GENOMIC VARIABILITY IN MAIZE CALLUS CULTURES OF LINE P 346 AND ITS DERIVATIVE SOMACLONAL LINES

Cal lus tis sues from in di vid ual maize plants of line P346 and de rived from it through in vi tro tis sue cul ture of somaclonal vari ants show ing in creased re gen er a tion ca pac ity have been an a lyzed by RAPD. As early as af ter two months in cul ture maize calli ex hib ited ge nome changes but fol low ing four months their level de creased al most twice. The emer gence of homotypic DNA changes in calli ob tained from dif fer ent plants of in di vid ual lines was reg is tered thus sug gest ing the ex is tence of vari abil ity hot-spots within the maize ge nome. The somaclonal lines were shown to vary by the level of the ge netic changes in cul ture in vi tro.

In tro duc tion.The ap pli ca tion of cell and tis sue culture in vi tro as an area of bio tech nol ogy is widely used in sci en tific re search and prac ti cal ex per i ments.Along with other meth ods of tra di tional se lec tion this ap proach is em ployed to ex pe dite the ob tain ing of new ge no types of tra di tional ag ri cul tural plants with cer tain char ac ter is tics.This ap proach is based on somaclonal vari abil ity, to be more pre cise, on variabil ity of phys i o log i cal, bio chem i cal, and ge netic features of plant cells in cul ture in vi tro [1].
This phe nom e non was used for ob tain ing new somaclonal maize lines with in creased ca pa bil ity to the for ma tion of totipotent cal lus and re gen er a tive poten tial through the method of di rect se lec tion of calli, pos sess ing spe cific fea tures.New maize lines UKCh-5, UKCh-6, UKCh-7, UKCh-8, UKCh-9, distin guished by in creased re gen er a tion ca pac ity, have been de vel oped by T.M. Checheneva at the In sti tute of Plant Phys i ol ogy and Ge net ics NAS of Ukraine us ing the method of ar ti fi cial se lec tion of in bred maize P346 line cal lus cul tures for fur ther ap pli ca tion in bio techno log i cal de vel op ments [2,3].Anal y sis, per formed at V.Ya.Yurjev In sti tute of Plant Pro duc tion of Ukrai nian Acad emy of Agrar ian Sci ences (UAAS), re vealed the pres ence of ge netic dif fer ences in var i ous ag ro nomic in di ces be tween de vel oped somaclonal lines and orig inal P346 line (Gurjeva, un pub lished data).
In the pre vi ous pa per we re vealed mo lec u lar and ge netic dif fer ences be tween in bred P346 line and its somaclonal de riv a tives.We also re vealed that somaclonal lines dif fer in both de gree of dif fer ences from orig i nal P346 line and the level of intraline heter o ge ne ity [4].
In the cur rent work, to eval u ate the bio tech no log ical po ten tial of de vel oped somaclonal lines their genetic sta bil ity in vi tro was as sayed by PCR with ar bitrary prim ers (RAPD-anal y sis).
Maize seeds were ster il ized in 70% eth a nol so lution (2 min) and then in 20% Domestos aque ous solu tion (20 min).Then the seeds were washed 6 times with ster ile dis tilled wa ter and put for ger mi na tion on agarose me dium with half amount of salts ac cording to Murashige-Skoog [5].Cal lus cul tures were ob tained from in di vid ual plants with spec i fied unique num bers.To in duce callusogenesis explants were placed on nu tri ent me dium of the fol low ing contents: salts ac cord ing to Murashige-Skoog [5], 7.7 mg/l glycine, 1.25 mg/l nic o tine am ide, 0.25 mg/l of thi a mine, 0.25 mg/l of pyridoxine, 0.25 mg/l calcium pantothenate, 2 mg/l 2,4-D, 30 g/l su crose, 100 mg/l mesoinosite, 1 g/l asparagine, 8 g/l agar; pH 5.7 (prior to autoclaving).Ob tained cal lus tis sues were trans ferred to fresh me dium ev ery 30 days and grown in dark room at 25°C.DNA sam ples were iso lated ac cord ing to method, de scribed in [6], from in di vid ual 14-day-old seed -lings (the same plants, do nors of explants for ob taining cal lus cul tures, were used) and cal lus tis sues af ter 2-and 4-month cul ti va tion in vi tro.Con cen tra tion of the ob tained prep a ra tions was as sessed vi su ally by the in ten sity of flu o res cence of DNA-ethidium bromide com plexes in UV light af ter elec tro pho re sis in 1.5% agarose gel in re la tion to the con trol with known con cen tra tion (phage l DNA).
