Patent Application: US-201615064435-A

Abstract:
described are methods for promoting increase in plant biomass and yield . this increase has its visible effects in organs such as leaf , stem , root and production of fruits and seeds . further described is the increase in tolerance of those plants to drought , generating plants better adapted to the environmental changes , improving their growth , biomass and yield .

Description:
described herein is the use of aip1 , or a variant thereof , to increase plant biomass and / or yield , and to increase drought tolerance . the use , as indicated here , is the use of the protein , and / or the use of a nucleic acid sequence ( polynucleotide ) encoding this protein , or the complement thereof the gene includes , but is not limited to , genomic dna , cdna , messenger rna ( including the 5 ′ and 3 ′ untranslated regions ) and rnai . “ variants ” as used herein , include , but are not limited to , homologues , orthologues and paralogues of seq id no : 1 and seq id no : 2 ( aip10 protein and its splicing variant , respectively ). “ homologues ” of a protein encompass peptides , oligopeptides , polypeptides , proteins and enzymes having amino acid substitutions , deletions and / or insertions relative to the unmodified protein in question and having similar biological and functional activity as the unmodified protein from which they are derived . paralogues are genes within the same species that have originated through duplication of an ancestral gene ; orthologues are genes from different organisms that have originated through speciation , and are also derived from a common ancestral gene . orthologues are genes from different organisms that have originated through speciation , and are also derived from a common ancestral gene . preferably , the homologue , orthologue or paralogue has a sequence identity at protein levels of at least 50 %, 51 %, 52 %, 53 %, 54 % or 55 %, 56 %, 57 %, 58 %, 59 %, preferably 60 %, 61 %, 62 %, 63 %, 64 %, 65 %, 66 %, 67 %, 68 %, 69 %, more preferably 70 %, 71 %, 72 %, 73 %, 74 %, 75 %, 76 %, 77 %, 78 %, 79 %, even more preferably 80 %, 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 % most preferably 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 % or more when compared with seq id no : 1 and seq id no : 2 , aligned or not , as measured using genomic analyses tools as , but not limited to , the programs blastp , clustal and cobalt . as a non - limited example , orthologues of aip10 ( seq id no : 1 and seq id no : 2 ) are selected from the list consisting of seq id no : 3 - seq id no : 23 . increase of plant growth and / or yield is measured by comparing the test plant , comprising a gene used according to the disclosure , with the parental , non - transformed plant , grown under the same conditions as control . preferably , increase of growth is measured as an increase of biomass production . “ yield ” refers to a situation where only part of the plant , preferably an economical important part of the plant , such as the leaves , roots or seeds , is increased in biomass . the term “ biomass ” as used herein means an increase in weight / mass of certain parts of the plant , and can result from an increase in the area and / or increase in the quantity of this part of the plant . the term “ increase ” as used herein means at least a 5 %, 6 %, 7 %, 8 %, 9 % or 10 %, preferably at least 15 % or 20 %, more preferably 25 %, 30 %, 35 % or 40 % more yield and / or growth in comparison to control plants as defined herein . increase of plant growth , as used herein , is preferably measured as increase of any one or more of leaf biomass , root biomass and seed biomass . increase of plant drought tolerance is measured by comparing the test plant , comprising a gene used according to the disclosure , with the parental , non - transformed plant , grown under the same conditions as control . the term “ increase in plant drought tolerance ” as used herein means that test plants are able to support longer periods in soils with deficit in water availability , producing higher yields than the parental , non - transformed plant , grown under the same conditions . the term “ increase ” as used herein means at least a 5 %, 6 %, 7 %, 8 %, 9 % or 10 %, preferably at least 15 % or 20 %, more preferably 25 %, 30 %, 35 %, 40 %, 45 % or 50 % more yield and / or growth under drought conditions in comparison to control plants as defined herein . in this disclosure , aip10 expression is repressed or completely eliminated . repression refers to the expression in the modified plant , compared with the non - modified parental plant , grown under the same conditions , and means a reduction or complete elimination of the mrna levels and protein of the target gene or variants . repression of gene