Improved Agrobacterium-mediated transformation protocol for VgDGAT1a gene based on shoot tip culture of cotton

Abstract

In past two decades,abundant researches have been published on transformation,regeneration,and genetic enhancement of cotton,especially Gossypium hirsutum.Generally,genes conferring agronomic advantages have been introduced into plants via Agrobacterium-mediation or particle bombardment,and then the transgenic plants are regenerated through somatic embryogenesis from callus.However,the embryogenic callus-based regeneration is difficult and time-consuming in cotton.Therefore,it is necessary to establish an effective system for the Agrobacterium-mediated genetic transformation of shoot tip in cotton.Triacylglycerols(TAG)are a heterogeneous group of molecules with a glycerol backbone and three fatty acids attached by ester bones.Diacylglycerol acyltransferase(DGAT;EC3.2.1.20)catalyzes the last step of TAG biosynthesis and it is the only key enzyme evolved in.DGAT1 and DGAT2,as two types of DGATin eukaryotes,belong to different gene families.And previous studies have reported that the expression of DGAT1 increased seed oil content and mass.In order to get new cotton germplasm with high oil content,we used the shoot tips of‘Zhongmiansuo 49’as explants and introduced an improved vector carrying a selection marker HptⅡ gene and a target VgDGAT1agene into cotton via Agrobacterium-mediated transformation.This improved Agrobacterium-mediated transformation and regeneration system were established by optimizing different parameters such as pre-culture period of seeds,concentration of Agrobacterium in solution,immersing time,co-cultivation period and components of MSB [Murashige and Skoog(MS)medium + vitamins of Gamborg’s(B5)medium].Cotton seeds(Gossypium hirsutum L.cv.‘Zhongmiansuo 49’)were decorticated manually and surface sterilized in 0.1% HgCl for 8min,followed by rinsing with sterile distilled water for five times.The cotyledons were removed from 3-day-old to 5-day-old in vitro germinated seedlings and the shoot tips were cut in lengthwise to decrease the damage of meristematic cells.The strain EHA105was grown overnight on a shaker at 200r/min and at 28℃ until the A600value of bacterial concentration reached 0.6-0.9.The suspension cells were centrifuged at 5 000 r/min for 10min and the pellets were resuspended in an equivalent volume of liquid co-cultivation medium [MSB+ 200μmol/L acetosyringone(AS)].The treated explants were immediately immersed into prepared Agrobacterium suspension containing pCAMBIA1301,a binary vector carrying the VgDGAT1agene,for 40-60minutes.The tips were blotted dry on sterile filter paper and transferred into the co-cultivation solid medium at 28℃in dark.After co-cultivation for 1day,shoot tips were transferred into root induction medium containing MSB,1g/L activated charcoal,400mg/L cephalothin(Cef)and solidified with 0.2%(W/V)phytagel,then grew in a growth chamber with stringent light,temperature and humidity control.In the following 3-4weeks,5-leaf-plants were transferred to plastic pots containing soil matrixes(1∶1of turf and vermiculite).Besides the optimized genetic transformation protocol of cotton shoot tip as stated above,it is found that 50mg/L hygromycin(Hyg)could accurately distinguish the resistant plants.In addition,reduplicated selections improved accuracy.In conclusion,this study describes an optimized transformation protocol for shoot tip of‘Zhongmiansuo 49’ with an A.tumefaciens strain EHA105harboring DGAT1 gene,and proves that it is an efficient and economical method to obtain transgenic plants based on the results of different and important parameters influencing the transformation efficiency.