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Abstract Factors affecting micro-graft in vitro were studied in persimmon lines of Jiro， Nishimarawase， Zenjiomaru， Okitsu-20， Xinqiu， Maekawa Jiro and Youhou. The results indicated that higher survival rate was obtained when using the plantlets sub-cultured for 30 d with vigorous adventitious shoots. The best moisture-preserving material was the tampon + parafilm. The optimal medium was （1/2N） MS + BA 3.0 mg/L + IAA 0.1 mg/L + sugar 30.0 g/L + agar 6.0g/L， and the graft survival rate was up to 40%.
Key words Persimmon； Micro-grafting； Survival rate
Micro-grafting of tissue culture plantlets （micro-grafting for short） is a kind of technique for grafting stock and scion in test tubes， which is the combination of tissue culture and grafting technique. This technique is widely applied in research about fruit tree， such as identification of graft compatibility[1]， grafting mechanism research[2]， rapid detection of virus， virus elimination， rapid raising of virus-free seedling[3]， identification of stock growth form and screening of stock.
Diospyros lotus is widely applied in northern China as a stock of persimmon. However， in recent years， sweet persimmon cultivars introduced from Japan and Korea are more strict in stock[4]， which is reflected by poor compatibility， inhibited growth at the later stage and reduced deastringent degree. Ma et al.[5] deemed that Zhejiang persimmon had the strong compatibility with Youhou sweet persimmon， and the grafted plantlets grew the best. However， Zhejiang persimmon is not suitable for planting in northern China， which restricts its development. In this study， the influencing factors and suitable conditions for micro-grafting of test-tube plantlets were discussed through the grafting of D. lotus with seven major sweet persimmon cultivars in production， so as to lay a foundation and provide a theoretical basis for the screening of stock suitable for micro-grafting of sweet persimmon.
Materials and Methods
Materials
Tissue culture plantlets were obtained from culture of D. lotus seeds， and sweet persimmon cultivars， Jiro， Nishimarawase， Zenjiomaru， Okitsu-20， Xinqiu， Maekawa Jiro and Youhou were used as scions. All the sweet persimmon cultivars were collected from the orchard of Yuandi Village， Hongmen County， Xinxiang City， Henan Province， and the trees were 9 years old.
Methods
Acquisition of aseptic plantlets of D. lotus
D. lotus seeds were obtained from mature fruit and cleaned with clear water. The seeds were soaked in 75% ethanol for 30 d under aseptic condition， and flushed with sterile water for 3 times. The seeds were then soaked with 0.1% HgCl2 for 10 min under continuous shaking， and flushed with sterile water for 5-6 times， and water on seed surface was absorbed with sterile filter. The treated seeds were cultured in medium， which was （1/2N）MS + GA 0.5mg/L+ PVP 0.6 g/L + sucrose 30 g/L + agar 6.0 g/L. Acquisition of aseptic plantlets of sweet persimmon
In dormant season （December-March）， annual branches growing strongly were selected and cut into segments， each having one dormant bud. The segments were cleaned and sterilized with 70% for 30 s and flushed with sterile water for 3 times. The segments were then soaked with 0.1% HgCl2 for 10 min under continuous shaking， and flushed with sterile water for 5-6 times， and water on the surface was absorbed with sterile filter. Bud scales and three layers of leaf primordium were stripped off from each bud， and about 3 mm of bud tip was cut and inoculated on medium， which was （1/2N）MS + 6-BA 4.0 mg/L + IAA 0.2 mg/L + PVP 0.6 g/L + sucrose 30 g/L +agar 6.0 g/L.
Test tube micro-grafting
The micro-grating was performed with reference to the grafting method of apple[1]. Strong stock and scion segment were selected， respectively. Under sterile condition， the head of each tissue culture plantlets of D. lotus was removed， leaving a stem segment with a length of about 1.5 cm. Lateral buds at the bottom were cut off longitudinally， with a length of about 0.5 cm. A stem segment of sweet persimmon with a length of about 1.5 cm and a thickness close to the stock were selected as a scion， with 2-4 leaves left at the top. The base of the scion was cut into wedge shape （with a length of about 0.5 cm）. The scion was inserted into the stock， and the graft union was tightened with aluminum foil. The grated test-tube plantlets were then cultured in medium. For each kind of stock and scion combination， 10 plants were grafted， and there were three replications.