DNAs were hy dro lysed by re stric tion en zymes for 3 hours at con di tions rec om mended by man u facturer (Fermentas, Lith u a nia).Re ac tion mix ture, volume 30 ml, con tained 10 mg of in ves ti gated DNA and 20 un of restrictase.Hy dro lysed DNA was sep a rated in 1% agarose gel in 1´TAE-buffer at elec tric field gra di ent of 2 V/cm.South ern blot ting was per formed ac cord ing to the method of cap il lary trans fer, described in [7].Poly mor phic amplicons, iso lated from agarose gel by sil ica gel ad sorp tion us ing DNA extrac tion kit (Fermentas, Lith u a nia), were used as probes.DNA was la belled with [a-32 P]-dCTP (GE Healthcare, UK) us ing the method of ran dom priming, hy bridi sa tion was per formed at stan dard con ditions [7].
Re sults of anal y sis of RAPD-electrophoregrammes were pre sented as a bi nary ma trix, in which the pres ence/ab sence of amplicons was in di cated by 1/0, re spec tively.Poly mor phism of amplicons spectra was eval u ated us ing un weight ed pair group method with arith me tic mean (UPGMA) us ing POPGENE soft ware v.1.31[8].Dendrogramme of ge netic dis tances ac cord ing to Nei [9] be tween the ana lysed ob jects was con structed us ing MEGA software v. 3.1 [10].
Re sults and Dis cus sion.To study ge netic sta bility of somaclonal maize lines in vi tro, as well as orig inal P346 line, com par a tive anal y sis of in di vid ual plants of these lines and 2-month-old cal lus tis sues, ob tained from these tis sues, was per formed.Al together, DNA from 19 plants and their cal lus tis sues, in par tic u lar, P346 line (num ber of in di vid ual plants n = 2); UKCh-5 (n = 3); UKCh-6 (n = 4); UKCh-7 (n = 5); UKCh-8 (n = 3); UKCh-9 (n = 2) were ana lysed.Genetic anal y sis was per formed us ing PCR with ar bi trary decanucleotide prim ers -RAPD-PCR [11].The primers used had been pre vi ously ap plied in the in ves ti gation of ge netic maize poly mor phism [12,13].To tal num ber of prim ers used is 10.Se lected prim ers provided the for ma tion of dis tinct prod ucts of am pli fi cation (amplicons), the num ber of which var ied from 2 to 12, de pend ing on primer.Char ac ter is tic of prim ers and their prod ucts is pre sented in Ta ble 1.To tal number of ana lysed amplicons is 77, six of which (7.7%) show vari a tion be tween the plants and their de riv a tive cul ti vated tis sues (Ta ble 1).Poly mor phic amplicons were ob served in spec tra of only 3 of 10 prim ers used, namely M-07 (4 amplicons), M-06 and OPA-04 (1 amplicon each).Poly mor phism of RAPD-spec tra was re vealed as the dif fer ences in the pres ence of cer tain frag ments and in vari a tions in the in ten sity of some ho mol o gous frag ments (ma jor ity of cases).Upon the com par i son of spec tra the quan ti ta tive vari a tions were  RAPD-anal y sis showed ge nome vari abil ity, induced by in vi tro cul ti va tion, in both somaclonal maize lines and in orig i nal P346 line.Somaclonal maize lines dif fered in the level of vari abil ity in culture of 2-month-old tis sues, namely, in num ber of calli, show ing poly mor phic amplicons, and in num ber of poly mor phic amplicons within spec tra of cal lus tissues of one line (Ta ble 1).Thus, UKCh-7 and UKCh-9 calli dis play no poly mor phism of PCR-frag ments.In P346 line, only one out of two calli ob tained from differ ent plants lost sin gle amplicon.Sim i lar level of vari abil ity was dis cov ered for somaclonal line UKCh-5, where an ad di tional frag ment, ab sent in the spec trum of do nor explant, was found in cal lus of one out of three plants.Cal lus cul tures of two out of three plants of somaclonal UKCh-8 line, showed three polymor phic frag ments.The high est level of poly morphism is spe cific to UKCh-6, i.e. one to three vari able amplicons were dis cov ered in 2-month-old cal lus tissues of all four in ves ti gated plants.
