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Abstract [Objectives]This study was conducted on tissueculture rapid propagation techniques of Spathiphyllum kochii Engl. et Krause.[Methods]With lateral buds of S. kochii as explants， the effects of such four basic media as MS， B5， Nitsch and Wpm and the ratio of two hormones （1， 2， 3 mg/L 6BA and 0.1， 0.3， 0.5 mg/L NAA） on bud proliferation of S. kochii were studied by the complete test method， and the effect of the ratio of the two hormones （0.25， 0.50， 1.00 mg/L NAA and 1.0， 1.5 mg/L IBA ） on rooting of S. kochii as well as the effect of substrate ratio （perlite and peat soil at 1≥9， 2≥8， 3≥7， 4≥6 and 5≥5） on its transplanting survival rate were also studied.[Results]The best basic medium for the rapid propagation of S. kochii was MS. The best hormone ratio suitable for bud proliferation was 2 mg/L6BA+0.1 mg/L NAA， and the average number of buds proliferated at 45 d was 3.04. The average bud height was 2.05 cm; the most suitable medium for rooting was 1/2MS+1 mg/L IBA+0.25 mg/L NAA， and its rooting rate was 100%; and the best transplanting substrate was 5≥5 perlite and peat. The soil ratio had a survival rate of 94%.[Conclusions]This study provides a theoretical basis for the improvement of the transplanting survival rate of testtube S. kochii plantlets.
Key words Spathiphyllum kochii Engl. et Krause; Tissue culture; Rapid propagation
Spathiphyllum kochii Engl. et Krause， also known as white palm， is a perennial herb in Spathiphyllum of Araceae. S. kochii has green leaves and white spathes. It is one of the most important flowering and foliage plants in the world， as well as a small expert in cleaning indoor exhaust gas because it has certain effects on ammonia， acetone， benzene and formaldehyde[1]. S. kochii is one of the most popular Spathiphyllum varieties in China in recent years. Generally， the plant height can reach 30-40 cm in 5-8 months. Its stem is short， and the leaves are long round or nearly lanceolate， with clear midrib and petiole; and when flowering in spring and summer， the spathe is large and obvious， higher than the leaf surface， white or slightly green. It is very beautiful when flowering. It is also a good indoor potted foliage plant when it is not blooming. It is very popular as a potted flower in the house.
At present， the propagation method of S. kochii is mainly division propagation， but the annual reproductive coefficient is low， and the survival rate is low， which is not suitable for commercial scale production. By means of tissue culture， a large number of tissue culture seedlings can be raised for production in a short time[2-5]. The study of S. kochii tissue culture began in the late 1970s. Fonnesbech et al.[6]first used buds and stem segments as explants， and successfully induced adventitious buds on MS medium supplemented with 2 mg/L PBA. Subsequently， research on the tissue culture of S. kochii has gradually started at home and abroad. In China， Zhang et al.[7]first used buds as explants， and induced adventitious buds on MS medium supplemented with 6 mg/L BA+0.2 mg/LNAA hormone combination. They successfully established a tissueculture rapid propagation system of S. kochii and found that the bud proliferation effect was the best at 30. In 1993， Ma et al.[8]found that under the same conditions， the liquid oscillation culture method can significantly improve the proliferation rate of S. kochii buds. In 1994， Zhou et al.[9]used young S. kochii inflorescences as explants， and induced somatic embryos for the first time on the MS medium supplemented with 4-5 mg/L NAA after 25 d of culture in the dark. Since the 21st century， research on the culture technology of S. kochii has increased rapidly， including explant selection[10]， disinfection treatment[7，11]， hormone ratio[12]， culture conditions[8，13]and seedling training and transplanting[14].
Materials and Methods
Experimental materials
The potted S. kochii plants with strong growth and no pests and diseases of Hubei Nong Qing Gardening Technology Co.， Ltd. in Zhongxiang City， Hubei Province was used as experimental materials， of which the lateral buds were cut out as explants to establish a sterile culture system.
Experimental methods
The donor plants were watered less， to keep the soil dry. Then， the lateral buds were cut from the plants， and the outer leaves were peeled off. The buds were placed in a beaker filled with saturated washing powder water， shaken for 5 min to make uniform contact， and then rinsed with tap water until there was no foam and with running water for 1 h. The treated buds were soaked for 30 s with 75% alcohol on a clean bench， then sterilized with 0.15% mercuric chloride for 8 min， and rinsed with sterile water for 5-6 times. Finally， the material was inoculated on the medium， to produce the sterile line.
