BACKGROUND
impatiens is a top selling floriculture crop, with an annual value of approximately $ <dig> million in the us reported in  <dig>  <cit> .
it grows throughout tropical africa, india, southwest asia, southern china, japan, as well as parts of europe, russia, and north america  <cit> , with impatiens walleriana the most commonly cultivated species throughout the world  <cit> .
impatiens necrotic spot virus  is a serious threat for the floriculture industry both in north america and in europe  <cit> .
because no naturally insv-resistant impatiens cultivars have been available the possibility of transgenic virus resistance is attractive.
this possibility depends, however, on the availability of a robust transformation system.
there are few reports in the literature dealing with impatiens tissue culture and transformation.
these include micropropagation  <cit> , in vitro germination of immature ovules  <cit> , growth of cotyledon sections  <cit> , callus culture  <cit>  and embryo and ovule culture  <cit> .
the majority of these publications reported callus induction from explants using new guinea, java impatiens and impatiens platypetala, with no de novo plant regeneration.
plantlet production has only been reported via micropropagation.
in terms of impatiens transformation reports, hairy root induction in vivo from cotyledons of impatiens balsamina and impatiens hawkerii was reported using a. rhizogenes  <cit> .
gus and pcr positive plants of i. balsamina were obtained from cotyledon explants using microprojectile bombardment  <cit> .
a patent relating to agrobacterium-mediated transformation of impatiens was issued, but no experimental data confirming transformation were reported in the patent  <cit> .
we report the development of a simple and efficient agrobacterium-mediated transformation and regeneration system for impatiens using in vitro maintained multiple bud cultures as transformation explants.
to the best of our knowledge, there have been no reports of agrobacterium-mediated transformation of impatiens with experimental evidence of stable integration of t-dna and of agrobacterium-mediated transformation method for plants using in vitro maintained multiple bud cultures as explants.
RESULTS
regeneration protocol development
cotyledonary node explants  were evaluated for shoot regeneration after  <dig> weeks on shoot induction media  <dig>  and  <dig> , supplemented with  <dig> μm 6-benzyladenine  and  <dig> μm thidiazuron , respectively.
on sim <dig> medium amended with tdz, 43% of explants produced multiple shoots  whereas only 18% of explants formed multiple shoots on sim <dig> containing ba.
once tdz had been shown to promote greater shoot regeneration than ba from cotyledonary nodes of impatiens, the concentration of tdz in the regeneration medium was optimized.
with i. walleriana cv.
accent red, tdz at  <dig>   <dig> and  <dig> μm had significantly  greater frequencies  of explants producing multiple shoots than  <dig> μm , but there was no significant difference for percent of explants producing multiple shoots among the three concentrations .
for i. walleriana cv.
salmon picote, the frequency  of explants producing multiple shoots was significantly  greater when using  <dig> μm tdz than the other  concentrations tested .
x treatments followed by different letters are significantly  different using the lsd all-pairwise comparison test .
to further optimize shoot regeneration frequency, we tested combinations of tdz with ba at different concentrations using hypocotyl segments containing cotyledonary nodes .
more than 95% of explants from both genotypes produced multiple shoots when using im <dig> and im <dig> with both ba and tdz , whereas tdz at  <dig> μm without ba  promoted only 40% response for accent red and 50% response for salmon picote .
the combination of  <dig>  μm tdz and  <dig>  μm ba  generated significantly  greater frequencies of explants  producing massive shoots, determined as production of  <dig> to  <dig> elongated shoots from a single explant  compared to other treatments .
im <dig> not only significantly increased the frequency of explants responding, but also increased number of shoots produced per explant and reduced time to produce the shoots.
multiple buds formed  <dig> to  <dig> weeks after culture  and up to  <dig> shoots were produced from a single explant on im <dig> .
shoots were separated 3- <dig> weeks after multiple shoot induction on im <dig> and transferred to shoot elongation medium im <dig> similar to im <dig> except with  <dig>  μm tdz and  <dig>  μm ba for several weeks.
for rooting,  <dig> % to 100% of shoots produced roots within  <dig> to  <dig> weeks after the shoots were transferred to the rooting medium im <dig>  kinetin at  <dig> μm produced the greatest number of shoots  from shoot tips of an interspecific hybrid, t63- <dig>  of a cross between impatiens platypetala and a new guinea impatiens  <cit> .
