| Sweetgums(Liquidambar spp.)is an important and worldwide forestry resource,especially Liquidambar formosana and L.styraciflua,both of them preformed high economic,ornamental and ecological values and attracting more and more attention from Chinese.The foundation of sweetgum breeding in China is insufficient.It is urgent to cultivate new germplasm to meet different social needs Polyploid breeding is an effective way to create new germplasms.However,no related studies have been carried out in sweetgum all over the world.In this study,L.styraciflua was used as the maternal parent and L.formosana was used as the paternal parent to generate hybrid seeds by the method of controlled pollination.Hybrid sweetgum seeds(L.formosana × L.styraciflua)were used to establish in vitro tissue culture system.The leaves and petioles obtained from in vitro cultured hybrid sweetgum plants were used for chromosome doubling.Mixoploids were then purified by direct shoot regeneration.In addition,transcriptome sequencing was carried out in order to analyze causes of variation of the in vitro regenerated tetraploid plantlets.It is of great theoretical and practical significance to carry out the above research in order to 1)make a breakthrough in sweetgum polyploid breeding;2)to analyze the phenotype variation mechanism of the regenerated tetraploid plants in hybrid sweetgum;and 3)to improve its propagation efficiency,The main results were concluded as the followings(1)in vitro tissue culture system suitable for rooting and regeneration of hybrid sweetgum plants in multi-genotypes was established.These works lay a solid foundation for further reserch on in vitro chromosome doubling.Concentrations of indolebutyric acid(IBA)affected rooting and plant height of hybrid sweetgum significantly.Half-strength WPM medium supplemented with 2.0 mg/L indole butyric acid(IBA)and 0.1 mg/L α-naphthalene acetic acid(NAA),30 g/L sucrose,2 g/L agar and 2 g/L polygel(pH 5.8～5.9)produced 100%rooted plantlets.The survival rate of plants transferred from in vitro to ex vitro conditions was 85.1%in green house and 75.0%in the field later.Thidiazuron(TDZ)concentrations significantly affected leaf and petiole regeneration rates,or the adventitious shoot induction rate.The highest regeneration rate of leaf and petiole,as explants,were 86.6%and 90.0%,respectively by using WPM basal medium supplemented with 0.2 mg/L TDZ,0.8 mg/L benzyladenine(BA),and 0.1 mg/L NAA.High concentrations of TDZ promoted adventitious lateral bud growth with little bud elongation,resulted in a large number of abnormal shoots.Optimal elongation medium for the induced buds from both leaf and petiole was WPM medium containing 0 mg/L TDZ,0.4 mg/L BA and 0.1 mg/L NAA.The highest induction rate of adventitious buds,with leaves and petioles were 58.33%or 68.33%respectively,while the average induced buds per explants was 3.55 or 2.39,respectively.(2)It is the first report that in vitro tetraploid induction from leaf and petiole explants of hybrid sweetgum via colchicine treatments,and a technique of purifying tetraploids by organ in vitro regeneration was proposed.The development of callus around the incision of leaf and petiole significantly affected the efficiency of chromosome doubling.the most suitable period for somatic chromosome doubling in three typical periods was that the vein and petiole wounds begin to expand,a small amount of callus appears,and a large number of meristematic tissues appear internally,The results of orthogonal experiment and range analysis showed that the induction rate of tetraploid was affeeted by genotype,pre-culture time,colchicine concentration and treatment time.The treatment time had the greatest influence on the survival rate and doubling efficiency of explants from leaves and petioles.The optimum conditions for leaves were as follows:pre-culture for 8 days,colchicine concentration was 200 mg/L for 3 days,for petioles,pre-culture for 6 days,and the others were the same as leaves.The petiole doubling efficiency of hybrid hybrid sweetgum was higher than that of leaf,and the tetraploid induction rate could reach 18%,respectively.The callus around the incision for most genotypes developed at the same speed during this period,and the pre-culture time was more conducive to chromosome doubling.In addition,when colchicine concentration was increased to 350 mg/L,the highest tetraploid induction rate was 8.33%,and the highest mixoploidy induction rate was 13.30%.Tetraploid can be purified by direct adventitious shoot regeneration in vitro from mixoploid.Genotypes significantly affect the efficiency of purification of mixoploid via in vitro regeneration.The highest percentage of tetraploids was 20.18%.In this study,7 genotype tetraploids,8 genotype mixoploids,Totally 15 new polyploid germplasms were obtained.(3)We found that chromosome doubling resulted in significant changes in morphology and histological of in vitro tetraploid hybrid sweetgum,especially the elongation ability of roots and stems was significantly different from that of diploids.After rooting 25 to 50 days,the diploid growthrapidly,the average plant height increased by 16.94 mm,while the tetraploid only increased by 1.93 mm.The thickness of tetraploid leaves and veins,palisade and spongy tissues,root epidermis and cortex were significantly thicker than that of diploid,but there were no significant difference in stem epidermis,cortex and vascular column between diploid plants and tetraploid plants.For most tetraploids,bud apex began to dormancy at 50 days,and bud apex completely dormancy at 70 days.Compared with diploid,the width and shape of tetraploid root cells increased but the length of tetraploid root meristematic zones decreased at 50 days in abnormal roots,and there was no obvious coleoptile sheath and complete coleoptile structure in tetraploid.However,the cross-sectional area of xylem cells,upper and lower epidermis cells,cortex cells,medullary cells,spongy tissue and palisade tissue cells of tetraploid were larger than that of diploid.Moreover,annual tetraploids grow in green house showed dwarfing characteristics too,the average plant height of diploids was 49.13 cm and 2.6 times that of tetraploids,and the average height of tetraploids was 21.74 cm.(4)The gene expression characteristics of in vitro tetraploid and diploid hybrid sweetgum were studied,Transcriptome sequencing results showed that differentially expressed genes were significantly enriched in biological pathways related to in vitro organs elongation,such as plant hormone biosynthesis and signal transduction,sugar and starch metabolism,cell cycle and elongation etc.Compared with diploid,majority genes positive regulation of organ elongation,such as YUCCA、TAA1、GH3、AUX1、SAUR、CPS、KO、KAO、GA20ox、GA3ox、BAS1 and CYCD3 etc were down-regulated expression.This may be the main reason for the reduced root and stem elongation of tetraploid plants.(5)It was proved that the difference of content of endogenesis auxin,gibberellin and brassinolide between tetraploid and diploid hybrids sweetgum showed similar trend with their genes expression.By adding exogenous hormones GA3 and LAA could significantly improve the growth of tetraploid plantlets.The results showed that the contents of IAA,GA3 and BR in tetraploid roots and stems were significantly lower than those in diploid.At the initial stage,exogenous GA3 and IAA could significantly promote the elongation of in vitro tetraploid stems and roots. |
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