Screening of basic medium
MS， B5， Nitsch and Wpm were tested under the same concentrations of hormones while keeping other factors consistent （2.0 mg/L 6BA+0.1 mg/L NAA+sucrose 3%+agar powder 0.6%， pH 5.8）， to screen out the best basic medium. The height of buds and the number of buds proliferated were recorded at 15， 30 and 45 d. Eight bottles were set per treatment， and three explants were inoculated per bottle.
Effects of hormone ratio on proliferation of lateral buds
After subculture in the medium， MS+2.0 mg/L 6BA+0.1 mg/L NAA+sucrose 3%+agar powder 0.6%， pH 5.8， for 2-3 generations， the lateral buds were taken out and inoculated to media containing 1， 2 and 3 mg/L 6BA and 0.1， 0.3 and 0.5 mg/L NAA， respectively， for proliferation culture. The two hormones were combined by the complete test method， forming nine groups of tests， from which the optimal hormone ratio was screened. At 15， 30 and 45 d of culture， the height of buds and the number of buds proliferated were recorded. Each bottle was inoculated with three lateral buds， and each treatment included eight bottles. The media contained 3% sucrose and 0.6% agar powder， and had a pH value of 5.8. Effects of hormone concentrations on rooting of testtube plantlets
After the proliferation culture， single buds of 4-5 cm on the same medium were inoculated into the rooting medium for rooting culture. The rooting used 1/2MS as the basic medium， which was supplemented with 0.25， 0.5 and 1 mg/L NAA and 1 and 2 mg/L IBA， respectively. Six groups of tests were formed according to the complete test method. The number of rooted buds， root length and rooting rate were counted after 30 and 45 d， and the best rooting medium was screened. Eight bottles were set per treatment， and three buds were inoculated per bottle. The media contained 3% sucrose and 0.6% agar powder， and had a pH value of 5.8.
Effect of substrate ratio on survival rate of testtube plantlets
After 45 d of rooting culture， the caps of the test tube plantlets were released， and they were moved to natural light for 2 d to train the plantlets. The bottle caps were opened and 3-5 mm of sterile water was added to the surface of the culture medium to train the plantlets for 7 d. Then， the tissue culture plantlets were gently removed with tweezers. After washing to remove the rootadhered medium， plantlets with uniform growth were transplanted to five groups of perlite and peat soil substrates which were at the ratio of 1≥9， 2≥8， 3≥7， 4≥6 and 5≥5， respectively， to screen for the best nutrient substrate. Each substrate was filled into 32hole trays and subjected to disinfection treatment with 0.1% potassium permanganate solution. The plantlets were watered thoroughly and covered with film to keep moisture. They were cultured under relative humidity over 65% and shade of 60%-70%. The film was uncovered 7 d later， and watering was performed at a higher frequency to keep the seedbed wet. Each treatment included 16 plantlets， and the survival rates of domesticated plants under different substrate ratios were recorded after 30 d of culture.
Culture conditions
The culture chamber had a culture temperature at （25÷2）， a light intensity of 2 000-3 000 Lx and illumination time of 12 h per day. The transplanting temperature was natural temperature in spring and autumn， at about 20-28.
Data analysis
The test data was statistically analyzed using Excel2007 and SAS9.2 software.
