Rootstock‐to‐scion transfer of transgene‐derived small interfering RNAs and their effect on virus resistance in nontransgenic sweet cherry

Small interfering RNAs (siRNAs) are silencing signals in plants. Virus‐resistant transgenic rootstocks developed through siRNA‐mediated gene silencing may enhance virus resistance of nontransgenic scions via siRNAs transported from the transgenic rootstocks. However, convincing evidence of rootstock‐to‐scion movement of siRNAs of exogenous genes in woody plants is still lacking. To determine whether exogenous siRNAs can be transferred, nontransgenic sweet cherry (scions) was grafted on transgenic cherry rootstocks (TRs), which was transformed with an RNA interference (RNAi) vector expressing short hairpin RNAs of the genomic RNA3 of Prunus necrotic ringspot virus (PNRSV‐hpRNA). Small RNA sequencing was conducted using bud tissues of TRs and those of grafted (rootstock/scion) trees, locating at about 1.2 m above the graft unions. Comparison of the siRNA profiles revealed that the PNRSV‐hpRNA was efficient in producing siRNAs and eliminating PNRSV in the TRs. Furthermore, our study confirmed, for the first time, the long‐distance (1.2 m) transfer of PNRSV‐hpRNA‐derived siRNAs from the transgenic rootstock to the nontransgenic scion in woody plants. Inoculation of nontransgenic scions with PNRSV revealed that the transferred siRNAs enhanced PNRSV resistance of the scions grafted on the TRs. Collectively, these findings provide the foundation for ‘using transgenic rootstocks to produce products of nontransgenic scions in fruit trees'.     

Conferring high‐temperature tolerance to nontransgenic tomato scions using graft transmission of RNA silencing of the fatty acid desaturase gene

We investigated graft transmission of high‐temperature tolerance in tomato scions to nontransgenic scions from transgenic rootstocks, where the fatty acid desaturase gene (LeFAD7) was RNA‐silenced. Tomato was transformed with a plasmid carrying an inverted repeat of LeFAD7 by Agrobacterium. Several transgenic lines showed the lower amounts of LeFAD7 RNA and unsaturated fatty acids, while nontransgenic control did not, and siRNA was detected in the transgenic lines, but not in control. These lines grew under conditions of high temperature, while nontransgenic control did not. Further, the nontransgenic plants were grafted onto the silenced transgenic plants. The scions showed less of the target gene RNA, and siRNA was detected. Under high‐temperature conditions, these grafted plants grew, while control grafted plants did not. Thus, it was shown that high‐temperature tolerance was conferred in the nontransgenic scions after grafting onto the silenced rootstocks.     

Effects of mutual grafting on the cadmium accumulation characteristics of two ecotypes of Solanum photeinocarpum

Abstract

The effects of mutual grafting on the cadmium (Cd) accumulation characteristics of two ecotypes (farmland and mining) of the potential Cd-hyperaccumulator Solanum photeinocarpum were studied through a pot experiment for one month. Four treatments were used in the experiment: ungrafted farmland ecotype (F-CK), ungrafted mining ecotype (M-CK), the farmland ecotype as the scion grafted onto rootstocks of the mining ecotype (F-Scion), and the mining ecotype as the scion grafted onto rootstocks of the farmland ecotype (M-Scion). Mutual grafting increased the rootstock biomass of both S. photeinocarpum ecotypes. However, mutual grafting decreased the scion biomass of F-Scion compared with F-CK and M-CK, and the scion biomass of M-Scion was higher than that of M-CK and lower than that of F-CK. The Cd content in the rootstock of M-Scion increased compared with F-CK, and the Cd content in the rootstock of F-Scion increased compared with M-CK, but mutual grafting decreased the Cd content in scions of both S. photeinocarpum ecotypes. Mutual grafting increased Cd extraction by rootstocks of both S. photeinocarpum ecotypes, but decreased extraction by scions. Therefore, mutual grafting can increase Cd accumulation in S. photeinocarpum rootstocks but not increase Cd accumulation in S. photeinocarpum scions in a short period.     