han and stephens  <cit>  reported that ba at  <dig> μm was most effective for stimulating shoot multiplication using shoot tips as explants for the same hybrid, for which  <dig>  shoots per explant were obtained.
also ba at  <dig> mg/dm <dig> and kinetin at  <dig> or  <dig> mg/dm <dig> induced multiple shoots from shoot tip explants of i. walleriana in micropropagation  <cit> .
x treatments followed by different letters are significantly  different using the lsd all-pairwise comparison test .
cotyledonary nodes have been the explants of choice for transformation of soybean and otherwise recalcitrant legumes  <cit> .
they have recently been used to transform a member of the bignoniaceae family  <cit> .
micropropagation of recalcitrant species of legumes, including common bean  <cit> , grasspea  <cit> , and sesbiana  <cit> , among others, has been facilitated by the use of cotyledonary nodes.
recently cothledonary nodes were identified as the only explants capable of regeneration for the ice plant, mesembranthemum crystallinum  <cit> .
tdz was reported to induce a high frequency of plant regeneration in stachys sieboldii  <cit> , lingonberry  <cit> , linum  <cit> , salicornia europaea  <cit> , cichorium intybus  <cit> , astragalus cicer  <cit> , carthamus tinctorius  <cit> , pisum sativum  <cit>  and ginger  <cit> .
determination an optimal concentration of kanamycin for transformation selection
for shoot selection, the percentage of multiple bud cluster explants  producing new growth was significantly  lower when using kanamycin at  <dig> mg/l than at  <dig>   <dig> or  <dig> mg/l  inhibited shoot growth and this non-lethal concentration may allow recovery of transgenic plants having low expression of a neomycin phosphotransferase ii .
for root selection of accent red, kanamycin at  <dig>   <dig> and  <dig> mg/l significantly inhibited the percentages of shoots producing roots  compared with no kanamycin.
however, there was no significant difference among the percentages of shoots producing roots when using kanamycin at  <dig>   <dig> and  <dig> mg/l.
kanamycin at  <dig> mg/l was chosen for root selection because it prevented 90% of shoots from producing roots, and this non-lethal concentration may allow the recovery of transgenic plants having low nptii expression.
effect of co-culture duration on agrobacterium infection
after  <dig> and  <dig> days of co-culture a significantly  greater percentage of explants  exhibited a green fluorescent protein  expression compared to those receiving  <dig> or  <dig> days of co-culture when there was no gfp expression was observed and there were only a few shoots with gfp expression when mbce were co-cultured for  <dig> or  <dig> days .
by using mbce and co-culture of the explants on filter paper dampened with an inoculation medium, we were able to control agrobacterium growth even  <dig> days after co-culture .
cheng et al.
<cit>  reported that placing explants on filter paper moistened with inoculation medium during co-culture significantly reduced agrobacterium overgrowth compared to solid co-culture medium.
regeneration of transgenic plants
six to seven days after co-culture, mbce were selected on im <dig> supplemented with  <dig> mg/l kanamycin and  <dig> mg/l carbenicillin.
approximately  <dig> to  <dig> weeks after selection, gfp positive buds were observed , with gfp positive shoots obtained  <dig> to  <dig> weeks after selection .
the percentage of explants producing gfp positive buds and/or shoots was 59%.
gfp positive shoots were transferred to im <dig> with  <dig> mg/l kanamycin and  <dig> mg/l carbenicillin to induce roots, and 79% of gfp positive shoots generated roots on im <dig>  <dig> to  <dig> weeks after culture .
transgenic plants expressing gfp were transplanted into soil.
the transformation cycle from inoculation to soil took approx.
<dig> to  <dig> months.
the percentage of explants producing transgenic plants was 59% and the frequency of initial explants that produced independent transgenic plant events confirmed by southern blot was  <dig> %.
transgenic plants expressed gfp in many tissues, including leaf, stem, root, flower , and immature seed .
transgenic plants appeared phenotypically normal and fertile .
taha et al  <cit>  reported that gus and pcr positive shoots of i. balsamina were regenerated from bombarded cotyledon explants on ms medium supplemented with  <dig> mg/l ba and  <dig> mg/l hygromycin; subsequently, gus and pcr positive plants were obtained on ms medium supplemented with  <dig>  mg/l indole-3-acetic acid .
