Development of an efficient Agrobacterium-mediated genetic transformation for an important ornamental plant, Phlox drummondii Hook

Development of an efficient Agrobacterium-mediated genetic transformation for an important ornamental plant, Phlox drummondii Hook | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Development of an efficient Agrobacterium-mediated genetic transformation for an important ornamental plant, Phlox drummondii Hook Anupama razdan tiku, M. V. Rajam, M. K. Razdan, S. N. Raina This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7162932/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract In ornamental plants, continuous attempts are being made to introduce new quality traits that have market value. Of various modes of plant propagation that have been followed for production and propagation new traits, the genetic transformation technology has reportedly played an important role in creating novel varieties of ornamentals of rose, dahlia, carnation, lily, tulips, gerbera, etc. Many of transformed varieties of these ornamentals reportedly produce plants that have improved aesthetic traits like flower colour, fragrance, variant floral architecture, and vase life. Phlox drummondii Hook., also known as “Drummond” Phlox, is a prized ornamental among 67 species of the genus Phlox . In the present investigation, an efficient Agrobacterium -mediated genetic transformation protocol was developed to study its impact on floral morphology, an important commercial trait, of P. drummondii . About 3-4-week-old embryogenic calli derived from zygotic embryo on callus regeneration and multiplication medium (CRMM) comprising MS basal medium + 3% Sucrose + BAP (5 µM) + NAA (10 µM) were pre-cultured on shoot regeneration medium (SRM) comprising MS medium with 3% sucrose + BAP (15 µM) + IAA (2.5 mµ M) for 2–3 days. A. tumefaciens strain GV2260 carrying the GUS gene construct (PPZP 200) was grown overnight in YEM medium and calli were infected with bacteria (A 600 0.3–0.5) for 10 min. Infected calli were subsequently co-cultured on the medium SRM + acetosyringone (100 µM) for 2- days. Selection of transformed calli was achieved by transfer of these co-cultured calli on the SRM I (SRM supplemented with kanamycin (30 mg/l) and augmentin (300 mg/l), which allowed the production of 76% transformed shoots. For shoot elongation SRM II MS basal salts + 3%sucrose + BAP 10 µM + augmentin 300 mg + kanamycin (30 mg/l) was used. Rooting of transformed shoots occurred on MS + IAA (7.5 µM). Transformation status of putative transgenics was confirmed by PCR using primers(Supplementary Table No 5) specific for GUS and NPT-II genes, and by Southern hybridization using NPT-II gene as probe. Genetic transformation protocol has been standardized in this ornamental species for the first time and transformants were observed having unique changes in floral architecture hitherto unknown. Ornamental plants Phlox drummondii Genetic transformation Agrobacterium Floral variants Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Key Messages This study showed that a novel agrobacterium mediated genetic transformation protocol established in important ornamental plant Phlox Drummondii along with mutagenic changes reported in transgenics. Introduction Ornamentals constitute an important component of horticulture industry with direct impact on human life because of their aesthetic and economic importance. Flower crop cultivation is considered as a lucrative and income generating venture. More importantly, ornamentals play an important role in economic strengthening of many countries particularly in Kenya, Ethiopia, Costa Rica, Colombia and Ecuador (Sharma and Messar 2017 ). For economic purposes, floral industry mostly utilizes cut flowers, loose flowers, potted- flowering and foliage plants. Further ornamental grasses, trees, shrubs and annuals are also grown, which fulfil the aesthetic needs and also form an integral part of ecosystem. Cut flowers in ornamentals like phlox have more market value, followed by flowering pot plants, nursery crops and trees (Shibata 2008 ). Phlox drummondii Hook. is an important ornamental species and a mark of eternal beauty in the genus Phlox , which belongs to family Polemoniaceae comprising Ca. 67 annual and perennial species. Plants of P. drummondii are grown all over the world because of its beautiful and attractive flowers, which occur in shades of purple, pink, Crimson, red, scarlet, violet and many other intermediate shades. Flowers are fragrant, trumpet shaped, 5- lobed with short narrow