| The cultivation range of pitaya(Hylocereus undulatus(Haw.)Britton&Rose)in China is more and more extensive,which makes the industry especially vulnerable to low temperature stress.Extreme weather is frequently encountered in Guizhou,Yunnan,Guangxi and other subtropical regions,such as low temperature or late spring coldness,etc.Cultivation of pitaya is prone to chilling and freezing,resulting in about 30%of the yield decreased.In recent years,some researches on physiological effects of low temperature stress on pitaya have been carried out,which provides a valid method for evaluating cold resistance of pitaya.Some seedlings with cold hardiness have been preliminarily achieved by the induction of low temperature with EMS or irradiation.The conventional breeding of pitaya is limited by the lack of cold hardiness resources.We found that it was difficult for calluses that induced from adult pitaya stems to redifferentiate in previous research,which affected the efficiency of genetic transformation.There is no relevant public report on pitaya genetic transformation research.The researchers want to find a breakthrough from genetic engineering,genetic transformation and so on,to provide new ways and methods for the breeding of cold resistant varieties of pitaya.In this study,construction of regeneration system for pitaya was developed by anther culture and pitaya stem culture.Some researches were carried out,such as construction of vector with exogenous LTP gene,anther callus and stem segments as the receptor,mediation by Agrobacterium tumefaciens,relevant molecular identification of resistance callus or resistant buds respectively,and the morphology and physiology of cold resistance for transgenic plants.The purpose of this study is to provide an important technical scheme and reference for the establishment of the genetic transformation system and the breeding of new varieties for pitaya.The main results are as follows:(1)The dynamic changes of "Zihonglong" pitaya flower buds were observed from its budding and the flower buds were collected from 1?13 days in squaring period.The characteristics of external morphology structure and changes of cytological features of pitaya microspore at different developmental stages were also observed by electron microscope,which provided cytological basis and optimal sampling time for the culture of anther and microspore.It was expected to help to build an efficient and stable tissue culture system for pitaya anther or microspore.The results indicated that the development of pitaya microspore went through tetrad stage,eraly-uninucleate stage,late-uninucleate stage,binucleate stage,and finally developed into the mature pollen grain,and each period had obvious morphological characteristics.The microspore development period of pitaya was closely related to bud size and anther length.It took 4 days for most microspores to develop into tetrad stage and the buds were 5.5?6.0 cm in longitudinal diameter and 2.5?3.2 cm in transverse diameter;it took 5 days for most microspores to develop into eraly-uninucleate stage and the buds were 6.8?7.6 cm in longitudinal diameter and 3.0?3.4 cm in transverse diameter;it took 6 days for most microspores to develop into binucleate stage and the buds were 8.1?8.6 cm in longitudinal diameter and 3.3?3.9 cm in transverse diameter;it took 7-8 days for most microspores to develop into binucleate stage and the buds were 8.9?10.5 cm in longitudinal diameter and 3.8?4.4 cm in transverse diameter.In conclusion,the standard of bud selection for optimal culture period of microspores could be determined according to the form,size and anther develpoment period of pitaya flower buds.(2)In this study,anthers at different stages of microsporogenesis were cultured,and low temperature pretreatments were compared,and the induction medium was optimized for callus induction,proliferation and differentiation,with abundant embryogenic callus generated and adventitious buds formed.The results indicated that without any appropriate pretreatments,pitaya anthers spanning stages from early-uninucleate to late-uninucleate showed a callus induction rate of up to 23.9%,but no embryoid formed.Low temperature pretreatment of 2 days duration resulted in an induction rate of 32.8%.The optimum medium for callus induction was MS + 2.0 mg/L 6-BA + 1.0 mg/L 2,4-D + 60 g/L sucrose,with an induction rate of 37.8%and the embryogenic callus was compact in yellowish-green color,with granular bulge on surface.The optimum medium for callus proliferation was MS + 0.4 mg/L TDZ + 0.8 mg/L KT,with a multiplication factor over 9.1.Culture medium for callus redifferentiation was MS + 0.4 mg/L TDZ + 0.5 mg/L 2,4-D + 0.6 mg/L NAA,with a redifferentiation rate of 36.7%.A large number of embryogenic calluses were formed through anther callus proliferation,then differentiated to form adventitious shoots,which had the same genetic stability with mother plants by SRAP molecular markers verification.The system developed herein could be applied in future pitaya rapid micropropagation and genetic engineering.