| As one of the most commonly used Chinese herbal medicines in China,the requirement and price of Bletillae Rhizoma have been increasing year by year.Wild Bletilla striata(Thunb.)Reichb.f.have been listed as a plant that needs to be protected in the Information System of Chinese Rare and Endangered Plants.It is necessary to carry out the research on artificial cultivation of B.striata.At present,there are three main ways of cultivation of B.striata,such as traditional tuber propagation,direct seeding method and the combination of tissue culture and field cultivation.The cultivation cycle of latter two methods is longer than the traditional cultivation,which is the key problem of them to spread out.The temperature has an important influence on the growth of B.striata,and in winter,both the tuber and bud of the plant stop growing and enter dormancy period.In this experiment,the seed germination and seedling growth of B.striata were carried out to study the effects of different maturity and plant growth regulator on them.And then,on the basis of optimization conditions,sterile seedlings were cultured and treated with different low temperatures,to study their physiological responses of temperatures.On the basis of the previous experiment,we carried out chilling stress and exogenous substances treatment to study the effects of different exogenous substances on cold resistance.The main results are as follows:1.The seeds of 19 weeks after pollination have the advantages of germination rate,germination time and embryo rate,so it’s better for 19 weeks seeds to be explant.The growth of B.striata in 1/2MS medium appeared in ’S’ cuve and it’s better to transfer to a new medium within 90 days of culture.2,4-D can induce seeds into calluses and the best concentration is 1.0 mg·L-1 with the induction rate of 100%.The other treatments show that NAA is best in promoting seedling growth with the concentration of 0.5 or 1.0 mg·L-1.2.The addition of exogenous MeJA,SA and endophytic fungi under certain concentrations can improve the resistance of B.striata and increase the content of total phenols at some degree.And the treatment of MeJA,H.koningii and T.koningiopsis can promote the growth of seedlings under certain concentrations.3.The temperature of 5℃ and below can significantly increase the permeability of plasma membrane and lead to the increase of membranaceous peroxide extent and inhibit the activity of protective enzymes increasing in seedlings.9’C and above only increase the extent of membranaceous peroxide and the activity of protective enzymes can be inspired at later period of treatments to deal with environmental changes.The MDA in leaves of 5.·strziata seedlings is more sensitive to the low temperature and the accumulation can ’be significant in the mild low temperature environment.The SOD can respond rapidly to low temperature,but sustained low temperature will inhibit the activity of SOD and the lower the temperature,the greater the inhibition.Low temperature can inhibit the activity of POD and CAT,but it changes a little with the variation of temperature,the response of POD to low temperature is delayed at some degrees.4.40 p,mol·L’1 of exogenous MeJA pretreatment can inhibit the increase in plasma membrane permeability,reduce the extent of membranaceous peroxides and improve the activity of SOD,POD and CAT under the chilling stress of 1℃to reduce the damage caused by low temperature and enhance the resistance of B.striata seedlings.Under the chilling stress of 5℃,exogenous MeJA pretreatment significantly reduces the relative conductivity,increase the activity of POD and CAT,but the content of MDA and soluble protein are not significantly reduced and SOD activity is not significantly different.The effect of exogenous MeJA pretreatment is more significant under 1℃ chilling stress.5.20 μmol·L-1 of exogenous SA pretreatment can inhibit the increase in plasma membrane permeability,reduce the extent of membranaceous peroxides and improve the activity of all the three protective enzymes under the chilling stress of 1℃,to reduce the damage caused by low temperature and enhance the resistance of B.striata seedlings.Under the chilling stress of 5℃,exogenous SA pretreatment significantly reduces the content of MDA and increase SOD activity,but there is no significant difference in relative conductivity,soluble protein content,POD and CAT activity.The effect of exogenous SA pretreatment is more significant under 1 ℃ chilling stress.6.31.4 mg·L-1 of T.Koningiopsis pretreatment significantly increases the activity of SOD,POD and CAT under 1℃ chilling stress and reduce the relative conductivity at recovery period.The pretreatment can alleviate the damage caused by low temperature,and enhance the resistance of B.striata seedlings.Under the chilling stress of 5℃,the pretreatment significantly increases POD activity only.The results showed that the effect of pretreatment is more significant under 1 ℃ chilling stress.7.74.5 mg·L-1 of H.Koningii pretreatment significantly increases the activity of SOD,POD and CAT under 1℃ chilling stress and inhibit the increase of plasma membrane permeability at later period of stress.The pretreatment can alleviate the damage caused by low temperature,and enhance the resistance of B.striata seedlings.Under the chilling stress of 5℃,the pretreatment significantly increases POD activity and inhibit the increase of plasma membrane permeability at later period of stress.The results showed that the effect of pretreatment is more significant under 1℃ chilling stress.But pretreatment significantly increases the content of MDA under certain periods,indicating that H.Koningii pretreatment may cause membrane peroxidation,leading to the accumulation of MDA. |
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