In vitro induction of tetraploids in Phlox subulata L.

In vitro induction of tetraploids in Phlox subulata L.Zhihong Zhang ÆHongyan Dai ÆMin Xiao ÆXin LiuReceived:19November 2006/Accepted:8May 2007/Published online:27May 2007ÓSpringer Science+Business Media B.V.2007Abstract Tetraploid plants of Phlox subulata L.were induced successfully by treating shoot tips in vitro with colchicine.Shoot tips excised from in vitro shoots were treated with four different concentrations of colchicine (0.005,0.01,0.02,0.04%)in solid MS medium supplemented with 4.54m M TDZ and 0.49m M IBA for 10,20or 30days,respectively.The survival rates of shoots tips were affected by the concentration of colchicine and the duration of treatment.High concentration and longer duration reduced survival of the shoot tips,but the effect of duration of colchicine was more than that of concentration.Tetraploid plants were obtained in all of the treatments,but the percentages of tetraploids varied among different treatments,from 25.0%to 75.0%.The most efficient condition for inducing tetraploids was to treat shoot tips with 0.005%colchicine for 20days,with 30.0%survival rate of shoot tips and 6tetraploid plants out of 10plants examined.The rooted tetraploid plants were transplanted successfully in a solar greenhouse.Under the same growing condition,significant vari-eties in flower bud and flower sizes were detectedbetween 2x and 4x plants.The flower diameters of tetraploid and diploid plants were 2.91cm and 2.24cm,respectively.Keywords Phlox subulata L.ÁTetraploid ÁInduction ÁColchicine ÁShoot tip ÁFlower diameter Abbreviations BA 6-Benzylaminopurine GA 3Gibberellic acid IBA Indole-3-butyric acid MS Murashige and Skoog (1962)NAA a -Naphthaleneacetic acid TDZ ThidiazuronIntroductionPhlox subulata L.,a species of the Phlox genus in the Polemoniaceae,is popularly used in home gardens and landscapes throughout the world as a mounded,low-growing perennial plant with needle-like,semi-evergreen foliage (Eom et al.2006).The plants of P.subulata grow no more that about 6-inches-tall forming dens mats which are suitable for rock gardens,ground covers or for planting on top of a garden wall (Wherry 1955).Clustered inflorescences occur at the termini of the very short flowering stems,covering the entire prostrate plant with a stunning carpet of small five-petaled flowers in early springZ.Zhang (&)ÁH.Dai ÁM.Xiao ÁX.Liu College of Horticulture,Shenyang AgriculturalUniversity,Dongling Road 120,Shenyang,Liaoning 110161,P.R.Chinae-mail:zhangz@Euphytica (2008)159:59–65DOI 10.1007/s10681-007-9457-8(Schnabelrauch and Sink1979;Li et al.2005).It makes a nice stabilizer for a sloping landscape.The genus Phlox comprises65species,all of which are native to North America except one,which is in Siberia(Ferguson and Jansen2002;Meyer 1944).The chromosomes of Phlox are relatively large and comparatively few in number.The plants of most species have the diploid somatic number of 2n=2x=14chromosomes,and polyploid plants only exist in several species.For P.subulata,almost all cultivars are diploid and a few tetraploid plants were found in North America(Meyer1944).Polyploidy is a prominent process and has been significant in the evolutionary history of plants (Adams and Wendel2005).It has been estimated that up70%of land plants and95%of ferns have some polyploidy in their evolutionary history(Leitch and Bennett1997;Otto and Whitton2000).Poly-ploidy can result in a wide range of effects on plants, including significant enlargement of fruits andflow-ers,longer lasting time forflowers,overcoming barriers to hybridization,no seed or fewer seed, enhancing pest resistance and stress tolerance(San-ford1983;Predieri2001).Specific effects will vary dramatically based on the species involved,the degree of heterozygosity and ploidy level.Colchicine,a compound that effectively arrests mitosis at the anaphase stage,is widely used to induce polyploidy in plants.In vivo treatment of shoots,smaller axillary or sub-axillary meristems, seeds or seedlings with colchicine is traditionally employed for generating polyploids.However,low efficiency of polyploidy plants production and a high frequency of chimeras are often associated with this method.With the development of adventitious organ regeneration techniques,in vitro induction of poly-ploidy has been became the main method in many plant species,including Pyrus pyrifolia(Kadota and Niimi2002),Musa acuminata(Van Duren et al. 1996),Morus alba(Chakraborti et al.1998),Bixa orellana(Carvalho et al.2005),etc.It shows