pcr, southern blot and r <dig> segregation analysis of transgenic i. walleriana
amplified pcr products corresponded to the expected size for the nptii gene in five gfp positive plants and in the agrobacterium, but not in the wild type plant  .
in addition, the pcr band for the pica gene was only present in the agrobacterium strain eha <dig> containing phb <dig> .
this indicated that the nptii gene had integrated into the genome of the transgenic plants, and was not due to agrobacterium contamination of the transgenic plants.
up to twenty transgenic plants that expressed gfp were analyzed by southern blot using probes of nptii and gfp genes.
southern blot hybridization with the nptii probe revealed the transgene copy number varying between one and three for ten t <dig> and two t <dig> transgenic plants .
an expected band size of  <dig>  kb, positioned within the t-dna for the gfp gene, was present in eleven t <dig> and six t <dig> transgenic plants .
segregation of gfp expression among the t <dig> plants for  <dig> transgenic t <dig> lines displayed the expected 3: <dig> ratio  for a single gene hemizygous in the t <dig> .
segregation in the remaining ten transgenic exhibited a disproportionate number of nontransgenics in the t <dig> .
no seedlings from the control impatiens plant displayed gfp.
for our transformation system, gfp expression was used to select transformed buds and/or shoots  <dig> to  <dig> weeks after selection.
the gfp positive buds and/or shoots were transferred to shoot elongation medium with  <dig> mg/l kanamycin for  <dig> to  <dig> weeks and the gfp positive shoots were transferred to rooting medium with  <dig> mg/l kanamycin for  <dig> to  <dig> weeks.
among selected gfp positive shoots tested, 100% of them produced transgenic plants that were confirmed by southern blot and r <dig> segregation analysis.
among mbce that were inoculated and plated on selection medium, 10% of them produced gfp positive buds and/or shoots.
approximately 40% to 94% of kanamycin resistant shoots have been reported to be non-transgenic  when using kanamycin as a selective agent in many plant species  <cit> .
for transformation of tomato cv.
microtom,  <dig> % of kanamycin resistant shoots after selection on  <dig> mg/l kanamycin were shoot escapes  <cit> .
however, additional selection of kanamycin resistant shoots on rooting medium with  <dig> mg/l kanamycin yielded  <dig> % transgenic plants  <cit> .
an advantage of visualizing gfp expression in our system was to enable us to select transformation events at an early stage thus avoiding the transfer of non-transgenic shoot that survived the kanamycin selection, saving both time and labor.
CONCLUSIONS
we report the development of a simple and efficient agrobacterium-mediated transformation and regeneration system for impatiens.
to the best of our knowledge, there have been no reports of agrobacterium-mediated transformation of impatiens with experimental evidence of stable integration of t-dna.
co-cultivation period, type of explants, a gfp gene as reporter marker to allow early stage selection of transformants and selection regime were critical factors for successful transformation of impatiens.
many agrobacterium-mediated transformation methods have used explants derived directly from in vivo plants or from pre-cultured embryogenic calli for inoculation, due to ease of infection by agrobacterium, but these methods have disadvantages such as expense of stock plant growth, detrimental sterilization procedures, time-consuming explant preparation, lack of flexibility, microbial contamination of explants, variation due to age and position of explants, all of which affect transformation efficiency.
our direct shoot regeneration system without an intervening callus phase is desirable for both nuclear and plastid transformation using agrobacterium-mediated and biolistics methods for several reasons, including: 1) efficient, simple and rapid regeneration and transformation , 2) flexibility  for in vitro manipulation, 3) uniform and desirable green tissue explants for both nuclear and plastid transformation using agrobacterium-mediated and biolistics methods and 4) no somaclonal variation.
in addition, impatiens cne derived from in vivo seedlings necrosed, rotted and died several days after agrobacterium inoculation  <cit> .
using in vitro mbce derived from the hsccn of impatiens overcame this problem and enabled transgenic plant regeneration.
maziah et al.