Long‐distance ABA transport can mediate distal tissue responses by affecting local ABA concentrations

Environmental stresses that perturb plant water relations influence abscisic acid (ABA) concentrations, but it is unclear whether long‐distance ABA transport contributes to changes in local ABA levels. To determine the physiological relevance of ABA transport, we made reciprocal‐ and self‐grafts of ABA‐deficient flacca mutant and wild‐type (WT) tomato plants, in which low phosphorus (P) conditions decreased ABA concentrations while salinity increased ABA concentrations. Whereas foliar ABA concentrations in the WT scions were rootstock independent under conditions, salinity resulted in long‐distance transport of ABA: flacca scions had approximately twice as much ABA when grafted on WT rootstocks compared to flacca rootstocks. Root ABA concentrations were scion dependent: both WT and flacca rootstocks had less ABA with the flacca mutant scion than with the WT scion under conditions. In WT scions, whereas rootstock genotype had limited effects on stomatal conductance under conditions, a flacca rootstock decreased leaf area of stressed plants, presumably due to attenuated root‐to‐shoot ABA transport. In flacca scions, a WT rootstock decreased stomatal conductance but increased leaf area of stressed plants, likely due to enhanced root‐to‐shoot ABA transport. Thus, long‐distance ABA transport can affect responses in distal tissues by changing local ABA concentrations.     

Effects of transgenic rootstocks on growth and development of non-transgenic scion cultivars in apple

Although cultivation of genetic modified (GM) annual crops has been steadily increasing in the recent 10 years, the commercial cultivation of GM fruit tree is still very limited and reports of field trials on GM fruit trees are rare. This is probably because development and evaluation of GM fruit trees require a long period of time due to long life cycles of trees. In this study, we report results from a field trial on three rolB transgenic dwarfing apple rootstocks of M26 and M9 together with non-transgenic controls grafted with five non-transgenic scion cultivars. We intended to investigate the effects of transgenic rootstock on non-transgenic scion cultivars under natural conditions as well as to evaluate the potential value of using the rolB gene to modify difficult-to-root rootstocks of fruit trees. The results showed that all rolB transgenic rootstocks significantly reduced vegetative growth including tree height regardless of scion cultivar, compared with the non-transgenic rootstocks. Flowering and fruiting were also decreased for cultivars grown on the transgenic rootstocks in most cases, but the fruit quality was not clearly affected by the transgenic rootstocks. Cutting experiment and RT-PCR analysis showed that the rolB gene was stably expressed under field conditions. PCR and RT-PCR analyses displayed that the rolB gene or its mRNA were not detectable in the scion cultivars, indicating no translocation of the transgene or its mRNA from rootstock to scion. Our results suggest that rolB modified rootstocks should be used in combination with vigorous scion cultivars in order to obtain sufficient vegetative growth and good yield. Alternatively, the rolB gene could be used to dwarf vigorous rootstocks of fruit trees or produce bonzai plants as it can significantly reduce the vegetative growth of plants.     

Modification of gibberellin biosynthesis in the grafted apple scion allows control of tree height independent of the rootstock

The availability of short stature apple scions that required minimal applications of chemical growth retardants and could be used with a range of rootstocks would be of considerable benefit to fruit growers. We have suppressed the expression of a gene encoding the gibberellin (GA) biosynthetic enzyme GA 20-oxidase to reduce the levels of bioactive GAs in a scion variety, resulting in significant reductions in stem height. Application of GA3 reversed the effect. The scion remained dwarfed after grafting on to normally invigorating rootstocks, whilst control plants of the same cultivar displayed the expected vigour when grafted on to these rootstocks. This approach could be applicable to any perennial crop variety, allowing dwarf trees to be obtained on any available rootstock or on their own roots without the need for chemical growth retardant application. In effect, seedlings that are well suited to local conditions (drought, salinity) could be employed as tree rootstocks, as could existing rootstocks valued for characters other than vigour control, such as pest and disease resistance.     

Improved watermelon quality using bottle gourd rootstock expressing a Ca2+/H+ antiporter

Bottle gourd (Lagenaria siceraria Standl.) has been commonly used as a source of rootstock for watermelon. To improve its performance as a rootstock without adverse effects on the scion, the bottle gourd was genetically engineered using a modified Arabidopsis Ca²⁺/H⁺ exchanger sCAX2B. This transporter provides enhanced Ca²⁺ substrate specificity and decreased Mn²⁺ transport capability. Our previous work demonstrated that sCAX2B bottle gourds were more robust and nutrient dense than controls. Here, several cucurbit crops were test-grafted onto the transgenic bottle gourd to determine its effect on the scions. The grafted watermelons and melons onto the transgenic rootstocks appeared to show more robust growth than the controls 35 days after greenhouse transplanting. Watermelon fruits with the watermelon/transgenic bottle gourd (scion/rootstock) combination demonstrated higher osmotic pressure and more soluble solids than controls. These results suggest that sCAX2B expression in the bottle gourd rootstock facilitates improved watermelon quality through the translocation of nutrients and/or water toward enhancing the biomass of scion.     

