Patent Application: US-63001205-A

Abstract:
the present invention relates to methods and compositions for increasing seed yield of a plant . the methods comprise expression of a cytokinin oxidase in the aleurone and / or embryo of a seed . the invention also relates to vectors comprising a nucleic acid encoding a cytokinin oxidase that is operably linked to a promoter capable of driving expression in the aleurone and / or embryo of a seed , and to host cells , transgenic cells and plants comprising such sequences . the use of these sequences for increasing yield is also provided .

Description:
unless otherwise stated , recombinant dna techniques are performed according to standard protocols described in ( sambrook & amp ; russell ( 2001 ) molecular cloning : a laboratory manual , 3rd edition cold spring harbor laboratory press , csh , new york ) or in volumes 1 and 2 of ausubel et al . ( 1994 ), current protocols in molecular biology , current protocols . standard materials and methods for plant molecular work are described in plant molecular biology labfax ( 1993 ) by r . d . d . croy , published by bios scientific publications ltd ( uk ) and blackwell scientific publications ( uk ). the arabidopsis ckx2 gene ( corresponding to seq id no : 42 ) was amplified by pcr using as template an arabidopsis thaliana seedling cdna library ( invitrogen , paisley , uk ). after reverse transcription of rna extracted from seedlings , the cdnas were cloned into pcmv sport 6 . 0 . average insert size of the bank was 1 . 5 kb , and original number of clones was 1 . 59 × 10 7 cfu . the original titer was determined to be 9 . 6 × 10 5 cfu / ml , and became after a first amplification 6 × 10 11 cfu / ml . after plasmid extraction , 200 ng of template was used in a 50 μl pcr mix . primers prm3769 ( seq id no : 39 ) and prm1526 ( seq id no : 40 ), which include the attb sites for gateway recombination , were used for pcr amplification . pcr was performed using hifi taq dna polymerase in standard conditions . a pcr fragment of 1506 bp was amplified and purified also using standard methods . the first step of the gateway procedure , the bp reaction , was then performed , during which the pcr fragment recombines in vivo with the pdonr plasmid to produce , according to the gateway terminology , an “ entry clone ”, p41 ( fig3 ). pdonr was purchased from invitrogen , as part of the gateway technology . the entry clone p41 was subsequently used in an lr reaction with p831 or p830 , both destination vectors according to the gateway ™ terminology , used for rice transformation . p831 contains as functional elements within the t - dna borders a plant selectable marker , a screenable marker and a gateway cassette intended for lr in vivo recombination with the sequence of interest already cloned in the donor vector . the pro0218 promoter for embryo and aleurone preferred expression is located upstream of this gateway cassette . similarly , p830 contains as functional elements within the t - dna borders : a plant selectable marker ; a screenable marker ; and a gateway cassette intended for lr in vivo recombination with the sequence of interest already cloned in the donor vector . the pro0090 promoter for endosperm - preferred expression is located upstream of this gateway cassette . after the recombination step , the resulting expression vectors p37 ( originating from p831 , fig4 ) and p35 ( originating from p830 , fig5 ) were transformed into agrobacterium strain lba4404 and subsequently into oryza sativa plants . mature dry seeds of the rice japonica cultivar nipponbare were dehusked . sterilization was done by incubating the seeds for one minute in 70 % ethanol , followed by 30 minutes in 0 . 2 % hgcl 2 and by 6 washes of 15 minutes with sterile distilled water . the sterile seeds were then germinated on a medium containing 2 , 4 - d ( callus induction medium ). after a 4 - week incubation in the dark , embryogenic , scutellum - derived calli were excised and propagated on the same medium . two weeks later , the calli were multiplied or propagated by subculture on the same medium for another 2 weeks . 3 days before co - cultivation , embryogenic callus pieces were sub - cultured on fresh medium to boost cell division activity . the agrobacterium strain lba4404 , harbouring t - dna vectors comprising a suitable selection marker , was used for co - cultivation . agrobacterium was cultured for 3 days at 28 ° c . on ab medium with the appropriate antibiotics . the bacteria were then collected and suspended in liquid co - cultivation medium at an od6 % of about 1 . the suspension was transferred to a petri dish and the calli were immersed in the suspension during 15 minutes . next , the callus tissues were blotted dry on a filter paper , transferred to solidified co - cultivation medium and incubated for 3 days in the dark at 25 ° c . hereafter , co - cultivated callus was grown on 2 , 4 - d - containing medium for 4 weeks in the dark at 28 ° c . in the presence of a selective agent at a suitable concentration . during this period , rapidly growing resistant callus islands developed . upon transfer of this material to a regeneration medium and incubation in the light , the embryogenic potential was released and shoots developed in the next four to five weeks . shoots were excised from the callus and incubated for 2 to 3 weeks on an auxin - containing medium from which they were transferred to soil . hardened shoots were grown under high humidity and short days in a greenhouse . finally seeds were harvested three to five months after transplanting . the method yielded single locus transformants at a rate of over 50 % ( aldemita and hodges , 1996 , chan et al ., 1993 , hiei et al ., 1994 ). approximately 15 to 20 independent t0 transformants were generated . the primary transformants were transferred from tissue culture chambers to a greenhouse for growing and harvest of t1 seed . four events ( for p37 transformants , pro0218 promoter ) or five events ( for p35 transformants , pro0090 promoter ) of which the t1 progeny segregated 3 : 1 for presence / absence of the transgene were retained . for each of these events , 10 t1 seedlings containing the transgene ( hetero - and homo - zygotes ), and 10 t1 seedlings lacking the transgene ( nullizygotes ), were selected by monitoring visual marker expression . the selected t1 plants were transferred to a greenhouse . each plant received a unique barcode label to link unambiguously the phenotyping data to the corresponding plant . the selected t1 plants were grown on soil in 10 cm diameter pots under the following environmental settings : photoperiod : 11 . 5 h , daylight intensity : 30 , 000 lux or more , daytime temperature : 28 ° c . or higher , night time temperature : 22 ° c ., relative humidity : 60 - 70 %. transgenic plants and the corresponding nullizygotes were grown side - by - side at random positions . from the stage of sowing until the stage of maturity the plants were passed several times through a digital imaging cabinet . at each time point digital images ( 2048 × 1536 pixels , 16 million colours ) were taken of each plant from at least 6 different angles . in a next step , the mature primary panicles were harvested , bagged , barcode - labelled and then dried for three days in the oven at 37 ° c . the panicles were then threshed and all the seeds were collected and counted . the filled husks were separated from the empty ones using an air - blowing device . the empty husks were discarded and the remaining fraction was counted again . the filled husks were weighed on an analytical balance and the cross - sectional area of the seeds was measured using digital imaging . this procedure allows deriving a set of seed - related parameters . the parameters described below were derived in an automated way from the digital images using image analysis software and were analysed statistically . a two factor anova ( analysis of variance ) corrected for the unbalanced design was used as statistical model for the overall evaluation of plant phenotypic characteristics . an f - test was carried out on all the parameters measured in all the plants and of all the events transformed with that gene . the f - test was carried out to check for an effect of the gene over all the transformation events and to verify for an overall effect of the gene , also named herein “ global gene effect ”. if the value of the f test showed that the data are significant , than it was concluded that there is a “ gene ” effect , meaning that not only presence or the position of the gene is causing the effect . the threshold for significance for a true global gene effect was set at 5 % probability level for the f test . to check for an effect of the genes within an event , i . e ., for a line - specific effect , a t - test was performed within each event using data sets from the transgenic plants and the corresponding null plants . “ null plants ” or “ null segregants ” or “ nullizygotes ” are the plants treated in the same way as the transgenic plant , but from which the transgene has segregated . null plants can also be described as the homozygous negative transformed plants . the threshold for significance for the t - test was set at a 10 % probability level . the results for some events can be under or below this threshold . this is based on the hypothesis that a gene might only have an effect in certain positions in the genome , and that the occurrence of this position - dependent effect is not uncommon . this kind of gene effect is also named herein a “ line effect of the gene ”. the p value was obtained by comparing the t value to the t distribution or alternatively , by comparing the f value to the f distribution . the p value gives the probability of the null hypothesis ( i . e ., that there is no effect of the transgene ) is correct . the threshold for significance was set at a 5 % p - value for the f test and a 10 % p - value for the t - test . vegetative growth and seed yield was measured according to the methods as described above . the inventors surprisingly found that the seed yield of transgenic plants was increased for both the endosperm - preferred and embryo and / or aleurone - preferred promoter