How many chromosomes in impatiens walleriana

Germinating seeds were treated with 0 control , 0. Louis, MO for 2. Each treatment consisted of three replicates with 40 seeds per replicate, and the different treatments were arranged in a completely randomized design. Subsequently, seedlings were transferred into 6 inch plastic containers filled with Fafard 3B Mix, with one seedling per container.

L ea ves were collected from colchicine treated impatiens plants, and co chopped, using a sharp bla d e, with a similar amount of leaf tissues of the original diploid impatiens in 1 mL of cold LB01 lysis buffer Dole el et al. Ploidy a nalysis was repeated two to three time s for each plant. Solid tetraploids were identified as those that showed only one peak in the flow cytometry analysis and whose peak of relative fluore scence was nearly twice of that of diploids.

Mature impatiens leaf segments about 0. Da kovsk i n 1 mL of cold LB01 lysis buffer Dole el et al. For each tetraploid and diploid, at least three replicates of leaf samples from different shoots were analyzed, and in each run, at least 3, nuclei were analyzed.

Chromosome counting was done following a protocol described by Cao et al. Briefly, root tips about 1 cm in length were excised from vigorously growing roots of impatiens plants grown in containers, pre treated in a 0. The root tips were rinsed with deionized water three times and then stained in an aceto carmine staining solution Carolina Biology Supply Company, Burlington, NC for 3 h The meristematic tissue of stained root tips was squashed in a drop of aceto carmine on a clean glass slide using a dissecting needle.

A glass coverslip was gently placed on the meristematic tip using a pair of tweezers and the meristematic tissue was fu rther squashed firmly with a thumb on the coverslip Slides were slightly heated over an alcohol lamp to increase the contrast between the cytoplasm and the chromosome s in cells All slides were examined under a bright field microscope BH 2, Olympus, Toky o, Japan Cells with darkly stained and well spread chromosomes were photographed under a BX41 microscope Olympus Tokyo, Japan at magnification using an Olympus Q Color 5 camera and the Q imaging software Olympus America Inc.

Mor phological C haracterization Three diploids and three tetraploids were characterized to reveal potential morphological differences between them C utting s were taken from each diploid and PAGE 25 25 tetraploid and rooted to produce at least three clonal plants of each C uttings were made from mature shoots and placed upright with the ir basal end s stuck into the commercial potting mix Fafard 3B mix in the Todd planter flats model TR72D All flower bud s on the cuttings were removed The flats were placed in a plastic tent until roots emerge d from the basal ends of the cuttings.

Approximately 4 weeks later, rooted cuttings were transplanted to 6 inch plastic containers filled with Fafard 3B mix. For each diploid and tetraploid, three uniform rooted cuttings were selected and used as three biological replicat e s. T en mature leaves were collected from each clonal plant 30 leaves total per diploid and per tetraploid and used to measure leaf length, width, and thickness. Leaf length and width were measured with a household stainless steel ruler as the distance from the tip to the bottom of the basal end of the leaf and the widest distance across the leaf bl ade area, respectively.

Nail polish imprints were taken from the abaxial surface of mature leaves and observed under the BX41 microscope at and magni fication for stomatal length and density. Digital images of the imprints were taken using an Olympus Q Color 5 camera and analyzed using t he Q imaging software to measure the length and density of stomata For each clonal plant, five leaves were sampled an d for each leaf, f ive fields o f the imprints were examined, resulting in a total of 75 fields on 15 leaves for a given diploid or tetraploid.

Days to flower for each clonal plant were recorded as the number of days from sticking cuttings into the potting mix to the opening of the first flower for the plant The number of fully opened flowers produced per plant was counted every 10 days during PAGE 26 26 the full bloom period of the plant F lower counting was repeated four times, and the number of flowers in each 10 day interval were averaged to obtain the number of flowers per clonal plant.

The size of flowers was measur ed at the widest distance between the two opposite lateral petals of a fully opened flower.

