Abstract Erianthus arundinaceum， a related species of the most important sugar crop Saccharum， and is an essential gene resource for stress and disease resistance in sugarcane breeding. Colchicine is the most common and effective chemical inducer in polyploid breeding at present. To detect the effects of colchicine on callus induction and shoot regeneration， E. arundinaceus ‘Yun 83183’， with the chromosome of 2n=60， was used as the material for studying the effect of different concentrations of 2，4D and colchicine， treatment time and culture methods on callus induction and shoot regeneration. The results indicated， when the callus was cultured for 7 d in solid medium with 0.07% colchicine or 0.5 d in liquid medium with 0.05% colchicine， callus had no ability of shoot regeneration， which meant that in liquid culture， colchicine had not only high inhibitory effect on callus， but also lower concentration and shorter treatment time than solid culture； when the concentration of colchicine was 0.03% and callus was cultured for 7 d in solid medium or 0.5 d in liquid medium， the resistant callus rate and resistant bud rate were the highest， although the resistant callus rates were all about 88.00% and had no significant difference between solid culture and liquid culture， the resistance bud rate was only 6.00% in liquid culture， 16.22% lower than in solid culture， which meant that solid culture was more effective than liquid culture at the same colchicine concentration. For E. arundinaceus ‘Yun 83183’， the best concentration of 2，4D was 2 mg/L， the best concentration of colchicine was 0.03%， and the best effect of callus induction and shoot regeneration was achieved within 7 d of solid culture. This study would provide fundamental information for chromosome doubling of E. arundinaceus in the future.
　　Key words Erianthus arundinaceum； Colchicine； Callus
　　Received： October 20， 2017 Accepted： January 13， 2018
　　Supported by Earmarked Fund for China Agriculture Research System （CARS170101）； Science and Technology Innovation Talent Program of Yunnan Province （2014HC015）； Science and Technology Project for Benefiting the People in Yunnan Province （Agriculture） （2014RA059）； Major Science and Technology Project of Yunnan Province （2015ZA001）.
　　Li YAO （1984-）， female， P. R. China， assistant research fellow， master， devoted to research about genetic breeding of sugarcane.
　　*Corresponding author. Email： [email protected]
　　Erianthus arundinaceum， a related species of the most important sugar crop Saccharum[1]， has the characteristics of good tolerance to drought and poor soil， strong tillering ability， high biological yield， good disease resistance， good ratoon property and wide adaptability， and is an important gene source for breeding of sugarcane for stress resistance and disease resistance， thereby attracting much attention from scholars devoted to sugacane breeding[2-5]. The hybridization of E. arundinaceum and sugarcane dates back to 1885[6]， when the wild parent was utilized for the first time， and BC5 and many breeding intermediate materials closely related to E. arundinaceum have been obtained so far[7]. However， backcross descendants usually have the disadvantages of thin stem， serious pith， more No. 57 pricklets and low brix[4，8]， so varieties， which are identified to be actual hybrid of E. arundinaceus， have not been found yet. The main reason might be that E. arundinaceum （2n=20， 40 and 60）[9] and sugarcane （2n=80-148）[10] differ greatly in chromosome number， the pollen fertility of hybrid offsprings is worse and mainly inherited in the n+n mode， and with the increase of backcross generation， the chromosome number of E. arundinaceum is halved in a generational sense. In a word， the crossbreeding utilization of E. arundinaceum has not entered the nobilization era yet， bringing great difficulty to the utilization of E. arundinaceum[11-15]. Research shows that performing chromosome doubling using one of the two parents with relatively lower chromosome ploidy or number is one of the effective means for improving the pollen fertility of the F1 generation[16]. 　　Colchicine is a kind of alkaloid extracted from Colchicum autumnale in Liliaceae family， which could inhibit the formation of spindle fiber and induce chromosome doubling， and serves as a chemical inducer most commonly used in polypliod breeding with the best effect[17-19]. Through the induction of polyploid with colchicine， plants are changed in shape， dissection， physiology and cultivation characteristics[18]， which is of great significance to germplasm innovation and breeding of new variety， and success application has been reported on grains[19-21]， vegetables[22-24]， flowers[25-27] and fruits[28-30]. However， different crops and different receptors differ greatly in the sensitivity to colchicine， while there were no reports about the research of mutation treatment on E. arundinaceum callus. This study was conducted to screen the optimal concentration of colchicine， treatment time and culture method for E. arundinaceum callus and to understand the effects of colchicine on the induction and root regeneration of E. arundinaceum callus， providing an important basis for chromosome doubling of E. arundinaceum with colchicine in the future.
