论文部分内容阅读
Abstract This study was carried out to optimize the culture media for the micropropagation of Taxodium hybrid Zhongshanshan （T. distichum ×T. mucronatum）. Using the tender stems of Zhongshanshan 301 as the explants， the effects of NAA， 6-BA， IBA and KT on the induction of differentiation， proliferation and rooting were evaluated on MS or 1/2 MS medium. The results showed that Zhongshanshan can be proliferated via tissue culture. The combined use of NAA and 6-BA in MS medium greatly promoted the differentiation of Zhongshanshan explants， and the optimal medium was MS + 0.2 mg/L NAA + 0.4 mg /L6-BA. The optimal medium for the proliferation of differentiated buds was MS + 0.3 mg/L NAA + 0.4 mg/L 6-BA， on which the proliferation rate was up to 3.8. 1/2 MS medium was more conducive than MS medium to the induction of rooting. The optimal medium for the rooting of tissue-cultured Zhongshanshan shoots was 1/2MS + 0.3 mg/L IBA + 0.2 mg/L NAA.
Key words Taxodium hybrid Zhongshanshan； Micropropagation； Differentiation； Proliferation； Rooting
Taxodium hybrid Zhongshanshan （T. distichum ×T. mucronatum）， a superior clone of Taxodium， grows fast， and has tolerance to salt and wide adaptability[1]. It is also widely grown as ornamental plants. However， sexual reproduction of Zhongshanshan is usually restricted by the quality and dormancy period of the seeds， which limits the rapid propagation of seedlings. In addition， seed propagation may cause mutation in seedlings， and thus loss the excellent traits of parental plants[2]. At present， cutting is the main method for proliferation of Zhongshanshan[3-5]， which can pass on the excellent traits of parental plants to seedlings. However， there are some disadvantages in this method， such as the low survival rate of cuttings and limited shoots that can be used as cuttings[6]. Micropropagation is not affected by time， season， weather， pests and diseases， by which excellent traits of parental plants can be inherited， and seedlings can be produced rapidly in a large scale. So， it has a promising prospect in breeding and propagation of plants[7]. So far， the proliferation of Zhongshanshan through micropropagation has been rarely reported. Therefore， in this study， the effects of different plant hormones and media on the differentiation， proliferation and rooting of Zhongshanshan explants were evaluated， to determine the optimal culture medium for the micropropagation of Zhongshanshan.
Materials and Methods Materials
The stems of a superior clone Zhongshanshan 301 in Nanjing Zhongshan Botanic Garden were collected and used as the explants in this experiment.
Methods
Disinfection of explants
The explants were washed with detergent， rinsed with tap water for 10 min， washed with distilled water for three times， soaked in saturated calcium hypochlorite solution for 10 min， and rinsed with distilled water again for three to five times. Then， the explants were cut into segments of about 2 cm， disinfected with 0.1% HgCl2 for 1-2 min， then with 75% alcohol for 10 s on a clean bench， rinsed with sterile water for three to five times， wiped with sterile filter paper， and finally inoculated onto different media.
Induction of differentiation of explants
Zhongshanshan explants were induced to differentiate on MS， supplemented with different concentrations of NAA and 6-BA. Five treatments of the hormones as shown in Table 1 were prepared， with three replicates for each treatment and 50 explants in each replicate. The browned explants were counted on Day 10 of culture， and differentiated explants were counted on Day 21.
Proliferation of explants
The differentiated buds were transferred to new media for proliferation. The media were MS supplemented with different concentrations of IBA， KT， 6-BA and NAA. Five treatments of the growth regulators as shown in Table 2 were prepared， with three replicates for each treatment and 50 explants in each replicates. The total number of differentiated buds was counted after 30 d of proliferation induction.
Induction of adventitious roots
IBA and NAA were added to MS medium or 1/2MS medium for the induction of adventitious roots. Six treatment as shown in Table 3 were established， with three replicates for each treatment， 50 buds in each replicate. The growth of adventitious roots was observed after 28 d of subculture.
To all above media， 6.0 g/L agar and 3% sucrose were added. The final pH of the media was maintained at 6.0. The explants were cultured at 22-25 ℃， with a photoperiod of 12 h light and a light intensity ranging from 1 600 to 1 900 lx.
Data analysis
Data processing and analysis was done using Excel 2003 software.