expression can be realized , as a non - limiting example , by gene silencing , antisense rna , rnai , artificial microrna , methodologies of genome editing ( zfn —“ zinc - finger nucleases ,” talens —“ transcription activator - like effector nuclease ,” crispr - cas , and others ), t - dna insertion , transposons and others . design of rnai and antisense rna is known to the person skilled in the art . as a non - limiting example , rnai can be designed with tools available on the internet . the rnai can be directed against a part of the 5 ′ untranslated terminal region , against a part of the coding sequence , and / or against the 3 ′ terminal region of the mrna . some non - limiting examples of target sequences are : seq id no : 3 , seq id no : 4 , seq id no : 5 , seq id no : 6 , seq id no : 7 , seq id no : 8 , seq id no : 9 , seq id no : 10 , seq id no : 11 , seq id no : 12 , seq id no : 13 , seq id no : 14 , seq id no : 15 , seq id no : 16 , seq id no : 17 , seq id no : 18 , and seq id no : 19 of the sequence listing . also described herein are genetically modified plants , containing rnai , or another method to decrease or eliminate gene expression , against a nucleic acid encoding aip10 or a variant thereof , as defined above , to increase plant growth , biomass and tolerance against drought stress . this rnai will target only a part of the nucleic acid , whereby the target sequence can be situated in the coding sequence , or in the 5 ′ or 3 ′ untranslated regions of the nucleic acid encoding aip10 or variant . a “ genetically modified plant ,” as used herein , is a plant which genome was modified by a recombinant dna construct and / or by genome editing , in which the referred recombinant dna can be introduced directly by transformation or indirectly by inbreeding or crossings . the rnai against a nucleic acid encoding aip10 or a variant thereof , or another method to decrease or eliminate gene expression , as defined above , means that the method is able to decrease or eliminate the expression of aip10 or a variant in a non - modified parental plant . repression of expression of a target gene can be obtained by transfer of a genetic construct . the transfer of foreign genes into the genome of a plant is called transformation . transformation of plant species is a fairly routine technique known to the person skilled in the art . advantageously , any of several transformation methods may be used to introduce the gene of interest into a suitable ancestor cell . the methods described for the transformation and regeneration of plants from plant tissues or plant cells may be utilized for transient or for stable transformation . transformation methods include , but are not limited to , agrobacterium - mediated transformation , “ floral dip ,” the use of liposomes , electroporation , chemicals that increase free dna uptake , injection of the dna directly into the plant , particle gun bombardment , transformation using viruses or pollen and microprojection . preferably , the plants used in this disclosure are from the group of commercially cultivated plants and arabidopsis thaliana . the term “ commercially cultivated plants ” as used herein means plants belonging to monocot , dicot and eudicot families , traditionally used in the agriculture , preferably , but not limited to : maize , soybean , cotton , sugarcane , sorghum , wheat , barley , millet , rye , oats , cocoa , beans , rice , grape , tomato , cassava , castor bean , papaya and poplar . described is a method of increasing plant biomass , plant yield and / or plant drought tolerance involves , but is not limited to : plant growth , rna extraction , dnase treatment , cdna synthesis and cloning in plant expression vector , plant transformation and generation of genetically modified plants with the rnai construct against aip10 or variant , or another method to decrease or eliminate gene expression , as defined above . described herein are methods of utilizing ( e . g ., by down - regulating or reducing the expression of ) the aip10 gene ( or a variant thereof ) in a plant , so as to promote increased biomass , plant yield , and / or to promote plant drought tolerance . in such a use , the aip10 may comprise a polynucleotide encoding a peptide of seq id no : 1 , seq id no : 2 , or a variant of either thereof . when a variant is used that is a homologue , or orthologue or paralogue variant of aip10 , it may have a sequence identity at the protein level of preferably at least 50 %, 51 %, 52 %, 53 %, 54 %, 55 %, 56 %, 57 %, 58 %, 59 %, 60 %, 61 %, 62 %, 63 %, 64 %, 65 %, 66 %, 67 %, 68 %, 69 %, more preferably 70 %, 71 %, 72 %, 73 %, 74 %, 75 %, 76 %, 77 %, 78 %, 79 %, even more preferably 80 %, 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, preferably more than 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 % or more , when compared to seq id no : 1 , seq id no : 2 . such use may take place in a plant species such as arabidopsis thaliana or any other species such as those selected from the group consisting of monocot , dicot , and eudicot , particularly commercially cultivated plants , preferably , but not limited to : maize , soybean , cotton , sugarcane , sorghum , wheat , barley , millet , rye , oats , cocoa , beans , rice , grape , tomato , cassava , castor bean , papaya , poplar , and / or others . to carry out the in vitro culture , arabidopsis thaliana ecotype columbia , plant knockouts for aip10 obtained from the salk bank of insertion mutants and rnai - aip10 plants were first grown in petri dishes with ms medium half strength . after 14 days growing in plates , the plants were transferred to pots containing autoclaved soil and vermiculite ( 3 : 1 ). for sterilization , seeds of a . thaliana , kept at 4 ° c ., were placed in microcentrifuge tubes containing 1 ml of 70 % ethanol for two minutes . after removal of the ethanol , the seeds were placed in a sodium hypochlorite solution with 5 % tween ® and 0 . 025 % for 10 minutes . the samples were washed five times with autoclaved distilled water . the sterilized seeds were placed on plates with solid culture medium with the aid of autoclaved sticks . the plants were grown in a greenhouse at 21 ° c . with a photoperiod of 10 hours of light and 14 hours of darkness . to carry out the in vivo cultivation of a . thaliana , the plants were transferred to pots containing autoclaved vermiculite and soil ( 3 : 1 ) after 14 days of in vitro growing on plates . the plants were grown in a greenhouse at 21 ° c . with a photoperiod of 16 hours of light and 8 hours of darkness . the plants were collected for molecular and phenotypic analysis , at varying times after cultivation and in some cases after different regimes of suspension and resumption of water irrigation . the collected plant material was immediately frozen in liquid nitrogen and stored in a freezer at − 80 ° c . for rna extraction , the material was macerated in liquid nitrogen and rapidly transferred to 1 . 5 ml microtubes containing 500 ul of tle buffer ( 200 mm tris - cl , 100 mm licl , 5 mm edta , 1 % sds , ph 7 . 5 ) 250 μl 250 μl of phenol and chloroform . the microtubes were stirred for 1 minute and centrifuged at 12000 g for 20 minutes at 4 ° c . after centrifugation , the aqueous phase was transferred to microcentrifuge tubes and was added to one volume of chloroform : isoamyl alcohol ( 24 : 1 ). the new mixture was stirred for 1 minute and centrifuged at 20000 g for 15 minutes at 4 ° c . the aqueous phase was transferred to microcentrifuge tubes and 1 volume of 6 m licl with diethyl pyro - carbonate ( depc ) 0 . 1 % was added . the tube was shaken for 1 minute and kept at 4 ° c . for 16 hours . the next day , tubes were centrifuged at 12000 g for 20 minutes at 4 ° c ., the supernatant discarded and the precipitate solubilized in 1 ml 3m licl , depc 0 . 1 %. the tubes were again centrifuged at 12000 g for 20 minutes at 4 ° c ., the supernatant discarded and the precipitate solubilized in 250 μl h 2 o with 0 . 1 % depc . 1 / 10 volume of 3 m naoac ph 4 . 8 with 0 . 1 % depc and 2 volumes ( considering the amount of added naoac ) of absolute ethanol were added to these samples . the samples were homogenized and incubated for 30 minutes at − 80 ° c . or for 2 hours at − 20 ° c . after incubation , the tubes were again centrifuged at 12000 g for 20 minutes at 4 ° c ., the supernatant was discarded and the precipitate was washed with 1 ml of 70 % ethanol with 0 . 1 % depc . again , the tubes were centrifuged at 12000 g for 20 minutes at 4 ° c ., the supernatant was discarded and the precipitated rna was solubilized in 20 μl h 2 o with 0 . 1 % depc . total rnas were treated with dnase i ( new england biolabs ) to eliminate any contamination with genomic dna . 0 . 5 u of dnase i was used for each 1 μg of total rna in enzyme buffer ( 200 mm tris - cl ph 8 . 3 , 500 mm kcl , 25 mm mgcl2 , 0 . 1 % depc ). rnas were incubated with buffer and dnase at 37 ° c . for 15 minutes . the rna was purified by adding 1 volume of phenol , the phases were mixed by vortexing and then centrifuged at 20000 g for 10 minutes . the aqueous phase was transferred to a fresh tube to which was added 1 v of chloroform . a new centrifugation at 20000 g for 10 minutes was carried out and the aqueous phase was transferred to a new tube . the rna was then precipitated by adding 1 / 10 v 3 m naoac 0 . 1 % depc and 2 v of absolute ethanol , followed by incubation at − 80 ° c . for 20 minutes , and centrifuged at 20000 g for 20 minutes . the supernatant was discarded and the precipitated rna was washed with a solution of 70 % ethanol with 0 . 