Investigation and statistics of survival rate were performed 50 d after grafting. Grafting survival rate was calculated according to following equation： Grafting survival rate=Number of survived plants/Total number of grafted plants×100%.
Data processing
Percentage values were subjected to anti-sine conversion. Statistical analysis was performed in DPS12.0. ， and the significant level was 0.05.
Results and Analysis
Effect of growth condition of tissue culture plantlets on grafting survival rate
The growth conditions of tissue culture plantlets of stock and scion directly affected the survival rate of test tube micro-grafting. When the tissue culture plantlets had strong stem and top growing vigorously， the survival of grafted plants was facilitated[6]， and long internode facilitated operation. However， tissue culture plantlets of most Diospyros plants grow slowly， have short internode and was prone to browning， which is not beneficial to grafting. It was found in the experiment that internodes could grow longer by performing 10 d of dark culture before subculture of tissue culture plantlets， then， the stems could grow strongly with long internodes by performing 30 d of culture under strong light， and finally， grafting operation was facilitated when obtaining a high grafting survival rate. Furthermore， the addition of 5 g/L PVP in subculture medium could effectively reduce browning of tissue culture plantlets of D. lotus and sweet persimmon. Effects of different scions on grafting survival rate and compatibility
Different sweet persimmon cultivars were grafted with D. lotus as stock. It could be seen from Table 1 that there were greater differences in grafting survival rate between different sweet persimmon varieties， among which Youhou had the highest survival rate， followed by Jiro and Zenjiomaru. This might be due to the tissue culture plantlets of Youhou grown strongly， and the performance in grafting compatibility still needs further observation.
Effect of sucrose concentration on grafting survival rate
Sucrose as carbon source could provide energy for plant cells， and also could induce the formation of phloem. Navarro et al. found during micro-grafting of citrus that sucrose concentration plays a key role in survival of micro-grafted plants. With D. lotus as stock， Jiro was grafted and cultured to medium with different sucrose concentrations. The results （Table 2） showed that with the increase of sucrose concentration， the grafting survival rate increased significantly， but there were no significant difference between the sucrose concentrations of 5% and 7%， and when the sucrose concentration was 7%， the base parts of tissue culture plantlets browned seriously， which might be due to that the improvement of sugar content improved osmotic pressure in medium. Comprehensively from the experimental results and experimental cost， the sucrose concentration of 5% was more suitable.
Effect of different BA concentrations of grafting survival rate
The tissue culture plantlets of Jiro were grafted to tissue culture plantlets of D. lotus as stock. In the medium without the addition of BA， the growth of tissue culture plantlets was inhibited， and the grafting survival rate was thus lower. The addition of BA into medium could effectively promote the growth of tissue culture plantlets and improve grafting survival rate. However， the BA concentration should not be too high， and when the BA concentration reached 5.0 mg/L， the stock grew vigorously and thickly， and thus competed with the scion for nutrition， the graft unions could not well recover， and the grafting survival rate decreased.
Effect of air permeability of culture bottle on grafting survival rate
Air permeability of culture bottles would affect the microenvironment in bottles， including gas and humidity， among which humidity is one of the important factors affecting the survival of grafted plants. In this study， kraft paper， tampon + kraft paper， tampon + parafilm and double-layer parafilm were adopted to perform sealing treatment. The results showed that sealing with kraft paper resulted in serious water loss of medium and serous pollution， and after 3 weeks of culture， massive pollution was observed. Under sealing with parafilm， humidity in bottles was higher， there were a lot of water drops on the walls of bottles in the morning， which affected illumination， and the tissue culture plantlets suffered from serious vitrification. As to sealing with tampon + kraft paper， pollution was hardly observed in culture bottles， air permeability in the bottle was strong， tissue culture plantlets grew better， but due to lower humidity， the grafting survival rate was not as high as treatment tampon + parafilm. Therefore， tampon + parafilm was a more suitable sealing method， which not only could keep the sterile environment in bottles， but also could ensure suitable humidity in bottles， and was thus beneficial to survival of grafted plants. Agricultural Biotechnology2018
Discussion
During micro-grafting of tissue culture plantlets， when scions have too many leaves thereon， not only grafting is very difficult， but also the transpiration area increases， and scions suffer from serious water loss， resulting in reduced survival rate. When the scions have no leaves thereon， power for absorbing water and nutrients is lowered， and the survival rate is also lower. Therefore， during grafting， scions should have leaves thereon， and 2-4 leaves are proper.