Results and Analysis
Effect of different concentrations of NAA and 6-BA on differentiation of Zhongshanshan explants
As shown in Table 4， there were significant differences in number of browned explants， number of differentiated explants， induction rate and bud growth on the media supplemented with different concentrations of NAA and 6-BA. The number of browned explants was the largest on the medium supplemented with NAA alone， followed by that on the medium supplemented with only 6-BA. There were fewest browned explants on the medium supplemented with both NAA and 6-BA （treatment T3）. In addition， both the number of differentiated explants and induction rate were the largest in treatment T3， followed by those in treatment T4 and T5 （with the addition of only 6-BA）. For 6-BA， 0.6 mg/L （treatment T4） showed better effect than 0.9 mg/L （treatment T5） in the induction of differentiation of Zhongshanshan explants， indicating that 0.9 mg/L 6-BA suppressed the differentiation of explants to a certain degree. The number of differentiated explants and induction rate in treatments T1 and T2 （with the addition of only NAA） were lower than those in other treatments， and were negatively correlated with the number of browned explants. The differentiated buds on the media supplemented with NAA or 6-BA alone were thin and delicate， and the buds on the medium supplemented with NAA and 6-BA in combination were robust， indicating there were an interaction effect between the two hormones. The optimal medium for the differentiation of Zhongshanshan explants was MS + 0.2 mg/L NAA + 0.4 mg /L6-BA. Effect of different growth regulators on the proliferation of Zhongshanshan explants
IBA， KT， NAA and 6-BA showed different effects on the number of proliferated shoots， proliferation rate and shoot growth （Table 5）. In detail， the number of proliferated shoots and proliferation rate in treatment T9 （0.3 mg/L NAA and 0.4 mg/L 6-BA） were much higher than those of other treatments， and the shoots grew well in this treatment. The number of differentiated shoots and proliferation rate on the medium supplemented with NAA and 6-BA （treatment T8） were higher than those on the medium supplemented with the same concentration of IBA and KT （treatment T6）， suggesting that NAA and 6-BA are more conducive to the proliferation of Zhongshanshan explants than IBA and KT. The number of proliferated shoots and proliferation rate began to decrease when 0.4 mg/L NAA and 0.8 mg/L 6-BA were added to the medium， indicating that high concentrations of the two growth regulators inhibited the proliferation of Zhongshanshan explants. The optimal medium for the proliferation of differentiated buds was MS + 0.3 mg/L NAA + 0.4 mg/L 6-BA， on which the proliferation rate was up to 3.8.
Effects of different media and hormones on the growth of adventitious roots
The tissue cultured shoots that grew well and were similar in size were subcultured on rooting media for 28 d to induce adventitious roots. The results revealed that the growth of adventitious roots was closely related to the type of medium and the concentration of IBA and NAA. The number of rooted shoots and rooting rate on 1/2 MS medium were both higher than those on MS medium， supplemented with the same concentration of IBA or NAA alone or combination of IBA and NAA， which indicated that 1/2MS medium was more conducive to the rooting of Zhaongshanshan explants than MS medium. The number of rooted shoots on MS and 1/2 MS supplemented with 0.4 mg/L NAA （treatments T12 and T15） was much higher than that on the same medium supplemented with 0.4 mg/L IBA （T11 and T14）， and the difference was significant. Moreover， the number of rooted shoots in the treatment supplemented with IBA and NAA in combination was much larger than that in the treatments supplemented with IBA or NAA alone. In summary， 1/2 MS medium + 0.3 mg/L IBA + 0.2 mg/L NAA （treatment T16） was optimal to induce the rooting of Zhaongshanshan explants， and the roots grew vigorously in this treatment.