T en flowers were sampled for each clonal plant, and 30 flowers total for each diploid and tetraploid. The total number of flowering shoots and the thickness of flowering shoots were used as parameters to evaluate plant architecture T he thickness of flowering shoots was measured at the midpoint of each shoot using an electronic digital caliper Pollen stainability was determined using the fluorochromatic procedure FCR described by Heslop Harrison and Heslop Harrison with minor modifications.

Fresh pollen grains in fully opened flowers were collected into a microcentrifuge tube 1. After staining at 25 C in dark for 1. Pollen grains emitting green fluorescence were counted as stainable. Five flowers were sampled for each clonal plant, 15 flowers for each dipl oid or tetraploid, and pollen grains in three microscopic fields were examined for each flower. The length of pollen grains was measured by analyzing digital images of stained pollen grains with the Q imaging software.

Fifteen pollen grains were measured f or each clonal plant, and 45 pollen grains total for each diploid and tetraploid. The isolate was maintained on live plants of I.

Super Elfin Lipstick in a growth room and transferred onto new live plants monthly. Two complemen tary resistance assays were used: leaf disc assay and in vivo inoculation assay. In leaf disc assays, fresh mature impatiens leaves were harvested from plants grown in the growth room and rinsed with autoclaved deionized water three times under a laminar h ood.

Leaf discs 1. Leaf discs were inoculated by pipetting 20 L of the P. The inoculated leaf discs in the Petri dishes were left in the hood at 22 C in dark for 24 h, and then the droplets of spore suspension if still present, were blotted dry with filter paper and the leaf discs were turned over so that the abaxial side was up.

Each Petri dish contained ten leaf discs and was considered as an experimental unit. For each diploid and tetraploid, at least four replicates 40 leaf discs total were examined at each point of time To determine sporangia densities on i noculated leaf discs, the leaf discs were immersed in L autoclaved deionized water containing 0.

Approx imatey 10 L of the suspension was loaded onto a hemocytometer, which was then observed under a BH 2 microscope Olympus to count sporangia Sporangia c ounts were converted into sporangia densities as the number of sporangia per square centimeter of leaf surface Sporangia counting was performed at 6 dpi 8 dpi, and 10 dpi. Two leaf disc assay experiments were conducted for the three diploids and three tetraploids In each experiment, t en leaf discs wer e inoculated and examined for each diploid or tetraploid.

In the in vivo inoculation assays, five randomly chosen mature leaves on each clonal plant were labeled and inoculated by pipetting L of a P. Two independent in vivo inoculation experiments were conducted using three replicates for each diploid and tetraploid, arranged in a completely randomized design. The pathogen structures of P. Briefly, inoculated leaf discs were collected at 1, 2, 3, 4, or 6 dpi and cleared in 50 m L centrifuge tubes first with 5 mL of the clearing solution A [ acetic acid EMD Chemicals Inc.

Cleared leaf discs were stained in 5 mL of 0. By approx imately 2 months after colchicine treatment, a number of the treated seedlings looked q uite different from the untreated seedlings with larger and thicker leaves and thicker stems. All the growing seedlings were analyzed for their p loidy levels, and eleven tetraploids were identified In addition to tetraploids, mixoploids and some octoploi ds were observed.

This study focused on characterizing the solid tetraploids. The average nuclear DNA content of impatiens diploids was 3. The average nuclear DNA content of tetraploids based on analyzing leaf tissue samples was 7.

Changes in M orphology The induced tetraploids showed significant changes in a number of morphological aspects compared to diploids Figure 2 3. Tetraploids produced significantly larger, thicker leaves Significantly thicker shoots Tetraploids took more days to flower 9.

The length of stomata on tetraploid leaves was on average Pollen grains of tetraploids were larger No significant differences were ultimately observed among tetraploids or diploids Table 2 4.