<cit>  inoculated a single bud isolated from in vitro multiple buds with agrobacterium to produce transgenic banana.
our use of in vitro mbce for agrobacterium inoculation circumvented the problem of necrotic explants and resulted in more efficient transformation than inoculation of single buds.
to our knowledge from literatures, there have been no reports of agrobacterium-mediated transformation method for plants using in vitro maintained multiple bud cultures as explants.
a major challenge associated with plant transformation technology is the death of agrobacterium-transformed tissues or cells, which severely limits the number of transgenic plants that can be regenerated, and the biotechnological exploitation of economically important crops.
hydrogen peroxide  plays an important role in programmed cell death  and stress  <cit> .
however, little is known about the molecular mechanisms regulating plant pcd, and the signaling pathway leading from h2o <dig> to the death of the tissues during transformation.
impatiens cne derived from in vivo seedlings necrosed, rotted and died several days after agrobacterium inoculation  <cit> .
using in vitro mbce derived from the hsccn of impatiens overcame this problem and enabled transgenic plant regeneration.
we can further investigate levels of cell death and the correlation between the cell death and h2o <dig> production of the both explant types and determine whether mbce reduces the h2o <dig> production and subsequently it decreases cell death.
a mechanistic knowledge of how the tissue death is triggered by the signaling molecule  during the transformation is required in order to solve this issue.
ornamental horticulture, and particularly floriculture, is well-suited to the application of genetic engineering technology because the product is not intended to be consumed, so commercialization may meet fewer obstacles without the need of food safety studies.
however, genetic engineering technologies including transformation have had limited impact in ornamental horticulture.
as the development of new varieties is an important driving force in the industry, there are, therefore, good prospects for the development of new genetically modified ornamental crops.
using the present protocol, thousands of transgenic impatiens could be produced within  <dig> to  <dig> months.
the transformation methodology developed in this study will thus be useful for future impatiens improvement.

 impatiens is a top selling floriculture crop, with an annual value of approximately $ million in the us reported in. it grows throughout tropical africa, india, southwest asia, southern china, japan, as well as parts of europe, russia, and north america, with impatiens walleriana the most commonly cultivated species throughout the world. impatiens necrotic spot virus is a serious threat for the floriculture industry both in north america and in europe. because no naturally insv-resistant impatiens cultivars have been available the possibility of transgenic virus resistance is attractive. this possibility depends, however, on the availability of a robust transformation system. there are few reports in the literature dealing with impatiens tissue culture and transformation. these include micropropagation, in vitro germination of immature ovules, growth of cotyledon sections, callus culture and embryo and ovule culture. the majority of these publications reported callus induction from explants using new guinea, java impatiens and impatiens platypetala, with no de novo plant regeneration. plantlet production has only been reported via micropropagation. in terms of impatiens transformation reports, hairy root induction in vivo from cotyledons of impatiens balsamina and impatiens hawkerii was reported using a. rhizogenes. gus and pcr positive plants of i. balsamina were obtained from cotyledon explants using microprojectile bombardment. a patent relating to agrobacterium-mediated transformation of impatiens was issued, but no experimental data confirming transformation were reported in the patent. we report the development of a simple and efficient agrobacterium-mediated transformation and regeneration system for impatiens using in vitro maintained multiple bud cultures as transformation explants. to the best of our knowledge, there have been no reports of agrobacterium-mediated transformation of impatiens with experimental evidence of stable integration of t-dna and of agrobacterium-mediated transformation method for plants using in vitro maintained multiple bud cultures as explants.  regeneration protocol development cotyledonary node explants were evaluated for shoot regeneration after weeks on shoot induction media and, supplemented with μm 6-benzyladenine and μm thidiazuron, respectively. on sim medium amended with tdz, 43% of explants produced multiple shoots whereas only 18% of explants formed multiple shoots on sim containing ba. once tdz had been shown to promote greater shoot regeneration than ba from cotyledonary nodes of impatiens, the concentration of tdz in the regeneration medium was optimized. with i. walleriana cv. accent red, tdz at and μm had significantly greater frequencies of explants producing multiple shoots than μm, but there was no significant difference for percent of explants producing multiple shoots among the three concentrations. for i. walleriana cv. salmon picote, the frequency of explants producing multiple shoots was significantly greater when using μm tdz than the other concentrations tested. x treatments followed by different letters are significantly different using the lsd all-pairwise comparison test. to further optimize shoot regeneration frequency, we tested combinations of tdz with ba at different concentrations using hypocotyl segments containing cotyledonary nodes. more than 95% of explants from both genotypes produced multiple shoots when using im and im with