Conclusions and Discussion
By using the tender stems of Zhongshanshan 301 as the explants， the effects of NAA， 6-BA， IBA and KT on micropropagation of Zhongshanshan were evaluated on MS or 1/2 MS medium in this study. The results showed that Zhongshanshan can be proliferated via tissue culture. The combined use of NAA and 6-BA in MS medium greatly promoted the differentiation of Zhongshanshan explants， and the optimal medium was MS + 0.2 mg/L NAA + 0.4 mg/L6-BA. The optimal medium for the proliferation of differentiated buds was MS + 0.3 mg/L NAA + 0.4 mg/L 6-BA， on which the proliferation rate was up to 3.8. 1/2 MS medium was more conducive than MS medium to the induction of rooting. The optimal medium for the rooting of tissue-cultured Zhongshanshan shoots was 1/2MS + 0.3 mg/L IBA + 0.2 mg/L NAA. There are various factors that affect browning of explants in tissue culture. It has been suggested that the part of plants that are used as explants， the timing of sampling and plant species all have an effect on the browning of explants[8]. Our results showed that the number of browned plants in the treatments with only NAA or 6-BA was higher than that in the treatments with both NAA and 6-BA. The reason may be that the concentration and proportion of hormones have an influence on browning of explants. Woody plants have a high content of phenolic compounds， and 6-BA can promote the synthesis of phenolic compounds， to elevate the activity of polyphenol oxidase （PPO）[9]， which is a critical enzyme catalyzing the oxidation of phenolic compounds to form grey or brown colors， leading to the browning of explants. The number of differentiated buds and induction rate were both increased by the addition of 0.1-0.4 mg/L NAA in medium， but decreased when the concentration of 6-BA was increased from 0.6 to 0.9 mg/L， proving that the concentrations of NAA and 6-BA had an effect on the induction of Zhongshanshan explants. Fang[10] found that 6-BA played a decisive role in the proliferation of Pinus elliottii × Pinus caribaea adventitious buds， while NAA was able to improve the proliferation efficiency and proliferation rate. Our results showed that the addition of 0.3 mg/L NAA and 0.4 mg/L 6-BA greatly increased the number of differentiated buds and proliferation rate， and the buds grew very well， suggesting that the combined treatment with NAA and 6-BA is critical for the proliferation of differentiated shoots/seedlings of Zhongshanshan.
After a large number of buds and shoots are produced from explants through tissue culture， induction of rooting is a vital step in determining whether the buds and shoots can survive[11]. Lin[12] believed that the optimal medium for the rooting of tissue cultured C. lanceoleta seedlings was 1/2MS + 30/L sucrose + 1.2 mg/L IBA + 0.4 mg/L NAA. Our results showed that 1/2MS medium +0.3 mg/L IBA +0.2 mg/L NAA was the optimal medium for the rooting of tissue culture seedlings of Zhongshanshan， which is slightly different from the findings in Juglans regia[8]. The reason may be that rooting of tissue cultured seedlings is affected by multiple factors including plant species， genotype， basal medium type and plant growth regulators.
References
[1] YIN YL， CHEN YH. The survey and evaluation to comparative plantation of Taxodium ''zhongshansa'' and T. ascendens， T. distichum and Metaseqoia glyptostroboides[J]. Journal of Plant Resources and Environment， 1997， 6（3）： 23-28. [2] TULECKE W， MCGRANAHAN G， AHMADI H. Regeneration bysomatic embryogenesis of triploid plant from endosperm of walnnt， Julans regia[J]. Plant Cell Reports， 1988（7）： 301-304.
[3] LIU YQ， YANG JM， PENG WX， et al. The anatomical study on the rooting of the green shoot cutting propagation of two kinds of Tilia[J]. Journal of Agricultural University of Hebei， 2004， 27（2）： 33-37.
[4] CAO B， GAO HD. Technology of cutting propagation of Simmondisa chinensis （Link） Schneider[J]. Journal of Nanjing Forestry University （Natural Science Edition）， 2003， 27（4）： 62-66.
[5] GUO SJ. Progress of study on rooting anatomy and physiology of forest tree cuttings[J]. Journal of Beijing Forestry University， 1997， 19（4）： 64-69.
[6] WANG HF， HE M， YU CG， et al. Review on research status of Taxodium ‘Zhongshansa’ clones[J]. Nanfang Forestry Science， 2015， 43（3）： 17-21， 29.
[7] GUO DY. Application of biotechnology in fruit trees[J]. Journal of Fruit Science， 1992， 9（3）： 173-178.
[8] LIU SL， HAN BW. Plant regeneration from excised embryos of Juglans regia[J]. Plant Physiology Journal， 1989， 15（1）： 78-100.
[9] GAO GX. Browning in plant tissue culture[J]. Plant Physiology Communications， 1999， 35（6）： 501-506.