The pathogen seemed to go through a similar germination and development process in side both diploid and tetraploid leaves. V esicles, hyphae and haustoria were observed in leaf epidermal cells at 2 dpi; more hyphae and haustoria appeared in the leaf tissues from 3 to 6 dpi. By 6 dpi, m onopodially branched sporangiophores began to emerge from the stomata on the abaxial side of the leaves, and white sporulation became visible to the naked eyes Figur e 2 7.

In the in vivo inoculation assays, DM sporulation was not visible until 10 dpi on diploid or tetraploid leaves, even though the leaves were challenged with a P. However, tetraploid 4x 1 showed a significantly lower DM incidence T etraploid 4x 3 showed a lower DM incidence than the three diploids at all three time points 10 dpi through 12 dpi. PAGE 33 33 Discussion Tetraploid Induction, I d entification and C onfirmation Chromosome doubling is a powerful ploidy manipulation tool for plant breeding and genetic improvement.

Colchicine is a chemical widely used to induce chromosome doubling It is an antimitotic agent that functions by destroying the spindle fiber s during mitosis C olchicine also inhibit s seed germination and shoot growth Peterse n et al. Thus, a decrease of seedling survival rate in impatiens following colchicine treatment was expected S imilar observation s have been reporte d in other plant s Majdi et al.

Tetraploid induction efficiency is a n important parameter to select the best colchicine treatment for tetraploid induction as it takes into account both the seedling survival rate and the t etraploid induction rate Lehrer et al. In this study, the application of 0. Morphological C hanges Results from this study showed that induced tetraploids produced thicker stems, larger leaves, and larger flowers, and had larger stomata but lower densities of stomata.

Th ese results agreed well with the morphological changes in New Guinea and Java impatiens reporte d by Arisumi The induced tetraploids produced larger flowers, which may be desirable, however, they produced fewer shoots and fewer flowers per PAGE 34 34 plant.

It will be interesting to see if these morphological changes affect the ornamental values and performance of induced tetraploids in the landscape Stoma tal size and density have been used as an indicator of plant ploidy levels Kadota and Niimi, ; Thao et al.

As observed in this study, stomata on induced impatiens tetraploids were significantly longer than those o n impatiens diploids, and the density of stomata o n impatiens tetraploids was significantly lower than that on diploids. The se differences may provide an efficient way to identify puta tive tetraploids in future impatiens studies and breeding should ploidy analyzers be un available The FCR test protocol has been widely used to assess pollen stainability Heslop Harrison et al.

In this study, pollen grains from impatiens tetraplo id s showed Lower pollen stainabilities have been reported in many tetraploids in other species For example, tetraploids of Lantana camara had lower pollen stainabilities than diploids Czarnecki et al. Simil arly, Cohen et al. L ower pollen stainabilities in induced autotetraploids are often associated with the formation of polyads instead of tetrads durin g meiosis.

However, a study i n Lippia alba revealed that large number s of meiotic abnormal cells triads and diads had no significant effects on the stainability of pollen grains Reis et al. PAGE 35 35 Further studies are necessary to understand why the ind uced impatiens tetraploids in this study showed higher pollen stainabilit ies Changes in DM R esistance Downy mildew d isease severity, disease incidence, and sporangia density of P.

Compared to diploids, all three tetraploids had significantly lower DM disease severity from 6 to 10 dpi and lower sporangia densities at 8 and 10 dpi. A significant reduction of DM disease incidence was also observed in one of the tetraploids 4x 3 in in vivo inoculation assays. Unlike the resistance to P. We noted similar pathogen development in the leaf tissues of diploids and induced tetraploids.

Increased disease resistance has been reported in induced polyploids of other plant species, however the mechanisms of such changes remain to be elucidated. Fort suggested that the increased resistance to pathogen s in tetraploid A. C uticles, trichomes, and stomata are anatomical leaf structures that can play an important role in disease defense. Thick cuticles have been shown to be important structures against the penetration of pathogens Archer and Cole, Levi n PAGE 36 36 discussed the role t ric h omes play in insect defense and suggest ed they may also protect plants against pathogen penetration.