1 % depc , after which it was solubilized in milli - q ® water with 0 . 1 % depc . after treatment with dnase , the first strand cdna was synthesized using the superscript ® iii reverse transcriptase with total rna samples ( invitrogen ). the following protocol enables cdna synthesis in a reaction where there is a range of 10 ng to 5 μg of total rna . it was added into a microtube of 0 . 5 ml : 1 μl oligonucleotide oligo ( dt ) 20 ( 50 mm ); 10 ng - 5 μg of total rna ; 1 μl dntp ( 10 mm ) and the volume was complete with sterile distilled h 2 o to 13 μl . the mixture was heated at 65 ° c . for 5 minutes and incubated on ice for 1 minute . after , 4 μl of 5 × first - strand buffer ( buffer first tape ); 1 μl dtt ( 0 . 1 m ); 1 μl of rnaseout recombinant rnase inhibitor ( 40 u / μl ) and 1 μl of superscript ® iii ( 200 u / μl ) were added to the microtube . the reaction was incubated at 50 ° c . for 60 minutes and then inactivated at 70 ° c . for 15 minutes . to remove remaining rna , 2 u of rnaseh ( usb , affymetrix ) were added to the microtube , and it was incubated at 37 ° c . for 15 minutes . the aip10 amplification pcr reactions were performed in a mj - research thermocycler ( ptc - 100 ) using the following conditions : after pcr amplification , the generated fragments were verified on a 1 % agarose gel . the bands with dna fragments with the size of interest were excised from the gel and placed in a microcentrifuge tube . the kit wizard sv gel and pcr clean - up system ( promega ) was used to make the purification of genes . after elution , the dna was quantified using a n ano d rop ® 2000 spectrophotometer ( thermo scientific ). after purification of the fragments of interest from the agarose gel , a reaction between the recombination sites attb1 and attb2 placed in the fragment using the primers and attp1 and attp2 sites present in the vector pdonr221 ( invitrogen ) was performed . this reaction , called bp is part of the invitrogen gateway technology , and utilizes the gateway kit bp clonase ii enzyme mix . to the reaction , 1 to 7 μl of the purified pcr product from the 1 % agarose gel to a final amount of 15 to 150 ng ; 1 μl of pdonr221 ( 150 ng / μl ); 2 μl bp clonase ii enzyme mix ; and autoclaved milli - q ® water to a final volume of 10 μl were pipetted in a microtube of 1 . 5 ml . the reactions were incubated at 25 ° c . for 1 hour and at the end of this time , the reaction was stopped by adding 1 μl of proteinase k and incubating at 37 ° c . for 10 minutes . after completion of the bp reaction , the samples were dialyzed for 3 hours in 0 . 025 μm membranes ( millipore ). for electroporation , 40 pl of electro - competent bacteria were used . the electroporation was done in an electroporation cuvette of 1 . 8 kv to 25 μf and 200ω in an eppendorf eletroporador as described by ausubel et al . ( 1992 ). after the formation of the entry clone , the transfer of aip10 gateway to another vector was made through a recombination reaction called lr . in this reaction , the gene of interest is transferred to other plasmids with attr recombination arms . this reaction also is part of the invitrogen gateway technology , and utilizes the gateway lr clonase ii kit enzyme mix . to the reaction , it was pipetted : 75 ng the destination vector , 150 ng entry clone , 1 μl lr buffer , 0 . 5 μl of enzyme lr and autoclaved milli - q ® water to a final volume of 5 μl . the reactions were incubated at 25 ° c . for 1 hour . the recombination reaction was dialyzed for three hours in a 0 . 025 μm membrane ( millipore ). agrobacterium tumefaciens containing the plasmid of interest was inoculated into lb medium without antibiotics and incubated at 28 ° c . under shaking conditions for eight hours . after this time , 9 ml of lb without antibiotic was added to the culture of these bacteria and were incubated at 28 ° c . under agitation until the od 2 . 0 at 600 nm . 40 ml of milli - q ® water containing 10 % sucrose and 0 . 05 % silwet ® were added to the a . tumefaciens culture and this mixture was used immediately . a . thaliana plants growing in soil with inflorescence stems of 7 to 10 cm were immersed in the mixture containing the bacteria for 2 to 3 seconds according to the established protocol ( clough ; bent , 1998 ). these plants were covered with plastic pvc and kept in a humid environment at 21 ° c . for 24 hours . after this time , the plastic was removed and the plants were kept in a greenhouse at standard growth conditions . the strains of transformed plants ( t0 ) had their seeds collected , and they were plated on selective medium ( vectors used in the selection was 50 μg / ml kanamycin ). the resistant plants ( t1 ) were transferred to the soil and the seeds were collected . ti seeds generated t2 plants that were analyzed for segregation . the first strand cdna was synthesized using the kit “ t aq m an ® first strand cdna synthesis ” and it was performed in reactions with a final volume of 25 μl , according to the manufacturer . for each reaction , 500 ng of total rna were added plus 2 . 