[10] FANG LJ. Influence of 6-BA and NAA on propagation of Pinus elliottii × Pinus caribaea[J]. Journal of Anhui Agricultural Sciences， 2012， 40（14）： 8180， 8183.
[11] CAO ZY， LIU GM. Plant tissue culture[M]. Lanzhou： Gansu Science and Technology Press， 1999： 85.
[12] LIN JQ. Studies on subculture and rooting culture of superior C. lanceoleta[J]. Journal of Anhui Agricultural Sciences， 2011， 39（15）： 8933-8934， 8937.
Key words Taxodium hybrid Zhongshanshan； Micropropagation； Differentiation； Proliferation； Rooting
Taxodium hybrid Zhongshanshan （T. distichum ×T. mucronatum）， a superior clone of Taxodium， grows fast， and has tolerance to salt and wide adaptability[1]. It is also widely grown as ornamental plants. However， sexual reproduction of Zhongshanshan is usually restricted by the quality and dormancy period of the seeds， which limits the rapid propagation of seedlings. In addition， seed propagation may cause mutation in seedlings， and thus loss the excellent traits of parental plants[2]. At present， cutting is the main method for proliferation of Zhongshanshan[3-5]， which can pass on the excellent traits of parental plants to seedlings. However， there are some disadvantages in this method， such as the low survival rate of cuttings and limited shoots that can be used as cuttings[6]. Micropropagation is not affected by time， season， weather， pests and diseases， by which excellent traits of parental plants can be inherited， and seedlings can be produced rapidly in a large scale. So， it has a promising prospect in breeding and propagation of plants[7]. So far， the proliferation of Zhongshanshan through micropropagation has been rarely reported. Therefore， in this study， the effects of different plant hormones and media on the differentiation， proliferation and rooting of Zhongshanshan explants were evaluated， to determine the optimal culture medium for the micropropagation of Zhongshanshan.
Materials and Methods Materials
The stems of a superior clone Zhongshanshan 301 in Nanjing Zhongshan Botanic Garden were collected and used as the explants in this experiment.
Methods
Disinfection of explants
The explants were washed with detergent， rinsed with tap water for 10 min， washed with distilled water for three times， soaked in saturated calcium hypochlorite solution for 10 min， and rinsed with distilled water again for three to five times. Then， the explants were cut into segments of about 2 cm， disinfected with 0.1% HgCl2 for 1-2 min， then with 75% alcohol for 10 s on a clean bench， rinsed with sterile water for three to five times， wiped with sterile filter paper， and finally inoculated onto different media.
Induction of differentiation of explants
Zhongshanshan explants were induced to differentiate on MS， supplemented with different concentrations of NAA and 6-BA. Five treatments of the hormones as shown in Table 1 were prepared， with three replicates for each treatment and 50 explants in each replicate. The browned explants were counted on Day 10 of culture， and differentiated explants were counted on Day 21.
Proliferation of explants
The differentiated buds were transferred to new media for proliferation. The media were MS supplemented with different concentrations of IBA， KT， 6-BA and NAA. Five treatments of the growth regulators as shown in Table 2 were prepared， with three replicates for each treatment and 50 explants in each replicates. The total number of differentiated buds was counted after 30 d of proliferation induction.
Induction of adventitious roots
IBA and NAA were added to MS medium or 1/2MS medium for the induction of adventitious roots. Six treatment as shown in Table 3 were established， with three replicates for each treatment， 50 buds in each replicate. The growth of adventitious roots was observed after 28 d of subculture.
To all above media， 6.0 g/L agar and 3% sucrose were added. The final pH of the media was maintained at 6.0. The explants were cultured at 22-25 ℃， with a photoperiod of 12 h light and a light intensity ranging from 1 600 to 1 900 lx.
Data analysis
Data processing and analysis was done using Excel 2003 software.
