In Vitro Polyploid Induction,Identification And Applicability Evaluation In Caladium

Caladiums（Caladium×hortulanum Birdsey）are perennial herbaceous foliage plants of the Araceae family,which is native to tropical South America.They are often forced in containers or utilized in the landscape for their beautiful leaves,veins,stripes,patches and long viewing period.Morphological characteristics are often used to describe and identify caladiums.During the past decades,interspecific or intraspecific hybridization between elite cultivars,breeding lines,and/or related species has been the primary breeding approach in caladium with the aim to improve the horticultural characters and increase the ornamental value of caladium.However;it might be in risk of losing genetic diversity in breeding populations due to intensive hybridization and selection.In addition,the flowers of caladium are less,the flowering period is not fixed,and the pollen and seed life are short,which reduced the efficiency of cross-breeding.Thersfore,it is necessary to adopt new breeding methods to cultivate new types of caladium.Polyploidization has profound effects on dosage-regulated gene expression,resulting in diverse phenotypic and genetic variation,and thus plays an important role in plant genetic improvement and breeding.Although several studies have been conducted on in vitro chromosome doubling in ornamental aroids including caladium in recent years,few reports in the literature are available regarding the effects of polyploidization on the ornamental values and stress tolerance of these plants.Herein,one-month-cultured leaf cultures of‘Red Flash’caladium were treated by different concentrations of colchicine or oryzalin for different time periods,respectively,and relative DNA content analysis,chromosome counting,morphological comparison,stomatal analysis,photosynthesis comparison as well as chilling tolerance evaluation were performed to characterize the regenerated morphological variants,with the aim to develop an efficient in vitro polyploidization technique and identify promising variants holding enhanced ornamental values and/or chilling tolerance for future caladium breeding.The main results are as follows:1. Screening of mutagenic methods for ploidy of caladium.Small amount of callus was observed around the cut surfaces of the leaf segments of‘Red Flash’Caladium after culture on the CIM（MS medium supplemented with 2 mg/L thidiazuron（TDZ）,1 mg/L6-Benzyladenine（6-BA）,4%（w/v）sucrose and 0.7%（w/v）agar,p H 5.8.）for one month.The in vitro pre-cultured leaf segments were treated with 0.1%,0.2%and 0.3%（w/v）colchicine,or 0.001%,0.002%and 0.003%（w/v）oryzalin for 2,4 and 6 days,respectively,and then placed horizontally on solidified MS medium supplemented with 1 mg/L 6-BA,1 mg/L 1-Naphthyl acetic acid（NAA）,3%（w/v）sucrose and 0.7%（w/v）agar（p H 5.8）for plantlet regeneration.Five months after transplantation from culture vessels,large quantities of plants with different leaf color patterns were established（Fig.1 E）.At last,a total of 723 plants were produced from all treatments,and 206 plants showed stable morphological changes in leaf shape,color,and/or coloration from the wild type were established in this study.Based on the differences in leaf shape,and color of main veins,leaf margins and leaf spots,the variants could be grouped into 10variant types,i.e.,5 somaclonal variation types（SVT）from SVT1 to SVT5,and 5tetraploid types（T）including T1 to T5.2. Identification of polyploid induction effects of caladium.As many as 93 plants among the 206 plants with stable morphological difference from the wild type were identified preliminary as tetraploids by flow cytometry.According to the preliminary ploidy analysis,all colchicine and oryzalin treatments yielded tetraploids,and the largest number of tetraploids（21）and the highest tetraploid induction rate（46.67%）were achieved when the leaf cultures were exposed to 0.002%oryzalin for six days,followed by the treatment of 0.003%oryzalin for 6 days.3. Analysis of DNA relative content and chromosome number of variation types.Results showed that extensive variation of mean fluorescence intensity（MFI）was recorded among the 10 variation types,and results showed that chromosome gains or losses occurred frequently in the established variants.By chromosome counting,the wild caladium had 30 chromosomes.The chromosome number was 2n=2x+2=32 in the SVT1,SVT2 and SVT3,as might be explained by their increased MFI compared with the wild type.The chromosome numbers of the SVT4 were similar to the wild type（2n=2x=30）,which was consistent with their MFI.The SVT5 had four more chromosomes than the wild type（2n=2x+4=34）,as might be attributed to its significantly increased MFI as compared to the wild type.The chromosome number of T1,T2 and T4 was 2n=4x=60,64 and 58 in the cells,respectively,which was largely consistent with their measured MFI.No data were obtained for the T3 and T5,as they grew poorly and could not provide enough vigorously growing roots for chromosome counting.4. Cytological identification of variation types.Generally speaking,the tetraploid caladium had a significantly higher plant height,leaf width,leaf thickness and petiole diameter,and a significantly lower leaf number per plant as well as leaf length/width ratio as compared to the wild type.No significant differences between the wild type and the SVT1 in terms of all the analyzed morphological traits besides the plant height.For the stomatal parameters,the stomatal density of the T1 was significantly lower than that of the wild type and the SVT1,while their stomatal guard cell length was significantly higher among the three types.These results indicated that polyploidization leaded to rounder and thicker leaves,thicker petiole diameter,lower leaf number per plant,and lower stomatal density in caladium.5. Analysis of dynamic distribution pattern and color parameters of the morphological variation types.Compared with the wild caladium,the color of tetraploid has changed significantly in morphology,and the color range is more extensive.Leaf lightness,saturation and hue angle of the induced caldium are all obviously different from wild type and diploid variantion types.The results showed that the lightness of the tetraploid was obviously decreased and the saturation was increased whether it was red or green leaves.6. Analysis of pigment content and photosynthetic parameters.Results showed that polyploidization of caladium resulted in the chlorophyll content of the leaf margin and the middle part was both higher than that of the diploid,while the anthocyanin content and flavonoid content in leaf margin and the middle part of the diploid plants were all higher than that of the tetraploids.Photosynthesis analysis showed that the tetraploid caladium had significantly increased photosynthetic parameters including the net photosynthesis rate,transpiration rate,intercellular CO₂ concentration and stomatal conductance as compared to the wild type.The results of correlation analysis showed that the net photosynthetic rate was positively correlated with the chlorophyll content,but negatively correlated with the flavonoids content and the anthocyanin content.7. Evaluation of chilling tolerance of tetraploid and diploid plants.Results showed that the tetraploid caladium（T1）always had a higher superoxide dismutase（SOD）activity,peroxidase（POD）activity and proline（Pro）content,and a lower Relative electrical conductivity（REC）and malondialdehyde（MDA）content as compared to those of the wild type and the SVT1 during chilling stress,indicating that the tetraploids（T1）had enhanced chilling tolerance as compared to the wild type and the diploid aneuploid plants（SVT1）.In summary,the present study demonstrates that tetraploids could be efficiently induced in caladium when the leaf cultures were treated with 0.002%oryzalin for 6days.Chromosome doubling resulted in significant changes in morphological,stomatal and photosynthetic characteristics,and enhanced chilling tolerance as compared to the wild caladium.Moreover,results of the DNA content analysis and chromosome counting revealed that chromosome gains or losses occurred frequently in the established morphological variants.These variants including the tetraploids,diploid variants,diploid aneuploids,and tetraploid aneuploids hold great potential for cultivar development,genetic study and chromosome engineering in caladium.