Stomata as natural surface openings on the leaves often serve as important entry sites for plant pathogens Melotto et al. Previous studies have shown the existence of complicated relationships between plant disease resistance and leaf morphology and anatomical structures. However, t he literature is contradictory.

Ramos and Volin revealed that Lycopersicon spp. P otato Solanum tuberosum cultivars resistant against the late blight disease Phytophthora infestans had thicker cuticles, smaller stomata, lower stomatal densities, and larger trichomes compared with susceptible cultivars Mahajan and Dhillon, However, i n grapevine genotypes Vitis vinifera and Vitis riparia , no clear relationships wer e observed between the density and morphology of trichomes and stomata and their resistance to P.

In this study impatiens leaves were inoculated on the adaxial side where no stomata existed, thus it seems unlikely that the di fference in DM resistance between diploids and tetraploids resulted from the differences in stomatal size and density. As shown by our histological survey, the sporangi o phores of P.

A question could be asked as to whether or not the much larger but much fewer stomata on the abaxial side of tetraploid leaves could reduce the emergence of P. Future studies may focus on understanding the changes in leaf architecture and other anatomical structures cuticle, trichome, etc. PAGE 38 38 Table 2 1. M orphological differences between diploid 2 x and tetraploid 4 x Impatiens walleriana Leaf length cm Leaf width cm Leaf thickness mm Stomata l length m Stomata l density no.

T hickness of shoots mm Flower diameter cm D ays to flower no. F lowers no. Differences in downy mildew severity between diploid 2 x and tetraploid 4 x Impatiens walleriana from 6dpi days post inoculation to 10 dpi.

PAGE 41 41 Table 2 4. Differences in s porangia density of P. PAGE 42 42 Table 2 5. Differences in downy mildew incidence between diploid 2 x and tetraploid 4 x Impatiens walleriana from 10 dpi to 12 dpi after in vivo inoculation of live plants 10 dpi 11 dpi 12 dpi 2 x 1 0. PAGE 43 43 Figure 2 1. Flow cytometric histograms of impatiens and rye. A Diploid impatiens and rye. B T etraploid impatiens and rye. The y axis indicates the nuclei counts and the x axis indicates the fluorescence peaks of impatiens left and rye internal reference right.

A Diploid impatien s B I nduced tetraploid impatiens. Nail polish imprints taken from the abaxial surface of mature impatiens leaves. A Diploid impatiens has a higher stomata l density and a smaller stomata l size B Induced tetraploid impatiens has a lower stomata l density and a larger stomata l size. Stained impatiens pollen grains under a stereomicroscope. A Pollen grains of a diploid impati ens. B Pollen grains of an induced tetraploid impatiens. Leaf discs of diploid and induced tetraploid impatiens in leaf disc inoculation assay showing white downy mildew sporulation emerging from the abaxial surface of impatiens leaf discs.

A, B, C downy mildew progression on diploid leaf discs; D, E, F downy mildew progres sion on tetraploid leaf discs. Histological survey of P lasmopara obducens causal agent of downy mildew development inside impatiens leaf discs from 1 dpi to 6 dpi by trypan blue staining A Sporangia inoculated on the adaxial s urface of a diploid impatiens leaf disc at 1 dpi B, C Vesicle, hypha e, and haustoria observed in s ide a diploid impatiens leaf disc from 2 to 6 dpi.

D Sporangi o phore emerging from stomata on the abaxial surface of diploid impatiens at 6 dpi with sporangia borne on sporangi o phore branches.

E Sporangia inoculated on the adaxial surface of tetraploid impatiens leaf discs at 1 dpi F, G Vesicle, hypha e, and haustoria observed in side tetraploid impatiens leaf discs from 2 6 dpi. H Sporangi o phore emerging from stomata on the abaxial surface of tetraploid impatiens leaf discs at 6 dpi with sporangia bor ne on sporangi o phore branches.