both ba and tdz, whereas tdz at μm without ba promoted only 40% response for accent red and 50% response for salmon picote. the combination of μm tdz and μm ba generated significantly greater frequencies of explants producing massive shoots, determined as production of to elongated shoots from a single explant compared to other treatments. im not only significantly increased the frequency of explants responding, but also increased number of shoots produced per explant and reduced time to produce the shoots. multiple buds formed to weeks after culture and up to shoots were produced from a single explant on im. shoots were separated 3- weeks after multiple shoot induction on im and transferred to shoot elongation medium im similar to im except with μm tdz and μm ba for several weeks. for rooting, % to 100% of shoots produced roots within to weeks after the shoots were transferred to the rooting medium im kinetin at μm produced the greatest number of shoots from shoot tips of an interspecific hybrid, t63- of a cross between impatiens platypetala and a new guinea impatiens. han and stephens reported that ba at μm was most effective for stimulating shoot multiplication using shoot tips as explants for the same hybrid, for which shoots per explant were obtained. also ba at mg/dm and kinetin at or mg/dm induced multiple shoots from shoot tip explants of i. walleriana in micropropagation. x treatments followed by different letters are significantly different using the lsd all-pairwise comparison test. cotyledonary nodes have been the explants of choice for transformation of soybean and otherwise recalcitrant legumes. they have recently been used to transform a member of the bignoniaceae family. micropropagation of recalcitrant species of legumes, including common bean, grasspea, and sesbiana, among others, has been facilitated by the use of cotyledonary nodes. recently cothledonary nodes were identified as the only explants capable of regeneration for the ice plant, mesembranthemum crystallinum. tdz was reported to induce a high frequency of plant regeneration in stachys sieboldii, lingonberry, linum, salicornia europaea, cichorium intybus, astragalus cicer, carthamus tinctorius, pisum sativum and ginger. determination an optimal concentration of kanamycin for transformation selection for shoot selection, the percentage of multiple bud cluster explants producing new growth was significantly lower when using kanamycin at mg/l than at or mg/l inhibited shoot growth and this non-lethal concentration may allow recovery of transgenic plants having low expression of a neomycin phosphotransferase ii. for root selection of accent red, kanamycin at and mg/l significantly inhibited the percentages of shoots producing roots compared with no kanamycin. however, there was no significant difference among the percentages of shoots producing roots when using kanamycin at and mg/l. kanamycin at mg/l was chosen for root selection because it prevented 90% of shoots from producing roots, and this non-lethal concentration may allow the recovery of transgenic plants having low nptii expression. effect of co-culture duration on agrobacterium infection after and days of co-culture a significantly greater percentage of explants exhibited a green fluorescent protein expression compared to those receiving or days of co-culture when there was no gfp expression was observed and there were only a few shoots with gfp expression when mbce were co-cultured for or days. by using mbce and co-culture of the explants on filter paper dampened with an inoculation medium, we were able to control agrobacterium growth even days after co-culture. cheng et al. reported that placing explants on filter paper moistened with inoculation medium during co-culture significantly reduced agrobacterium overgrowth compared to solid co-culture medium. regeneration of transgenic plants six to seven days after co-culture, mbce were selected on im supplemented with mg/l kanamycin and mg/l carbenicillin. approximately to weeks after selection, gfp positive buds were observed, with gfp positive shoots obtained to weeks after selection. the percentage of explants producing gfp positive buds and/or shoots was 59%. gfp positive shoots were transferred to im with mg/l kanamycin and mg/l carbenicillin to induce roots, and 79% of gfp positive shoots generated roots on im to weeks after culture. transgenic plants expressing gfp were transplanted into soil. the transformation cycle from inoculation to soil took approx. to months. the percentage of explants producing transgenic plants was 59% and the frequency of initial explants that produced independent transgenic plant events confirmed by southern blot was %. transgenic plants expressed gfp in many tissues, including leaf, stem, root, flower, and immature seed. transgenic plants appeared phenotypically normal and fertile. taha et al reported that gus and pcr positive shoots of i. balsamina were regenerated from bombarded cotyledon explants on ms medium supplemented with mg/l ba and mg/l hygromycin; subsequently, gus and pcr positive plants were obtained on ms medium supplemented with mg/l indole-3-acetic acid. pcr, southern blot and r segregation analysis of transgenic i. walleriana amplified pcr products corresponded to the expected size for the nptii gene in five gfp positive plants and in the agrobacterium, but not in the wild type plant. in addition, the pcr band for the pica gene was only present in the agrobacterium strain eha containing phb. this indicated that the nptii gene had integrated into the genome of the transgenic plants, and was not due to agrobacterium contamination of the transgenic plants. up to twenty transgenic plants that expressed gfp were analyzed by southern blot using probes of nptii and gfp genes. southern blot hybridization with the nptii probe revealed the transgene copy number varying between one