　　Materials and Methods
　　Materials
　　Plant material
　　E. arundinaceum ‘Yun 83183’ was provided by the National Germplasm Repository of Sugarcane of Sugarcane Research Institute， Yunnan Academy of Agricultural Sciences.
　　Reagents
　　Plant hormones， 2，4D， NAA， 6BA and KT were purchased from Yunke Biotechnology Company. Colchicine was purchased from Sigama. The experiment was carried out in Yunnan Key Laboratory of Sugarcane Genetic Improvement from September 2010 to October 2012.
　　Methods
　　Medium
　　Induction medium M1： MS+2，4D （1， 2， 3， 4 mg/L）+0.2 g/L AC； differentiation medium M2：MS+ 6BA 1 mg/L+KT 0.5 mg/L. Above media （except liquid medium） were added with agar 4.0 g/L and sucrose 30 g/L， with a pH value of 5.8.
　　Induction of E. arundinaceum callus
　　Tender leaf sheaths of sugarcane were selected as explants， which were scrubbed with 70% ethanol. Old leaves were stripped off， and the young leaves with a distance 2-8 cm from the top growing point were transversely cut into thin sheets with a thickness of 0.1 mm， which were inoculated on induction medium.
　　Effect of colchicine on growth of E. arundinaceum callus
　　The embryonic calli were inoculated to medium M1 （2，4D 2 mg/L） containing colchicine. Colchicine was designed with 4 concentrations， i.e.， 0.00% （CK）， 0.03%， 0.05% and 0.07%， respectively. Solid culture was designed with 4 culture time， i.e.， 7， 14， 21 and 28 d， and liquid culture was designed with 5 culture time， i.e.， 0.5， 1， 2， 3 and 4 d. Each treatment had 3 replicates. After the treatment， the calli were transferred to medium M2 without colchicine， to allow differentiation and budding. After 30 d of culture， observation and statistics were performed. The treatment with the most resistant calli （buds） was the optimal treatment. 　　Induction rate=Total number of embryonic calliTotal number of explants×100%（1）
　　Differentiation rate=Total number of differentiated budsTotal number of inoculated explants×100%（2）
　　Resistant callus rate=Total number of resistant calliTotal number of inoculated calli×100%（3）
　　Resistant bud rate=Total number of resistant budTotal number of inoculated calli×100%（4）
　　Data processing
　　The data was subjected to variance analysis and multiple comparisons by Duncans new multiple range method in SPSS 13.0.
　　Results and Analysis
　　Effects of different 2，4D concentrations on induction rate of E. arundinaceum ‘Yun 83183’
　　As shown in Table 1， the induction rate was about 50% under the 2，4D concentration of 2-4 mg/L， but the highest of 69.62% under the concentration of 1 mg/L， without a significant difference. The quality of calli was the best under the 2，4D concentration of 2 mg/L， and the calli were fresh yellow， granular with loose structure. Therefore， the best 2，4D concentration for the induction of E. arundinaceum ‘Yun 83183’ was 2 mg/L.
　　Table 1 Effects of different 2，4D concentrations on induction rate of E. arundinaceum%
　　2，4D
　　Replicate
　　IIIIIIMean
　　Significant level
　　0.050.01Quality
　　1 mg/L59.6288.4660.7869.62aA++
　　2 mg/L43.7550.0075.0056.25aA++++
　　3 mg/L24.4946.0069.3946.63aA+++
　　4 mg/L72.0059.6221.1550.92aA+
　　"+" represents gray sticky callus； "++" represents light yellow callus； "+++" represents callus with white close structure； "++++" represents granular callus with bright yellow loose structure.