Moisture is one of the main factors affecting survival rate of test-tube micro-grafting. During micro-grating， union should be bound with foil， while foil could hardly keep the humidity at the union， so the humidity at the union is the same as the environmental humidity in bottles. In order to improve environmental humidity in bottles and improve grafting survival rate， humidity could be regulated by sealing. The results of this study showed that tampon + parafilm not only could maintain sterile environment in bottles， but also could keep proper humidity in bottles， thereby facilitating survival of grafted plants.
There are also various factors influencing test-tube micro-grafting. In this study， factors including different sweet persimmon cultivars， growth status of test-tube plantlets， sucrose concentration， BA concentration and air permeability of culture environment were investigated. Overall. The grafting survival rate is not high， and various factors influencing micro-grafting of sweet persimmon should be symmetrically studied， so as to obtain higher grafting survival rate. This study laid a foundation for stock screening and breeding of sweet persimmon.
References
[1] CHENG YQ， HAN ZH， XU XF， et al. Early testing of grafting compatibilities between Malus xiaojinensis and two apple cultivars by micrografting in vitro[J]. Chinese journal of agricultural biotechnology， 2003， 11（5）： 472-476. （in Chinese）
[2] FAN SY， LIU YS. Effect of water stress on photosynthetic of Chinese dwarf cherry[C]// Proceedings of the Fourth Youth Colloquium of Chinese Society for Horticultural Science， 2000. （in Chinese）
[3] LIU Y， YANG YS， TONG X， et al. Development of an in vitro micrografting method for the enhancement for growth of isolated shoots and buds in Jatropha curcas L.[J]. Acta Botanica Boreali-Occidentalia Sinica， 2014， 34（10）： 2118-2124. （in Chinese）
[4] GONG BC， WANG JF. Study on different biological characteristics of stocks of persimmon[J]. Nonwood Forest Research， 1997， 15（ 1） ： 9-12. （in Chinese）
[5] MA P， GONG BC， JIANG XB， et al. Evaluation on affinity， growth trait and physiology indicators of persimmon grafted on different stocks[J]. Forest Research， 2015， 28（4）： 518-523. （in Chinese）
[6] ZHOU RJ， DU GQ， SHI XX， et al. Studies on factors affecting survival rate of micro-graft in vitro in transgenic apple[J]. Journal of Fruit Science， 2007， 24（2）： 215-217. （in Chinese）
Key words Persimmon； Micro-grafting； Survival rate
Micro-grafting of tissue culture plantlets （micro-grafting for short） is a kind of technique for grafting stock and scion in test tubes， which is the combination of tissue culture and grafting technique. This technique is widely applied in research about fruit tree， such as identification of graft compatibility[1]， grafting mechanism research[2]， rapid detection of virus， virus elimination， rapid raising of virus-free seedling[3]， identification of stock growth form and screening of stock.
Diospyros lotus is widely applied in northern China as a stock of persimmon. However， in recent years， sweet persimmon cultivars introduced from Japan and Korea are more strict in stock[4]， which is reflected by poor compatibility， inhibited growth at the later stage and reduced deastringent degree. Ma et al.[5] deemed that Zhejiang persimmon had the strong compatibility with Youhou sweet persimmon， and the grafted plantlets grew the best. However， Zhejiang persimmon is not suitable for planting in northern China， which restricts its development. In this study， the influencing factors and suitable conditions for micro-grafting of test-tube plantlets were discussed through the grafting of D. lotus with seven major sweet persimmon cultivars in production， so as to lay a foundation and provide a theoretical basis for the screening of stock suitable for micro-grafting of sweet persimmon.
Materials and Methods
Materials
Tissue culture plantlets were obtained from culture of D. lotus seeds， and sweet persimmon cultivars， Jiro， Nishimarawase， Zenjiomaru， Okitsu-20， Xinqiu， Maekawa Jiro and Youhou were used as scions. All the sweet persimmon cultivars were collected from the orchard of Yuandi Village， Hongmen County， Xinxiang City， Henan Province， and the trees were 9 years old.