and three for ten t and two t transgenic plants. an expected band size of kb, positioned within the t-dna for the gfp gene, was present in eleven t and six t transgenic plants. segregation of gfp expression among the t plants for transgenic t lines displayed the expected 3: ratio for a single gene hemizygous in the t. segregation in the remaining ten transgenic exhibited a disproportionate number of nontransgenics in the t. no seedlings from the control impatiens plant displayed gfp. for our transformation system, gfp expression was used to select transformed buds and/or shoots to weeks after selection. the gfp positive buds and/or shoots were transferred to shoot elongation medium with mg/l kanamycin for to weeks and the gfp positive shoots were transferred to rooting medium with mg/l kanamycin for to weeks. among selected gfp positive shoots tested, 100% of them produced transgenic plants that were confirmed by southern blot and r segregation analysis. among mbce that were inoculated and plated on selection medium, 10% of them produced gfp positive buds and/or shoots. approximately 40% to 94% of kanamycin resistant shoots have been reported to be non-transgenic when using kanamycin as a selective agent in many plant species. for transformation of tomato cv. microtom, % of kanamycin resistant shoots after selection on mg/l kanamycin were shoot escapes. however, additional selection of kanamycin resistant shoots on rooting medium with mg/l kanamycin yielded % transgenic plants. an advantage of visualizing gfp expression in our system was to enable us to select transformation events at an early stage thus avoiding the transfer of non-transgenic shoot that survived the kanamycin selection, saving both time and labor.  we report the development of a simple and efficient agrobacterium-mediated transformation and regeneration system for impatiens. to the best of our knowledge, there have been no reports of agrobacterium-mediated transformation of impatiens with experimental evidence of stable integration of t-dna. co-cultivation period, type of explants, a gfp gene as reporter marker to allow early stage selection of transformants and selection regime were critical factors for successful transformation of impatiens. many agrobacterium-mediated transformation methods have used explants derived directly from in vivo plants or from pre-cultured embryogenic calli for inoculation, due to ease of infection by agrobacterium, but these methods have disadvantages such as expense of stock plant growth, detrimental sterilization procedures, time-consuming explant preparation, lack of flexibility, microbial contamination of explants, variation due to age and position of explants, all of which affect transformation efficiency. our direct shoot regeneration system without an intervening callus phase is desirable for both nuclear and plastid transformation using agrobacterium-mediated and biolistics methods for several reasons, including: 1) efficient, simple and rapid regeneration and transformation, 2) flexibility for in vitro manipulation, 3) uniform and desirable green tissue explants for both nuclear and plastid transformation using agrobacterium-mediated and biolistics methods and 4) no somaclonal variation. in addition, impatiens cne derived from in vivo seedlings necrosed, rotted and died several days after agrobacterium inoculation. using in vitro mbce derived from the hsccn of impatiens overcame this problem and enabled transgenic plant regeneration. maziah et al. inoculated a single bud isolated from in vitro multiple buds with agrobacterium to produce transgenic banana. our use of in vitro mbce for agrobacterium inoculation circumvented the problem of necrotic explants and resulted in more efficient transformation than inoculation of single buds. to our knowledge from literatures, there have been no reports of agrobacterium-mediated transformation method for plants using in vitro maintained multiple bud cultures as explants. a major challenge associated with plant transformation technology is the death of agrobacterium-transformed tissues or cells, which severely limits the number of transgenic plants that can be regenerated, and the biotechnological exploitation of economically important crops. hydrogen peroxide plays an important role in programmed cell death and stress. however, little is known about the molecular mechanisms regulating plant pcd, and the signaling pathway leading from h2o to the death of the tissues during transformation. impatiens cne derived from in vivo seedlings necrosed, rotted and died several days after agrobacterium inoculation. using in vitro mbce derived from the hsccn of impatiens overcame this problem and enabled transgenic plant regeneration. we can further investigate levels of cell death and the correlation between the cell death and h2o production of the both explant types and determine whether mbce reduces the h2o production and subsequently it decreases cell death. a mechanistic knowledge of how the tissue death is triggered by the signaling molecule during the transformation is required in order to solve this issue. ornamental horticulture, and particularly floriculture, is well-suited to the application of genetic engineering technology because the product is not intended to be consumed, so commercialization may meet fewer obstacles without the need of food safety studies. however, genetic engineering technologies including transformation have had limited impact in ornamental horticulture. as the development of new varieties is an important driving force in the industry, there are, therefore, good prospects for the development of new genetically modified ornamental crops. using the present protocol, thousands of transgenic impatiens could be produced within to months. the transformation methodology developed in this study will thus be useful for future impatiens improvement. 