that in vitro induction increases efficiency and decreases occurrence of chimeras than in vivo.In our previous research of in vitro propagation of P.subulata,we found that multiple bud clumps were differentiated on calli induced from shoot tips of P. subulata at high frequency(Dai et al.2004),so the present work aimed to establish a methodology for in vitro induction of tetraploid in P.subulata by treating shoot tips with colchicine.Materials and methodsPlant materials and establishment of shoot culture Phlox subulata cv.Emerald Pink was used in this study.The shoots were collected from plants actively growing in an experimentalfield plot at the Shenyang Agricultural University,China.The terminal shoots were washed with running water for1h,then surface-sterilized by immersion in70%(v/v)ethanol for30s, followed by soaking in a solution of HgCl2(0.1%,w/ v)for10min.After the terminal shoots were rinsed three times with sterile distilled water,the shoot tips (1mm)were excised and inoculated on MS medium (Murashige and Skoog1962)supplemented with 1.78m M BA,0.20m M IBA and2.89m M GA3to establish shoot proliferation in vitro.For shoot multiplication,stem cuttings(1cm in length)from the in vitro shoots were placed vertically on MS medium supplemented with0.89m M BA,0.20m M IBA and0.58m M GA3and subcultured at5-week intervals.Colchicine treatmentShoot tips(1mm)were excised from in vitro shoots and inoculated on MS medium supplemented with 4.54m M TDZ,0.49m M IBA and different concen-trations of colchicine(0.005,0.01,0.02and0.04%). The duration of treatment was10,20or30days. After the treatment,the explants were transferred on the fresh medium of similar composition without colchicine.Experiments were set up in a completely randomized design and repeated two times with15 explants per treatment.The survival rate[(number of survival shoot tips/total number of shoot tips)·100%] was assessed after the shoot tips had been cultured for 60days.Medium preparation and culture conditionsAll of media were gelled with0.7%agar(w/v). After adjusting the pH to 5.8,the medium was sterilized by autoclaving at104kPa at1218C for20min.Filter-sterilized colchicine was added into the medium before it gelled.Each100-ml bottle con-tained35ml medium,and15explants were inocu-lated.After15days culture in darkness,the explants were transferred under cool,whitefluorescent light (60m mol mÀ2sÀ1)with a14-h photoperiod light.The temperature of the culture room was maintained at 23±28C.Chromosome observationThe verification of chromosome number was per-formed after the regenerated plants reached3cm in height.Actively growing shoot tips about8–10mm were excised from in vitro shoots and pre-treated with 0.05%colchicine at208C for4h.Then they were washed and transferred tofixative solution—95% ethanol:acetic acid(3/1,v/v)–for1week at room temperature.Thefixed shoot tips were hydrolyzed in 5mol/l HCl for8min at room temperature.After hydrolysis,shoot tips were rinsed three times with distilled water and then stained with Carbol fuchsin. At least20well-spread metaphases per shoot were observed under the microscope.Rooting and transplantationRegeneration shoots were excised from the original explants and then transferred to MS medium supple-mented with0.89m M BA,0.20m M IBA and0.58m M GA3for subculture.Elongated shoots>3cm were transferred to MS/2medium supplemented with 0.25m M IBA and0.27m M NAA for rooting.Rooted plantlets were transferred to plastic pots containing substrate composed with peat moss and vermiculite (2/1,v/v).The pots were covered with plasticfilm, placed in a solar greenhouse kept at15–258C and a 12-h photoperiod light,and irrigated with a solution of1/8-strength MS inorganic salts at2-to3-day intervals.The percentage of shoots rooted[(number of shoots with roots/total number of shoots)·100%] was scored after the shoots were cultured in rooting medium for4weeks.The percentage of transplanta-tion plants survived[(number of plantlets survived/ total number of plantlets transplanted)·100%]was recorded after the rooted plantlets grew in vivo for 90days.Analysis of morphological characteristicsThe investigation of the morphological characteristics of leaf,stem,flower bud andflower was performed after the rooted plantlets grew in vivo for4months. The sizes of leaf,flower buds andflowers were measured with a vernier caliper.Two-sample t-test was used for significance analysis.ResultsSurvival and growth of colchicine-treated shoottipsAfter the shoot tips excised from in vitro shoots were incubated on the regeneration