Compared to traditional plant breeding techniques, genetic transformation can overcome the limitation that gene transfer only occurs in closely related plant species and can avoid the introgression of undesirable genes, thus providing a better control of the gene modification and expression process Gepts, Genetic transformation has offered a wide range of possibilities for crop improvement in terms of insect resistance Vaeck et al.

It takes advantage of the ability of Agrobacterium tumefaciens a soil dwelling bacteri um to infect a wide range of host plants primarily eudicots through wo und sites and transfer part of its DNA known as the transfer DNA or T DNA into plant genomes. T DNA is part of A grobacterium tumor inducing Ti plasmid and contains gene s that code for enzymes synthesizing opine s and phytohormones.

Opines are utilize d by the Agrobacterium as a source of nitrogen and carbon Raven et al. The synthesis of plant hormones auxin and PAGE 51 51 cytokinin cause s plant cells to grow uncontrollably and form crown gall tumor s In gene tic engineering, the gene s in t he T DNA that confer the crown gall formation are replaced by desireable genes which are incorporated into the plant genome for desired phenotypes Dale and von Schantz, ; Gelvin, Relative to other transformation systems, such as the microprojectile bombardment and electroporation, Agrobacterium mediated transformation is more efficient and less cost ly and the foreign genes will be stably integrated into the plant genome and transmitted on to progenies De Block, To use Agrobacterium mediated transformation in a targeted plant, it is necessary to have an efficient tissue culture and plant regeneration system and an effective selection scheme that will suppre ss the growth and multiplication of non transformed cells.

An efficient and practica l plant regeneration system and sel ection scheme are of particular importance to plant breeding and large scale production of transgenic plant Impatiens R egener ation and T ransformation A reliable and efficient system for culturing plant tissue and regenerating plants is required.

Although a few in planta protocols exist that allow Agrobacterium mediated transformation to be conducted in vivo Rohini and Rao, , ; Seol et al. Unfortunately, this regeneration system could not be readily utilized in impatiens transformation as the inoculated Agrobacterium often PAGE 52 52 damaged the cotyledonary node explant CDE and prevented the regeneration of transformation protocol by using the hypocotyl segment containing cotyledonary nodes HSCCN as explant s to regenerat e plants and a combination of 2.

These changes resulted in a much higher frequency of responding explants and a larger number of shoots produced per explant. Subsequently, th e multiple bud culture explants MBCE derived from HSCCN were used in Agrobacterium mediated transformation of impatiens This system overcame the problem of explant necrosis and rotting and produced transgenic impat iens at a transformation frequency up t o Several studies in other plants have shown that true leaf nodes may also serve as good explants for plant regeneration and Agrobacterium mediated transformation Agrawal et al.

In our preliminary experiments, impatiens nodal explants grew into multiple bud cultures MBC that were morphologically similar to those induced from impatiens cotyledonary nodes. Therefore, it was decided to assess the regeneration capacity of impatiens nodal explants and their potential for use in impatiens transformation. Select able Markers and Reporter Genes In Agrobacterium mediated transformation, selectable marker genes are fre quently used. In reality, however, kanamycin based selection often result s in regeneration of non This situation was alleviated in some studies where kanamycin was replaced by G4 18, a more stringent antibiotic Maziah et al.

Dan et al. The green fluorescent protein GFP gene isolated from jellyfish Aequorea victoria has become one of the most commonly used reporter genes.

The GFP gene is advantageous over other reporter genes as it requires no exogenous substrate for detection and does not cause damage to plant tissues Molinier et al. Reporter genes are extremely important especially when selection can not provide adequate suppression of untransforme d cells. In impatiens, transformation events could be selected at early stages by visualizing GFP expression, thus saving both time and labor Dan et al.