Results and Analysis
Screening of basic medium
The sterile plantlets obtained by cultivating the lateral bud explants with uniform growth were inoculated on the basic media of different treatments. After a few days， the lateral buds began to germinate， and the bud height and the number of proliferated buds were recorded at 15， 30 and 45 d， as shown in Table 1. It can be seen from Table 1 that there was no significant difference in the growth and proliferation of S. kochii in the four basic media at 15 and 30 d， but with the prolongation of the culture time， the growth and proliferation of the buds were obviously different at 40 d. The proliferation effects of buds in B5 and MS media were both better， but the buds in MS medium had better growth vigor. In summary， the optimal basic medium for the proliferation of S. kochii buds was determined to be MS. Effect of hormone ratio on the proliferation of adventitious buds
After subculture of the same treatment for 2-3 generations， the buds with relatively uniform growth were inoculated onto the proliferation media with different hormone concentrations. After a few days， the lateral buds began to germinate， and the bud height and the number of buds proliferated were recorded at 15， 30 and 45 d， as shown in Table 2. It can be seen from Table 2 that in different hormone ratio treatments， the differences in the growth and proliferation of S. kochii buds were more and more significant with the prolongation of the culture time. There were differences in the growth of bud height and bud number between the nine groups at 15 d， but the differences between them were not particularly significant. At 30 d， the differences in the growth of bud height and bud number between the nine groups increased significantly， and especially the significance of the difference in bud height was enhanced faster. At 45 d， the significance of the differences in bud height and bud number between the nine groups of treatments was significantly enhanced， and especially the significance of the difference in bud number was enhanced faster， reaching a level of significant difference. On the 45th day， the buds proliferated fastest on the medium of 2.0 mg/L 6BA+0.1 mg/L NAA， and the bud proliferation quantity was the highest， reaching about 3.04; and the bud height increased also faster， reaching about 2.05 cm， second only to treatment 3.0 mg/L 6BA+0.5 mg/L NAA. In summary， the optimal medium for the proliferation of buds in the subculture of S. kochii was MS+2.0 mg/L 6BA+0.1 mg/L NAA.
Effects of hormone ratios on the rooting of testtube plantlets
Single buds of 4-5 cm with the best growth in the same culture medium were selected for rooting culture. The rooting conditions of the test tube plantlets in the six different hormone treatments are shown in Table 3. It can be seen from Table 3 that the rooting speed of S. kochii was slightly lower， and many plantlets did not rooted at 30 d when there were no obvious differences in root length and rooting rate between the six different treatments， but the number of roots differed， though the difference did not reach a particularly significant level. There were significant difference in root length， root number and rooting rate between the six different treatments at 45 d. However， there was no significant difference between treatments 1 mg/L IBA+0.25-0.5 mg/L NAA and 1.5 mg/L IBA+1 mg/L NAA. The rooting rate in treatment 1 mg/L IBA+0.25-0.5 mg/L NAA was the highest， reaching 100%， while in treatment 1.5 mg/L IBA+1 mg/L NAA， the roots grew best. The differences in root length， root number and rooting rate were not significant between any two of the three treatments mentioned above. Taking into account the principle of saving， it was determined that the suitable rooting medium for the S. kochii testtube plantlets was 1/2MS+1.00 mg/L IBA+0.25 mg/L NAA. Effects of substrate ratio on the survival rate of transplanted testtube seedlings
The domesticated rooted testtube plantlets were transplanted to substrates with different ratios of perlite and peat soil for cultivation. The results recorded after 30 d are shown in Table 4. It can be seen from Table 4 that the ratio of 5≥5 （perlite to peat soil） in the five different substrate ratios was most suitable for the domestication and transplanting of the S. kochii testtube plantlets， and the survival rate can reach about 94%.
Conclusions and Discussion
Conclusions
In rapid propagation of S. kochii by tissue culture， the proliferation of adventitious buds induced from buds is easier than that of adventitious buds produced from calli induced with leaves and petioles with a lower mutation rate. The number of top buds is relatively smaller than that of lateral buds， so lateral buds were chosen in this experiment as explants.
In plant tissue culture， different plants require different basic medium types， and the addition of hormones is an important factor affecting plant proliferation and rooting. In the rapid propagation of S. kochii by tissue culture， MS was determined as the basic medium more suitable for the growth and proliferation of S. kochii buds by screening four different kinds of basic media. Due to the accumulation of hormones， the types and concentrations of hormones required by S. kochii in different culture stages are different. In this study， the appropriate bud proliferation medium was determined by hormone ratio treatment as MS+2.0 mg/L 6BA+0.1 mg/L NAA， and the medium suitable for rooting was 1/2MS+1 mg/L IBA+0.25 mg/L NAA.
In addition， the type of transplanting substrate also affected the transplanting survival rate of the S. kochii testtube plantlets. In this study， the mixed substrate of perlite and peat soil at the ratio of 5≥5 was determined through experimental comparison to be a suitable substrate， and the survival rate of transplanting was about 94%.