Root and stem hydraulic conductivity as determinants of growth potential in grafted trees of apple (Malus pumila Mill.)

The anatomy of the graft tissue between a rootstock and its shoot (scion) can provide a mechanistic explanation of the way dwarfing Malus rootstocks reduce shoot growth. Considerable xylem tissue disorganization may result in graft tissue having a low hydraulic conductivity (k(h)), relative to the scion stem. The graft may influence the movement of substances in the xylem such as ions, water and plant-growth-regulating hormones. Measurements were made on 3-year-old apple trees with a low-pressure flow system to determine k(h) of root and scion stem sections incorporating the graft tissue. A range of rootstocks was examined, with different abilities of dwarfing; both ungrafted and grafted with the same scion shoot cultivar. The results showed that the hydraulic conductivity (k(hroot)) of roots from dwarfing rootstocks was lower compared with semi-vigorous rootstocks, at least for the size class of root measured (1.5 mm diameter). Scion hydraulic conductivity (k(hs)) was linked to leaf area and also to the rootstock on to which it was grafted, i.e. hydraulic conductivity was greater for the scion stem on the semi-vigorous rootstock. Expressing conductivities relative to xylem cross-sectional areas (k(s)) did not remove these differences suggesting that there were anatomical changes induced by the rootstock. The calculated hydraulic conductivity of the graft tissue was found to be lower for grafted trees on dwarfing rootstocks compared to invigorating rootstocks. These observations are discussed in relation to the mechanism(s) by which rootstock influences shoot growth in grafted trees.     

Phytophthora collar rot of apple: influence of the rootstock on scion variety resistance

In field experiments with young trees great differences were found in the resistance to Phytophthora cactorum of Cox's Orange Pippin apple scions grafted on different clonal rootstocks. The rootstock effect on scion resistance was inversely related to the effect on tree vigour: the rootstocks inducing high resistance were dwarfing (M. 9) or semi-dwarfing (M. 7, M. 26, MM. 106), and those inducing low resistance were vigorous or very vigorous (M. 25, MM. 104, MM. 109). Mean lesion lengths in Cox on MM. 104 were five to eight times greater than those in Cox on M. 9. The rootstock influence on scion resistance was associated primarily with effects on the rate of lesion extension: during the early stages only of host colonization there appeared to be threshold extension rates below which host resistance factors effectively suppressed a large proportion of infections. The influence of the root-stock on scion resistance was apparently unrelated to inherent rootstock resistance. On all rootstocks Cox showed diminished resistance to infection during the period from the swelling of buds to the early stages of shoot growth. Although most susceptible during the ‘mouse-ear’ and ‘pink bud’ stages of development, suscpetibility was not associated with flowering per se. Rootstock type did not affect the resistance of Cox scions to P. syringae, for which the period of susceptibility to infection occurred in the dormant season.     

柑桔接穗对砧木生长及若干生理生化特性的影响

以体细胞杂种红桔+枳、红桔+粗柠檬和有性杂种Troyer枳橙、Swingle枳柚以及枳作砧木的"国庆4号"温州蜜柑、脐橙(无枳砧)和不嫁接的砧木幼树为试材,通过两年盆栽试验研究接穗对砧木生长、根系活力和根系抗氧化酶活性的影响。结果表明,Swingle枳柚和红桔+粗柠檬的砧粗易受接穗影响,接穗显著地影响砧木根系体积。在一般情况下,同一种砧木嫁接后砧木根系活力、超氧化物歧化酶(SOD)和过氧化物酶(POD)活性低于未嫁接砧木,或与其差异不显著;同时存在接穗促进根系过氧化氢酶(CAT)活性升高的现象。     

Scion and Rootstock Effects on ABA-mediated Plant Growth Regulation and Salt Tolerance of Acclimated and Unacclimated Potato Genotypes

