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Abstract In order to solve the problems including low fruit??bearing rate and low yield caused by blind plantation in production， the medium formula for pollen germination of Lonicera caerulea L. var. edulis Turcz. ex Herd.， its pollination habit， self??compatibility and open pollination compatibility between different superior individuals were investigated with superior individuals from superior wild individuals of L. caerulea in Changbai Mountains as experimental materials. Self??compatibility and open pollination compatibility among different fine plants provide theoretical basis for its further application and further selection of new varieties. The results showed the optimal medium for germination of L. caerulea pollen was 27.5% sucrose + 100 PPM boric acid with a pH value of 6.0. On this medium， the superior individuals L1， L2 and L3 had the pollen germination rates of 53.4%， 50.9% and 51.6%， respectively. The three superior individuals had no significant differences in the quantity of germinated pollen tubes， which ranged from 1 to 4. The three excellent single plants were the most likely to germinate a single pollen tube， accounting for 83.3% of the pollen germinated and 89.6% of the pollen germinated. Therefore， in production， it is necessary to plant L1 with a certain amount of pollination trees or plant it together with multiple species， to improve its yield. On the contrary， L2 and L3 have no need for pollination trees theoretically， but whether the fruits obtained by self??pollination and outcrossing differ in quality still needs further study.
Key words Lonicera caerulea L. var. edulis Turcz. ex Herd.； Pollen germination； Pollination compatibility
Lonicera caerulea L. var. edulis Turcz. ex Herd. is a variant of L. caerulea L. of Caprifoliaceae family [1-2]， and is a kind of perennial shrub. Its fruit contains very abundant bioactivators， with very high utilization value. L. caerulea is an edible wild berry type plant， the fruit of which tastes delicious with slight sweetness and sour with the characteristics of rich nutrition and high juice yield， and could serve as a raw material for wine， beverages and natural pigments [3]. However， its wild resources are limited， in scattered distribution， and the fruit is smaller and hard to collect. Furthermore， its artificial cultivation has not been industrialized， and good new varieties cannot be well extended. Existing resources can hardly satisfy the aggregate demand day by day. Jiao et al. [4] performed hybridization using cold??region berry L. caerulea， and the results showed that the wide hybridization using it could achieve a high success rate of breeding. Huo et al. [5] observed and measured the flower part of four wild L. caerulea populations in Jilin Province and Helongjiang Province. The size and morphology of the flower part were also observed with a scanning electron microscope， and the results showed that there were no big differences in pollen shape between different populations， but these populations differed significantly in pollen size， indicating that superior individuals in Northeast China region are genetically closer， but significant genetic differentiation exists due to different geographical types.
L. caerulea is widely distributed in Northeast China region as a newly??developing small berry tree species. Its fruit contains very rich bioactivators， and this species has strong cold tolerance and good disease resistance， and has been concerned by more and more people due to its high development potential. Though the basic research on L. caerulea has been strengthened in China in recent years， research on the pollination compatibility of L. caerulea is in a blank state relatively. In this study， in order to solve the problems including low fruit??bearing rate and low yield caused by blind plantation in production， the medium formula for pollen germination of L. caerulea， its pollination habit， self??compatibility and open pollination compatibility between different superior individuals were investigated with superior individuals from wild L. caerulea in Changbai Mountains as experimental materials. This study will provide a theoretical basis for further extension and further breeding of new varieties.
Materials and Methods
Experimental materials
The experimental materials were three superior individuals （L1， L2 and L3） selected from the wild L. caerulea in Changbai Mountains by the L. caerulea Scientific Research Team of Yanbian Academy of Forestry Sciences. The experiment was carried out at Longjing L. caerulea Base of Yanbian Academy of Forestry Sciences. The experimental trees were five year old.