Discussion
In rapid propagation of S. kochii by tissue culture， the number of buds induced by lateral buds as explants was relatively limited. The average number of buds proliferated after 45 d of culture was only about 3， which might be related to the action of hormones. The explants had a certain adaptation process for invitro culture， and an increase in the number of proliferated buds was observed in the same medium in subsequent experiments. Three suitable rooting media appeared in the rooting experiment of S. kochii， which might be related to the shorter days recorded and the limited levels of IBA and NAA. According to Liu et al.[15]， activated carbon plays a very important role in plant tissue culture. It can not only adsorb toxic substances in the medium， but also can provide a dark environment to avoid the inhibition of root growth by strong light. In further experimental research， the effect of activated carbon on the rooting of testtube S. kochii plantlets can be tested.
References
[1]HUANG ZM. Precious flower cultivation[M]. Guangzhou： Guangdong Science and Technology Press， 1996. （in Chinese）
[2]HU TY， LI CK. Study on the selection of provenances of Eucalyptus grandis[J]. Journal of Sichuan Agricultural University， 1999， 17（01）： 44-493. （in Chinese）
[3]QIU JQ， LAN HS， XIE GY， et al. A primary report on the provenance/family trial on Eucalyptus grandis[J]. Acta Agriculturae Universitis Jiangxiensis， 2002， 24（04）： 517-521. （in Chinese）
[4]LIANG KN. Study on trial of Eucalyptus species/provenance[J]. Forest Research，2000，13（02）： 203-208. （in Chinese）
[5]WANG HR. The provenance test of Eucalyptus grandis and research on its suitable range in China[J]. Forest Research， 1989， 2（05）： 411-419. （in Chinese）
[6]FONNESBECH M， FONNESBECH A. In vitro propagation of Spathiphyllum[J]. Scientia Horticculturae，1979，10（1）： 21-25.
[7]ZHANG YK， ZHU XR， HUANG HQ. Rapid propagation and transplanting test of test tube plantlets of Spathiphyllum[J]. Guangdong Agricultural Sciences， 1991（4）： 29-31. （in Chinese）
[8]MA GH， ZHU XR， ZHANG YK， et al. High speed propagation of Spathiphyllum shoots in liquid shake culture[J]. Acta Horticulturae Sinica， 1993， 20（3）： 307-308. （in Chinese）
[9]ZHOU LN， KUANG ZS， CHEN JQ， et al. Establishment of somatic embryo clones of Spathiphyllum[J]. Guangdong Landscape Architecture， 1994（4）： 18-20. （in Chinese）
[10]YANG ST， WANG HX， LI N， et al. Preliminary research on explant selection of Spathiphyllum floribundum[J]. Mod Agric Sci Technol， 2011（16）： 187. （in Chinese）
[11]ZHU FX， GUO L. Study on tissue culture technology of Spathiphyllum[J]. Agriculture of Henan， 2014（6）： 40-42. （in Chinese）
[12]YANG PJ， ZHAO H， LI HN， et al. Medium in subculture and rapid propagation of green jiant[J]. Journal of Hanzhong Teachers College： Natural Science， 2001（3）： 75-78. （in Chinese）
[13]DEWIR YH， CHAKRABARTY D， HAHN EJ， et al. A simple method formass propagation of Spathiphyllum cannifolium using an airlift bioreactor[J]. In Vitro Cellular & Developmental Biology Plant， 2006， 42 （3）： 291-297.
[14]CHEN HX， ZHANG LS， WAN XF， et al. Study of Spathiphyllum ‘Vicki’ shoot tip culture in vitro and rapid propagation[J]. Fujian Journal of Agricultural Sciences， 2013， 28（5）： 486-489. （in Chinese）
[15]LIU GL， LIANG HZ， ZHU J. Effects of activated charcoal on plant tissue cultures： A review[J]. Journal of Jiangsu Forestry Science & Technology， 2001， 28（05）： 46-48. （in Chinese）
Key words Spathiphyllum kochii Engl. et Krause; Tissue culture; Rapid propagation
Spathiphyllum kochii Engl. et Krause， also known as white palm， is a perennial herb in Spathiphyllum of Araceae. S. kochii has green leaves and white spathes. It is one of the most important flowering and foliage plants in the world， as well as a small expert in cleaning indoor exhaust gas because it has certain effects on ammonia， acetone， benzene and formaldehyde[1]. S. kochii is one of the most popular Spathiphyllum varieties in China in recent years. Generally， the plant height can reach 30-40 cm in 5-8 months. Its stem is short， and the leaves are long round or nearly lanceolate， with clear midrib and petiole; and when flowering in spring and summer， the spathe is large and obvious， higher than the leaf surface， white or slightly green. It is very beautiful when flowering. It is also a good indoor potted foliage plant when it is not blooming. It is very popular as a potted flower in the house.
