generated 5 , 704 morphological candidates , of which 5 , 601 were phenotypically abnormal and 103 were twin - seeded . in this screen , therefore , 99 . 95 % of seedlings were classed as normal and 0 . 05 % as aberrant prior to transfer to the nursery house ( fig3 ). the protocol applied to perform a molecular prescreen of seedlings showing abnormal phenotypes to discard heterozygotes comprised the following stages : 1 . dna extraction 2 . amplification of microsatellite markers by pcr 3 . separation of pcr products by agarose gel electrophoresis 4 . scoring of results to discard individuals with one or more heterozygous loci around 0 . 5 cm of the radicle ( around 50 mg ) was removed from the seedling and used to extract dna using the qiagen 96 dneasy extraction kit according to the manufacturer &# 39 ; s instructions as described below , although other systems for dna extraction could also be used . 1 . for new kits , add 100 % ethanol to ap3 / e buffer and aw buffer 4 . if ap1 buffer has a cloudy appearance , heat to 65 ° c . and shake until the solution becomes clear 1 . add 50 mg plant material into each tube in two collection microtube racks . retain the clear cover . 3 . prepare the lysis solution : ( 400 μl ap1 + 1 μl rnase + 1 μl reagent dx )/ reaction plus 15 % of each component . 4 . disrupt the sample using mm 300 , 30 hz for 1 . 5 minutes . 6 . remove and discard caps , add 130 μl ap2 buffer into each collection microtube . 7 . close the microtubes with new caps . place a clear cover ( from step 1 ) over the 96 well plate . shake the plate vigorously for 15 s . pulse centrifuge to 3000 rpm . 9 . remove and discard the caps . transfer 400 μl of each supernatant to new plate of collection microtubes ( provided ). do not transfer pellet and floating particles . hold the strips and use the lowest pipette speed . recover the tungsten beads . 14 . transfer 1 ml of sample into each well of the 96 well plate . 15 . seal with airpore tape sheet and centrifuge for 4 min at 6000 rpm . 18 . add 100 μl of buffer ae to each sample and seal with new airpore sheets . in all cases , 10 μl a pcr reaction mixture contained the following reagents ; 1 . 0 μl of 10 × pcr buffer ( bioline ), 0 . 3 μl mgcl 2 ( 10 mm ), 0 . 4 μl dntps ( 10 mm of each ), 0 . 2 μl of each primer pair ( 10 μm ), 1 - 5 ng of dna ( extracted as above ) and 1 u of tag dna polymerase ( 5 u μl − 1 bioline ). the following conditions were used for the polymerase chain reaction for all microsatellite markers : an initial 94 ° c . denaturing step for 2 min followed by 35 cycles of ; 94 ° c . for 30 sec , 52 ° c . for 30 sec and 72 ° c . for 45 sec , with a final extension step of 72 ° c . for 7 min . agarose gel electrophoresis and ethidium bromide staining were routinely used to fractionate and visualise products generated by microsatellite pcr . 1 . 0 - 1 . 5 % ( w / v ) agarose ( was prepared in 1 × tbe buffer and subjected to heating in a microwave ( 700 w ) for 2 × 1 min at full power to create a gel solution . the gel solution was cooled to approximately 55 ° c . prior to the addition of ethidium bromide ( 3 . 5 μl per 100 ml gel ). the ends of a suitable gel tray rig ( midi - gel tray for 100 ml gels , maxi - gel tray for 250 ml gels ) were sealed with masking tape and an appropriate number and type of combs placed in position . combs with 16 × 20 μl wells were most often employed . the gel solution was carefully poured into the prepared tray and allowed to cool for at least 20 min . combs and tape were then removed and the gel tray submerged into a tank containing 1 × tbe buffer . generally , 5 μl of sample were mixed with 2 μl of bromophenol blue buffer prior to loading . the loading buffer serves two functions : first , it increases the specific gravity of the sample thereby preventing diffusion of dna from the top of the well into the surrounding buffer , and second , it indicates the progress of product as they migrate through the gel by electrophoresis ( the blue dye migrates at approximately the same position as dna fragments 200 bp in length ). to estimate the size of the amplicons , 4 μl of 100 by gibco &# 39 ; s ladder ( gibco life science brl ) were loaded together with the analysed samples . electrophoresis of mid - gels ( 100 ml ) was performed at 120 volts in 1 × tbe buffer for approximately 1 h . following electrophoresis , gels were removed from the rig and post - stained in 5 mg / l aqueous ethidium bromide solution for 40 min , destained in distilled water for 2 min and then viewed under ultra violet illumination using a uvp bio - doc - system . images of the gels were captured by the uvp bio - doc system as jpeg format and used for scoring . 