R esistance R genes in a plant species encode hundreds of resistance R proteins that can recognize corresponding avirulence avr factors from pathogens. This R avr recognition between plant and pathogen often triggers a localized resistance reaction known as the hypersensitive response HR characterized by rapid cell death at the site of infection.

HR prevents the spread of the pathogen to other parts of the plant, leading to host pathogen incompatibility and disease resistance Dangl and Jones, Concomitant with this resistance mechanism, plants have also developed a secondary resistance respons e known as the systemic acquired resist ance SAR , which confers protection against a broad spectrum of pathogens Sticher et al.

This defense response requires salicylic acid SA as a signal molecule and is associated with elevated express ion of pathogenesis related PR genes Durrant and Dong Due to the limited protection provided by a single R gene or PR gene in terms of spectrum, degree and duration Jach et al. Such mechanism has been well elucidated in the model species Arabidopsis thaliana where the NPR1 non expresso r of PR gene gene product proves to be an essential regulator of SAR Cao et al.

It has been reported that overexpression of Arabidopsis NPR1 or its orthologs could enhance resistance to biotrophic and necrotrophic fungal, bacterial and oomycete pathogens in a number of plants including Arabidopsis rice, grape, tomato, citrus a nd strawberry Cao et al. These results suggest that the NPR1 mediated resistance may involve a conserved signal transduction pathway that may be shared by monocot and dicot plants Chern et al.

These results also suggest that Arabidopsis NPR1 and its orthologs may be good candidate genes for transgenic manipulation for enhanced disease resistance in c rops Cao et al. The reported success in utilization of the NPR1 in increasing disease resistance in other plants prompted the idea of introducing the NPR1 gene into impatiens and determin ing if it can enhance impatiens resistance to DM.

The objectives of this study were to: 1 Develop a plant regeneration system using impatiens true leaf nodes as initial explants; 2 Test the efficacy of G as an alternative selective agent to assist impatiens transf ormation; and 3 Introduce the Arabidopsis NPR1 into impatiens and characterize the resistance of transgenic impatiens to DM.

Seeds were germinated in glass baby food jars that each contained 30 mL of a germination medium GM Table 3 1 Fig ure 3 2 A. The baby food jars were capped and sealed with Parafilm Parafilm M, Chicago, IL and placed on a shelf in the tissue culture room set at 24 C, 16 h photoperiod and light intensity of approx imately mol m 2 s 1 Cotyledonary node and true le af node explant s TLNE were prepared from 28 day old seedlings by removing cotyledons and true leaves and hypocotols and hypercotols above and below the cotyledon ary nodes or true leaf nodes Figure 3 2 B.

The nodes were cut through vertically using a scalpel blade Feather Safety Razor Co. LTD Medical Division, Japan to produce 2 explants from each cotyledonary node or true leaf node 4 explants from each seedling. S ix weeks post inoculation the fresh weight of the induced MBC and the number o f shoots produced by each explant were recorded. One Magenta culture vessel contain ing four explants was an experimental unit T reatment s were replicated 5 times a total of 5 vessels per treatment , and all Magenta vessels were arranged on the same shelf following a completely randomized design.

The e xperiment was repeated one more time. The vector was constructed by Dr. T en colonies of the Agrobacterium w ere pooled and inoculated into a glass flask containing 1 00 mL of 2. The bacteri al culture was transferred to a 50 mL centrifuge tube and centrifuged at rpm for 8 min. The bacteria l pellet at the bottom of the centrifuge tube was re suspended in t he inoculation medium IM Table 3 1 T he OD value of the bacterial suspension was adjusted to approximately 0. Subsequently, the bacteria l suspension was decanted and the MBC s were blotted dry The inoculated MBCs were then co cultivated in sterile 15 mm Petri di sh es Fisher Scientific each contain ing two pieces of filter paper Fisher Scientific moistened with L of the IM The Petri dish es w ere sealed and kept at 24 C in dark for 5 days.