Experimental methods
Collection and preservation of pollen At full??bloom stage （May 2， 2017）， flowers at big bud stage were collected from Longjing L. caerulea Base. Anthers were taken out at laboratory and naturally dried in a shady and cool place for 1-2 d， and when the anthers ruptured and pollen was released， they were wrapped with parchment and preserved in a refrigerator for later use. Detection of pollen germination rate
Screening of optimal medium for germination of L. caerulea pollen Screening of optimal sucrose： the pH value was fixed at 6.0， and the concentration of sucrose was 10%，12.5%， 15%， 17.5%， 20%， 25%， 27.5% and 30%. The medium was composed of pH 6.0 + 100 PPM + boric acid with different concentrations of sucrose. Pollen of L1 was sown onto the media， which were then placed in an incubator at 25 ?? for 2-4 h with controlled humidity. Finally， the germination rate was observed under a microscope.
Screening of optimal pH value： the optimum sucrose concentration was used as the sucrose concentration of pollen germination medium， and pH value of medium was adjusted to 0.5， 1.0， 1.5， 2.0， 2.5， 3.0， 3.5??4.0， 4.5， 5.0， 5.5， 6.0， 6.5， 7.0， 7.5， 8.0， 8.5， 9.0， 9.5 and 10.0， i.e.， Other components in the media and the investigation method of pollen germination rate were the same as above.
Detection of pollen germination rate of superior L. caerulea individuals The collected pollen was subjected to germination rate detection on the optimal medium screened above before being used for selfing. The pollen was sown onto the medium， which was then placed in an incubator at 25 ?? for 2-4 h with controlled humidity. The germination rate， number of germinated pollen tubes and its proportion were observed under a microscope.
Investigation of self??compatibility， pollination habit and open pollination fruiting rate Self??compatibility investigation： Well??developed fruited mother branches were selected and subjected to bagging isolation after self??pollination at big bud stage. Self??pollination fruiting rate was investigated preliminary when removing the bags 10 d later， and the final fruiting rate was investigated 20 d later. In total， 100 flowers were bagged with 10 bags.
Pollination habit investigation： Well??developed fruited mother branches were selected and subjected to bagging isolation at big bud stage. The self??pollination fruiting rate was investigated according to the method the same as above. In total， 100 flowers were bagged with five bags. The pollination habit （anemophilous pollination and entomophilous pollination） was investigated combining with the self??compatibility during bagging isolation following pollination and direct bagging isolation.
Open pollination fruiting rate investigation： Ten well??developed fruited mother branches were selected and marked at large bud stage for the investigation of total number of flowers. Open pollination fruiting rate was investigated 20 d after full bloom. Results and Analysis
Effect of sucrose concentration on pollen germination of superior L. caerulea individuals
The effect of sucrose concentration on pollen germination of L. caerulea under the pH of 6.0 and 100 PPM boric acid is shown in Table 1. It could be seen from Table 1 that there were significant differences in the germination rate of L. edulis pollen with different sucrose concentrated. In the range of 10%-15% sucrose concentration， no germinated pollen tube could be observed. In the range of 17.5%- 30.0% sucrose concentration， the germination rate of L. edulis pollen increased with the increase of sucrose concentration. When sucrose concentration was 27.5%， pollen germination rate was the highest （51.2%）. It can be seen from the table that the pollen germination rate is higher when sucrose concentration is 30%， but the length and growth speed of pollen tube is much lower than that of the former. Therefore， 27.5% sucrose concentration is more suitable for pollen germination of L. edulis. Reportedly， the apple， pear， large cherry， plum， jujube and strawberry were in the range of 5%-15% [9]， all lower than the optimal sucrose concentration for the pollen germination of L. caerulea.