　　Effects of colchicine concentration and treatment time on growth of callus
　　Effect of colchicine concentration in solid medium on growth of callus
　　As shown in Table 2， with the continuous increase of the concentration， the resistant callus rate and resistant bud rate both decreased to different degrees over time. There were no significant differences in resistant callus rate and resistant bud rate between any two concentrations of 0.03%， 0.05% and 0.07%， but at the concentration of 0.00% （CK）， the resistant callus rate was the highest， up to 36.34%， which was significantly higher than those of the colchicine treatments， and the resistant bud rate of the CK was also the highest （17.53%）， but was not significantly from the 0.03% colchicine treatment. Therefore， the optimal colchicine concentration for the solid culture of ‘Yun 83183’ was 0.03%. 　　In addition， from the point o f culture time， the resistant callus rate at the colchicine treatment time of 7 d was significantly higher than those at the treatment time of 14， 21 and 28 d， and the resistant bud rate at 7 d was not significantly different from that at 14 d， but significantly higher than those at 21 and 28 d， while between any two of the time of 14， 21 and 28 d， there were no significant differences in the resistant callus rate and resistant bud rate. Therefore， the optimal treatment time for ‘Yun 83183’ was 7 d in solid medium.
　　Effect of colchicine concentration on callus growth in liquid medium
　　It could be seen from Table 3 that the treatment with colchicine did not affected resistant callus significantly， but had a significant effect on resistant bud rate. The resistant bud rate under no addition of colchicine （9.44%） was significantly higher than the resistant bud rate with the addition of colchicine， and with the colchicine concentration increasing， the resistant bud rate decreased continuously over time， and when reaching 0.05%， all the calli died. Therefore， the optimal colchicine treatment concentration for ‘Yun 83183’ was 0.03% in liquid medium.
　　Table 2 Effects of different colchicine concentrations on the induction and differentiation rates of E. arundinaceum callus under solid culture%
　　Colchicine concentrationItem7 d14 d21 d28 dMean
　　Significant level （concentration）
　　0.050.01
　　0（CK） Resistant callus rate100.0038.576.780.0036.34aA
　　Resistant bud rate42.4224.293.390.0017.53aA
　　0.03Resistant callus rate88.890.000.000.0022.22bA
　　Resistant bud rate22.222.220.000.006.11abA
　　0.05Resistant callus rate81.820.000.000.0020.45bA
　　Resistant bud rate4.550.000.000.001.14bA
　　0.07Resistant callus rate77.780.000.000.0019.44bA
　　Resistant bud rate0.000.000.000.000.00bA
　　MeanResistant callus rate87.129.641.700.00
　　Resistant bud rate17.306.630.850.00
　　Significant level （time）0.05Resistant callus rateabbb
　　Resistant bud rateaabbb
　　0.01Resistant callus rateABBB
　　Resistant bud rateAAAA
　　Li Yao et al. Effects of Colchicine on Callus Induction and Differentiation of Erianthus arundinaceus
　　Table 3 Effects of different colchicine concentrations on the induction and differentiation rates of E. arundinaceum callus under liquid culture%
　　Colchicine concentrationItem0.5 d1 d2 d3 d4 dMean
　　Significant level（concentration） 　　0.050.01
　　0（CK）Resistant callus rate115.2262.533.3335.2937.7456.82aA
　　Resistant bud rate23.9110.007.415.880.009.44aA
　　0.03Resistant callus rate88.0020.93100.0063.1615.5657.53aA
　　Resistant bud rate6.002.330.000.000.001.67bAB
　　0.05Resistant callus rate66.6767.526.0982.3513.9551.31aA
　　Resistant bud rate0.000.000.000.000.000.00bB
　　0.07Resistant callus rate69.4458.8218.0080.0012.7747.81aA
　　Resistant bud rate0.000.000.000.000.000.00bB
　　MeanResistant callus rate84.8352.4444.3665.2020.01
　　Resistant bud rate7.483.081.851.470.00
　　Significant level （time）0.05Resistant callus rateaababab
　　Resistant bud rateaabababb
　　0.01Resistant callus rateAABABABB
　　Resistant bud rateAAAAA
　　From the point of treatment time， both the resistant callus rate and resistant bud rate decreased continuously with treatment time increasing. There were no very significant differences in resistant callus rate， and no significant differences in resistant bud rate， between any two of the treatment time of 0.5， 1， 2 and 3 d， but the resistant callus rate at the treatment time of 0.5 d was very significantly higher than that at the treatment time of 4 d， and the resistant bud rate was significantly higher than that at the treatment time of 4 d. Furthermore， after 4 d of treatment， even the CK showed no buds. Therefore， 0.5 d was the optimal time for the liquid culture of ‘Yun 83183’.