At present， the propagation method of S. kochii is mainly division propagation， but the annual reproductive coefficient is low， and the survival rate is low， which is not suitable for commercial scale production. By means of tissue culture， a large number of tissue culture seedlings can be raised for production in a short time[2-5]. The study of S. kochii tissue culture began in the late 1970s. Fonnesbech et al.[6]first used buds and stem segments as explants， and successfully induced adventitious buds on MS medium supplemented with 2 mg/L PBA. Subsequently， research on the tissue culture of S. kochii has gradually started at home and abroad. In China， Zhang et al.[7]first used buds as explants， and induced adventitious buds on MS medium supplemented with 6 mg/L BA+0.2 mg/LNAA hormone combination. They successfully established a tissueculture rapid propagation system of S. kochii and found that the bud proliferation effect was the best at 30. In 1993， Ma et al.[8]found that under the same conditions， the liquid oscillation culture method can significantly improve the proliferation rate of S. kochii buds. In 1994， Zhou et al.[9]used young S. kochii inflorescences as explants， and induced somatic embryos for the first time on the MS medium supplemented with 4-5 mg/L NAA after 25 d of culture in the dark. Since the 21st century， research on the culture technology of S. kochii has increased rapidly， including explant selection[10]， disinfection treatment[7，11]， hormone ratio[12]， culture conditions[8，13]and seedling training and transplanting[14].
Materials and Methods
Experimental materials
The potted S. kochii plants with strong growth and no pests and diseases of Hubei Nong Qing Gardening Technology Co.， Ltd. in Zhongxiang City， Hubei Province was used as experimental materials， of which the lateral buds were cut out as explants to establish a sterile culture system.
Experimental methods
The donor plants were watered less， to keep the soil dry. Then， the lateral buds were cut from the plants， and the outer leaves were peeled off. The buds were placed in a beaker filled with saturated washing powder water， shaken for 5 min to make uniform contact， and then rinsed with tap water until there was no foam and with running water for 1 h. The treated buds were soaked for 30 s with 75% alcohol on a clean bench， then sterilized with 0.15% mercuric chloride for 8 min， and rinsed with sterile water for 5-6 times. Finally， the material was inoculated on the medium， to produce the sterile line.
Screening of basic medium
MS， B5， Nitsch and Wpm were tested under the same concentrations of hormones while keeping other factors consistent （2.0 mg/L 6BA+0.1 mg/L NAA+sucrose 3%+agar powder 0.6%， pH 5.8）， to screen out the best basic medium. The height of buds and the number of buds proliferated were recorded at 15， 30 and 45 d. Eight bottles were set per treatment， and three explants were inoculated per bottle.
Effects of hormone ratio on proliferation of lateral buds
After subculture in the medium， MS+2.0 mg/L 6BA+0.1 mg/L NAA+sucrose 3%+agar powder 0.6%， pH 5.8， for 2-3 generations， the lateral buds were taken out and inoculated to media containing 1， 2 and 3 mg/L 6BA and 0.1， 0.3 and 0.5 mg/L NAA， respectively， for proliferation culture. The two hormones were combined by the complete test method， forming nine groups of tests， from which the optimal hormone ratio was screened. At 15， 30 and 45 d of culture， the height of buds and the number of buds proliferated were recorded. Each bottle was inoculated with three lateral buds， and each treatment included eight bottles. The media contained 3% sucrose and 0.6% agar powder， and had a pH value of 5.8. Effects of hormone concentrations on rooting of testtube plantlets
After the proliferation culture， single buds of 4-5 cm on the same medium were inoculated into the rooting medium for rooting culture. The rooting used 1/2MS as the basic medium， which was supplemented with 0.25， 0.5 and 1 mg/L NAA and 1 and 2 mg/L IBA， respectively. Six groups of tests were formed according to the complete test method. The number of rooted buds， root length and rooting rate were counted after 30 and 45 d， and the best rooting medium was screened. Eight bottles were set per treatment， and three buds were inoculated per bottle. The media contained 3% sucrose and 0.6% agar powder， and had a pH value of 5.8.
