| Plumbago auriculata is a perennial evergreen shrub,native to South Africa.It is introduced and cultivated in many countries and regions around the world due to its high ornamental and medicinal value.P.auriculata introduced and cultivated in South China is diploid(2n=12),which is mainly used for landscaping.However,its application is single,the flower is small and the cold resistance is not high.When the temperature is lower than 0 ℃,P.auriculata started wilting,which limits the cultivation area of P.auriculata.Polyploid plants often have the advantages of increased stress resistance,large organs and are used as an important means to increase genetic variation.At present,no polyploid induction technique and successful polyploid species about P.auriculata have been reported,so it is not clear whether the polyploid has improved in cold resistance compared with diploid.Based on this,in this study,the sterile stem segments and single buds of P.auriculata were used as explants.The concentration of pendimethalin and trifluralin is 400μmol/L,600μmol/L,800μmol/L,the concentration of colchicine is 0.10%,0.15%,0.20%,and the treatment time gradient is 7d,14 d,21d.We studied the best polyploid induction technique of P.auriculata.Combining morphological identification,flow cytometry and chromosome identification,a rapid and efficient polyploid identification system was explored.After obtaining the tetraploid of P.auriculata,the difference in morphology and anatomy between tetraploid and diploid was compared.The tetraploid and diploid were treated with different low temperature(5 ℃,0 ℃,-5 ℃)for 24 h,and the chlorophyll fluorescence parameters,relative conductivity(REC)and malondialdehyde content(MDA)were compared.It answers whether tetraploid is more resistant compared to diploid.The results can fill the gap of the polyploid induction technology of P.auriculata,enrich its germplasm resources,and lay an important foundation for the improvement varieties and the cultivation of cold-resistant varieties of P.auriculata.The research results are as follows:(1)Comparing the survival rate,morphological variation rate and polyploidization rate of explants treated with three mutagens,the results showed that the sterile single bud treated by MS medium supplemented with 800 μmol/L pendimethalin(p H 5.8)for 7 days was the best treatment.The culture conditions were as follows: temperature 25 ± 1 ℃,photoperiod 12 h / d,light intensity1500-2000 lx.The survival rate is 60%,the morphological variation rate is 79.63%,and the polyploidization rate is 23.26%.In addition,compared with the mutagen type,pendimethalin had better induction effect than trifluralin and colchicine;single bud had better induction effect than stem segment.(2)Purified P.auriculata tetraploids were obtained.The surviving mutagen material was cultured in MS medium,regenerated into whole plants,and then the apical buds were cut off,inoculated on new MS medium,and the step was repeated 6 times,and the purified tetraploids were screened by flow cytometry.The effectiveness of obtaining purified autotetraploids by in vitro shoot bud subculture was validated.(3)Exploring the procedure for rapid and accurate identification of polyploids: firstly screening the mutagenic materials by morphological variation,and then initially identifying the ploidy of morphological variants by flow cytometry,and finally verifying the results of flow cytometry by chromosome counting method.This identification system can save a lot of time,cost and workload.In addition,the effectiveness of flow cytometry as a means of identifying plant ploidy was verified based on chromosome count results.(4)Compared with diploid,the tetraploid morphology and anatomical structure are bigger.The length and width of tetraploid leaf were increased by 17.84% and 34.51%,stem diameter increased by22.99%,corolla diameter increased by 29.38%,stem length and leaf shape index were not significantly different from diploid.The stomata size of the tetraploid plants was 10.29 μm × 4.18 μm,the diploid was 10.0 μm × 3.51 μm,which was increased by 22.54%,and the length and width of the guard cells were increased by 10.66 and 9.71%,respectively.The tetraploid leaves were 86.36%thicker than the diploid,and the leaf sponge tissue was 68.80% thicker.The diameter of the vascular bundle of the blade is slightly reduced,but the number is increased.The root diameter increased by26.78%,and the root parenchyma thickness increased by 22.36%.The tetraploid stem epidermal cell thickness and root epidermal cell thickness were not significantly different from diploid.(5)Compared with diploid,tetraploid has strong cold tolerance.The three-month seedlings can last for 24 hours and the extreme minimum temperature is-5 ℃,and then resume growth at room temperature with a recovery rate of 80%.The results of specific cold tolerance indicators were as follows: low temperature of 5 ℃ had little effect on tetraploid of P.auriculata,and diploid leaves were slightly wilted;there was no significant difference between Fo,Fv/Fm and Fv/Fo.At 0 ℃,the tetraploid leaves were slightly wilted,the diploid leaves and some stems were wilted,the diploid Fo was 12.93% higher than the tetraploid;the diploid Fv / Fm was 29.17% lower than the tetraploid;The Fv / Fo of the diploid is 45.98% lower than that of the tetraploid,indicating that the diploid photosystem II is more damaged than the tetraploid.-5 ℃ low temperature makes tetraploid leaves and stem tips wilting,diploid leaves and most stems wilted;diploid Fo is 16.76% higher than tetraploid;diploid Fv / Fm is lower than tetraploid 50 %;diploid Fv / Fo is 60.35% lower than tetraploid;compared with the control group,diploid REC increased twice as much as tetraploid;diploid MDA content increased more than twice.In summary,800μmol/L pendimethalin treatment of sterile single bud 7d is the best mutagenesis treatment;the homozygous tetraploid can be effectively obtained by in vitro shoot bud subculture;combined with morphological variation,flow cytometry and chromosome count can quickly and accurately screen tetraploid;tetraploid have larger leaves and thicker stems;tetraploid anatomical changes may lead to plant photosynthetic system enhancement and plant cells more stable,resulting in tetraploid cold resistance is better than diploid.Low temperature at 5 ℃ has no effect on tetraploid of P.auriculata,low temperature at 0 ℃ has some stress on P.auriculata,and low temperature at-5 ℃is the extreme low temperature that it endures. |
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