Effects of pH value on pollen germination of superior L. caerulea individuals
Table 2 shows the germination rate of pollen from superior L. caerulea individuals in media with different pH values. It could be seen from data in the table that different pH values greatly affected the germination rate of L. caerulea pollen. Specifically， at the pH value of 1， L. caerulea pollen had the highest germination rate of 91.7%. With the increase of pH value， the germination rate of L. caerulea pollen decreased sharply. When pH was equal to or higher than 2.5， the germination rate of L. caerulea pollen was 0. Pollen of most plants requires the pH in the range of 4-7 for germination [6-7]. Vaccinium uliginosum grows in habitats similar to L. caerulea， Liu et al. [8] showed that it had the highest pollen germination rate at pH 6.5， which was higher than the optimal pH 6.0 obtained in this study. Though V. uliginosum is a plant accustomed to acid soil， the germination of its pollen still requires a pH value in the normal range.
Comprehensively from Table 1 and Table 2， the optimal medium for pollen germination of L. caerulea was 27.5% sucrose + 100 PPM boric acid + pH 6.0. On this medium， the superior individuals L1， L2 and L3 had the pollen germination rates of 53.4%， 50.9% and 51.6%， respectively. Quantities of pollen tubes of superior L. caerulea individuals germinated on the optimal medium
The quantities of L. caerulea pollen tubes germinated on the optimal medium were in the range of 1-4， and the number of pollen tubes produced was not significantly different among the three superior plants. It was the most in one pollen tube， accounting for 83.3% and 89.6% of the germination pollen. It was found in the experiment that more than one pollen tube germinated， the pollen tube showed only the sign of germination， the length of pollen tube was shorter than the diameter of pollen grain， moreover， the less suitable medium was， the more pollen grains germinated by more than one pollen tube. Zhang et al. [10] collected L. caerulea from Changchun Botanical Garden， and observed the micromorphology of pollen. The results showed that L. caerulea pollen grains were in the shape of a flat sphere with thorns and had three germinal apertures， which were in the special shape of the orifice is corniculate collapse [11-12]. The results of this study showed that the pollen of the three superior L. caerulea individuals all had four germinal apertures. Therefore， different sources or species of L. caerulea might differ in germinal aperture quantity， which needs further verification.
Self??compatibility， pollination habit and open pollination fruiting rate of superior L. caerulea individuals
In order to investigate the self??compatibility， pollination habit and open pollination fruiting rate of superior L. caerulea individuals， this study designed three treatments： direct bagging， bagging following pollination （selfing） and open pollination， and the results are shown in Table 4. It could be seen from Table 4 that the three superior individuals all had self??pollination fruiting rate of 0 in the direct bagging treatment； and the bagging following pollination treatment had the self??pollination fruiting rates of 0， 100% and 85% for the three superior individuals， respectively. In the experimental process， it was found that L. caerulea pollen had a higher sugar content， and the pollen was attached to the anther shell， not prone to falling， and even difficult to dipped with a pollenator. It could thus been that it is difficult for wind to blow pollen off， and therefore， L. caerulea only could depend on entomophilous pollination. Because L1 is completely self??incompatible， it is necessary to plant a certain amount of pollination trees or plant it together with multiple species， to improve its yield. On the contrary， L2 and L3 have no need for pollination trees theoretically， but whether the fruits obtained by self??pollination and outcrossing differ in quality still needs further study. It could be seen from Table 4 that the open pollination fruiting rates of the three individuals in mixed form were 94%， 96% and 98%， respectively， suggesting that different L. caerulea individuals have good pollination compatibility therebetween， and could serve as a pollination tree mutually. Discussion
During the screening of the optimal medium for germination of L. caerulea pollen， it was found that L. caerulea pollen required a special pH for germination， i.e.， the optimal pH of 6.0. Xu et al. [16] searched the optimal pH required by the germination of L. maackii （Rupr.） Maxim. pollen in the range of 3.9-8.0， and the results showed that the optimal pH value was 6.7. Most plant pollen requires pH values in the range of 4-7 for germination [6-7]， which are all higher than that of L. caerulea. The experiment for screening the optimal medium for the germination of L. caerulea pollen was carried out for two years. It was failed in the first year because the searching was performed in a conventional sucrose， but germinated pollen tubes were observed once by chance， and the sucrose of the very medium was analyzed to be 27.5. After much trial and error， the optimal sucrose for the germination of L. caerulea pollen was finally determined to be 27.5. Why the germination of L. caerulea pollen needs so low a sucrose still needs further study.