Tolerance of salt stress in potato (Solanum tuberosum L.) increased when the plants were pre-exposed to low concentrations of salt (salt acclimation). This acclimation was accompanied by increased levels of abscisic acid (ABA) in the shoot. To further study the role of roots and shoots in this acclimation process, reciprocal grafts were made between a salt-tolerant (9506) and salt-sensitive ABA(−) mutant and its ABA(+) normal sibling potato genotype. The grafted plants were acclimated with 75 or 100 mM NaCl for 3 weeks and then exposed to 150–180 mM NaCl, depending on the salt tolerance of the rootstock. After 2 weeks of exposure to the salt stress, the acclimated and unacclimated plants were compared for physiologic and morphologic parameters. The response to the salt stress was strongly influenced by the rootstock. The salt-tolerant 9506 rootstock increased the salt tolerance of scions of both the ABA-deficient mutant and its ABA(+) sibling. This salt tolerance induced by the rootstock was primarily modulated by salt acclimation and manifested in the scion via increased plant water content, stem diameter, dry matter accumulation, stomatal conductivity, and osmotic potential, and is associated with a reduction in leaf necrosis. There was also a pronounced scion effect on the rootstock. Using 9506 as a scion significantly increased root fresh and dry weights, stem diameter, and root water content of ABA(−) mutant rootstocks. Specific evidence was found of the role of exogenous ABA in the enhancement of water status in grafted plants under salt stress beyond that of grafting alone. This was verified by more positive stomatal conductivity and upward water flow in ABA-treated grafted and nongrafted plants and the absence of upward water flow in nontreated grafted plants through NMR imaging. Grafting using either salt-tolerant scions or rootstocks with inherently high ABA levels may positively modify subsequent responses of the plant under salt stress.     

Changes in the anatomical structure of tree rings of the rootstock and scion in the heterografts of Siberian pine

The analysis of the anatomical characteristics of tree rings of the Pinus sibirica scions and Pinus sylvestris rootstocks is presented. The main anatomical features maintain the seasonal dynamics characteristic of the reference (ungrafted) trees of scion and rootstock, pointing to the stability of the genetic control of the xylem differentiation. However, various anatomical characteristics are reduced for rootstocks and increased for scions relative to the reference trees. A mechanism consistent with our results is that the graft union zone creates a barrier for the ascending and descending transport of substances, including phytohormones. The reaction of the scion and rootstock to climatic factors is weakened relative to the reaction of the reference trees. Thus, the presumed shift towards phytohormone disbalance reduces the influence of external factors on xylem differentiation. The interannual variation of the individual characteristics of the scions and rootstocks is increased relative to the reference trees, reflecting either the influence of the graft partners on each other or the non-uniform distribution of the growth regulators across the graft union. Thus, heterografts can be used to evaluate the interaction of the internal and external mechanisms of xylem differentiation.     

Transmission of RNA silencing signal through grafting confers virus resistance from transgenically silenced tobacco rootstocks to non-transgenic tomato and tobacco scions

We examined the transmission of RNA silencing signal in non-transgenic tomato and tobacco scions grafted onto the tobacco Sd1 rootstocks, which is silenced in both NtTOM1 and NtTOM3 required for tobamovirus multiplication. When the non-transgenic tomato scions were grafted onto the Sd1 rootstocks, RT-PCR analysis of the scions showed the reduced level of mRNA compared with that before grafting in both LeTH3 and LeTH1, tomato homologs of NtTOM1 and NtTOM3, respectively. siRNAs from both genes were detected in the scions after grafting but not before grafting. Further tomato scions were inoculated with Tomato mosaic virus (ToMV) and used for virus infection. They showed very low level of virus accumulation. Necrotic responding tobacco to tobamovirus was grafted onto the rootstock of Sdl. RT-PCR analysis showed low level expression of both NtTOM1 and NtTOM3 in the scions but siRNA was detected after grafting. When the leaves of scions were inoculated with ToMV or Tobacco mosaic virus, they produced very few local necrotic lesions (LNLs) while the control scions did many LNLs. These results suggest that RNA silencing was transmitted to non-transgenic tomato and tobacco scions after grafting onto the Sd1 rootstocks and that virus resistance was induced in the scions.     