transgenic impatiens plants, with a frequency up to %, were obtained within to weeks from inoculation to transfer of transgenic plants to soil. transgenic plants were confirmed by southern blot, phenotypic assays and t segregation analysis. the transgenic plants were fertile and phenotypically normal. in a first step towards the development of transgenic insv-resistant impatiens, we developed an efficient plant regeneration system using hypocotyl segments containing cotyledonary nodes as explants. rooting efficiency was high, and 100% of shoots produced roots within days under optimal conditions, allowing plant regeneration within approximately weeks. impatiens is a top selling floriculture crop. this transformation system has the advantages of 1) efficient, simple and rapid regeneration and transformation, 2) flexibility for in vitro manipulation, 3) uniform and desirable green tissue explants for both nuclear and plastid transformation using agrobacterium-mediated and biolistics methods, 4) no somaclonal variation and 5) resolution of necrosis of agrobacterium-inoculated tissues. using this regeneration system, we developed an efficient agrobacterium-mediated impatiens transformation method using in vitro multiple bud cultures as explants and a binary plasmid bearing gfp and nptii genes. to the best of our knowledge, there have been no reports of agrobacterium-mediated transformation of impatiens with experimental evidence of stable integration of t-dna and of agrobacterium-mediated transformation method for plants using in vitro maintained multiple bud cultures as explants. with this regeneration system, 80% of explants produced an average of elongated shoots per initial explant plated, with up to elongated shoots produced per explant. we have established a regeneration and transformation system for impatiens that provides new alternatives to genetic improvement of this crop. we report the development of a simple and efficient agrobacterium-mediated transformation system for impatiens.
 impatiens is a top selling floriculture crop. the potential for genetic transformation of impatiens to introduce novel flower colors or virus resistance has been limited by its general recalcitrance to tissue culture and transformation manipulations. we have established a regeneration and transformation system for impatiens that provides new alternatives to genetic improvement of this crop.  in a first step towards the development of transgenic insv-resistant impatiens, we developed an efficient plant regeneration system using hypocotyl segments containing cotyledonary nodes as explants. with this regeneration system, 80% of explants produced an average of elongated shoots per initial explant plated, with up to elongated shoots produced per explant. rooting efficiency was high, and 100% of shoots produced roots within days under optimal conditions, allowing plant regeneration within approximately weeks. using this regeneration system, we developed an efficient agrobacterium-mediated impatiens transformation method using in vitro multiple bud cultures as explants and a binary plasmid bearing gfp and nptii genes. transgenic impatiens plants, with a frequency up to %, were obtained within to weeks from inoculation to transfer of transgenic plants to soil. transgenic plants were confirmed by southern blot, phenotypic assays and t segregation analysis. transgene expression was observed in leaves, stems, roots, flowers, and fruit. the transgenic plants were fertile and phenotypically normal.  we report the development of a simple and efficient agrobacterium-mediated transformation system for impatiens. to the best of our knowledge, there have been no reports of agrobacterium-mediated transformation of impatiens with experimental evidence of stable integration of t-dna and of agrobacterium-mediated transformation method for plants using in vitro maintained multiple bud cultures as explants. this transformation system has the advantages of 1) efficient, simple and rapid regeneration and transformation, 2) flexibility for in vitro manipulation, 3) uniform and desirable green tissue explants for both nuclear and plastid transformation using agrobacterium-mediated and biolistics methods, 4) no somaclonal variation and 5) resolution of necrosis of agrobacterium-inoculated tissues. 