constructs ( expressed as total weight of seeds , number of ( filled ) seeds and harvest index ), when compared to null plants , and that transgenic plants with the embryo and / or aleurone - preferred promoter constructs additionally had an increased thousand kernel weight compared to transgenics with the endosperm - preferred promoter construct . the inventors furthermore observed that the yield increase was higher for plants transformed with the embryo and / or aleurone - preferred promoter constructs than for plants with the endosperm - preferred promoter construct . details are given in paragraphs e and f . the data obtained in the experiment with t1 plants were then confirmed in a further experiment with t2 plants . seed batches from the positive plants ( both hetero - and homozygotes ) in t1 , were screened by monitoring marker expression . for each chosen event , the heterozygote seed batches were then retained for t2 evaluation . within each seed batch an equal number of positive and negative plants were grown in the greenhouse for evaluation . in particular , four events of p37 t2 transformants and three events of p35 t2 transformants were selected for further analysis . for both p37 t2 transformants and p35 t2 transformants , a total of 120 plants were tested , evenly distributed over each event . upon analysis of the seeds as described above , the inventors found that plants transformed with the atckx2 gene under control of the embryo and / or aleurone - preferred promoter had a higher total weight of seeds , a higher number of filled seeds , a higher harvest index and a higher thousand kernel weight than plants lacking the ckx2 transgene . these findings were consistent over 2 independent experiments with t1 plants as well as in an experiment with t2 plants , as shown in table 5 . in addition to these yield parameters , 3 lines in t1 scored also positive for the total number of seeds . this increase in total seed number was confirmed in t2 , where the effect was shown to be a significant global gene effect ( mean increase + 24 %, p - value from the f - test 0 . 0032 ). the total seed weight was measured by weighing all filled seeds harvested from a transformed rice plant . the number of filled seeds was determined by counting the number of filled seeds harvested from a transformed rice plant . the total seed number was determined by counting the number of seeds harvested from a plant . the harvest index is defined as the ratio between the total seed weight and the above ground area ( mm 2 ), multiplied by a factor 106 . thousand kernel weight ( tkw ) was derived from the number of filled seeds that were counted , and their total weight . the figures gave the mean increase ( in %) of each parameter calculated from transgenes versus corresponding nullizygotes of 4 independent events in t1 generation , each event comprising 10 plants carrying the transgene and 10 nullizygotes , and of 4 independent events in the t2 generation , each event comprising 20 plants carrying the transgene and 20 nullizygotes . the p - values of the f - test listed for the data of the t2 generation demonstrate that the obtained increases for the various seed yield parameters are all significant and that there is clearly an overall gene effect . plants transformed with the atckx2 gene under control of the endosperm - preferred promoter also had a better yield compared to the control nullizygous plants , in particular for total seed weight , number of filled seeds and harvest index . the total seed weight , number of filled seeds and harvest index are defined as above . in a first experiment , plants of the t1 generation of five independent events were compared , for each event 10 t1 plants carrying the transgene versus 10 corresponding control t1 plants . for the parameter “ total seed weight ”, two out of the five events had a significant increase ( 58 % and 67 %, with a p - value of the t - test of 0 . 0551 and 0 . 0211 respectively ). similar results were obtained for the number of filled seeds , for which these two lines showed in increase of 47 % and 68 % with a p - value of respectively 0 . 0846 and 0 . 0166 . the two lines also scored positive for harvest index ( increases of 41 % ( p - value of 0 . 0223 ) and 31 % respectively ). besides these two lines , a third line also scored significantly higher than the corresponding nullizygous control plants (+ 41 %, p - value of 0 . 035 ). the positive data for seed yield observed in the t1 generation were confirmed in the t2 generation . data are given in table 6 . the figures give the mean increase ( in %) of each parameter calculated from transgenes versus corresponding nullizygotes of 3 independent events in the t2 generation , each event comprising 20 plants carrying the transgene and 20 nullizygotes . the p - values of the f - test listed for the data of the t2 generation demonstrate that the obtained increases for the various seed yield parameters are all significant and that there is clearly an overall gene effect . aldemita , r . r . and hodges , t . k . 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