The prepared reactions were first denatured at 94 C for 5 min, followed by 35 cycles of amplification at 95 C for 30 s, 56 C for 1 min, and 68 C for 1 min, and a 5 min final extension at 68 C. T ransgenic impatiens line s and the wildtype impatiens w ere propagated by cuttings refer to Chapter 2 to produce at least six clonal plants per transgenic line To reveal potential differences between the transgenic lines and the wildtype, leaf length, leaf width, leaf thickness, stoma tal length, stoma tal density, number of shoots on each plant, thickness of shoots, days to flower, flower diameter, number of open flowers on each plant, pollen size, and pollen stainability were measured or recorded as described in Chapter 2.

Leaf disc assays in vivo inocul ation assays and histological staining were conducted as described in Chapter 2 to assess the resistance of transgenic lines to DM. In leaf disc assays, the DM severity and sporangia density of P. Statistical analyses were conducted on all collected data, as described in Chapter 2.

In this study, three concentrations of BA 0. CNEs cultured under 1. The fresh weight of MBCs under 1. When cultured with 1. The fresh weight of the MBCs under 1. These shoots readily formed roots after they were transferred onto the RIM. Five concentrations of IBA 0. At this concentration of IBA, impatiens shoots formed multiple roots and developed into complete plantlets Figure 3 2, I, J that were ready for transplanting into containers for acclimati on.

At the end of the six week culture, the average size of the MBCs and the number of shoots produced by the MBCs were recorded. MBCs cultured under these selection conditions were significantly smaller and produced significantly fewer shoots Figur e 3 3. These concentrations were non lethal and allowed the recovery of transgenic shoots expressing the NPTII gene but inhibited the growth of non transformed shoots.

When calcu lated on the basis of the percentage of MBCs showing GFP positive cell masses, the observed transformation efficiency ranged from Up to GFP positive shoots of 1 1. These species can be distinguished by the following vegetative and floral traits Christenhusz, :. Impatiens balsamina : flowers usually solitary in leaf axils; lateral petals completely united, only shallowly incised, unevenly bilobed; fruits are hairy.

Impatiens sodenii : larger plant often up to 1. Impatiens walleriana : flowers usually in pairs or with three to five flowers, rarely solitary; lateral petals almost free, equal in size, deeply incised and united only at the very base; fruits are glabrous.

Due to the variable regulations around de registration of pesticides, your national list of registered pesticides or relevant authority should be consulted to determine which products are legally allowed for use in your country when considering chemical control. Pesticides should always be used in a lawful manner, consistent with the product's label.

There is no information available for the control of I. Adamowski, W. Balsams on the offensive: the role of planting in the invasion of Impatiens species.

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Wegulo, S. F, G Vesicle, hyphae, and haustoria observed inside tetraploid leaf discs from 2 to 6 dpi. H Sporangiophore emerging from stomata on the abaxial surface of tetraploid leaf discs, and sporangia borne on sporangiophore branches, 6 dpi. In the in vivo inoculation assays, IDM sporulation was not visible until 10 dpi on diploid or tetraploid leaves, even though the leaves were inoculated with a spore suspension containing two times more spores than the spore suspension used in leaf disc assays.

No significant differences in IDM incidence were observed among diploids or tetraploids in any of the three evaluations Table 5. Colchicine has been widely used to induce chromosome doubling. Tetraploid induction efficiency has been used as an important parameter to select colchicine treatments for tetraploid induction Lehrer et al. In this study, the application of 0. Induced tetraploids produced thicker stems, larger leaves, and larger flowers, and larger stomata but at lower densities.

These results agreed with the morphological changes in New Guinea and Java impatiens tetraploids reported by Arisumi b. The induced tetraploids produced larger flowers but fewer shoots and fewer flowers per plant in container trials.