Some of the poly mor phic amplicons were pres ent in cal lus tis sues of dif fer ent lines of plants (Ta ble 1), e.g.poly mor phic frag ment (~800 b.p. long), am pli fied with OPA-04, ab sent in do nor plants was dis cov ered in 2-month-old cal lus of sev eral plants of UKCh-5, UKCh-6, and UKCh-8 lines.The frag ment of 1600 b.p. (primer M-06) re vealed the dif fer ences be tween P346 and UKCh-6.How ever, the men tioned frag ment dis ap peared in spec tra of cal lus tis sues of P346 line, at the same time it arose within spec tra of three UKCh-6 plants.Note wor thy is the fact that UKCh-6 is a somaclonal vari ant of P346 line, ob tained by se lec tion from in vi tro cul ture.In ge nome of UKCh-6 this amplicon was lost (see pre vi ous pub li ca tion [4]).In cur rent work we also ob serve the loss of this amplicon in cal lus of one of P346 plants.On the other hand, in cal lus tis sues of sev eral plants of UKCh-6 we observed the re ap pear ance of this amplicon.Such nonrandomness in vari abil ity of poly mor phic RAPD-frag ments al lows sup pos ing the ex is tence of cer tain mech a nisms, de ter min ing changes in un sta ble ge nome re gions, re vealed us ing afore men tioned amplicons in tis sues of cul tures in vi tro.
To study the con tri bu tion of cul ture du ra tion to genetic vari abil ity of in ves ti gated maize lines in the culture of tis sues in vi tro we re peated the anal y sis of obtained cal lus cul tures once again af ter 4-month-cul tiva tion.Anal y sis dem on strated that in the ma jor ity of cases (9 of 15 poly mor phism man i fes ta tions), the differ ences, re vealed be tween do nor plant and their 2-month-old calli, dis ap peared in the course of fur ther cul ti va tion.These un sta ble poly mor phic frag ments are pre sented in pa ren the sis in Ta ble 1. Af ter four months of cul ti va tion, plant-cal lus dif fer ences persisted in one plant of P346 line, two plants of UKCh-6, and two UKCh-8 plants, while the num ber of polymor phic amplicons de creased to three (3.9%).
On the ba sis of RAPD-anal y sis con ducted we calcu lated ge netic dis tances ac cord ing to Nei [9] and constructed a dendrogramme of re la tions be tween the inves ti gated ob jects us ing POPGENE soft ware v. 1.31 [8].The ob jects on dendrogramme are grouped into clus ters cor re spond ing to each spe cific line (Fig. 2).The ex cep tion is in tact plant and cal lus tis sues of UKCh-5 No.2 and UKCh-7 No.8, which formed a sepa rate subcluster, rel a tively closer to UKCh-5.Line UKCh-5 was shown to be the clos est to the orig i nal line of P346, fol lowed by UKCh-7 and UKCh-9.UKCh-8 and UKCh-6 were most re mote.It has to be men tioned also that ac cord ing to the level of dif ferences be tween cul ti vated tis sues and do nor plants, somaclonal lines can be lo cated in ap prox i mately the same suc ces sion.In cul ture of plant tis sues of UKCh-7 and UKCh-9 re ar range ments were not discov ered at all; in UKCh-5 line there was only one poly mor phic frag ment re vealed in cal lus of one plant out of three in ves ti gated.The high est level of polymor phism is spe cific for 2-month-old tis sue cul tures of UKCH-6 and UKCh-8.De spite the fact that cal lus tis sues were ob tained from dif fer ent types of explants (seed lings and air-roots), the de pend ence be tween the type of explant and vari abil ity in cul ture in vi tro was not found.
