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Abstract This study was conducted to screen and breed Azolla varieties with strong resistance. Four Azolla samples （CD， XT， HY， YZ） were collected from Hunan Province， and the growth characteristics such as propagation coefficient， temperature， light intensity， pH value and humidity were analyzed using artificial culture method. The results showed the four Azolla varieties all had the largest reproduction coefficients after cultured for 21 d， and the optimum culture conditions were temperature at 24-28 ℃， illumination intensity of 15 000-20 000 lx， pH 6.0， and humidity of 60%-80%. Among the four Azolla varieties， YZ has strong reproductive performance and high temperature resistance， and XT has strong acid resistance.
Key words Azolla; reproduction coefficient; culture temperature; illumination intensity
Azolla imbricate is an annual aquatic fern， commonly known as Hongping or Luping. The plant is small in size and triangular in shape. It grows rapidly in paddy field in the south， and grows into a piece on water surface. The plant is green when young， and is red after maturity because it contains anthocyanins， which is also the reason for its name Manjianghong[1-3]. After a long evolutionary process， Azolla and nitrogenfixing Anabeana are able to grow symbiotically and synchronously[4]. Azolla has strong application potential in agricultural production in the south. Azolla has higher nitrogen fixation efficiency than clover， soybean and other legumes， and can serve as excellent green manure for rice; it is rich in crude protein and crude fiber， and can serve as feed for fish and livestock; and it can reduce the content of eutrophic nitrogen and phosphorus in water. Furthermore， it has the effect of enriching heavy metal ions， and thus could degrade heavy metal pollution in rice field to a certain extent when it is cultured in rice field[5-6]. However， the growth characteristics of Azolla limit its application in agricultural production to a certain extent. For instance， the growth of Azolla requires strict temperature and light conditions， and the plant has difficulties in surviving in summer and winter， and needs artificial culture and breed conservation[7-8]. Studies have shown that fernlike Azolla has strong adaptability in Xinjiang， and can grow at a temperature of 5-40 ℃ and pH of 4-10. The optimum temperature for its growth is in the range of 25-30 ℃， and it is resistant to low temperature[9]. A. imbricate introduced from East Germany has strong cold resistance and low reproductive temperature， and is more suitable for spring multiplication in early spring in the north. A. imbricate starts to grow at 5 ℃， with a growth rate higher than that in the south at 10 ℃ by 8 times and that at 15-20 ℃ by 2 times[10]. If the temperature in the south is too high in the summer， it will lead to the death of Azolla， and it is difficult for Azolla to survive for a long time when light intensity decreases due to the closure of rice. At present， the traditional Azolla varieties have weak adaptability to the climate in the south， so it is urgent to screen Azolla varieties that have high resistance to high temperature and strong shade tolerance and grow fast. Therefore， with four Azolla samples collected from Hunan as experimental materials， the biological characteristics such as reproductive coefficient， suitable growth temperature， light intensity， pH and humidity were studied by artificial culture method， aiming at screening and breeding Azolla varieties having higher stress resistance. Materials and Methods
Test materials
Azolla Azolla samples were collected from Shimenqiao Town of Changde City （CD）， Yisuhe Town of Xiangtan City （XT）， Qimenzhou Town of Hengyang City （HY） and Sanxi Town of Yizhang County （YZ）. The samples were washed repeatedly with double distilled water and precultured with modified Hoagland nutrient solution[11] for 2 weeks before the experiment.
Others
Square plastic pots （30 cm×20 cm×13 cm）; artificial climate chamber.
Test methods
The effects of culture time， temperature， humidity， light intensity and pH on the growth of Azolla were investigated. Among them， culture time， temperature and pH were set with 5 treatments， and light intensity and humidity were set with 4 treatments （Table 1）.
Culture time
The four Azolla samples from different sources were inoculated separately into square plastic pots containing 4 L of modified Hoagland medium， with three replicates. Each pot was inoculated with 4 g of fresh Azolla which was cultured in an artificial climate incubator. The basic culture parameters were temperature at 24 ℃ （19 ℃ in the dark）， illumination intensity of 10 000 lx， 14 h in light， pH of 7.0， and humidity of 60%. Double distilled water was replenished every 3 to 4 d. Azolla was collected at 7 d， 14 d， 21 d， 28 d and 35 d， respectively， and the collected plants were washed with double distilled water for 3-4 times， and then wiped with filter paper， followed by weighing. The accumulation of Azolla biomass was expressed with reproduction coefficient K.
K=（InW1-InW0）/t
Wherein W1 is the weight of Azolla in different culture periods; W0 is the initial weight of Azolla; and t is the number of cultivation days.
Temperature
Azolla was cultured at 18， 21， 24， 27 and 30 ℃， respectively， and other culture conditions were the same as "Culture time". The plants were weighed after 21 d of culture.