Effect of substrate ratio on survival rate of testtube plantlets
After 45 d of rooting culture， the caps of the test tube plantlets were released， and they were moved to natural light for 2 d to train the plantlets. The bottle caps were opened and 3-5 mm of sterile water was added to the surface of the culture medium to train the plantlets for 7 d. Then， the tissue culture plantlets were gently removed with tweezers. After washing to remove the rootadhered medium， plantlets with uniform growth were transplanted to five groups of perlite and peat soil substrates which were at the ratio of 1≥9， 2≥8， 3≥7， 4≥6 and 5≥5， respectively， to screen for the best nutrient substrate. Each substrate was filled into 32hole trays and subjected to disinfection treatment with 0.1% potassium permanganate solution. The plantlets were watered thoroughly and covered with film to keep moisture. They were cultured under relative humidity over 65% and shade of 60%-70%. The film was uncovered 7 d later， and watering was performed at a higher frequency to keep the seedbed wet. Each treatment included 16 plantlets， and the survival rates of domesticated plants under different substrate ratios were recorded after 30 d of culture.
Culture conditions
The culture chamber had a culture temperature at （25÷2）， a light intensity of 2 000-3 000 Lx and illumination time of 12 h per day. The transplanting temperature was natural temperature in spring and autumn， at about 20-28.
Data analysis
The test data was statistically analyzed using Excel2007 and SAS9.2 software.
Results and Analysis
Screening of basic medium
The sterile plantlets obtained by cultivating the lateral bud explants with uniform growth were inoculated on the basic media of different treatments. After a few days， the lateral buds began to germinate， and the bud height and the number of proliferated buds were recorded at 15， 30 and 45 d， as shown in Table 1. It can be seen from Table 1 that there was no significant difference in the growth and proliferation of S. kochii in the four basic media at 15 and 30 d， but with the prolongation of the culture time， the growth and proliferation of the buds were obviously different at 40 d. The proliferation effects of buds in B5 and MS media were both better， but the buds in MS medium had better growth vigor. In summary， the optimal basic medium for the proliferation of S. kochii buds was determined to be MS. Effect of hormone ratio on the proliferation of adventitious buds
After subculture of the same treatment for 2-3 generations， the buds with relatively uniform growth were inoculated onto the proliferation media with different hormone concentrations. After a few days， the lateral buds began to germinate， and the bud height and the number of buds proliferated were recorded at 15， 30 and 45 d， as shown in Table 2. It can be seen from Table 2 that in different hormone ratio treatments， the differences in the growth and proliferation of S. kochii buds were more and more significant with the prolongation of the culture time. There were differences in the growth of bud height and bud number between the nine groups at 15 d， but the differences between them were not particularly significant. At 30 d， the differences in the growth of bud height and bud number between the nine groups increased significantly， and especially the significance of the difference in bud height was enhanced faster. At 45 d， the significance of the differences in bud height and bud number between the nine groups of treatments was significantly enhanced， and especially the significance of the difference in bud number was enhanced faster， reaching a level of significant difference. On the 45th day， the buds proliferated fastest on the medium of 2.0 mg/L 6BA+0.1 mg/L NAA， and the bud proliferation quantity was the highest， reaching about 3.04; and the bud height increased also faster， reaching about 2.05 cm， second only to treatment 3.0 mg/L 6BA+0.5 mg/L NAA. In summary， the optimal medium for the proliferation of buds in the subculture of S. kochii was MS+2.0 mg/L 6BA+0.1 mg/L NAA.
Effects of hormone ratios on the rooting of testtube plantlets
Single buds of 4-5 cm with the best growth in the same culture medium were selected for rooting culture. The rooting conditions of the test tube plantlets in the six different hormone treatments are shown in Table 3. It can be seen from Table 3 that the rooting speed of S. kochii was slightly lower， and many plantlets did not rooted at 30 d when there were no obvious differences in root length and rooting rate between the six different treatments， but the number of roots differed， though the difference did not reach a particularly significant level. There were significant difference in root length， root number and rooting rate between the six different treatments at 45 d. However， there was no significant difference between treatments 1 mg/L IBA+0.25-0.5 mg/L NAA and 1.5 mg/L IBA+1 mg/L NAA. The rooting rate in treatment 1 mg/L IBA+0.25-0.5 mg/L NAA was the highest， reaching 100%， while in treatment 1.5 mg/L IBA+1 mg/L NAA， the roots grew best. The differences in root length， root number and rooting rate were not significant between any two of the three treatments mentioned above. Taking into account the principle of saving， it was determined that the suitable rooting medium for the S. kochii testtube plantlets was 1/2MS+1.00 mg/L IBA+0.25 mg/L NAA. Effects of substrate ratio on the survival rate of transplanted testtube seedlings
The domesticated rooted testtube plantlets were transplanted to substrates with different ratios of perlite and peat soil for cultivation. The results recorded after 30 d are shown in Table 4. It can be seen from Table 4 that the ratio of 5≥5 （perlite to peat soil） in the five different substrate ratios was most suitable for the domestication and transplanting of the S. kochii testtube plantlets， and the survival rate can reach about 94%.