Cer tain poly mor phic amplicons -RAPD-fragments ~1600 b.p. long (primer M-06) and ~800 b.p. (primer OPA-04) -vari abil ity of which was ob served in cal lus tis sues of plants of dif fer ent lines, were used as probes for ad di tional in ves ti ga tion by South ern blot hy bridi sa tion.These frag ments were iso lated from PCR-spec tra of 2-month-old cal lus tis sues of plant No.8 of UKCh-6 and 2-month-old cal lus plant No.4 of UKCh-6 re spec tively.South ern blot hy bridi sa tion of Fig. 2 Dendrogramme, con structed us ing UPGMA on the ba sis of ge netic dis tances ac cord ing to Nei [9] be tween in tact plants (P) and 2-month-old calli (K) of in ves ti gated maize lines poly mor phic RAPD-spec tra with cor re spond ing amplicons re vealed the pres ence of se quences, ho molo gous to probes on all lanes, even on those where PCR prod ucts were not ob served af ter stain ing with ethidium bro mide (Fig. 3).Thus, in this case, polymor phism of ex am ined amplicons is most likely involved in changes in PCR prod ucts num ber but not their for ma tion de novo.The re sults ob tained do not al low con clud ing with cer tainty to what ex tent such changes re flect the real ge netic re ar range ments.One of the pos si ble rea sons is that cul ture cul ti va tion results in amplicon copy num ber changes within the cell ge nome or point mu ta tions in the re gion of primer hybridi sa tion, lead ing to the in crease in its homology to the primer.
In ad di tion, we per formed South ern blot hy bridisa tion of poly mor phic amplicons with genomic DNA of P346, treated by re stric tion endonucleases to analyse the con tents and or gani sa tion of se quences of inves ti gated poly mor phic amplicons within the maize ge nome.The re sults of this anal y sis are pre sented in Fig. 4, i.e. the pres ence of sev eral large frag ments in the hy bridi sa tion spec tra, sug gest the mod er ate or high copy num ber of se quences of in ves ti gated amplicons.Thus, we have ana lysed ge netic vari abil ity in culture tis sue in vi tro of five somaclonal maize lines with in creased ca pac ity to the for ma tion of totipotent cal lus and re gen er a tive po ten tial.The ne ces sity of this inves ti ga tion re sult from pos si ble de sta bi li sa tion of genome as a con se quence of cul ti va tion of plant cells and tis sues in vi tro, which is one of the rea sons of somaclonal vari abil ity.
RAPD anal y sis was cho sen as a method of in ves tiga tion due to its sim plic ity and high ef fi ciency.This method is suc cess fully ap plied in the in ves ti ga tion of ge nome changes, in duced by cul ti va tion of plant tissues in vi tro.For in stance, Osipova et al. [12] revealed the changes in RAPD-spec tra of plant regenerants, maize line A188; Godwin et al. found them in PCR-spec tra of rice regenerants [14], Linacero et al. [15] -in PCR-spec tra of rye regenerants.The ap pli ca tion of RAPD-PCR al lowed de ter min ing genomic changes in cal lus tis sues of Cereus peruvianus [16] and to ma toes [17], grown on nutri ent me dia dif fer ing in con tent.At the same time there were no changes in some cases [18,19].
In the cur rent work we stud ied genomic re ar rangements in cal lus tis sues of maize P346 line and its somaclonal vari ants.Note wor thy is the fact that the changes were ob served as early as af ter two months of in vi tro cul ti va tion.At that mo ment the re ar rangements were de tected in calli of more than a half of inves ti gated plants -10 of 19.The changes were predom i nantly re vealed in the form of ad di tional fragments in the amplicons spec tra of cal lus tis sues.Fur ther cul ti va tion (af ter 4 months of in vi tro cul ti vation) re sulted in al most 2-fold de crease in the num ber of both poly mor phic frag ments and calli with polymor phic spec tra.Pos si ble rea sons for that may be quan ti ta tive changes in am pli fied se quences in cell genome or shift in the pro por tion of cells, car ry ing these se quences.In both cases this phe nom e non is most likely to be of phys i o log i cal ba sis and it ei ther re flects epigenetic changes, tak ing place dur ing cal lus for mation, or it is con nected with the dif fer ences in pro lif era tion rate of var i ous types of cells within the explant at dif fer ent stages of in vi tro cul ti va tion.
Sim i lar ity of re vealed genomic changes in cul tivated tis sues of in ves ti gated maize lines at tracts special at ten tion.In par tic u lar, poly mor phism of some PCR-frag ments was ob served in calli of sev eral plants (2-5 plants, Ta ble 1).In some cases (frag ment ~1 600 b.p. long, primer M-06, frag ment ~800 b.p. long, primer OPA-04) the same amplicons poly mor phism was ob served in plant of var i ous lines.The ap pearance of frag ments, not dis cov ered in calli of other plants, was de tected within the spec tra of one plant only -UKCh-6 No.7.These data sug gest the pres ence of re gions with in creased vari abil ity in maize ge nome.These "hot spots" of ge nome in sta bil ity were ob served in cul ture in vi tro of other plant spe cies, namely, rice [20], rye [15], Populus deltoides [21], and arabidopsis [22].