It will be interesting to determine if these morphological changes will affect the ornamental value and performance of induced tetraploids in the landscape. Stomatal size and density have been used as an indicator of plant ploidy levels Kadota and Niimi, ; Thao et al. As observed in this study, stomata on impatiens tetraploid leaves were significantly longer than those on diploid leaves, and the density of stomata on tetraploids was significantly lower than that on diploids.

These differences may provide a simple way to identify putative tetraploids in future impatiens studies and breeding, in case ploidy analyzers are not available.

In this study, pollen grains from impatiens tetraploids showed This differs from the results reported by Arisumi b , where New Guinea and Java impatiens tetraploids had higher percentages of aborted pollen grains. Lower pollen stainabilities have been reported in many tetraploids in other species.

Additional studies are needed to understand why the induced impatiens tetraploids in this study showed higher pollen stainabilities. In this study, all three tetraploids had significantly lower IDM disease severity from 6 to 10 dpi and lower sporangia densities at 8 and 10 dpi, compared with diploids.

Increased disease resistance has been reported in induced polyploids of other plant species; however, the mechanisms of such changes remain to be elucidated. Fort suggested that the increased resistance to pathogens in tetraploid A. Thick cuticle and trichomes can be important structures that guard against the penetration of pathogens Archer and Cole, ; Levin, because stomata often serve as important entry sites for plant pathogens Melotto et al.

Ramos and Volin revealed that Lycopersicon spp. Potato Solanum tuberosum cultivars resistant to the late blight disease Phytophthora infestans had thicker cuticles, smaller stomata, lower stomatal densities, and larger trichomes compared with susceptible cultivars Mahajan and Dhillon, In this study, impatiens leaves were inoculated on the adaxial side where no stomata existed, thus it seems unlikely that the difference in IDM resistance between diploids and tetraploids resulted from the differences in stomatal size and density.

As shown by our histological survey, the sporangiophores of P. The question remains whether the much larger but much less dense stomata on the abaxial side of tetraploid leaves could reduce the emergence of P. Future studies may focus on understanding the changes in leaf architecture and other anatomical structures cuticle, trichome, etc. Archer, K. Arisumi, T. Bouvier, L. Brown, M. Burdon, J. Cao, Z. Ellwood, S. Medicago truncatula handbook.

Noble Res. Fort, A. National Univ. Ireland, Galway, PhD Diss. Hamill, S. Heslop-Harrison, J. Jones, K. Balsaminaceae Kew Bull. Kadota, M. Hosui Plant Cell Rpt.

Lehrer, J. Atropurpurea through exposure to colchicine and oryzalin Scientia Hort. Levin, D. Mahajan, M. Melotto, M. Morgan, R. Timber Press, Portland, OR. Oswald, B. Ramos, L. Vesicatori Phytopathology 77 Thao, N.

Uchneat, M. In: N. Anderson ed. Flower breeding and genetics. Issues, challenges and opportunities for the 21st century. Springer, London. PhD Diss. Warfield, C. Gloeckner Foundation, Inc. We thank Joyce Jones and Gail Bowman for their technical support, Yinghong Wang for germinating impatiens seeds and treating seedlings with colchicine, Dr.

Aaron Palmateer for confirming the identity of Plasmopara obducens , and Gaomin Deng for reviewing this manuscript. We greatly appreciate the guidance and valuable comments that Drs. Aaron Palmateer and David Clark provided. E-mail: zdeng ufl. User Account Login to save searches and organize your favorite content. Not registered? Recently viewed 0 Save Search. Advanced Search Help. Free access. Get Citation Alerts Get Permissions. Download PDF. Abstract Garden impatiens Impatiens walleriana , a very important floricultural crop in the United States, has been devastated by impatiens downy mildew IDM in recent years.

Keywords: colchicine ; ploidy manipulation ; Plasmopara obducens. Materials and Methods Polyploid induction. Identifying putative tetraploids. Determining nuclear DNA contents.

Chromosome counting. Morphological characterization. Determining IDM resistance. Statistical analysis. Results Polyploid induction, identification and confirmation. Table 1.