Establishment Of Rapid Propagation And Agrobacterium-mediated Transformation In Gypsophila Paniculata

Gypsophila paniculata L.is a perennail herb,which can be cultivated for viewing,and it is the most expensive variety among the three major flowers of fresh cut flowers.The number of transactions of Gypsophila ranked fourth among various fresh cut flowers,which demonstrated that its market demand is large and its commercial value is high.Because the multi-petal gypsophila is sterile,in traditional production we usually reproduce it by cutting.There are some disadvantages of this way,like the low reproductive coefficient,disable to provide flowers in the winter,so the market demand cannot be satisfied.Furthermore,there are a few studies on genetic transformation of gypsophila.In order to supplement the traditional production and breeding methods of gypsophila,in this study we carried out the following experiments:（1）The management of the planting process through sowing and cultivate in the greenhouse was established using the gypsophila single-petal material.The observation of the flower organs and the cuttage experiments of the branches were carried out using the multi-petal flower material,and it was found that the roots grown by the roots of the cuttings were weak.（2）To use stem segments,leaves and inflorescences of multi-petal gypsophila to induce callus.It demonstrated that the best disinfection effect is achieved by treating 0.1%HgCl₂ solution for 8 min,and the combination of 0.5 mg/L 6-BA and 0.3 mg/L NAA has the best effect on callus induction.More than half of the callus-induced callus is vitrified,while leaf-induced callus is difficult to differentiate into buds,so both of them are not suitable for rapid propagation.The unflowereded inflorescence has a better induction effect and is suitable for rapid propagation.（3）We established a rapid propagation system through inducing stem segments to produce buds,and inducing buds to produce roots.During this process,1.0 mg/L 6-BA shows the best induction effect on producing shoot buds,and we can obtain 9¹6 buds from each explant.Then we induced the plantlet to produce roots,and it demonstrate that auxins had no obvious effect on rooting.（3）Using stem segments as explants,we transformed gypsophila with Agrobacterium EHA105 strains carrying plasmid pTCK303（hygromycin resistance）and pCAM3301（glufosinate resistance）,and improve the conditions of cultivation and infection,like the size of explant,pre-cultivation or recovery,the negative pressure,and time of co-cultivation,etc.We established Agrobacterium-mediated transgenic system of gypsophila,and obtain four positive plants with glufosinate resistance.The conversion efficiency is 3.3%.The protocol of transformation is as follow:The stem sections of the shoots were cut into 5 mm,and pre-cultivated for 2 days in MS+0.5 mg/L 6-BA medium.Agrobacterium was resuspended in MS liquid medium with 200μM AS,and put the stem sections into liquid,keep it for 20 min,after blowing to dry,transfer the stem sections to 200μM AS-containing MS solid medium for 2 to 3 days.The explants were washed 4 to 5 times with steriled water until the water was completely clear.Transferred the explants to firstly-selective medium（1 mg/L glufosinate）and cultivated for 14 days,when the new buds appearred.Then transferred the buds to secondly-selective medium（2 mg/L glufosinate）and cultivated for21 days.Transferred the buds to medium without glufosinate for proliferation.When the seedlings growed to 3⁴ cm,they were transferred to rooting medium which contains1.0 mg/L IBA.