Conclusions
The optimal medium for germination of L. caerulea pollen was 27.5% sucrose + 100 PPM boric acid with a pH value of 6.0. On this medium， the superior individuals L1， L2 and L3 had the pollen germination rates of 53.4%， 50.9% and 51.6%， respectively. The three superior individuals had no significant differences in the quantity of germinated pollen tubes， which ranged from 1 to 4. The three excellent single plants were the most likely to germinate a single pollen tube， accounting for 83.3% of the pollen germinated and 89.6% of the pollen germinated. Therefore， in production， it is necessary to plant L1 with a certain amount of pollination trees or plant it together with multiple species， to improve its yield. On the contrary， L2 and L3 have no need for pollination trees theoretically， but whether the fruits obtained by self??pollination and outcrossing differ in quality still needs further study.
References
[1] Editorial Board of Flora of China， Chinese Academy of Sciences. Flora of China[M]. Beijing： Science Press， 1988. （in Chinese）
[2] ZHAO GH， YAO FL. Study on extraction conditions of natural red pigment from Lonicera caerulea[J]. Food Research and Development， 2005， 26（3）：106-107. （in Chinese）
[3] BAO YH， WANG EF， YING HZ. The study on the slending of Lonicera edulis wine[J]. Liquor Making， 2007， 34（1）：92-94. （in Chinese）
[4] JIAO L， JIANG SJ， HAN XD， et al. Cross breeding of Lonicera caerulea[J]. Protection Forest Science and Technology， 2015（6）： 35-38. （in Chinese） [5] HUO JW， SUI W， YANG GH， et al. Flower pattern variation of wild Lonicera caerluea var. edulis in Northeast China[J]. Journal of Northeast Agricultural University， 2008， 39（7）： 21-24
[6] QI XJ， ZHANG SL， FANG JB. Effect of culture condition on pollen germination of kiwifruit[J]. Acta Agriculturae Zhejiangensis， 2011， 23（3）：528-532. （in Chinese）
[7] WU Q， CAO L， ZHANG C， et al. Effects of extracellular pH on orange pollen germination rate and distribution of Ca2+ in pollen tubes[J]. Journal of Shanxi Agricultural University： Nature Science Edition， 2012， 32（4）：337-340. （in Chinese）
[8] LIU Z. Study on characteristics of the propagating system of Vaccinium uliginosum Linn. and factors influencing in??vitro germination of its pollen[D]. Yanji： Yanbian University， 2017： 10-11. （in Chinese）
[9] ZHAO CX， LIU CL. Elementary studies on measuration of life??ability of fruit tree pollen[J]. Hebei Journal of Forestry and Orchard Research， 2001（3）： 240-243. （in Chinese）
[10] ZHANG SM， et al. Observation of micro??morphology of pollen of several woody flowers[J]. Journal of Jilin Agricultural University， 1996（4）： 56-59. （in Chinese）
[11] LUAN ZH， ZHANG AN， LIU LP， et al. Research on pollen morphology of Lonicea Caerulea L.[J]. Journal of Tonghua Normal University， 2007（2）： 47-49. （in Chinese）
[12] ZHU L， XIE P， WANG GJ， et al. Observation of micro??morphology of pollen of 4 Loniceres[J]. Jilin Forestry Science and Technology， 2007（4）： 10-11， 47. （in Chinese）
[13] ZHANG LJ. The morphological diversity and fruit quality of blue honesuckle （Lonicera caerulea L.）[D]. Harbin： Northeast Agricultural University， 2012. （in Chinese）
[14] LI SQ. Study on Lonicera caerulea nutritional components[J]. Journal of Northeast Agricultural University， 1994， 25（4）： 401-404. （in Chinese）
[15] ZHOU F， ZHAO HF， YANG Y， et al. Comparitive study on anthocyanin level and antioxidant activity among the tall??stemmed blueberry cultivars[J]. Journal of Southwest Forestry University： Natural Science， 2011， 31（5）： 53-57. （in Chinese）
[16] XU K， GU S， JIANG S. A preliminary study of pollen germination in vitro of Lonicera maackii （Rupr.） Maxim.[J]. Journal of Tropical and Subtropical Botany， 2008， 16（2）： 109-115. （in Chinese）
Key words Lonicera caerulea L. var. edulis Turcz. ex Herd.； Pollen germination； Pollination compatibility