4 . scoring of results to discard individuals with one or more heterozygous loci pcr products generated by each microsatellite - genotype combination were evaluated for the presence of one or two distinct bands after fractionation by agarose gel electrophoresis ( stages 1 - 3 above ). any genotype that yielded two products for any of the microsatellite loci was deemed to be heterozygous and so discarded as a possible candidate haploid or doubled haploid plant . the remaining individuals were sent forward to step ( d ) of the pipeline ( flow cytometry ) there were over 2000 phenotypically abnormal seedlings identified from the two morphological screens described above that were randomly selected for the molecular screen : in addition , there were a further 150 individuals with the normal phenotype . there were also 24 diploid tenera clones used as controls ( all diploid heterozygotes ). when these were screened using up to 15 of microsatellite markers ( 1 - 15 ), 117 genotypes ( see table in flow cytometry section for identification codes ) exhibited a single allele for all loci ( an example is shown in fig4 , using marker 09 ) and so were deemed to be highly homozygous . accordingly , these individuals were considered as candidate haploids / doubled haploids and progressed to step d ( flow cytometry ). fig4 shows band profiles generated by marker 09 across 25 oil palm genotypes . individuals showing two alleles ( marked ‘ 2 ’) were discarded from the screen . individuals identified as morphologically abnormal and highly homozygous ( stages b and c ) were subjected to flow cytometry to establish their ploidy level using the following protocol . the cell nuclei were isolated from fresh plant material ( leaves or roots ), by chopping the plant material ( a few cm 2 / 20 - 50 mg )) with a sharp razor blade in an ice - cold buffer , in a plastic petri dish . the dna buffer ( stored at 4 ° c .) is based on : arumuganathan , k . and earle , e . d . estimation of nuclear dna content of plants by flow cytometry . plant molecular biology reporter , vol 9 ( 3 ) 1991 , pages 229 - 233 . dapi , a fluorescent dye that selectively binds to form a complex with double - stranded dna and give a product that fluoresces at 465 nm , was introduced to the solution . dapi has specific dna - binding properties , with preference for adenine - thymine ( ad - rich sequences . after chopping , the buffer ( ca . 2 ml . ), containing cell constituents and large tissue remnants , is passed through a nylon filter of 40 micrometer mesh . this method will produce thousands of nuclei from a leaf piece of a few cm 2 . the solution containing stained nuclei was passed through the flow cytometer . controls are required of known ploidy ( dna content ) as reference — for oil palm , tissue from diploid tenera palms were used because the shell thickness must be heterozygous and therefore the palm cannot be haploid . the fluorescence of the stained nuclei , passing through the focus of a light beam from a high - pressure mercury lamp , was measured by a photomultiplier and converted into voltage pulses . these voltage pulses were electronically processed to yield integral and peak signals that can be processed by a computer . when the samples are run with the appropriate filter - settings for excitation and emission , dna histograms can be produced . flow cytometer : cyflow ml ( partec gmbh , otto hahnstrasse 32 , d - 4400 munster , germany ) with a high pressure mercury lamp , osram hbo 100 long life . objective : 40 × n . a . 0 . 