Illumination intensity
Azolla was cultured at 5 000， 10 000， 15 000 and 20 000 lx， respectively， and other culture conditions were the same as those in "Culture time". The plants were weighed after 21 d of culture.
pH
Azolla was cultured at pH of 4.0， 5.0， 6.0， 7.0 and 8.0， respectively， and other culture conditions were the same as those in "Culture time". The plants were weighed after 21 d of culture.
Humidity
Azolla was cultured under humidity of 50%， 60%， 70% and 80%， respectively， and other culture conditions were the same as those in "Culture time". The plants were weighed after 21 d of culture. Data statistics and analysis
Data differences were compared using the Students t test， and plotting was performed with GraphPad software.
Results and Analysis
Growth and reproduction coefficient of Azolla at different culture time
It could be seen from Fig. 1 that during the culture period （35 d）， the reproductive coefficients of the four Azolla varieties increased at first and then decreased， and all of them could maintain positive growth of biomass. Among them， YZ had the strongest growth performance， with a reproductive coefficient higher than 0.03 in the culture period. The reproductive coefficients of the four Azolla varieties were all the highest when cultured for 21 d， and except HY， other three Azolla varieties all had a reproduction coefficient higher than 0.06， suggesting that the four Azolla varieties all had stronger growth performance when cultured for 21 d.
Growth of Azolla under different temperatures， humidity， light intensities and pH
It could be seen from Fig. 2 that with the increase of temperature， the biomass of four Azolla varieties increased at first and decreased then. Among them， CD had the largest biomass at 24 ℃， which was 13.5 g; and XT， HY and YZ showed the largest biomass at 27 ℃， which was 12.8， 13.3 and 14.8 g， respectively. YZ was resistant to high temperature， and it still had stronger growth performance at 30 ℃， with a fresh weight of 13.4 g. In terms of humidity， with the increase of humidity， the biomass of the four Azolla varieties increased， and their fresh weights were over 10 g under the humidity of 60%-80%， indicating better growth performance. There were no significant differences in fresh weight between the four Azolla varieties. As to illumination intensity， within the illumination intensity of 5 000-15 000 lx， the biomass of the four Azolla varieties all increased， and within the illumination intensity of 15 000-20 000 lx， the four Azolla varieties showed no significant differences in fresh weight. Forthe last parameter pH， the biomass of the four Azolla varieties all showed a trend of increasing at first and decreasing then within the pH range of 4.0-8.0， and reached their maximum values at pH 6.0， which were 13.6， 13.8， 13.1 and 14.5 g， respectively. In the pH range of 5.0-7.0， the fresh weights were all over 10 g， indicating better growth performance. Among the four varieties， XT had stronger acid tolerance， and its fresh weight was over 10 g in the pH range of 4.0-8.0 all the time.
Key words Azolla; reproduction coefficient; culture temperature; illumination intensity
Azolla imbricate is an annual aquatic fern， commonly known as Hongping or Luping. The plant is small in size and triangular in shape. It grows rapidly in paddy field in the south， and grows into a piece on water surface. The plant is green when young， and is red after maturity because it contains anthocyanins， which is also the reason for its name Manjianghong[1-3]. After a long evolutionary process， Azolla and nitrogenfixing Anabeana are able to grow symbiotically and synchronously[4]. Azolla has strong application potential in agricultural production in the south. Azolla has higher nitrogen fixation efficiency than clover， soybean and other legumes， and can serve as excellent green manure for rice; it is rich in crude protein and crude fiber， and can serve as feed for fish and livestock; and it can reduce the content of eutrophic nitrogen and phosphorus in water. Furthermore， it has the effect of enriching heavy metal ions， and thus could degrade heavy metal pollution in rice field to a certain extent when it is cultured in rice field[5-6]. However， the growth characteristics of Azolla limit its application in agricultural production to a certain extent. For instance， the growth of Azolla requires strict temperature and light conditions， and the plant has difficulties in surviving in summer and winter， and needs artificial culture and breed conservation[7-8]. Studies have shown that fernlike Azolla has strong adaptability in Xinjiang， and can grow at a temperature of 5-40 ℃ and pH of 4-10. The optimum temperature for its growth is in the range of 25-30 ℃， and it is resistant to low temperature[9]. A. imbricate introduced from East Germany has strong cold resistance and low reproductive temperature， and is more suitable for spring multiplication in early spring in the north. A. imbricate starts to grow at 5 ℃， with a growth rate higher than that in the south at 10 ℃ by 8 times and that at 15-20 ℃ by 2 times[10]. If the temperature in the south is too high in the summer， it will lead to the death of Azolla， and it is difficult for Azolla to survive for a long time when light intensity decreases due to the closure of rice. At present， the traditional Azolla varieties have weak adaptability to the climate in the south， so it is urgent to screen Azolla varieties that have high resistance to high temperature and strong shade tolerance and grow fast. Therefore， with four Azolla samples collected from Hunan as experimental materials， the biological characteristics such as reproductive coefficient， suitable growth temperature， light intensity， pH and humidity were studied by artificial culture method， aiming at screening and breeding Azolla varieties having higher stress resistance. Materials and Methods
Test materials
Azolla Azolla samples were collected from Shimenqiao Town of Changde City （CD）， Yisuhe Town of Xiangtan City （XT）， Qimenzhou Town of Hengyang City （HY） and Sanxi Town of Yizhang County （YZ）. The samples were washed repeatedly with double distilled water and precultured with modified Hoagland nutrient solution[11] for 2 weeks before the experiment.