Lonicera caerulea L. var. edulis Turcz. ex Herd. is a variant of L. caerulea L. of Caprifoliaceae family [1-2]， and is a kind of perennial shrub. Its fruit contains very abundant bioactivators， with very high utilization value. L. caerulea is an edible wild berry type plant， the fruit of which tastes delicious with slight sweetness and sour with the characteristics of rich nutrition and high juice yield， and could serve as a raw material for wine， beverages and natural pigments [3]. However， its wild resources are limited， in scattered distribution， and the fruit is smaller and hard to collect. Furthermore， its artificial cultivation has not been industrialized， and good new varieties cannot be well extended. Existing resources can hardly satisfy the aggregate demand day by day. Jiao et al. [4] performed hybridization using cold??region berry L. caerulea， and the results showed that the wide hybridization using it could achieve a high success rate of breeding. Huo et al. [5] observed and measured the flower part of four wild L. caerulea populations in Jilin Province and Helongjiang Province. The size and morphology of the flower part were also observed with a scanning electron microscope， and the results showed that there were no big differences in pollen shape between different populations， but these populations differed significantly in pollen size， indicating that superior individuals in Northeast China region are genetically closer， but significant genetic differentiation exists due to different geographical types.
L. caerulea is widely distributed in Northeast China region as a newly??developing small berry tree species. Its fruit contains very rich bioactivators， and this species has strong cold tolerance and good disease resistance， and has been concerned by more and more people due to its high development potential. Though the basic research on L. caerulea has been strengthened in China in recent years， research on the pollination compatibility of L. caerulea is in a blank state relatively. In this study， in order to solve the problems including low fruit??bearing rate and low yield caused by blind plantation in production， the medium formula for pollen germination of L. caerulea， its pollination habit， self??compatibility and open pollination compatibility between different superior individuals were investigated with superior individuals from wild L. caerulea in Changbai Mountains as experimental materials. This study will provide a theoretical basis for further extension and further breeding of new varieties.
Materials and Methods
Experimental materials
The experimental materials were three superior individuals （L1， L2 and L3） selected from the wild L. caerulea in Changbai Mountains by the L. caerulea Scientific Research Team of Yanbian Academy of Forestry Sciences. The experiment was carried out at Longjing L. caerulea Base of Yanbian Academy of Forestry Sciences. The experimental trees were five year old.
Experimental methods
Collection and preservation of pollen At full??bloom stage （May 2， 2017）， flowers at big bud stage were collected from Longjing L. caerulea Base. Anthers were taken out at laboratory and naturally dried in a shady and cool place for 1-2 d， and when the anthers ruptured and pollen was released， they were wrapped with parchment and preserved in a refrigerator for later use. Detection of pollen germination rate
Screening of optimal medium for germination of L. caerulea pollen Screening of optimal sucrose： the pH value was fixed at 6.0， and the concentration of sucrose was 10%，12.5%， 15%， 17.5%， 20%， 25%， 27.5% and 30%. The medium was composed of pH 6.0 + 100 PPM + boric acid with different concentrations of sucrose. Pollen of L1 was sown onto the media， which were then placed in an incubator at 25 ?? for 2-4 h with controlled humidity. Finally， the germination rate was observed under a microscope.