8 air ( partec ) of the 117 genotypes identified as highly homozygous in step c , 83 were identified as haploid by flow cytometry , with remaining 34 individuals being diploid ( see table 1 below ) genome characterisation was used for two purposes . first , to confirm the lack of heterozygosity among plants identified as haploids by the morphological assessment , molecular screen and by flow cytometry . second , to identify doubled haploids on the basis of being diploid and lacking any detectable heterozygosity . the method used to assess both sets of plants was identical and is described below . genome characterisation ( for heterozygosity ) was performed using 96 microsatellite loci . rather than screening all primers against all candidate samples using labelled primers , a pooling strategy ( as proposed by cryer et al ., 2005 ) was adopted that avoids the need for large numbers of expensive , labelled ssr primers . the method involves amplifying each microsatellite locus for all haploid candidates using unlabelled primers , bulking and ligating the products together into a vector , and then performing a second amplification using a fluorescently labelled vector primer to expose allelic forms . the number of alleles at each locus for all individuals could then be assessed by fractionation on the capillary sequencer . validity of the results obtained by this method was verified by comparing profiles generated using the pooled strategy with those obtained on 10 representative samples ( diploid and haploid ) using a subset of 24 labelled microsatellite markers fractionated and detected by conventional capillary electrophoresis . the first step in the screen involved amplifying 12 candidate samples ; using 96 microsatellite markers listed in table 2 ( below ). for all reactions , the 10 μl microsatellite reaction mixture contained the following reagents ; 1 . 0 μl of 10 × pcr buffer ( bioline ), 0 . 3 μl mgcl 2 ( 10 mm ), 0 . 4 μl dntps ( 10 mm of each ), 2 . 0 μl of each primer pair ( 1 μm ), 1 - 5 ng of dna ( extracted at blrs ) and 1 u of taq dna polymerase ( 5 u μl bioline ). the thermal cycler was programmed with an initial 94 ° c . denaturing step for 2 min followed by 35 cycles of ; 94 ° c . for 30 s , 52 ° c . for 30 sec and 72 ° c . for 45 sec , with a final extension step of 72 ° c . for 7 min . pcr products were assessed for size by electrophoresis through a 1 % w / v agarose gel for 30 min at 120 v . two bulks were constructed for each of the 12 individuals with each bulk containing 48 markers . the bulked pcr products were then purified using qiaquick pcr purification columns ( qiagen ) as per manufacturer &# 39 ; s instructions . the purified products were then ligated into a pdrive cloning vector ( qiagen ) to allow a universal binding site for the second round pcr . the pdrive vector was selected because of its high efficiency for ligation and due to the fact that it contains the m13 forward and m13 reverse primer - binding sites . the linear vector is designed to exploit the behaviour of taq polymerase , which produces a single adenosine nucleotide overhang on resulting pcr fragments , by containing a complementary base ( u - base ) at the points of insertion . with a simple ligation reaction the adenosine base from the pcr product and the u - base from the vector ligate together resulting in the recircularisation of the plasmid . this was achieved by adding 5 μl of 2 × ligation master mix , 4 μl of pcr product and 1 μl of the pdrive vector ( 50 ng μl − 1 ) into a 0 . 2 ml eppendorf tube . reagents were collected by pulse centrifugation and the ligation reaction was performed at 4 ° c . for approximately 15 h . the ligation product was diluted 1 : 10 with nanopure water and this formed the template for the second pcr involving a single microsatellite locus specific primer in combination with a fluorescently labelled universal primer m13 ( either forward or reverse ). the forward m13 (− 40 ) was labelled with the fluorescent dye ( fam ) and the reverse with hex ( both supplied by sigma aldrich ). the pcr conditions were the same as in the initial amplification step this time using the diluted ligation product as the dna template . products were diluted 1 in 5 and arranged in bulks based on the expected size of fragment and the fluorescent dye used , which allowed numerous samples to be assessed in a single run of the capillary sequencer . these products were separated by capillary electrophoresis on an abi prism 3100 sequencer . the sequencer uses a linear flowing media , namely pop - 6 ™ polymer ( applied biosystems ), to separate fragments in the capillaries and the fluorescence emitted from the incorporated labelled primer is recorded by the software program genescan version 3 . 1 ™ ( applied biosystems ). the output file allows comparisons of the genetic profiles of individuals by portraying peaks that represent the aflp - dna fragments . this fragment analysis was performed using abi prism genotyper ® 3 . 