Conclusions and Discussion
Conclusions
In rapid propagation of S. kochii by tissue culture， the proliferation of adventitious buds induced from buds is easier than that of adventitious buds produced from calli induced with leaves and petioles with a lower mutation rate. The number of top buds is relatively smaller than that of lateral buds， so lateral buds were chosen in this experiment as explants.
In plant tissue culture， different plants require different basic medium types， and the addition of hormones is an important factor affecting plant proliferation and rooting. In the rapid propagation of S. kochii by tissue culture， MS was determined as the basic medium more suitable for the growth and proliferation of S. kochii buds by screening four different kinds of basic media. Due to the accumulation of hormones， the types and concentrations of hormones required by S. kochii in different culture stages are different. In this study， the appropriate bud proliferation medium was determined by hormone ratio treatment as MS+2.0 mg/L 6BA+0.1 mg/L NAA， and the medium suitable for rooting was 1/2MS+1 mg/L IBA+0.25 mg/L NAA.
In addition， the type of transplanting substrate also affected the transplanting survival rate of the S. kochii testtube plantlets. In this study， the mixed substrate of perlite and peat soil at the ratio of 5≥5 was determined through experimental comparison to be a suitable substrate， and the survival rate of transplanting was about 94%.
Discussion
In rapid propagation of S. kochii by tissue culture， the number of buds induced by lateral buds as explants was relatively limited. The average number of buds proliferated after 45 d of culture was only about 3， which might be related to the action of hormones. The explants had a certain adaptation process for invitro culture， and an increase in the number of proliferated buds was observed in the same medium in subsequent experiments. Three suitable rooting media appeared in the rooting experiment of S. kochii， which might be related to the shorter days recorded and the limited levels of IBA and NAA. According to Liu et al.[15]， activated carbon plays a very important role in plant tissue culture. It can not only adsorb toxic substances in the medium， but also can provide a dark environment to avoid the inhibition of root growth by strong light. In further experimental research， the effect of activated carbon on the rooting of testtube S. kochii plantlets can be tested.
References
[1]HUANG ZM. Precious flower cultivation[M]. Guangzhou： Guangdong Science and Technology Press， 1996. （in Chinese）
[2]HU TY， LI CK. Study on the selection of provenances of Eucalyptus grandis[J]. Journal of Sichuan Agricultural University， 1999， 17（01）： 44-493. （in Chinese）
[3]QIU JQ， LAN HS， XIE GY， et al. A primary report on the provenance/family trial on Eucalyptus grandis[J]. Acta Agriculturae Universitis Jiangxiensis， 2002， 24（04）： 517-521. （in Chinese）
[4]LIANG KN. Study on trial of Eucalyptus species/provenance[J]. Forest Research，2000，13（02）： 203-208. （in Chinese）
[5]WANG HR. The provenance test of Eucalyptus grandis and research on its suitable range in China[J]. Forest Research， 1989， 2（05）： 411-419. （in Chinese）
[6]FONNESBECH M， FONNESBECH A. In vitro propagation of Spathiphyllum[J]. Scientia Horticculturae，1979，10（1）： 21-25.
[7]ZHANG YK， ZHU XR， HUANG HQ. Rapid propagation and transplanting test of test tube plantlets of Spathiphyllum[J]. Guangdong Agricultural Sciences， 1991（4）： 29-31. （in Chinese）
[8]MA GH， ZHU XR， ZHANG YK， et al. High speed propagation of Spathiphyllum shoots in liquid shake culture[J]. Acta Horticulturae Sinica， 1993， 20（3）： 307-308. （in Chinese）
[9]ZHOU LN， KUANG ZS， CHEN JQ， et al. Establishment of somatic embryo clones of Spathiphyllum[J]. Guangdong Landscape Architecture， 1994（4）： 18-20. （in Chinese）
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