Screening of optimal pH value： the optimum sucrose concentration was used as the sucrose concentration of pollen germination medium， and pH value of medium was adjusted to 0.5， 1.0， 1.5， 2.0， 2.5， 3.0， 3.5??4.0， 4.5， 5.0， 5.5， 6.0， 6.5， 7.0， 7.5， 8.0， 8.5， 9.0， 9.5 and 10.0， i.e.， Other components in the media and the investigation method of pollen germination rate were the same as above.
Detection of pollen germination rate of superior L. caerulea individuals The collected pollen was subjected to germination rate detection on the optimal medium screened above before being used for selfing. The pollen was sown onto the medium， which was then placed in an incubator at 25 ?? for 2-4 h with controlled humidity. The germination rate， number of germinated pollen tubes and its proportion were observed under a microscope.
Investigation of self??compatibility， pollination habit and open pollination fruiting rate Self??compatibility investigation： Well??developed fruited mother branches were selected and subjected to bagging isolation after self??pollination at big bud stage. Self??pollination fruiting rate was investigated preliminary when removing the bags 10 d later， and the final fruiting rate was investigated 20 d later. In total， 100 flowers were bagged with 10 bags.
Pollination habit investigation： Well??developed fruited mother branches were selected and subjected to bagging isolation at big bud stage. The self??pollination fruiting rate was investigated according to the method the same as above. In total， 100 flowers were bagged with five bags. The pollination habit （anemophilous pollination and entomophilous pollination） was investigated combining with the self??compatibility during bagging isolation following pollination and direct bagging isolation.
Open pollination fruiting rate investigation： Ten well??developed fruited mother branches were selected and marked at large bud stage for the investigation of total number of flowers. Open pollination fruiting rate was investigated 20 d after full bloom. Results and Analysis
Effect of sucrose concentration on pollen germination of superior L. caerulea individuals
The effect of sucrose concentration on pollen germination of L. caerulea under the pH of 6.0 and 100 PPM boric acid is shown in Table 1. It could be seen from Table 1 that there were significant differences in the germination rate of L. edulis pollen with different sucrose concentrated. In the range of 10%-15% sucrose concentration， no germinated pollen tube could be observed. In the range of 17.5%- 30.0% sucrose concentration， the germination rate of L. edulis pollen increased with the increase of sucrose concentration. When sucrose concentration was 27.5%， pollen germination rate was the highest （51.2%）. It can be seen from the table that the pollen germination rate is higher when sucrose concentration is 30%， but the length and growth speed of pollen tube is much lower than that of the former. Therefore， 27.5% sucrose concentration is more suitable for pollen germination of L. edulis. Reportedly， the apple， pear， large cherry， plum， jujube and strawberry were in the range of 5%-15% [9]， all lower than the optimal sucrose concentration for the pollen germination of L. caerulea.
Effects of pH value on pollen germination of superior L. caerulea individuals
Table 2 shows the germination rate of pollen from superior L. caerulea individuals in media with different pH values. It could be seen from data in the table that different pH values greatly affected the germination rate of L. caerulea pollen. Specifically， at the pH value of 1， L. caerulea pollen had the highest germination rate of 91.7%. With the increase of pH value， the germination rate of L. caerulea pollen decreased sharply. When pH was equal to or higher than 2.5， the germination rate of L. caerulea pollen was 0. Pollen of most plants requires the pH in the range of 4-7 for germination [6-7]. Vaccinium uliginosum grows in habitats similar to L. caerulea， Liu et al. [8] showed that it had the highest pollen germination rate at pH 6.5， which was higher than the optimal pH 6.0 obtained in this study. Though V. uliginosum is a plant accustomed to acid soil， the germination of its pollen still requires a pH value in the normal range.
