| Pitaya, a member of cactus family, is a perennia, climbing and succulent plant, the fruit of which is rich in phytalbumin and betalains, a variety of vitamins, minerals and water-soluble dietary fiber. It has very high nutritious, economic, social and ecological value. Temperature is one of the main limiting factors that affect the commercial cultivation of pitaya. According to the research, when the temperature is lower than 5℃, pitaya will be suffered from damage, their shoots, twigs, branches and even some mature plants may be frosted or frozen to death.0℃ is a minimum threshold of most cultivated varieties that can stand. Therefore, physiological responses of pitaya to chilling stress and synthetic evaluation of their cold resistance, and cold-tolerant mutant screening in vitro by means of biotechnology were investigated in the study to provide theoretical foundation and references for physiological mechanism of cold hardiness, screening of cold materials and looking for a new approach to increasing the cold hardiness. The main results were as follows.(1) Researches were carried out into the physiological responses of annual branches on the adult pitaya trees to chilling stress and synthetic evaluation of their cold resistance The REC was determined in branches (fleshy stem) of pitaya and their semilethal temperatures (LT50) were calculated after the plants had been treated under a series of low temperature for 12 h, respectively. The cold resistance of pitaya was evaluated by LT50 and verified through recovery growth experiments. Then the contents of soluble sugar, soluble protein, Pro and MDA were analyzed under LT50 after a serial treatment period. The results indicated that the REC of the four types of pitaya increased following an S-curve with the temperature droping and LT50 was between 0.347 and -1.031℃. The soluble sugar, soluble protein and MDA increased with the extension of time, and the proline increased during 0-24 h, then decreased during 24-48 h when treated at LT50. Based on LT50 and related cold resistance physiological indexes of pitaya, the cold resistance ability of four types was evaluated as Liang Tianchi> Jing Honglong> Fen Honglong> Zi Honglong, which was in close agreement with the test result of recovery growth experiment.(2) The dynamic changes of REC, soluble sugar, soluble protein, Pro and MDA in cold acclimation, chilling stress and recovery growth were investigated using the pitaya cutting seedlings of a wild type and three main cultivars. The results indicated that REC increased with the temperature decrease, and there was no significant difference between control and recovery growth experiment. The contents of soluble sugar, soluble protein, Pro and MDA increased in low temperature treatment and decreased in recovery growth in all materials. Furthermore, the variation of soluble sugar and Pro contents were remarkable in different treatments, but MDA and soluble protein contents had no apparent changes in the whole processes. The cold resistance of these pitaya materials was evaluated as Liang Tianchi> Jing Honglong> Fen Honglong> Zi Honglong by using fuzzy mathematics method, which was in agreement with the results of recovery growth experiment.(3) A plant regeneration technique was successfully developed using in vitro culture of spring twig explants (10-20 cm), sprouting from Zihonglong adult trees. The effects of sterilization method, time of sampling, basal medium, plant growth regulators and culture conditions on the callus induction, proliferation and differentiation were studied. The result indicated that a satisfactory sterilization was achieved by treating the explants with 75% alcohol for 30 s, then 0.1% mercuric chloride for 6-8 min before culture. The optimum initial medium for callus induction was MS+TDZ 0.4 mg/L+KT 0.8 mg/L, and the callus was superior in quality in dark culture for 7 days followed by light culture. The best medium for callus proliferation was MS+TDZ 0.4 mg/L+KT or ZT 0.8 mg/L, giving rise a multiplication coefficient of more than 8 times. Adding coconut water of 20-30% had a certain promotion effect on differentiation of callus, but the differentiation rate was quite low as a whole.(4) Adventitious buds were induced directly in vitro from branches of Zi Honglong adult trees. The results showed that the stem with small splinter and nap was easy to induce adventitious buds, and the induction rate of adventitious buds was 0 without stabs; the optimum medium was MS+6-BA 2.0 mg/L+NAA 0.1-0.2 mg/L, the best medium for adventitious buds proliferation was MS+6-BA 0.5 mg/L+NAA 0.1-0.2 mg/L+Tryptone 750 mg/L.(5) With aseptic plantlets induced from Zi Honglong adult trees as explants, the optimum medium for callus induction and adventitious bud differentiation was MS+TDZ 0.4 mg/L+2,4-d 1.0 mg/L+0.5 mg/L NAA, resulting in callus induction and adventitious bud differentiation rate of 89.1% and 94.0%, respectively. The most suitable rooting medium was 6-BA 1.0 mg/L+0.1-0.2 mg/L NAA+0.5-1.0 mg/L CCC+0.03-0.05% AC, resulting in a high rooting rate of more than 98%. The rooted plantlets were easy to transplant, and the survival rate was up to 100% with a good growth on the media of pearlite+humus soil (2:1) and vermiculite+humus soil (1:1).(6) The effect of EMS,60Co γ-rays and HYP on survival and growth of aseptic seedlings were studied. The results showed that seedlings had a good adaptability to EMS when subcultured for 15 to 30 d. The suitable concentration and optimum time for EMS mutation was 3.71-3.95% and 8.26-9.32 h, respectively. There was no significant effect of low irradiation dose (≤15 Gy) on survival and differentiation of seedlings. The survival and differentiation rate decreased significantly with the increase of irradiation does, and the higher the irradiation dose, the more obvious the effect. The feasible irradiation dose range determined by LD50 and LD40 for 60Co y mutation was 38.5-42.4 Gy. The seedlings of pitaya in vitro can tolerate HYP at low concentration (≤8 mmol/L). Compared with control, seedlings had a significant higher survival rate under high concentration of HYP (20 mmol/L) when treated by EMS.(7) The REC and MDA content of seedlings increased with the temperature decreasing and the time extending no matter using EMS or 60Co y mutagens or not, while the contents of chlorophyll and carotenoid were just the opposite. The lower the temperature and the longer the treating time, the greater of the increase or decrease. There was a remarkable influence of low temperature on soluble sugar, soluble protein and proline, especially in the soluble protein which was more sensitive to changes in temperature than the others. Most changes of osmotic regulation substances completed in a relatively short period of time. All kinds of protective enzymes (SOD, POD, CAT and APX) activity changed inconsistently with the fall of temperature and extension of time. Most of the enzyme activity increased notably in the early stage of low temperature treatment, but declined gradually with the temperature decline and treating time prolongation.(8) Seedlings treated with EMS or irradiated with 60Co y, then selected with different concentration of HYP, showed better cold hardiness under the different temperatures or under different treating time at the same temperature in comparison with the control in the preliminary tests of physiological and biochemical indexes.(9) REC and proline of mutants screened from materials induced by EMS and 60Co γ irradiation were measured and their molecular marker analyzed by ISSR. The results indicated that REC of mutant decreased compared with the control, and among them No. 5 mutant had the biggest drop of REC. Proline content of mutants was significantly increased, and No.5 was 6.5 times higher than that of the control, which was consistent with REC. A total of 67 bands were yielded from the 9 selected primers, among which 71.64% polymorphism was obtained, which showed that the variation of the germplasms were more abundant. There was a specific band in No.5 mutant detected by primer M06. Analyzed by NTSYS pc 2.1, the genetic distances among the tested materials were 0.0120 to 0.3597, with an average of 0.2836. The UPGMA clustering analysis demonstrated that 15 germplasm could be divided into 6 groups at a cutpoint of genetic distance 0.11, and among them No.5,6,10,12 and 14 warranted separate categorization, which showed that they had more differences in tested germplasm. |
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