media with colchicine for10days,they began to expand on the middle of explant.On the contrary,the explants incubated on the regeneration medium without colchicine did not expand on the middle,but formed calli on the base of explants.After the explants treated with colchicine were cultured on media for15days,shoot tip expansion in the middle of explant was visible,in a way that most explants appeared to be balls(Fig.1A). Some explants treated with colchicine leaded to browning and death after50days of induction culture,but many calli and buds were induced on the survival explants(Fig.1B).After4months of induction culture,some buds induced on the survival explants developed into shoots.The tetraploid shoots regenerated from shoot tips treated with colchicine were strong(Fig.1C),and the diploid shoots regenerated from shoot tips without colchicine treat-ment were slim(Fig.1D).The survival rates of shoots tips were affected by the concentration of colchicine and the duration of treatment(Table1).In general,high concentration and longer duration reduced survival of the shoot tips. The mortality was100%when shoot tips were treated with0.04%colchicine for30days.The effect of duration of colchicine was more than that of concentration,for example,the survival rate de-creased from30.0%to10.0%when the duration at 0.01%colchicine was prolonged from10days to 20days,but for10days treatment,the survival rate only decreased from30.0%to20.0%when the concentration of colchicine increased from0.01%to 0.02%.Verification of chromosome number and evaluation of polyploidy induction capacity Cytological analysis established that the chromosome number of diploid plants of P.subulata was 2n =2x =14and that of tetraploid plants was 2n =4x =28(Fig.2).Seven cultivars of P.subulata ,including ‘Emerald Pink’,which were planted in Shenyang Agricultural University,were examined in ploidy level,and all of them were diploid.All plants regeneration from shoot tips of ‘Emerald Pink’without colchicine treatments were diploid,but the tetraploid plants were found among plants regener-ated from the shoot tips treated with colchicine.The same doubling event was not found more than once.In total,82plants regenerated from shoot tips treated with colchicine were investigated in ploidy level,and 38tetraploid plants were obtained.Although the tetraploid plants were found in all of the treatments,the percentages of tetraploids were varied acutely among different treatments,from 25.0%to 75.0%.When shoot tips were treated with colchicine for 10days,the rates of chimeras were high (52.6%).With 30days treatment,the rates of chimeras were low (27.8%),but the survival rates of explants decreased also,so the number of tetraploid plants was low.The most efficient condition forinducing tetraploids seemed to be treatment with 0.005%colchicine for 20days,with 30.0%survival rate of shoot tips and 6tetraploid plants out of 10plants examined (Table 1).Rooting,transplantation and morphological characteristicsAfter reaching a height of 3cm,shoots of P.subulata were transferred to MS/2medium supplemented with 0.25m M IBA and 0.27m M NAA for rooting.About 70%of the shoots rooted successfully,and there was no obvious difference in rooting ability between the diploid and tetraploid shoots.Rooted plantlets were transplanted into plastic pots containing peat moss and vermiculite (2/1,v/v)for further growth in the solar greenhouse.After 90days,more than 60%plants were survived.Four months after transplantation,the plants began to flower.Variants in the morphological characteris-tics of leaf,stem,flower bud and flower were observed between 2x and 4x plants under the same growing condition.Tetraploid plants had shorter leaves with increased leaf width,and the leaf index (leaf length/leaf width)was decreased from 9.29(diploid)to 8.63(tetraploid)(Table 2).There was no significant difference in flowing time betweendiploidFig.1Tetraploid plant regeneration from shoot tips of Phlox subulata cv.Emerald Pink treated with colchicine.Bars =3mm.(A )Shoot tip expansion in the middle of explant.(B )Callus and buds induced on the survival explant and the browning and dying explants.(C )Tetraploid shoots formed from explants treated with colchicine.