(3)The concentrations of four endogenous hormones,such as abscisic acid(ABA),indole acetic acid(IAA),gibberellin(GA3)and zeatin(ZT)in fresh stems or different calluses induced from pitaya anthers or stems were mensurated by high performance liquid chromatography-mass spectrometry(HPLC-MS/MS).The results showed that the higher callus induction rate of stem segments of was closely related to the higher content of endogenous IAA and ZT.The content of GA3 in fresh stem segments of pitaya was significantly lower than that in stem or anther callus,the content of ABA was higher than that in callus from stem segments,but lower than that in anther callus.The content of IAA in anther callus was significantly lower than that in the callus from stem segments,but the content of ABA was higher than that in the stem callus.The ratio of ABA/GA3,ABA/IAA in anther callus was significantly higher than that in the stem callus,the ratio of ABA/GA3 in the undifferentiated anther callus was 6.71 times higher than that in stem callus,is 3.82 times higher than that in differentiated anther callus.Most importantly,the ratio of ABA/IAA in the undifferentiated anther callus was 43.68 times and 16.02 times than that in stem callus and differentiated anther callus respectively,which was the critical factor for the lower redifferentiation of undifferentiated anther callus.The higher ZT and IAA contents were beneficial to the differentiation of adventitious buds in anther callus,while the high content of GA3 could inhibit the occurrence of adventitious buds,and the low level of GA3/ZT ratio was very important for the differentiation of adventitious buds.(4)Embryogenic calluses obtained from pitaya anther culture were used as receptors,and cold resistant LTP gene was mediated by Agrobacterium tumefaciens.Researches on construction of expression vector,preparation of Agrobacterium tumefaciens,selection pressure of Kan screening,the mode and time of calluses infection by Agrobacterium tumefaciens,co-culture time,culture medium,PCR detection of transformed calluses were carried out in the study.The results showed that the LTP gene was successfully constructed by CaMV35S promoter and plant expression vector pCAMBIA 3301-LTP,and into the cell by the competent cells of Agrobacterium tumefaciens LBA4404.75mg/L Kan was the suitable pressure for pitaya callus;suitable dip-dye time for callus was 8 min.The suitable co-culture medium was MS +TDZ 0.4 mg/L + 2,4-D 0.5 mg/L + NAA 0.6 mg/L + sucrose 30g/L+ agar 7.5g/L;suitable selective medium was MS + TDZ 0.4 mg/L + 2,4-D 0.5 mg/L + NAA 0.6 mg/L + sucrose 30 g/L+ agar 7.5 g/L+ Kan 75 mg/L + Cef 375 mg/L.Using this transformation system for genetic transformation,finally 106 Kan resistant calli were obtained.Specific amplification of LTP gene from 5 resistant calli's DNA randomly and the expected target band were amplified from all the 5 resistant calli,and positive rate was 100%.However,the subsequent resistant calli did not differentiate adventitious buds.Using calli of pitaya as receptors,a genetic transformation system mediated by Agrobacterium tumefaciens was established preliminarily.(5)By using seedling stems obtained from pitaya aseptic tissue culture as receptors,cold resistant LTP gene was trying to mediated by Agrobacterium tumefaciens.Researches on selection pressure of Kan screening,the mode and time of Agrobacterium tumefaciens infection,co-culture time,selective medium,PCR detection of resistant buds and southern blot were carried out.The results showed that:50mg/L Kan was the suitable selection pressure for pitaya stems;the suitable dip-dye time for stem segment was 15min;suitable medium for co-culture was MS +6-BA 2.0 mg/L + NAA 0.1 mg/L + Tryptone 250 mg/L + sucrose 30 g/L + agar 7.5 g/L;suitable selective medium was MS + 6-BA 2.0 mg/L+ NAA 0.1 mg/L + Tryptone 250 mg/L + sucrose 30 g/L + agar 7.5 g/L + Kan 50 mg/L + Cef 375 mg/L.Using this transformation system for genetic transformation,63 Kan resistant buds were obtained finally.Specific amplification of Bar gene of DNA extracted from 8 random resistant buds and the expected target band were amplified from 6 resistant buds,and positive rate was 75.0%.Further hybridization detection by Southern blot showed that 3 materials of 8 resistant buds appeared significant hybridization signals,suggesting that the LTP gene had been successfully imported to pitaya receptors,and the conversion rate reached 37.5%.The following antagonistic shoots were rooted and 25 regenerated plants were obtained.Agrobacterium tumefaciens mediated genetic transformation system of pitaya's stem segaments was established,which could provide the technical basis for the subsequent transgenic research.(6)The results of morphological observation and correlation analysis of cold resistance for the LTP transgenic seedlings indicated that under the same condition of low temperature,the non transgenic seedlings(CK)appeared wilting and yellowing branches significantly compared with transgenic seedlings.In the process of continuous low temperature,REC and MDA content of the transgenic plants gradually increased,but significantly lower than the control;while the contents of Pro,SOD,POD,CAT activity were first increased and then decreased,and also significantly higher than the control.Comprehensive morphological feature identification and resistance analysis showed that the resistance of LTP transgenic plants to low temperature was significantly higher than that of the control.Cold resistance increased significantly by the genetic transformation of LTP gene. |
PDF Full Text Request