Results and Analysis
Effect of different concentrations of NAA and 6-BA on differentiation of Zhongshanshan explants
As shown in Table 4， there were significant differences in number of browned explants， number of differentiated explants， induction rate and bud growth on the media supplemented with different concentrations of NAA and 6-BA. The number of browned explants was the largest on the medium supplemented with NAA alone， followed by that on the medium supplemented with only 6-BA. There were fewest browned explants on the medium supplemented with both NAA and 6-BA （treatment T3）. In addition， both the number of differentiated explants and induction rate were the largest in treatment T3， followed by those in treatment T4 and T5 （with the addition of only 6-BA）. For 6-BA， 0.6 mg/L （treatment T4） showed better effect than 0.9 mg/L （treatment T5） in the induction of differentiation of Zhongshanshan explants， indicating that 0.9 mg/L 6-BA suppressed the differentiation of explants to a certain degree. The number of differentiated explants and induction rate in treatments T1 and T2 （with the addition of only NAA） were lower than those in other treatments， and were negatively correlated with the number of browned explants. The differentiated buds on the media supplemented with NAA or 6-BA alone were thin and delicate， and the buds on the medium supplemented with NAA and 6-BA in combination were robust， indicating there were an interaction effect between the two hormones. The optimal medium for the differentiation of Zhongshanshan explants was MS + 0.2 mg/L NAA + 0.4 mg /L6-BA. Effect of different growth regulators on the proliferation of Zhongshanshan explants
IBA， KT， NAA and 6-BA showed different effects on the number of proliferated shoots， proliferation rate and shoot growth （Table 5）. In detail， the number of proliferated shoots and proliferation rate in treatment T9 （0.3 mg/L NAA and 0.4 mg/L 6-BA） were much higher than those of other treatments， and the shoots grew well in this treatment. The number of differentiated shoots and proliferation rate on the medium supplemented with NAA and 6-BA （treatment T8） were higher than those on the medium supplemented with the same concentration of IBA and KT （treatment T6）， suggesting that NAA and 6-BA are more conducive to the proliferation of Zhongshanshan explants than IBA and KT. The number of proliferated shoots and proliferation rate began to decrease when 0.4 mg/L NAA and 0.8 mg/L 6-BA were added to the medium， indicating that high concentrations of the two growth regulators inhibited the proliferation of Zhongshanshan explants. The optimal medium for the proliferation of differentiated buds was MS + 0.3 mg/L NAA + 0.4 mg/L 6-BA， on which the proliferation rate was up to 3.8.
Effects of different media and hormones on the growth of adventitious roots
The tissue cultured shoots that grew well and were similar in size were subcultured on rooting media for 28 d to induce adventitious roots. The results revealed that the growth of adventitious roots was closely related to the type of medium and the concentration of IBA and NAA. The number of rooted shoots and rooting rate on 1/2 MS medium were both higher than those on MS medium， supplemented with the same concentration of IBA or NAA alone or combination of IBA and NAA， which indicated that 1/2MS medium was more conducive to the rooting of Zhaongshanshan explants than MS medium. The number of rooted shoots on MS and 1/2 MS supplemented with 0.4 mg/L NAA （treatments T12 and T15） was much higher than that on the same medium supplemented with 0.4 mg/L IBA （T11 and T14）， and the difference was significant. Moreover， the number of rooted shoots in the treatment supplemented with IBA and NAA in combination was much larger than that in the treatments supplemented with IBA or NAA alone. In summary， 1/2 MS medium + 0.3 mg/L IBA + 0.2 mg/L NAA （treatment T16） was optimal to induce the rooting of Zhaongshanshan explants， and the roots grew vigorously in this treatment.
