Optimization Of Kiwifruit Genetic Transformation System And Establishment Of Protoplast Preparation System

Kiwifruit（Actinidia chinensis Planch.）is a deciduous vine fruit tree of the genus Kiwifruit in the family Actinidiaceae.Its fruit is rich in nutrients and is one of the newest fruits of the 20th century with good market prospects.The kiwifruit industry has flourished worldwide in recent years,however,basic research on kiwifruit has lagged relatively behind,with a large number of excellent resources and their valuable genes still unexplored and their functions yet to be elucidated.Stable genetic transformation is a powerful means for plants to carry out gene function,but it is difficult to establish efficient genetic transformation systems for most woody plants.Establishing a genetic transformation system for protoplasts and healing tissues first became an alternative,which is more time and effort efficient than stable transformation system.In this paper,we established a genetic transformation system for guaiac tissue in’Hongyang’kiwifruit and optimized a stable transformation system;we established an efficient protoplast isolation and transient transformation system for’Hayward’kiwifruit,which provides a technical basis for functional identification of kiwifruit genes and molecular We also established an efficient protoplast isolation and transient transformation system for’Hayward’kiwifruit,providing a technical basis for gene function identification and molecular genetic breeding of kiwi.1.The results are as follows:（1）Screening of the best explants.The leaves,cotyledons,petioles,stem tips,hypocotyls,roots,’Maohua’kiwifruit and’Hongshi2’kiwifruit were used as test materials for the induction of healing tissues from’Hongyang’kiwifruit seedlings.Tissue.The results showed that healing tissues were induced on MS+30 g/L sucrose+7 g/L agar+0.2 mg/L ZT+1 mg/L 2,4-D medium in all explants except for’Hongshi 2’fruit,which did not induce healing tissues.In terms of time to healing,petioles were the earliest,followed by leaves,and the slowest by fruits of kiwifruit;in terms of amount of healing,leaves induced the most healing tissue,1-2 times the size of the leaves themselves,followed by cotyledons,fruits of kiwifruit and stem tips.Therefore,leaves were the best explants,the advantages include easy to take,short induction time and large amount of healing.（2）Healing tissue induction and proliferation culture.The leaves were used as explants for the induction of healing tissues in different hormone combinations in the medium.The results showed that MS+30 g/L sucrose+7 g/L agar+0.5 mg/L ZT+0.5 mg/L 2,4-D was the best medium for healing induction;the above-mentioned induced healing tissues were used as the material to screen the healing tissue proliferation medium by designing different hormone combinations,and the results showed that the highest healing value added was achieved on N6+20 g/L sucrose+6g/L agar+1 mg/L 2,4-D+0.5 mg/L 6-BA+0.5 mg/L NAA culture.The results showed that N6+20 g/L sucrose+6 g/L agar+1 mg/L 2,4-D+0.5 mg/L 6-BA+0.5 mg/L NAA culture had the highest value added and the most suitable healing type as the subsequent transformation material.（3）Screening and validation of transformation conditions.The above-mentioned healing tissues were transformed by Agrobacterium-mediated method after 2-3 stable passages of culture.By screening the conditions of healing tissue growth time,pre-culture time,co-culture time and antibiotic concentration,we found that the healing wounds cultured for 10-20 d with a bacterial solution concentration(OD₆₀₀)of0.8 and 30 min of infestation,some resistant healing wounds grew under screening3-5 times conditions,and overexpressed Ac MYB110 healing wounds were obtained by DNA level detection.2.The stable genetic transformation system of’Hongyang’kiwifruit was optimized to obtain the Ac GST1 transgenic strain.Agrobacterium-mediated transformation was performed using leaves and petioles as explants,and the Ac GST1transgenic strain was obtained by DNA and RNA level detection under the conditions of OD₆₀₀=0.6,10 min of infestation time,3 d of co-culture and 4-5 times of screening culture,and the red phenotype appeared on the leaf edge of transgenic kiwifruit.Gene expression was analyzed using q RT-PCR,and the expression of GST1 in transgenic kiwifruit was found to be significantly up-regulated compared to wild-type kiwifruit.3.A system for the isolation and purification of kiwifruit healing tissue protoplasts was established.The optimal protoplasmic preparation conditions were obtained by screening the succession time of guava tissue,different enzyme combinations and concentrations,mannitol concentration,cell filter sieve pore size,and enzymatic digestion time using’Hayward’leaf guava tissue as the material.The optimized conditions were:2%cellulase and 0.5%macerozyme enzyme combination（0.7 M mannitol concentration）,7 h enzymatic digestion under dark conditions,yield of 2.83×10⁶g/FW and viability of 86.94%.The transfer of GFP-MYB61 into protoplasts by PEG-mediated method proved to be useful for transient genetic transformation of kiwifruit.