Comprehensively from Table 1 and Table 2， the optimal medium for pollen germination of L. caerulea was 27.5% sucrose + 100 PPM boric acid + pH 6.0. On this medium， the superior individuals L1， L2 and L3 had the pollen germination rates of 53.4%， 50.9% and 51.6%， respectively. Quantities of pollen tubes of superior L. caerulea individuals germinated on the optimal medium
The quantities of L. caerulea pollen tubes germinated on the optimal medium were in the range of 1-4， and the number of pollen tubes produced was not significantly different among the three superior plants. It was the most in one pollen tube， accounting for 83.3% and 89.6% of the germination pollen. It was found in the experiment that more than one pollen tube germinated， the pollen tube showed only the sign of germination， the length of pollen tube was shorter than the diameter of pollen grain， moreover， the less suitable medium was， the more pollen grains germinated by more than one pollen tube. Zhang et al. [10] collected L. caerulea from Changchun Botanical Garden， and observed the micromorphology of pollen. The results showed that L. caerulea pollen grains were in the shape of a flat sphere with thorns and had three germinal apertures， which were in the special shape of the orifice is corniculate collapse [11-12]. The results of this study showed that the pollen of the three superior L. caerulea individuals all had four germinal apertures. Therefore， different sources or species of L. caerulea might differ in germinal aperture quantity， which needs further verification.
Self??compatibility， pollination habit and open pollination fruiting rate of superior L. caerulea individuals
In order to investigate the self??compatibility， pollination habit and open pollination fruiting rate of superior L. caerulea individuals， this study designed three treatments： direct bagging， bagging following pollination （selfing） and open pollination， and the results are shown in Table 4. It could be seen from Table 4 that the three superior individuals all had self??pollination fruiting rate of 0 in the direct bagging treatment； and the bagging following pollination treatment had the self??pollination fruiting rates of 0， 100% and 85% for the three superior individuals， respectively. In the experimental process， it was found that L. caerulea pollen had a higher sugar content， and the pollen was attached to the anther shell， not prone to falling， and even difficult to dipped with a pollenator. It could thus been that it is difficult for wind to blow pollen off， and therefore， L. caerulea only could depend on entomophilous pollination. Because L1 is completely self??incompatible， it is necessary to plant a certain amount of pollination trees or plant it together with multiple species， to improve its yield. On the contrary， L2 and L3 have no need for pollination trees theoretically， but whether the fruits obtained by self??pollination and outcrossing differ in quality still needs further study. It could be seen from Table 4 that the open pollination fruiting rates of the three individuals in mixed form were 94%， 96% and 98%， respectively， suggesting that different L. caerulea individuals have good pollination compatibility therebetween， and could serve as a pollination tree mutually. Discussion
During the screening of the optimal medium for germination of L. caerulea pollen， it was found that L. caerulea pollen required a special pH for germination， i.e.， the optimal pH of 6.0. Xu et al. [16] searched the optimal pH required by the germination of L. maackii （Rupr.） Maxim. pollen in the range of 3.9-8.0， and the results showed that the optimal pH value was 6.7. Most plant pollen requires pH values in the range of 4-7 for germination [6-7]， which are all higher than that of L. caerulea. The experiment for screening the optimal medium for the germination of L. caerulea pollen was carried out for two years. It was failed in the first year because the searching was performed in a conventional sucrose， but germinated pollen tubes were observed once by chance， and the sucrose of the very medium was analyzed to be 27.5. After much trial and error， the optimal sucrose for the germination of L. caerulea pollen was finally determined to be 27.5. Why the germination of L. caerulea pollen needs so low a sucrose still needs further study.