Variation in the content of bioflavonoids of orange as affected by scion,rootstock, and fruit part

Fruits of citrus cultivars contain bioflavonoids and some other important secondary metabolites in pharmaceutical and nutritional industries. The present experiment was designed to investigate the correlation between the content of flavonoid components like naringin, hesperidin, and neohesperidin of the scions fruits and the same parameters in rootstocks fruits. Six-year-old trees including four citrus cultivars of ‘Moro’, ‘Mars’, ‘Salustiana’, and ‘Italian’ which were previously grafted on the four different rootstocks including ‘Yuzu’, ‘Shelmahalleh’, ‘Citromelo’, and ‘Sour orange’ were selected as experimental trees. The content of the mentioned flavonoids was investigated in the peel and pulp of the fruits of both scions and rootstocks. The results showed that the measured parameters were significantly influenced by scions, rootstocks, and tissues. Based on the obtained results, it can be suggested that the accumulation of chemicals in citrus fruit depends on genetic and inherent abilities of the scion, more than what was previously believed, while the rootstock can also play an important role in the accumulation of these substances.     

Anatomic changes due to interspecific grafting in cassava (Manihot esculenta)

Cassava rootstocks of varieties UnB 201 and UnB 122 grafted with scions of Manihot fortalezensis were prepared for anatomic study. The roots were cut, stained with safranin and alcian blue, and examined microscopically, comparing them with sections taken from ungrafted roots. There was a significant decrease in number of pericyclic fibers, vascular vessels and tyloses in rootstocks. They exhibited significant larger vessels. These changes in anatomic structure are a consequence of genetic effects caused by transference of genetic material from scion to rootstock. The same ungrafted species was compared. This is the first report on anatomic changes due to grafting in cassava.     

转基因逃逸及其环境生物安全评价研究进展——抗虫水稻案例分析

转基因技术研发为提高我国水稻产量和减少劳动力投入提供了巨大机遇。我国对转基因水稻研发进行了大量的投入,目前已培育了具有不同新性状的转基因水稻品系,许多品系已进入生物安全评价阶段。风险评价对转基因水稻的安全生产至关重要,是其商品化生产之前必须解决的问题,其中包括转基因逃逸及其潜在环境影响。对水稻抗虫转基因逃逸及其潜在环境风险的评价包括3个重要环节：（1）通过田间试验和模型模拟检测转基因漂移到非转基因栽培稻及其野生近缘种的频率;（2）检测转基因在栽培稻和野生近缘种后代中的表达;（3）确定转基因对野生近缘种群体适合度和进化潜力的影响。大量研究表明,在近距离的空间范围内栽培稻品种之间的基因漂移频率很低（〉0.1%）,但栽培稻与其野生近缘种的基因漂移频率变异很大。进一步研究还表明,Bt抗虫转基因在栽培稻与普通野生稻后代中均能正常表达,但在其不同生长阶段,表达量有很大变异。在有较高水平的害虫虫压下,含有抗虫转基因的栽培稻及野生近缘种杂交后代与不含转基因的对照相比,抗虫性显著提高且适合度利益明显;但是,在虫害发生水平较低时,含有抗虫转基因的群体与不含抗虫转基因的群体相比没有显著的适合度优势。综上,转基因逃逸到非转基因水稻的频率极低,并且可以通过空间隔离阻断其逃逸。虽然抗虫转基因向杂草稻以及与栽培稻距离较近的野生稻群体的逃逸无法避免,但是野生稻和杂草稻群体周围环境中的总体虫压较低,所以基因漂移带来的环境影响应十分有限。     

Analysis of transitive RNA silencing after grafting in transgenic plants with the coat protein gene of <Emphasis Type="Italic">Sweet potato feathery mottle virus</Emphasis>

We have previously reported the graft transmission of target specificity for RNA silencing using transgenic Nicotiana benthamiana plants expressing the coat protein gene (CP, including the 3′ non-translated region) of Sweet potato feathery mottle virus. Transgenic plants carrying the 5′ 200 and 400 bp regions of CP were newly produced. From these plants, two silenced and two non-silenced lines were selected to investigate the manifestation of transitive RNA silencing by graft experiments. Non-silenced scions carrying the entire transgene were grafted onto either 5′ or 3′ silencing inducer rootstocks. When non-silenced scions were grafted onto 5′ silencing inducer rootstocks, RNA silencing was induced in the non-silenced scions and spread toward the 3′ region of the transgene mRNA. Similarly, when non-silenced scions were grafted onto 3′ silencing inducer rootstocks, RNA silencing was induced in the non-silenced scions, but was restricted to the 3′ region of the transgene and did not spread to the 5′ region. In addition, results from crossing experiments, involving non-silenced and 3′ silencing inducer plants, confirmed the above finding. This indicates that RNA silencing spreads in the 5′–3′ direction, not in the 3′–5′ direction, along the transgene mRNA.