The determination of the optimal mutagenesis time and screening salinity
The time-lethal curve (a) of the ARTP tetraspores of G·lemaneiformis was shown in figure 1. It was shown that with the increase of the mutagenesis time, the mortality of the spores gradually increased. A formula Y=1.8227X-3.3156 and R2=0.9778 was obtained. The half-lethal action time of the ARTP mutagenesis was 29S, and when the death rate of tetraspores was 80%, the action time of the ARTP mutagen was 46S. To obtain a higher mutation rate, treatment time of 46S was used in subsequent mutagenesis in the experiment.
The survival rate (b) of tetraspores decreased successively with the increasing of salinity. When the salinity was 60‰, the survival rate of tetraspores was about 4.10%. When the salinity was 58‰, the survival rate of tetraspores was 22.67%. In subsequent experiments, salinity of 58‰ as well as 60‰ were chosen as the osmotic pressure screening condition for the mutants.
Mutagenesis and series salinity screening
Mutagenesis time of 46 seconds was used to treat spores with a scale about 2.7×105, and then artificial seawater with salinity of 58‰ was exploited for screening for one week. 8,680 survived spores were obtained and transferred to normal sea water for culture. The spores entered the vertical stage after two weeks in normal culture.
In order to further reduce the number of tetraspores, 60‰ salinity artificial seawater were used for the second hypertonic screening, and the screening time was 3 weeks. 17 dominant individuals were selected from the surviving individuals for subsequent cultivation.
The third hypertonic screening of the spores started after they had branched out with an artificial seawater treatment of 60‰ salinity for three weeks. A total of 9 hypertonic tolerant mutants were screened. After two months of cultivation, four mutants were selected which displayed better growth trends.
Confirmation of the high osmotic pressure tolerant of screen-out strains
4 screen-out strains were further validated for their tolerance aganist the high salinity treatment in 60‰ salinity within a treatment time of 21 day (table 1). In the control group, only about 3 algae tips remained in good condition showing bright red color and a complete body. About 15 algae tips in HAGL-X5 remained bright red. HAGL-X3 maintained a bright red state for 9 algae tips, followed by HAGL-X4 with 5, and finally HAGL-X2 with 3. Compared with the control group, t HAGL-X5 and HAGL-X3 displayed superior osmotic pressure resistance properties.
Tab.1 Number of algae tips survived after 60‰ salinity treatment for 21 days
strains
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Control
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HAGL-X2
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HAGL-X3
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HAGL-X4
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HAGL-X5
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Surviving tips
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3
|
3
|
9
|
5
|
15
|
The actual photosynthetic efficiency (Y(PSII)), photochemical quenching QL, non-photochemically quenched NPQ and QN
The actual photosynthetic efficiency (Y(PSII)) and photochemical quenching QL of Ctr as well as the mutants treated in artificial seawater with a salinity of 60‰ for 24 days were shown in figure 2 (a and c). On day zero, the Y(PSII) and QL values of all mutants were significantly higher than those of the control (P<0.05), and those for X3 and X4 were significantly higher than those of X2 and X5 (P<0.05). When entering the high salinity treatment (after the second day), the Y(PSII) and QL values of all strains decreased sharply to the lowest values, and gradually recovered between the second and tenth days. Among them, the Y(PSII) of X5 on the 2nd, 6th, 8th and 10th days was significantly higher than that of Ctr (P<0.05), and the Y(PSII) of X3 on the 2nd and 8th days was significantly higher than that of Ctr (P<0.05). The QL values of X5 on the 2nd, 4th, 8th, and 10th days were significantly higher than Ctr (P<0.05), and the QL values of X2, X3, and X4 on the second day were all significantly higher than Ctr (P<0.05). After the tenth day, between the 12th and the 16th day, the Y(PSII) of all the strains fluctuated around 0.20, and they were all very close, with no significant difference (P>0.05). From day 18 to day 24, Y(PSII) of all strains started to decline slowly. At this stage, only Ctr was significantly higher than that of X4 on the 24th day (P<0.05), there was no significant difference in Y(PSII) between Ctr and other mutants at this stage (P>0.05). From the 12th to the 22nd day, the QL of all the strains fluctuated around 0.65, and they were also very close, with no significant difference (P>0.05). On day 24, the QL values of all strains began to decline. There was no significant difference in QL between Ctr and X2, X3 and X5 on the 24th day (P>0.05).
The NPQ (b) of HAGL-X5 and HAGL-X3 were higher than those of the control group before 16 days. The NPQ of HAGL-X3 at the 2nd, 8th day was significantly higher than that of the control group. The NPQ of HAGL-X5 at the 16th day was significantly higher than that of the control group (P<0.05). After 16 days, the NPQ of the control and HAGL-X5 were close to each other (P>0.05). The NPQ of HAGL-X2 was not significantly different from that of the control group, while the NPQ of HAGL-X4 was even lower than that of the control group as a whole (P<0.05). From a trend point of view, the NPQ values of the mutant groups HAGL-X5 and HAGL-X3 were first reduced to the lowest value after being stressed, and then the NPQ value quickly returned to the maximum value (on the sixth day). The control group also decreased. Then it rose again, returning to a higher level on the sixth day. Both HAGL-X5 and the control decreased slightly, and rose to a higher point again at twelve days, and then slowly decreased. The overall trend was similar, but the value of HAGL-X5 was higher than the control overall. After HAGL-X3 rose to its highest point on the sixth day, it declined at a faster rate, but overall it was also higher than the control group.
D in Figure 2 shows the measurement results of non-photochemically quenched QN in artificial seawater with a salinity of 60‰ for 24 days. On day zero, the QN values of X3 and X4 were significantly lower than those of Ctr, X2 and X5 (P<0.05). After entering the high salinity treatment (after the second day), the QN values of Ctr, X2, and X5 also decreased sharply, while those of X3 and X4 increased. The QN values of the mutants and Ctr increased gradually and reached the maximum between the second and sixth days. The QL value of X5 on the 6th day was significantly higher than that of Ctr (P<0.05), and the QL value of X3 on the 2nd and 6th day was significantly higher than that of Ctr (P<0.05). After the rising period, between days 12 and 24, the QN values of all lines began to decline slowly. After 12 days, the QN values of X2 and X5 were higher than Ctr. The QN value of X5 on the 16th and 18th day was significantly higher than that of Ctr (P<0.05), and the QN value of X2 on the 16th and 22nd day was significantly higher than that of Ctr (P<0.05). The values of the mutants were higher than those of the control, though the difference was insignificant.
The growth rate
Fig.3 showed the results of the growth rate measurement of each strain. The growth rate of all mutants was higher than that of the control group, which suggested that the selected mutants had obvious growth advantages. The maximum growth rate of mutant HAGL-X4 was 0.45 cm per week, followed by HAGL-X3 at 0.37 cm per week, followed by HAGL-X5 at 0.32 cm per week, and HAGL-X2 at 0.30 cm per week. The growth rate of HAGL-X3 and HAGL-X4 was significantly higher than that of the control group (P<0.05), while those of HAGL-X2 and HAGL-X5 had no significant difference with control.
Agar content
Fig.4 showed the measurement results of the agar content of each strain. The highest agar content was evidenced in HAGL-X5 with 7.74%, followed by HAGL-X4 which was 6.01%. The agar content of HAGL-X5 and HAGL-X4 was about 1.5 and 1.2 times that of control. The agar content of HAGL-X5 was also significantly higher than that of other mutants (P<0.05), while those of HAGL-X2 and HAGL-X3 was even lower than that of the control. Therefore, HAGL-X5 is a mutant that meets the expectations of the screen.