Methods
Acquisition of aseptic plantlets of D. lotus
D. lotus seeds were obtained from mature fruit and cleaned with clear water. The seeds were soaked in 75% ethanol for 30 d under aseptic condition， and flushed with sterile water for 3 times. The seeds were then soaked with 0.1% HgCl2 for 10 min under continuous shaking， and flushed with sterile water for 5-6 times， and water on seed surface was absorbed with sterile filter. The treated seeds were cultured in medium， which was （1/2N）MS + GA 0.5mg/L+ PVP 0.6 g/L + sucrose 30 g/L + agar 6.0 g/L. Acquisition of aseptic plantlets of sweet persimmon
In dormant season （December-March）， annual branches growing strongly were selected and cut into segments， each having one dormant bud. The segments were cleaned and sterilized with 70% for 30 s and flushed with sterile water for 3 times. The segments were then soaked with 0.1% HgCl2 for 10 min under continuous shaking， and flushed with sterile water for 5-6 times， and water on the surface was absorbed with sterile filter. Bud scales and three layers of leaf primordium were stripped off from each bud， and about 3 mm of bud tip was cut and inoculated on medium， which was （1/2N）MS + 6-BA 4.0 mg/L + IAA 0.2 mg/L + PVP 0.6 g/L + sucrose 30 g/L +agar 6.0 g/L.
Test tube micro-grafting
The micro-grating was performed with reference to the grafting method of apple[1]. Strong stock and scion segment were selected， respectively. Under sterile condition， the head of each tissue culture plantlets of D. lotus was removed， leaving a stem segment with a length of about 1.5 cm. Lateral buds at the bottom were cut off longitudinally， with a length of about 0.5 cm. A stem segment of sweet persimmon with a length of about 1.5 cm and a thickness close to the stock were selected as a scion， with 2-4 leaves left at the top. The base of the scion was cut into wedge shape （with a length of about 0.5 cm）. The scion was inserted into the stock， and the graft union was tightened with aluminum foil. The grated test-tube plantlets were then cultured in medium. For each kind of stock and scion combination， 10 plants were grafted， and there were three replications.
Investigation and statistics of survival rate were performed 50 d after grafting. Grafting survival rate was calculated according to following equation： Grafting survival rate=Number of survived plants/Total number of grafted plants×100%.
Data processing
Percentage values were subjected to anti-sine conversion. Statistical analysis was performed in DPS12.0. ， and the significant level was 0.05.
Results and Analysis
Effect of growth condition of tissue culture plantlets on grafting survival rate
The growth conditions of tissue culture plantlets of stock and scion directly affected the survival rate of test tube micro-grafting. When the tissue culture plantlets had strong stem and top growing vigorously， the survival of grafted plants was facilitated[6]， and long internode facilitated operation. However， tissue culture plantlets of most Diospyros plants grow slowly， have short internode and was prone to browning， which is not beneficial to grafting. It was found in the experiment that internodes could grow longer by performing 10 d of dark culture before subculture of tissue culture plantlets， then， the stems could grow strongly with long internodes by performing 30 d of culture under strong light， and finally， grafting operation was facilitated when obtaining a high grafting survival rate. Furthermore， the addition of 5 g/L PVP in subculture medium could effectively reduce browning of tissue culture plantlets of D. lotus and sweet persimmon. Effects of different scions on grafting survival rate and compatibility
Different sweet persimmon cultivars were grafted with D. lotus as stock. It could be seen from Table 1 that there were greater differences in grafting survival rate between different sweet persimmon varieties， among which Youhou had the highest survival rate， followed by Jiro and Zenjiomaru. This might be due to the tissue culture plantlets of Youhou grown strongly， and the performance in grafting compatibility still needs further observation.
Effect of sucrose concentration on grafting survival rate
Sucrose as carbon source could provide energy for plant cells， and also could induce the formation of phloem. Navarro et al. found during micro-grafting of citrus that sucrose concentration plays a key role in survival of micro-grafted plants. With D. lotus as stock， Jiro was grafted and cultured to medium with different sucrose concentrations. The results （Table 2） showed that with the increase of sucrose concentration， the grafting survival rate increased significantly， but there were no significant difference between the sucrose concentrations of 5% and 7%， and when the sucrose concentration was 7%， the base parts of tissue culture plantlets browned seriously， which might be due to that the improvement of sugar content improved osmotic pressure in medium. Comprehensively from the experimental results and experimental cost， the sucrose concentration of 5% was more suitable.