5 μl 10 × t aq m an ® rt buffer , 25 mm of mgcl2 5 . 5 μl dntps mix , 1 . 25 μl of random hexamer , 0 . 5 μl rnase inhibitor , 0 . 625 μl of m ulti s cribe ™ reverse transcriptase ( 50 u / μl ). the samples were incubated at 25 ° c . for 10 minutes , followed by 48 ° c . for 30 minutes and a final step at 95 ° c . for 5 minutes . samples were diluted four times with 10 mm tris - cl ph 8 . 0 and stored at − 20 ° c . or used immediately . the real - time pcr was performed with sybr green pcr master mix kit of the company applied biosystems , according to manufacturer &# 39 ; s recommendations . the reaction was performed in a 96 - well plate ( microamp optical 96 - well reaction plate , applied biosystems company ). in each well , 1 μl of the first strand reaction was placed , 5 μl of the kit mix solution , and 4 μl of a mixture of the two oligonucleotides at 10 mm each . for each reaction , which used the specific oligonucleotides for each gene , another reaction with primers specific for the ubiquitin 14 gene ( ubi14 ) was taken as a constitutive gene in all plant cells . this reaction was used as a positive control and to normalize the amount of first strand cdna used in the experiments . as a negative control , a reaction was made in the same positive control conditions without the first strand cdna . to normalize the samples by the amount of first strand cdna used , the fluorescence value of each sample with primers specific for the gene in question was then divided by the fluorescence value of the sample with the ubi14 primer . ubi ct is the average number of cycles in the chosen point of the control with ubi14 . ct gene is the average number of cycles in the chosen point of the gene in question . to calculate the relative expression of the experiments with the experimental control , the following relationship was made : fl . rel . exp / fl . rel cont . to verify the biomass of a . thaliana rosettes , aip10 knockout plants and rnai - aip10 , and control plants , of 27 days were collected and immediately weighed on a precision balance . data were statistically analyzed and considered different ( t - test ). measurements of height , silique production and seed yield were made with aip10 knockout plants and rnai - aip10 , and compared with control plants . the height of the main axis of the plants was measured with the aid of a measuring tape and the number of siliques produced was counted throughout the development . data were statistically analyzed and considered different ( t - test ). the production of seeds of each plant was individually evaluated . seeds were collected , and the total number of seeds produced by each individual was weighed on a precision scale and the values obtained were analyzed and considered statistically different ( t - test ). to evaluate the tolerance to water stress , plants were cultivated and watered normally for 25 days . then watering was suspended for 7 days in a group of plants , and for 12 days in the other group . after this period , watering was resumed and after 7 days , plant survival rate was evaluated . at the end of development , the number of siliques produced by each individual was counted . data were statistically analyzed and considered different ( t - test ). in order to better understand the function of the aip10 gene , knockout plants for the gene were obtained in the salk collection of insertion mutants . confirmation of the t - dna insertion was identified by pcr ( fig1 ) and confirmation of absence of aip10 mrna was made by qrt - pcr ( fig2 ). to evaluate the effect of silencing the expression of aip10 on the development of a . thaliana , knockout and wild plants were grown under the conditions described above . fig3 a - 3e show the phenotypical differences in plants with reduced levels of aip10 . these plants have larger rosette leaves and more developed roots . to understand which developmental processes aip10 is controlling in plants , phenotype of plants silenced for aip10 ( aip10ko ) was characterized along development . the analyses showed that root length is longer in aip10ko plants all over development ( fig4 a , 4b ), and they develop more lateral roots ( fig4 a , 4c ) compared to wild - type plants . in the aerial part , aip10ko plants develop more leaves , and in a higher speed ( fig5 a - 5c ), demonstrating that aip10 controls the timing of development of new organs . kinematics studies of leaf development showed that aip10ko leaves have higher rates of cell divisions during a prolonged period ( fig6 a ), higher number of cells ( fig6 b ) and larger area of leaves ( fig6 c ). it shows that aip10 controls the growth and final size of organs in plants , by regulating cell division rates and final cell numbers . during the reproductive phase , aip10ko plants produce inflorescence main axis longer and with more branches , compared with wild - type plants ( fig7 a - 7c ). all together , the data shows that silencing of aip10 increases cell division rates in all main plant indeterminate meristems ( root apical meristem , root pericycle , shoot apical meristem and inflorescence ), leading to meristems formed with more cells . by speeding cell divisions and the provision of differentiating cells , aip10ko organogenesis might be accelerated , developing plants with increased cell number and biomass . also , aip10 silencing prolongs cell proliferation activity , delaying senescence , which effectively increases organs sizes and lengthens the lifespan of the plant . to quantify aip10ko biomass , within 27 days after germination , rosettes of control and mutant plants were collected for measuring the fresh weight ( fig8 a ). knockout plants have greater fresh weight than control plants . the production of siliques and seeds in control and aip10 knockout plants was also quantified . silique yield quantified in inflorescence main axis was higher in aip10ko plants , compared with wild - type plants ( fig8 b ). the amount of seed produced by each plant was weighed separately ( fig8 c ) in order to quantify the yield of plants . plants knockout to aip10 produced about 30 % more seeds than control plants . these results demonstrate that the disclosure leads to an increase in plant biomass and an increase in fruit and seed yield . wild - type and aip10 knockout plants were cultivated and watered normally until they are 25 days old . after this period , a group was subjected to water stress for 7 days and another for 12 days . after these periods , the plants returned to be watered noilnally . the survival rates of aip10 knockout and wild - type plants were measured after water deficit conditions ( fig9 a and b ). after a shorter period ( 7 days ) without water , aip10ko and wild - type col0 - 0 plants had the same survival rates following irrigation ; while after a prolonged period of 12 days without water , a higher percentage of aip10ko plants survived . it demonstrated that the disclosure produces plants more tolerant to drought stress . to assess the productivity of these plants after the water stress , the number of siliques produced by plants that survived the stress was evaluated ( fig1 a and 10b ). aip10ko plants produced more siliques than wild - type plants after growing under drought stress , even after shorter periods of drought stress ( 7 days without irrigation ), when the survival rates of aip10ko and wild - type plants was similar . it demonstrated that the disclosure produces plants with higher productivity when cultivated under drought stress . increase in biomass and yield in plants with reduced levels of aip10 ( aip10 rnai construct ) to evaluate the effect of aip10 rnai construct on the development of a . thaliana , wild - type plants transformed with the construct were grown under the conditions described above and their phenotype was evaluated . it was confirmed by qrt - pcr that plants transformed with the construct have reduced levels of aip10 ( fig1 ). as can be seen in fig1 , plants with reduced levels of aip10 ( containing the rnai construct ) have more leaves than control plants , as well as larger leaves , showing increased final biomass , in a phenotype similar to that of aip10 knockout plants . in fig1 , it can be seen that inflorescence of plants with reduced levels of aip10 are taller and have more branches , in a phenotype similar to that of aip10 knockout plants . the results show that the reduction in aip10 levels is responsible for increasing plant biomass and / or yield and / or drought tolerance . the data also show that different technologies can be used as a method for reducing or eliminating aip10 levels . among them , the insertion of t - dna , transposons , artificial microrna , rnai , antisense rna , methodologies of genome editing , among others , can be used . although particular embodiments of the disclosure have been shown and described , various combinations , changes and modifications may be made in this disclosure to meet specific needs without departing from the disclosure and from its broader aspects . in addition , while a particular feature of the disclosure may have been disclosed with respect to only one of several embodiments , such feature may be combined with one or more other features of other embodiments , as far as may be desired and advantageous for any particular application . 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