Conclusions and Discussion
By using the tender stems of Zhongshanshan 301 as the explants， the effects of NAA， 6-BA， IBA and KT on micropropagation of Zhongshanshan were evaluated on MS or 1/2 MS medium in this study. The results showed that Zhongshanshan can be proliferated via tissue culture. The combined use of NAA and 6-BA in MS medium greatly promoted the differentiation of Zhongshanshan explants， and the optimal medium was MS + 0.2 mg/L NAA + 0.4 mg/L6-BA. The optimal medium for the proliferation of differentiated buds was MS + 0.3 mg/L NAA + 0.4 mg/L 6-BA， on which the proliferation rate was up to 3.8. 1/2 MS medium was more conducive than MS medium to the induction of rooting. The optimal medium for the rooting of tissue-cultured Zhongshanshan shoots was 1/2MS + 0.3 mg/L IBA + 0.2 mg/L NAA. There are various factors that affect browning of explants in tissue culture. It has been suggested that the part of plants that are used as explants， the timing of sampling and plant species all have an effect on the browning of explants[8]. Our results showed that the number of browned plants in the treatments with only NAA or 6-BA was higher than that in the treatments with both NAA and 6-BA. The reason may be that the concentration and proportion of hormones have an influence on browning of explants. Woody plants have a high content of phenolic compounds， and 6-BA can promote the synthesis of phenolic compounds， to elevate the activity of polyphenol oxidase （PPO）[9]， which is a critical enzyme catalyzing the oxidation of phenolic compounds to form grey or brown colors， leading to the browning of explants. The number of differentiated buds and induction rate were both increased by the addition of 0.1-0.4 mg/L NAA in medium， but decreased when the concentration of 6-BA was increased from 0.6 to 0.9 mg/L， proving that the concentrations of NAA and 6-BA had an effect on the induction of Zhongshanshan explants. Fang[10] found that 6-BA played a decisive role in the proliferation of Pinus elliottii × Pinus caribaea adventitious buds， while NAA was able to improve the proliferation efficiency and proliferation rate. Our results showed that the addition of 0.3 mg/L NAA and 0.4 mg/L 6-BA greatly increased the number of differentiated buds and proliferation rate， and the buds grew very well， suggesting that the combined treatment with NAA and 6-BA is critical for the proliferation of differentiated shoots/seedlings of Zhongshanshan.
After a large number of buds and shoots are produced from explants through tissue culture， induction of rooting is a vital step in determining whether the buds and shoots can survive[11]. Lin[12] believed that the optimal medium for the rooting of tissue cultured C. lanceoleta seedlings was 1/2MS + 30/L sucrose + 1.2 mg/L IBA + 0.4 mg/L NAA. Our results showed that 1/2MS medium +0.3 mg/L IBA +0.2 mg/L NAA was the optimal medium for the rooting of tissue culture seedlings of Zhongshanshan， which is slightly different from the findings in Juglans regia[8]. The reason may be that rooting of tissue cultured seedlings is affected by multiple factors including plant species， genotype， basal medium type and plant growth regulators.
References
[1] YIN YL， CHEN YH. The survey and evaluation to comparative plantation of Taxodium ''zhongshansa'' and T. ascendens， T. distichum and Metaseqoia glyptostroboides[J]. Journal of Plant Resources and Environment， 1997， 6（3）： 23-28. [2] TULECKE W， MCGRANAHAN G， AHMADI H. Regeneration bysomatic embryogenesis of triploid plant from endosperm of walnnt， Julans regia[J]. Plant Cell Reports， 1988（7）： 301-304.
[3] LIU YQ， YANG JM， PENG WX， et al. The anatomical study on the rooting of the green shoot cutting propagation of two kinds of Tilia[J]. Journal of Agricultural University of Hebei， 2004， 27（2）： 33-37.
[4] CAO B， GAO HD. Technology of cutting propagation of Simmondisa chinensis （Link） Schneider[J]. Journal of Nanjing Forestry University （Natural Science Edition）， 2003， 27（4）： 62-66.
[5] GUO SJ. Progress of study on rooting anatomy and physiology of forest tree cuttings[J]. Journal of Beijing Forestry University， 1997， 19（4）： 64-69.
[6] WANG HF， HE M， YU CG， et al. Review on research status of Taxodium ‘Zhongshansa’ clones[J]. Nanfang Forestry Science， 2015， 43（3）： 17-21， 29.
[7] GUO DY. Application of biotechnology in fruit trees[J]. Journal of Fruit Science， 1992， 9（3）： 173-178.
[8] LIU SL， HAN BW. Plant regeneration from excised embryos of Juglans regia[J]. Plant Physiology Journal， 1989， 15（1）： 78-100.
[9] GAO GX. Browning in plant tissue culture[J]. Plant Physiology Communications， 1999， 35（6）： 501-506.
[10] FANG LJ. Influence of 6-BA and NAA on propagation of Pinus elliottii × Pinus caribaea[J]. Journal of Anhui Agricultural Sciences， 2012， 40（14）： 8180， 8183.
[11] CAO ZY， LIU GM. Plant tissue culture[M]. Lanzhou： Gansu Science and Technology Press， 1999： 85.
[12] LIN JQ. Studies on subculture and rooting culture of superior C. lanceoleta[J]. Journal of Anhui Agricultural Sciences， 2011， 39（15）： 8933-8934， 8937.