Conclusions
The optimal medium for germination of L. caerulea pollen was 27.5% sucrose + 100 PPM boric acid with a pH value of 6.0. On this medium， the superior individuals L1， L2 and L3 had the pollen germination rates of 53.4%， 50.9% and 51.6%， respectively. The three superior individuals had no significant differences in the quantity of germinated pollen tubes， which ranged from 1 to 4. The three excellent single plants were the most likely to germinate a single pollen tube， accounting for 83.3% of the pollen germinated and 89.6% of the pollen germinated. Therefore， in production， it is necessary to plant L1 with a certain amount of pollination trees or plant it together with multiple species， to improve its yield. On the contrary， L2 and L3 have no need for pollination trees theoretically， but whether the fruits obtained by self??pollination and outcrossing differ in quality still needs further study.
References
[1] Editorial Board of Flora of China， Chinese Academy of Sciences. Flora of China[M]. Beijing： Science Press， 1988. （in Chinese）
[2] ZHAO GH， YAO FL. Study on extraction conditions of natural red pigment from Lonicera caerulea[J]. Food Research and Development， 2005， 26（3）：106-107. （in Chinese）
[3] BAO YH， WANG EF， YING HZ. The study on the slending of Lonicera edulis wine[J]. Liquor Making， 2007， 34（1）：92-94. （in Chinese）
[4] JIAO L， JIANG SJ， HAN XD， et al. Cross breeding of Lonicera caerulea[J]. Protection Forest Science and Technology， 2015（6）： 35-38. （in Chinese） [5] HUO JW， SUI W， YANG GH， et al. Flower pattern variation of wild Lonicera caerluea var. edulis in Northeast China[J]. Journal of Northeast Agricultural University， 2008， 39（7）： 21-24
[6] QI XJ， ZHANG SL， FANG JB. Effect of culture condition on pollen germination of kiwifruit[J]. Acta Agriculturae Zhejiangensis， 2011， 23（3）：528-532. （in Chinese）
[7] WU Q， CAO L， ZHANG C， et al. Effects of extracellular pH on orange pollen germination rate and distribution of Ca2+ in pollen tubes[J]. Journal of Shanxi Agricultural University： Nature Science Edition， 2012， 32（4）：337-340. （in Chinese）
[8] LIU Z. Study on characteristics of the propagating system of Vaccinium uliginosum Linn. and factors influencing in??vitro germination of its pollen[D]. Yanji： Yanbian University， 2017： 10-11. （in Chinese）
[9] ZHAO CX， LIU CL. Elementary studies on measuration of life??ability of fruit tree pollen[J]. Hebei Journal of Forestry and Orchard Research， 2001（3）： 240-243. （in Chinese）
[10] ZHANG SM， et al. Observation of micro??morphology of pollen of several woody flowers[J]. Journal of Jilin Agricultural University， 1996（4）： 56-59. （in Chinese）
[11] LUAN ZH， ZHANG AN， LIU LP， et al. Research on pollen morphology of Lonicea Caerulea L.[J]. Journal of Tonghua Normal University， 2007（2）： 47-49. （in Chinese）
[12] ZHU L， XIE P， WANG GJ， et al. Observation of micro??morphology of pollen of 4 Loniceres[J]. Jilin Forestry Science and Technology， 2007（4）： 10-11， 47. （in Chinese）
[13] ZHANG LJ. The morphological diversity and fruit quality of blue honesuckle （Lonicera caerulea L.）[D]. Harbin： Northeast Agricultural University， 2012. （in Chinese）
[14] LI SQ. Study on Lonicera caerulea nutritional components[J]. Journal of Northeast Agricultural University， 1994， 25（4）： 401-404. （in Chinese）
[15] ZHOU F， ZHAO HF， YANG Y， et al. Comparitive study on anthocyanin level and antioxidant activity among the tall??stemmed blueberry cultivars[J]. Journal of Southwest Forestry University： Natural Science， 2011， 31（5）： 53-57. （in Chinese）
[16] XU K， GU S， JIANG S. A preliminary study of pollen germination in vitro of Lonicera maackii （Rupr.） Maxim.[J]. Journal of Tropical and Subtropical Botany， 2008， 16（2）： 109-115. （in Chinese）