Others
Square plastic pots （30 cm×20 cm×13 cm）; artificial climate chamber.
Test methods
The effects of culture time， temperature， humidity， light intensity and pH on the growth of Azolla were investigated. Among them， culture time， temperature and pH were set with 5 treatments， and light intensity and humidity were set with 4 treatments （Table 1）.
Culture time
The four Azolla samples from different sources were inoculated separately into square plastic pots containing 4 L of modified Hoagland medium， with three replicates. Each pot was inoculated with 4 g of fresh Azolla which was cultured in an artificial climate incubator. The basic culture parameters were temperature at 24 ℃ （19 ℃ in the dark）， illumination intensity of 10 000 lx， 14 h in light， pH of 7.0， and humidity of 60%. Double distilled water was replenished every 3 to 4 d. Azolla was collected at 7 d， 14 d， 21 d， 28 d and 35 d， respectively， and the collected plants were washed with double distilled water for 3-4 times， and then wiped with filter paper， followed by weighing. The accumulation of Azolla biomass was expressed with reproduction coefficient K.
K=（InW1-InW0）/t
Wherein W1 is the weight of Azolla in different culture periods; W0 is the initial weight of Azolla; and t is the number of cultivation days.
Temperature
Azolla was cultured at 18， 21， 24， 27 and 30 ℃， respectively， and other culture conditions were the same as "Culture time". The plants were weighed after 21 d of culture.
Illumination intensity
Azolla was cultured at 5 000， 10 000， 15 000 and 20 000 lx， respectively， and other culture conditions were the same as those in "Culture time". The plants were weighed after 21 d of culture.
pH
Azolla was cultured at pH of 4.0， 5.0， 6.0， 7.0 and 8.0， respectively， and other culture conditions were the same as those in "Culture time". The plants were weighed after 21 d of culture.
Humidity
Azolla was cultured under humidity of 50%， 60%， 70% and 80%， respectively， and other culture conditions were the same as those in "Culture time". The plants were weighed after 21 d of culture. Data statistics and analysis
Data differences were compared using the Students t test， and plotting was performed with GraphPad software.
Results and Analysis
Growth and reproduction coefficient of Azolla at different culture time
It could be seen from Fig. 1 that during the culture period （35 d）， the reproductive coefficients of the four Azolla varieties increased at first and then decreased， and all of them could maintain positive growth of biomass. Among them， YZ had the strongest growth performance， with a reproductive coefficient higher than 0.03 in the culture period. The reproductive coefficients of the four Azolla varieties were all the highest when cultured for 21 d， and except HY， other three Azolla varieties all had a reproduction coefficient higher than 0.06， suggesting that the four Azolla varieties all had stronger growth performance when cultured for 21 d.
Growth of Azolla under different temperatures， humidity， light intensities and pH
It could be seen from Fig. 2 that with the increase of temperature， the biomass of four Azolla varieties increased at first and decreased then. Among them， CD had the largest biomass at 24 ℃， which was 13.5 g; and XT， HY and YZ showed the largest biomass at 27 ℃， which was 12.8， 13.3 and 14.8 g， respectively. YZ was resistant to high temperature， and it still had stronger growth performance at 30 ℃， with a fresh weight of 13.4 g. In terms of humidity， with the increase of humidity， the biomass of the four Azolla varieties increased， and their fresh weights were over 10 g under the humidity of 60%-80%， indicating better growth performance. There were no significant differences in fresh weight between the four Azolla varieties. As to illumination intensity， within the illumination intensity of 5 000-15 000 lx， the biomass of the four Azolla varieties all increased， and within the illumination intensity of 15 000-20 000 lx， the four Azolla varieties showed no significant differences in fresh weight. Forthe last parameter pH， the biomass of the four Azolla varieties all showed a trend of increasing at first and decreasing then within the pH range of 4.0-8.0， and reached their maximum values at pH 6.0， which were 13.6， 13.8， 13.1 and 14.5 g， respectively. In the pH range of 5.0-7.0， the fresh weights were all over 10 g， indicating better growth performance. Among the four varieties， XT had stronger acid tolerance， and its fresh weight was over 10 g in the pH range of 4.0-8.0 all the time.