　　Conclusions
　　The study showed that though the optimal colchicine concentration was 0.03% for both solid culture and liquid culture of ‘Yun 83183’， the optimal time for solid culture was 7 d， and the resistant callus rate and resistant bud rate were 88.89% and 22.22%， respectively， while during liquid culture， the optimal culture time was 0.5 d， and the resistant callus rate and resistant bud rate were 88.00% and 6.00%， respectively. In short， though liquid culture was not significantly different from solid culture in resistant callus rate， and consumed a shorter time， its resistant bud rate decreased by 16.22% compared with solid culture. Furthermore， after 28 d of solid culture， all the calli died even in the no colchicine treatment， and to the colchicine concentration of 0.07%， even if cultured for only 7 d， with a resistant callus rate up to 77.78%， all the calli lost their differentiation ability； and after 4 d of liquid culture， there were few calli， which all lost their differentiation ability even in the no colchicine treatment， and to the colchicine concentration of 0.05%， even only cultured for 0.5 d， with a resistant callus rate up to 66.67%， all the calli lost their differentiation ability. The results showed that the inhibitory effect of colchicine on callus was higher during liquid culture than during solid culture， so the used concentration was lower， and the time was shorter； and under the same colchicine concentration， solid culture was better than liquid culture. This might be due to that during liquid culture， callus directly and closely contacted colchicine， while during solid culture， the contact was relatively less. After all， colchicine has certain inhibitory effect on the growth of callus. Therefore， except the control of colchicine concentration and treatment time， the selection of suitable culture mode is also very important. The results of this study showed that for the induction of E. arundinaceum ‘Yun 83183’callus， the optimal 2，4D concentration was 2 mg/L， the colchicine concentration was 0.03%， and 7 d of solid culture was the best for the growth and differentiation of callus. 　　Discussion
　　Chromosome doubling of plant is one of the effective means for overcoming pollen abortion of the filial generation of distant hybridization and improving some single characters. Colchicine is the chemical inducer used most commonly with the best effect， and has achieved success in the induction of chromosome doubling of multiple crops， but colchicine has the disadvantages of high price， high toxicity so much to inhibit the regeneration of adventitious bud， high usage concentration， low mutagenesis， and great potential safety hazard to human， livestock and environment. In this study， it was also found that E. arundinaceum browned seriously during the induction of callus， and colchicine had stronger inhibitory effect on the growth and differentiation of E. arundinaceum callus. In recent years， related studies showed herbicide type mutagenic agents， such as amiprophose methyl （APM）[31]， oryzalin and trifluralin[32]， have the advantages of low price， low usage concentration， low toxicity， short treatment time and low environmental pollution compared with colchicine， with the doubling effect not poorer than colchicine， and it is deemed that they are the most promising to take the place of the inducer colchicine.
　　In this study， the effects of colchicine concentration， treatment time and culture method on the induction and differentiation of callus were studied and analyzed， and the optimal concentration and time and culture mode for the mutation treatment of ‘Yun 83183’ callus was preliminarily selected， thereby providing a reference basis for the research on chromosome doubling of E. arundinaceum. However， how to screen regenerated plants and perform ploidy identification analysis still needs further experimental study.
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