6nt software ( applied biosystems ), which allows analysis of the size of the fragments ( in base pairs ) and can also assess the strength of the amplified product . allele sizes were assessed using abi prism genotyper ® 3 . 6nt software ( applied biosystems ). any individual that generated two allelic peaks for any microsatellite marker was deemed partly heterozygous and so discarded as not being a possible haploid or doubled haploid ( depending of flow cytometry results ). a subset of 8 of the 24 candidates identified after the molecular screen including both diploid and haploid individuals ( listed in the table below ) was subjected to more extensive molecular characterization with 80 additional microsatellite markers using the bulked ligation technique described above ( e ). as expected , all individuals identified as haploids by flow cytometry contained only a single allele across all 80 loci surveyed . thus , these individuals are hemizygous for 95 loci in total ( including the 15 markers used in the screen ) and this was deemed to confirm their haploid status . in contrast , the two diploids were heterozygous for many of the loci screened . when profiles of ten individuals were subjected to microsatellite analysis by conventional capillary electrophoresis and using labeled microsatellite primers , the profiles obtained indicated identical scores for allelic status across 24 markers . here , we screened all 34 diploid candidates that were homozygous for all 15 markers as described above and applied a further 32 fluorescent labeled microsatellite markers ( listed below ) using conventional capillary electrophoresis through a abi prism 3100 dna sequencer . there were two genotypes ( 65 - 0409034 mc - 144 and 65 - 0409034 mc - 114 ) that were homozygous for all markers . thus , these plants were homozygous for a total of 47 microsatellite markers and so were deemed to be doubled haploid oil palm plants . fig7 b shows images of a typical diploid heterozygous oil palm ( bottom ) and two doubled haploids ( top ) sown on the same day . haploid cells will sometimes undergo “ spontaneous doubling ” whereby failure of complete mitosis gives a doubling of the chromosomes . if this occurs early in development , the seed , plantlet and plant derived is a doubled haploid . if no such doubling occurs then a haploid is obtained and in most circumstances , such haploid plants are intrinsically infertile , in that the process of meiosis is unable to generate gametes capable of fertilisation . in order to produce a fertile plant from which sexual progeny can be produced it is necessary either to double the chromosome number of a haploid by application of an external stimulus , or to rely on the rare process by which a haploid cell can spontaneously double . the former method is the most usually adopted , and usually involves the application of a chemical agent capable of inhibiting mitosis and thereby inducing the formation of a diploid cell . there are several chemicals known to induce such a chromosome doubling process and of these colchicine is the best known , and most commonly utilised . other similar agents include microtubule inhibitors such as the herbicides trifluralin , and oryzalin . such chemicals can either be applied to a whole plant and fertile seeds may be produced on that plant , or they can be applied in vitro to isolated cells from which an intact plant can be regenerated using conventional tissue culture techniques . for a full description of available chemical and other methods and their means of application see kasha ( 2005 ) and references therein . an alternative to the external application of stimuli is the exploitation of spontaneous doubling . for example , in a haploid , the nucleus of an individual cell may occasionally fail to divide normally at mitosis and thus form a diploid cell that ultimately gives rise either to a diploid sector ( s ) that may encompass most or all of the main shoot axis or ( if it occurs in the first embryonic division ) a doubled haploid plant . in either case , the selfed seed secured from such individuals will be completely homozygous and genetically identical to the parent . this process can occur during the formation of reproductive cells and in this case it is possible that fertile gametes ( pollen or egg cells ) may be produced . if both male and female gametes form on the same plant then successful fusion of gametes can take place and an embryo will develop . such an embryo will be a homozygous diploid , and