Development Of Agrobacterium Rhizogenes Mediated Transformation And Canker Disease Resistance Analysis In Citrus

Citrus is one of the most important fruit-bearing trees in the world and plays a pivotal role in local economics. A hairy root induction and plant regeneration system in ’early gold’ sweet orange [Citrus sinensis (L.) Osbeck] and trifoliate orange [Poncirus trifoliata (L.) Raf.] was developed via Agrobacterium rhizogenes mediated transformation. The DR5::GUS-YFP fusion gene was successfully introduced into trifoliate orange. Based on the auxin reporter system, the auxin distribution was observed and elucidated in hairy roots and their regenerated plants. Besides, molecular identification and canker disease resistance assay were carried out in transgenic ’Anliu’ sweet orange [Citrus sinensis (L.) Osbeck] expressing the rice Xa21gene. The main results are as follows:1. Hairy root induction and plant regeneration system was developed in’early gold’ sweet orange and trifoliate orange via Agrobacterium rhizogenes MSU440mediated transformation. Hairy roots harboring DsRED1, a red fluorescence protein gene were obtained. Epicotyls were immersed in Agrobacterium rhizogenes solution for20min and co-cultured at21℃for3d before transferred to MT medium for hairy root induction. Hairy roots emerged at the cut sites of epicotyls after about2-3weeks’culture on the plant hormone-free MT medium supplemented with400mg/L cef. The integration of the rol B、rol C and the DsREDl genes was identified by PCR and fluorescence observation in the transgenic hairy roots. Transgenic plants were also obtained from the hairy roots. A dramatic dwarf phenotype with shortened internode and prosperous roots was observed in the plantlets regenerated from hairy roots of’early gold’sweet orange. However, the phenotype of plantlets regenerated from hairy roots of trifoliate orange was normal except for more adventitious roots produced in some transgenic lines.2. Based on Agrobacterium rhizogenes mediated transformation system, the transgenic regenerants of trifoliate orange with DR5::GUS-YFP fusion gene were successfully obtained. The presence of rol B, GUS and YFP genes in co-transformed hairy roots was confirmed by fluorescence observation, GUS staining and PCR analysis. The co-transformation rate based on GUS staining was as high as68.3%. The DR5::GUS-YFP reporter system was used to study the auxin response maxima in hairy roots. The results indicated that auxin maxima were present primarily in the quiescent center (QC) and columella cells of the root apex of hairy roots and their lateral roots. The spatial pattern of auxin maxima during lateral root primordium development in co-transformed hairy roots was further analyzed. Auxin maxima were present at specific pericycle cells at the early stage of lateral root primordium initiation; then the auxin gradient was gradually developed in the newly-emerging lateral roots. The activity of DR5::GUS-YFP in the apex of hairy roots increased under exogenous application of1-naphthalene acetic acid (NAA) and decreased when2,3,5-triiodobenzoic acid (TIBA) was applied both in a concentration dependent manner.A procedure of plant regeneration from transgenic hairy roots was also established. Tumor like calluses were indued from the hairy root segments on MT medium plus1mg/L6-BA. Multiple shoots then developed from the calluses and whole transgenic plants were obtained after cultured successively on shoot elongating and root inducing MT medium. The presence and expression of YFP gene in the regenerated co-transformed plants were further confirmed by Southern blot hybridization and qRT-PCR. The hairy roots obtained by A. rhizogenes mediated transformation were confirmed to be a promising system for root development study in P. trifoliata. Furthermore, the genetic transformation and plant regeneration system via A. rhizogenes provides an alternative method for genetic engineering in Citrus.3. The pattern of auxin distribution in the root, stem and leaf in transgenic plants of Poncirus trifoliata was observed based on the DR5::GUS-YFP reporter system. The results indicated that auxin maxima were located in root apex and the initiative site of lateral primordium. The auxin distribution pattern in the adventitious roots of regenerated plantlets was in accordance with that in the former hairy roots. So far as stem was concerned, the auxin maxima were observed in the primary phloem which indicated it could be the main pathway for auxin transportation from the shoot tips to the roots. No auxin maxima were observed in the epidermis, cortex and pith. Auxin maxima were also located in the sites of developing stomatal apparatuses and oil vacuoles in young leaves. Those results indicated that the auxin plays a pivotal role for the development of plant organs. The auxin response pattern was also observed in the root and stem after treated with NAA or TIBA. The results showed that the activity of DR5::GUS-YFP was increased in the root apex after treatment by NAA and decreased by TIBA. However, the sensitivity to NAA differed in different transgenic lines. The R54line was more sensitive to NAA than the R28line and higher expression of DR5::GUS-YFP was observed after treated with NAA at same condition.4. The transgene integration of recovered plantlets was identified by PCR and Southern blot hybridization. A putative1.4kb fragment of Xa21gene was amplified from all the transgenic plants tested. The8independent transgenic plantlets tested had undergone single copy integration, the expression of Xa21gene in eight different transgenic lines was detected by qRT-PCR and could be divided into three levels, high for T5and T6, middle for T4and low for the rest. The tolerance to citrus canker disease of the three recovered transgenic lines T2, T4and T6was assessed by in vitro pin-puncture inoculation with leaves. The results showed that all the three transgenic lines conferred improved resistance to citrus canker bacterium infection and the T4transgenic line displayed the highest resistance. The mechanism and feasibility of the rice Xa21gene to trigger innate immunity in citrus was briefly discussed.