| Citrus is one of the most important fruit crops in the world. In the past tens of years, obtaining dwarf characteristics was one of the main goals of citrus genetic improvement. There are no useful dwarfing rootstocks for citrus (Deng 1994; Grosser et al.,1988). Citrus traditional hybridization has made slow progress due to long juvenile period, high heterozygosity, sexual incompatibility and nucellar embryony. Biotechnology methods allow the introduction of one or few genes of dwarf genes into a citrus genome relatively quickly, and offer an alternative way to promote citrus rootstock dwarfing breeding.The rol A, rol B and rol C genes of Agrobacterium rhizogenes A4 are located on the TL-DN A of the Ri plasmid. rol genes have been introduced into a range of crops, and a wide of morphological and physiological changes occurred, these include dwarfing effects on plant height due to short internodes, wrinkled leaves and delayed flower timing, increased branching associated with loss of apical dominance, and inducing full hairy-root syndrome. Recently, rol genes were used to transform fruit trees for dwarfing breeding, especially for creating dwarfing rootstock with tolerance to environmental stress.Troyer citrange [Citrus sinensis (L.) Osb.?Poncirus trifoliata (L.) Raf.] is an important hybridization rootstock in citriculture. Which were find widespread use in European and America countries due to resist citrus tristeza virus (CTV) and citrus foot rot. To obtain dwarf and resistance by genetic improvement could enhance the value of Troyer citrange to a certain extent.Previously, rol ABC genes were transferred into Troyer citrange and three clones were obtained. These transgenic clones were propagated by tissue culture during the past six years. Many years observations showed that transgenic clones had typical dwarfing characters (up to 50% height reduction), and increased rooting ability. In the present study, morphological characteristics of rol ABC transgenic citrange clones were observed, molecular confirmation and expression of transgenes were investigated at different time intervals and in various tissues using qRT-PCR, many aspects of physiological property were determined, and the rootstock-scions interaction were surveyed, with the aim of evaluating the potential of transgenic Troyer citrange and understanding the induction mechanism of dwarfing by rol genes. 1. PCR. southern blot analysis revealed that rol A, rol B and rol C were integrated into the citrange genome, exactly two copies in clones B and D, and one in clone E. Semi RT-PCR and quantitative RT-PCR results indicated that rol A and rol C were over-expressed, with the highest expression for rol C, while rol B had a very weak expression signal.2. The transgenic citrange clones B, D and E showed typical dwarfing characteristics of rol gene. E clone showed stronger growth vigour and better environmental resistance, also the lateral branches growth thickly compare with B, D clones. Three clones have a narrow and intensive vessel structure, the cell number per cm2 was not decreased, all transgenic citranges reveal good grafting compatibility with Dahong sweet orange and Shatian pumelo, POD, SOD, Pro and chlorophyll content all dramatically increased compare to wild type citrange, and transgenic citrange show an stronger photosynthesis and environmental resistance.3. Developing suitable qRT-PCR method for analysising the expression of rol A, rol B, rol C genes in transgenic citrange. From 15 June to 15 September in 2009. seven plant tissues including buds, tender leaves, young stems, functional leaves, old leaves, bark and roots were used as materials for q RT PCR analysis, at the same stage, 10 plants per transgenic clone and wild-type citrange were chosen for morphological observations. Try to study the relation between rol genes'expression and plant growth. The result indicated that rol C gene had the highest relative expression in all tested tissues except old leaves, rol B had the lowest expression, and the expression of rol A was intermediate of the three genes. The expression of rol A and rol C showed two peaks on 30 June and 30 July, and one lowest point on 15 July. The temporal changes of rol genes'expression were found to be influenced by environmental condition. On July 15, the highest temperature was supressed rol genes'expression and restrained plant growth activity. The expression of rol A was mainly in old leaves, buds and functional leaves. Expression of rol B was highest in roots, and very low in other tissues. Expression of rol C was primarily in buds, tender stems and functional leaves, The summed expressions of the rol genes at four different sampling times (15 June, 15 July,15 August and 15 September), the relationship between the expression of rol genes in single tissues and RPHRR were showed the expression of rol A and rol C in bark was significantly positively correlated with RPHRR (r=0.75 and r=0.74, respectively; P<0.01). The expression of rol A and rol C in buds, tender stems, tender leaves, functional leaves and old leaves was significantly correlated with their expression in bark. This indicates that expression of rol A and rol C in these tissues was also involved in the induction of dwarfing.4. Transgenic citrange was neither defective genotype of GA3, nor unsensitive genotype of GA3. However, the spraying of GA3 elongated the internode of transgenic citrange, it was still shorter than wild type citrange. The content of IAA. GA] and GA4 decreased (P<0.05) while peroxidase activity increased markedly in the tender buds of transgenic citrange (P<0.05). The GA20ox1 expression quantity markedly decreased in apical bud (P?0.01), the expression of GA20oxl in tender leaves was higher than wild type. The expression of rol C gene and GA20oxl gene reveal negative correlation in tender bud, tender stems, we supposed that rol genes' expression could inhibit GAs'synthesis by down regulate the expression of GA20??l gene in bud, lower bioactive GAs could suppress citrange growth.5. The seed of transgenic citrange was smaller than CK, the shape was turned into ellipse from wedge-shape. Germination was later than CK about seven days, the germination rate of T1 seed was decreased partly, dwarf character was exhibited in young seedling.99% of the seedlings were originated from nucellus and growing congruously, However, the trunk diameter of transgenic citrange was smaller compare with CK plants, rol genes were steadily inherited to next generation, which was detected by PCR and RT-PCR.6. Taking transgenic citrange as inter-rootstock, we grafted Shatian pummel (Citrus grandis [L.] Osbeck cv. Shatian) and Dahong sweet orange on it, rol gene weren't detected in the leaves of scions by PCR and qRT PCR after 4 years, the result indicated rol gene didn't transfer into scions through grafting. The photosynthesis (Pn) ability of Shatian Pummelo scions enhanced compared to control rootstocks after grafting for one year, but the Pn difference of scions wasn't observed after 4 years. The dwarfing rate of scions were from 12%-30% after grafted on transgenic rootstock or inter-rootstock. The fruit quality improved dramatically, the total sugar of E-Dahong was increased 14.2% and 19.3% compare to CK-Dahong in 2009 and in 2010, respectively. |
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