will breed true in all future selfed generations ; all its selfed progeny will be genetically identical . in oil palm , the inflorescences are usually either male or female ( though hermaphrodite inflorescences are known to occur occasionally ) and therefore selfing of a particular haploid plant may require the storage of pollen from a male inflorescence until a suitable female inflorescence is available for . pollination . such procedures are commonly used in oil palm breeding . in the present example , we have found that haploid oil palm plants obtained by the process of the invention produce their first inflorescences after approximately two years of vegetative growth , and it is likely that such plants will produce a low , but usable frequency of fertile gametes , from which homozygous progeny can be isolated . one haploid plant has now started to flower fig1 shows confirmed haploid 50 - 03060260 — 0002 with first inflorescence two years and seven months after planting ( left photograph of inflorescence and right photograph of haploid seedling ). six oil palms identified as haploid from flow - cytometry were screened to confirm homozygous status . this was achieved by identifying heterozygous markers from each candidate &# 39 ; s maternal parent and recording the number of these markers that were also homozygous in the candidate . for a true haploid , the expectation is that all individual loci should contain one allele ( hemizygous ). a total of 96 markers ( listed in table 5 ) were screened on each of the mother palms and those that were shown to be heterozygous were then assessed on the progeny candidate palms . the markers used consisted of a universal anchor sequence that was used to incorporate a fluorescent dye into the pcr products , allowing the allele sizes to be assessed by fractionation through capillary electrophoresis . all six candidate palms were shown to be 100 % homozygous over all the heterozygous loci identified in the parent ( table 3 ). the palms were therefore deemed to be completely homozygous , as expected for haploid plants . roots of one haploid confirmed above were pretreated in iced water for 24 h and fixed in 3 : 1 alcohol : glacial acetic acid . roots were then stored at 4 ° c . for 24 h or until required . roots were then washed in water , softened in 1n hcl for 20 min at room temperature , washed in water ( 2 min ) and stained in saturated aceto - orcein for 1 minute . the root tip was then isolated , squashed and mounted onto a glass slide . mounted root squash preparations were then examined on a compound microscope . several unbroken cells were identified that contained the expected 16 chromosomes ( see photomicrograph , fig1 ) twenty - six palms previously scored homozygous for 15 markers at blrs ( bah lias research station , indonesia ) and identified as diploid from flow - cytometry were screened to confirm that they are homozygous and thus doubled haploids . this was achieved by identifying at least 20 unlinked heterozygous markers from each candidate &# 39 ; s maternal parent and assessing the level of homozygosity of these markers in the candidate . a total of 111 markers were screened on each of the mother palms ( 96 markers were screened in one laboratory and 15 markers in another ) and those that were shown to be heterozygous were then assessed on the progeny candidate palms . from this , one palm ( candidate 5 ; id - 0644 / 219 ; 05049082 ; 0003 ) proved to be entirely homozygous over all 35 markers identified heterozygous in the parent ( palm number bl013 / 12 - 06 ). taking linkage into account , the probability of this event occurring naturally is less than 1 in 2 million and could be less than 1 in 16 million ( considering the unmapped markers ). abdullah r ( 2005 ). a decade of oil palm gene manipulation . where are we now ? in : 9th international conference on agricultural biotechnology : ten years after organized by the : international consortium on agricultural biotechnology research ( icabr ) and the : catholic university of leuven ceis - university of rome “ tor vergata ” centre of sustainable resource development , university of california at berkeley economic growth centre , yale university ravello ( italy ), jul . 6 - 10 , 2005 . pp . 25 . abdullah r , zainal a , heng w y , li l c , beng y c , phing l m , sirajuddin s a , ping w y s , joseph j l , jusoh s a . 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