(D )Diploid shoots and bud clumps formed from explants without colchicine treatment (CK)and tetraploid plants.However,the tetraploid plants had larger flower buds and flowers than the diploid plants (Fig.3).Flower diameters of tetraploid and diploid plants were 2.91cm and 2.24cm (Table 2),respectively,and the difference was significant (P <0.05).DiscussionFlower size is an important character for ornamental plants.This paper first describes the methodology for efficient in vitro induction of tetraploid in P.subulata by treating shoot tips with colchicine.It is feasible to induce tetraploid plants with larger flowers in P.subulata by treating the shoot tips with colchicine in vitro,based on the multiple bud clumps differen-tiation from shoot tips.The percentages of tetraploid plants in P.subulata ,25.0–75.0%(Table 1),are higher than that of other plant species,such as Zizyphus jujuba Mill.(0–5.0%)(Gu et al.2005)and Alocasia (0–10.5%)(Thao et al.2003).The reasons for this may arise from two aspects.One is that theTable 1Effect of different concentrations and durations of colchicine on the survival rate of shoot tips of Phlox subulata cv.Emerald Pink Treatmentduration (days)Colchicine concentration (%)Survival rate a (%)No.of plants examined in cytological analysis Ploidy Diploid Tetraploid Mixploid 100(CK)1001010(100)b 0(0)0(0)0.00540.0154(26.7)4(26.7)7(46.6)0.0130.0101(10.0)3(30.0)6(60.0)0.0220.080(0)2(25.0)6(75.0)0.0416.751(20.0)3(60.0)1(20.0)Total 386(15.8)12(31.6)20(52.6)200(CK)1001010(100)0(0)0(0)0.00530.0100(0)6(60.0)4(40.0)0.0110.081(12.5)5(62.5)2(25.0)0.0216.751(20.0)2(40.0)2(40.0)0.046.730(0)2(66.7)1(33.3)Total 262(7.7)15(57.7)9(34.6)300(CK)1001010(100)0(0)0(0)0.00520.082(25.0)4(50.0)2(25.0)0.0110.060(0)4(66.7)2(33.3)0.02 6.7403(75.0)1(25.0)0.04––––Total182(11.1)11(61.1)5(27.8)a Survival rate was assessed after the shoot tips had been cultured for 60days bFigures in parentheses are the percentages of diploids,tetraploids ormixploidsFig.2(A )Chromosome number of shoot tip in diploid Phlox subulata (2n =2x =14).(Bar =4m m).(B )Chromosome number of shoot tip in tetraploid P.subulata (2n =4x =28).(Bar =4m m)shoot tips of P.subulata were treated with colchicine in solid medium at low concentration for long time but not in colchicine solution at high concentration for short time.The other is that the calli of induced from shoot tips of P.subulata had the strong ability for adventitious bud clumps regeneration (Dai et al.2004).In vitro propagation of P.subulata was first achieved through auxiliary bud culture by Schnabelr-auch and Sink (1979).Recently,seven cultivars of P.subulata were micropropagated successfully in our laboratory,and we found that calli induced from shoot tips of all of seven cultivars exhibited bud differentiation at high frequency on MS medium supplemented with 0.91–4.54m M TDZ.So,the methodology for induction of tetraploid would be suitable for all cultivars of P.subulata .Chromosome duplication in plants has been mainly accomplished by the use of antimitotic substances,such as colchicine,oryzalin,trifluralin,etc (Hansen et al.1998;Petersen et al.2003;Eeckhaut et al.2004).In vitro treatment of shoots or shoot tips with colchicine is one of prevalent methods of polyploidy induction,which was suc-cessfully applied to a number of plant species,including Pyrus pyrifolia (Kadota and Niimi 2002),Zingiber officinale (Adaniya and Shirai 2001),Punica granatum (Shao et al.2003),Alocasia (Thao et al.2003),and Zizyphus jujuba Mill.(Gu et al.2005).It is easy to induce buds from shoots or shoot tips,which is the main advantage of this induction method and is very important to the plants that are recalcitrant to adventitious organ regeneration.How-ever,a higher frequency of chimeras (mixploids)is associated with this induction method (Thao et al.2003;Gu et al.2005).The results in this research showed that the percentage of mixploids decreased with protracting the duration of treatment.The efficient method of separation of chimeral plants into pure types relies on adventitious organ regeneration (Abu-Qaoud et al.1990).The methodology of adventitious buds regeneration from internode seg-ments of P.subulata was developed in our laboratory recently (unpublished),so we will attempt to separate the chimeras obtained in this research through adventitious buds regeneration technique in future.ReferencesAbu-Qaoud H,Skirvin RM,Chevreau E (1990)In vitro sepa-ration of chimeral pears into their component genotypes.Euphytica 48:189–196Adams KL,Wendel JF (2005)Polyploidy and genome evolu-tion in plants.Curr Opin Plant Biol 8(2):135–141Table 2Morphological characteristics of diploid and tetraploid plants of P.subulata Ploidy Leaf length (cm)Leaf width (cm)Leaf index (length/width)Flower diameter (cm)Diploid 1.580.179.29 2.24Tetraploid 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