On the whole, the study per formed re vealed that somaclonal lines dif fered in the level of ge netic variabil ity in cul ture in vi tro.UKCh-7 and UKCh-9 calli fail to show any RAPD-frag ments poly mor phism at all.The high est level of poly mor phism was re vealed in the tis sue cul ture of UKCh-6 and UKCh-8 plants.Em ploy ment of UKCh-7 and UKCh-9 in gene en gineer ing will pro vide the low level of un de sir able muta tions at the stage of in vi tro cul ti va tion due to genomic in sta bil ity of explant.On the other hand, the lines with in creased genomic vari abil ity -UKCh-6 and UKCh-8 -may be used in breed ing prac tice for estab lish ing of new cultivars.
Ear lier we have per formed mo lec u lar and ge netic anal y sis of plants of in ves ti gated lines and de ter mined that somaclonal vari ants dif fer in the val ues of ge netic dis tances from P346, as well as in the level of intralinear het er o ge ne ity [4].UKCh-6 and UKCh-8 ex hib ited the high est intralinear het er o ge ne ity whereas UKCh-5 -the low est one.The most ge net ically dis tant from orig i nal P346 are the lines of UKCh-6 and UKCh-8, the least dis tant was UKCh-5.UKCh-7 and UKCh-9 oc cupy the in ter me di ate po sition by the val ues of ge netic dis tance from P346.There fore, it can be sup posed that there is a pos i tive re la tion ship be tween these three in di ces, namely level of ge netic vari abil ity in vi tro, intralinear het er o ge neity, and ge netic dis tance from orig i nal line.How ever, ver i fi ca tion of this re la tion ship as well as clar i fi ca tion of un der ly ing mech a nisms re quire some in-depth anal y sis with the greater sam pling of ob jects.
Con clu sions.Anal y sis of cal lus tis sues, ob tained from in di vid ual plants of in bred maize lines, us ing RAPD-PCR anal y sis, re vealed ge netic changes, induced by in vi tro cul ti va tion.Poly mor phism of RAPD-spec tra was ob served as early as in 2-month-old cal lus cul tures, but af ter 4 months of culti va tion the level of poly mor phism de creased al most 2-fold.The pres ence of ho mo ge neous changes in DNA in calli of dif fer ent lines may in di cate the pos sible ex is tence of hot spots of vari abil ity in maize genome.It has been de ter mined that among somaclonal lines dis play ing in creased re gen er a tion ca pac ity there were some to ex hibit high genomic sta bil ity in cul ture in vi tro, i.e.UKCh-7 and UKCh-9.
Ac knowl edge ments.The au thors are grate ful to T.M. Checheneva (In sti tute of Plant Phys i ol ogy and Ge net ics of NAS of Ukraine) and I.A. Gurjeva (V.
taken into ac count as pres ence/ab sence of frag ment only in the case of sig nif i cant (3-4 times) dif fer ences in the in ten sity of flu o res cence of amplicons.Spec tra of RAPD-frag ments of in tact plants and 2-month-old cal lus tis sues of P346 line, UKCh-5 and UKCh-6, obtained us ing primer M-06, which dem on strate the differ ences in cul ti vated tis sues from the plants, do nors of explants, are pre sented in Fig.1.

Fig. 1
Fig.1Spec tra of DNA ampli fi ca tion prod ucts of intact plants (P) and 2-month-old cal lus tis sues (K) of in bred maize lines with primer M-06.Numbers in di cate plants.Ar row marks the poly mor phic frag ment, which dif fer en tiates cal lus tis sues from plant -explant do nor.

Fig. 3
Fig.3 Changes in poly mor phic amplicons in tis sue cul ture of somaclonal maize lines: top panel -results of elec tro pho retic sep a ra tion of ampli fi ca tion prod ucts of plant DNA (P) and their cul ti vated tis sues of 2 and 4 month old (K2 and K4 re spec tively); bottom panel -South ern hy brid iza tion with se quences of cor re spond ing vari able amplicons (marked by ar rows on top panel); names of prim ers used in PCR are at the bottom.