Study On The Function Of Melatonin In The In Vitro Regeneration And Cryopreservation Of Shoot Tips Of Chrysanthemum Morifolium Cv. Chuju

Chuju is a perennial herb of Chrysanthemum in Compositae.It has high medicinal and health value.Therefore,it is necessary to establish a long-term and efficient method for germplasm conservation of Chuju excellent strains.Based on the selection of excellent strains of Chuju,an efficient in vitro regeneration protocol of Chuju was established,and an efficient procedure for cryopreservation of the shoot tips of Chuju excellent strains was established for the first time.In addition,the effects of N-acetyl-5-methoxytryptamine(MLT)on the the cryopreservation efficiency was investigated,and the mechanism of melatonin in the cryopreservation of Chuju was further studied by combining with physiological and biochemical analysis.The main conclusions of this research are listed as following:(1)An efficient in vitro regeneration protocol of Chuju was established.The main conclusions are as follows:The best disinfection effect was obtained when the shoot stems was disinfected in 0.1%Hg Cl₂ solution for 3 min.When the shoot stems were cultured on MS basal medium supplemented with 0.5 mg l^-1 6-BA(N-(Phenylmethyl)-9H-purin-6-amine),the highest adventitious bud induction frequency(100%)with average number of adventitious buds per explant(4.67)was obtained,and the proliferation coefficient of adventitious buds was 7.76 after sub-culture.The elongation of adventitious buds was the best when 0.2 mg L^-1 GA3 was added to the shoot induction medium.The highest adventitious root induction rate with average root length(5 cm)was obtained when the elongated shoots were inoculated on 1/2 MS medium supplemented with 0.1 mg L^-1 IBA(1h-indole-3-butanoic acid).Further study showed that 1/2 MS medium supplemented with50?M melatonin could improve the length of adventitious roots and seedling growth,and the growth of seedlings treated with melatonin was better than that of IBA under drought conditions.(2)The establishment of the regeneration procedure of Chuju provides a lot of materials for the development of the cryopreservation technology system.Through optimizing a series of key parameters that affecting the cryopreservation process,the cryopreservation system of Chuju was established for the first time.The specific process was as follows:The shoot stems of Chuju was cultured on the MS medium with 0.5 mg L^-1 6-BA to induce adventitious buds.the regeneration buds with the following generation within 5 times,gr Ew for 30-50 days and grew evenly for 4 days at 4?for 4 days of low temperature exercise.Then,the shoot tips(2.0-2.5 mm in length)were precultured on MS medium containing 0.4 M sucrose for 4 days in the darkness at 4?.The PVS2 solution was diluted into 60%PVS2 solution with MS basal medium and used as loading solution,the shoot tips were loading in this solution at0?for 20 minutes.Then,the stem tip was treated at 0?with PVS2 solution at 0?for 60minutes.After that,the shoot tips were placed in the frozen storage tube containing PVS2solution,and then put into liquid nitrogen for freezing and storage for more than 1 hour.After that the shoot tips were thawed quickly in 39±1?water bath.The MS liquid medium containing 1.2 M sucrose was used as the unloading solution for 15 min.Then,the unloaded shoot tips were inoculated on the medium supplemented with 0.1 mg L^-1 6-BA and 0.1 mg L^-1IBA for 3 days.Finally,The cultures were maintained at 25±2?under a 14-h photoperiod with a light intensity of 40?mol s^-1 m^-2 provided by cool-white fluorescent tubes(Philips,India).The regeneration rate of cryopreservation of shoot tips of Chuju was 58%.(3)In this study,the effect of MLT on the cryopreservation efficiency of shoot tips of Chuju was investigated.The results showed that the regeneration rate of the shoot tips of Chuju could be increased to 65.7%by adding MLT to the medium during the low temperature exercise stage.On this basis,we investigated the effect of MLT on the physiological and biochemical indexes of shoot tip cells during the process of cryopreservation.These indexes include MDA,SOD,OPC,OPC and·OH scanning ability.The results showed that MLT could promote the accumulation of antioxidant substances,reduce the formation of peroxide,improve the antioxidant activity of shoot tip cells and then improve the regeneration rate of cryopreservation.Meanwhile,the results of histological section of shoot tips of Chuju showed that MLT could maintain the normal physiological structure of shoot tip cells at the cell level,and accelerate the recovery of normal physiological function after cryopreservation.(4)We further adopted ISSR Molecular marker technology to analyze the genetic stability of the regenerated plants.Among the 10 ISSR primers tested,the single primer results in a variety of monotonic bands ranging from 4 to 9,and a total of 72 bands were obtained.According to ISSR results,no polymorphism bands were observed in the plantlets regenerated from cryopreserved shoot tips of Chuju and the mother plant,which confirmed the genetic fidelity between the regenerated plantlets and the mother plant.