Effect of different BA concentrations of grafting survival rate
The tissue culture plantlets of Jiro were grafted to tissue culture plantlets of D. lotus as stock. In the medium without the addition of BA， the growth of tissue culture plantlets was inhibited， and the grafting survival rate was thus lower. The addition of BA into medium could effectively promote the growth of tissue culture plantlets and improve grafting survival rate. However， the BA concentration should not be too high， and when the BA concentration reached 5.0 mg/L， the stock grew vigorously and thickly， and thus competed with the scion for nutrition， the graft unions could not well recover， and the grafting survival rate decreased.
Effect of air permeability of culture bottle on grafting survival rate
Air permeability of culture bottles would affect the microenvironment in bottles， including gas and humidity， among which humidity is one of the important factors affecting the survival of grafted plants. In this study， kraft paper， tampon + kraft paper， tampon + parafilm and double-layer parafilm were adopted to perform sealing treatment. The results showed that sealing with kraft paper resulted in serious water loss of medium and serous pollution， and after 3 weeks of culture， massive pollution was observed. Under sealing with parafilm， humidity in bottles was higher， there were a lot of water drops on the walls of bottles in the morning， which affected illumination， and the tissue culture plantlets suffered from serious vitrification. As to sealing with tampon + kraft paper， pollution was hardly observed in culture bottles， air permeability in the bottle was strong， tissue culture plantlets grew better， but due to lower humidity， the grafting survival rate was not as high as treatment tampon + parafilm. Therefore， tampon + parafilm was a more suitable sealing method， which not only could keep the sterile environment in bottles， but also could ensure suitable humidity in bottles， and was thus beneficial to survival of grafted plants. Agricultural Biotechnology2018
Discussion
During micro-grafting of tissue culture plantlets， when scions have too many leaves thereon， not only grafting is very difficult， but also the transpiration area increases， and scions suffer from serious water loss， resulting in reduced survival rate. When the scions have no leaves thereon， power for absorbing water and nutrients is lowered， and the survival rate is also lower. Therefore， during grafting， scions should have leaves thereon， and 2-4 leaves are proper.
Moisture is one of the main factors affecting survival rate of test-tube micro-grafting. During micro-grating， union should be bound with foil， while foil could hardly keep the humidity at the union， so the humidity at the union is the same as the environmental humidity in bottles. In order to improve environmental humidity in bottles and improve grafting survival rate， humidity could be regulated by sealing. The results of this study showed that tampon + parafilm not only could maintain sterile environment in bottles， but also could keep proper humidity in bottles， thereby facilitating survival of grafted plants.
There are also various factors influencing test-tube micro-grafting. In this study， factors including different sweet persimmon cultivars， growth status of test-tube plantlets， sucrose concentration， BA concentration and air permeability of culture environment were investigated. Overall. The grafting survival rate is not high， and various factors influencing micro-grafting of sweet persimmon should be symmetrically studied， so as to obtain higher grafting survival rate. This study laid a foundation for stock screening and breeding of sweet persimmon.
References
[1] CHENG YQ， HAN ZH， XU XF， et al. Early testing of grafting compatibilities between Malus xiaojinensis and two apple cultivars by micrografting in vitro[J]. Chinese journal of agricultural biotechnology， 2003， 11（5）： 472-476. （in Chinese）
[2] FAN SY， LIU YS. Effect of water stress on photosynthetic of Chinese dwarf cherry[C]// Proceedings of the Fourth Youth Colloquium of Chinese Society for Horticultural Science， 2000. （in Chinese）
[3] LIU Y， YANG YS， TONG X， et al. Development of an in vitro micrografting method for the enhancement for growth of isolated shoots and buds in Jatropha curcas L.[J]. Acta Botanica Boreali-Occidentalia Sinica， 2014， 34（10）： 2118-2124. （in Chinese）
[4] GONG BC， WANG JF. Study on different biological characteristics of stocks of persimmon[J]. Nonwood Forest Research， 1997， 15（ 1） ： 9-12. （in Chinese）
[5] MA P， GONG BC， JIANG XB， et al. Evaluation on affinity， growth trait and physiology indicators of persimmon grafted on different stocks[J]. Forest Research， 2015， 28（4）： 518-523. （in Chinese）
[6] ZHOU RJ， DU GQ， SHI XX， et al. Studies on factors affecting survival rate of micro-graft in vitro in transgenic apple[J]. Journal of Fruit Science， 2007， 24（2）： 215-217. （in Chinese）