corolla tube appearing in clusters (broad flat-topped cymes) at the stem ends. Additionally, central eye of the flower emerging from corolla tube differs in colour from the rest of petals making it more attractive. Stigma and style with ovary emerge from the base of five epipetalous anthers growing up to the upper wall of corolla tube (Grant and Grant 1965 ). Leaves as well as stem are soft, hairy, and sticky. P. drummondii adores border clumps, central flower beds of lawns, window boxes, flower vases, hanging baskets, and sold as excellent cut flowers (Bailey 1950 ; Hay and Synge 1969 ; Herman 1973 ). A winter annual P . drummondii is native to grasslands and open woods of Central and Eastern Texas. The genus name Phlox is derived from the Greek word “phlox”, meaning flame, in context of its intense flower colours. In 1835, Thomas Drummond originally collected seeds of this plant in Texas and subsequently sent them to England from where they were distributed to nurserymen in different European and other countries world over (Levin 1977 ). Considering the striking features of P. drummondii , its ornamental potential has not been exploited to full potential owing to sensitivity towards water stress, temperature, and fast maturing habit thus imparting limitations in its multiplication and floriculture trade. Even conventional practices like selection, sexual crossing, back crossing and mutation breeding could overcome these impediments as well as widen its genetic base only to a certain extent. Attempts needed to be initiated for achieving the horticultural excellence in P. drummondii using other non-conventional procedures (Razdan et al. 2008 , Razdan-Tiku et al. 2014 ). Application of genetic transformation procedures is one of the biotechnological approaches that could be utilized to supplement the efforts of existing conventional methods. Through transformation procedures the potential to incorporate novel characters in plants is possible which may otherwise appear recalcitrant using conventional breeding procedures (Ali et al. 2017 , Sharma et al. 2017). Molecular-based gene transformation technology in ornamentals has brought changes in plant morphology (Chandler and Lu 2005 , Casanova et al.2005), flower shape (Casanova et al.2005), flower colour (Meyer et al.,1987, Tanaka et al. 2005 , Debener and Winkelmann 2010 , Chandler and Brugliera 2011 , Umemoto and Toguri 2012 ), flower fragrance (Mori et al., 2004 ., Dudareva 2002 ., Underwood and Clarke 2011 ), pest as well as disease resistance (Milosevic et al., 2011a, 2011 b, Milosevic et al., 2015 ), stress tolerance ( Lojic at al., 2015), and increased vase or post-harvest life necessary for cut flowers (Chandler and Sanchez 2012 ). Transgenic breeding in major cut flower crops like roses (Kim et al. 2004 , Katsumoto et al. 2007 ), gladiolus (Graves and Goldman 1987 , Kamo et al. 2009 , 2010 ) carnations (Lu et al. 1991 , Holton et al. 1993 a, Meng et al. 2009 ) and chrysanthemums (Kazeroonian et al. 2018 ) was reported to have improved important traits in these ornamentals thus providing huge profits and avenues (Sharma et al. 2017). In the present study, Agrobacterium tumefaciens -mediated genetic transformation procedure has been developed with an objective of producing P. drummondii plants with new improved floral traits since this technology acts as powerful tool for delivery of alien gene(s) of interest inside the host plant for integration with the host plant nucleus. Needless to state that a wide range of plant species have been transformed through application of this procedure for improvement of traits of agronomic, horticultural and ornamental value, including important traits in tree species. Moreover, an added advantage in this technology is that transfer of small copy number of T- DNA is good enough to result in stable integration of foreign DNA into host plant genome (Hwang et al., 2017 ). Considering these aspects an efficient Agrobacterium -mediated genetic transformation protocol was developed in present study for first time in P. drummondii or the genus Phlox . Material and Methods Plant material found suitable for genetic transformation For genetic transformation of P. drummondii 3-week-old callus induced from zygotic embryo and multiplied by repeated subculturing on CRMM (callus regeneration and multiplication medium) was found suitable (Fig. 1) although calli derived from cotyledonary leaves were also tried. CRMM constituted of MS basal medium + 3% sucrose + BAP (5 µM) + NAA (10 µM). Prior to transformation the zygotic embryo induced callus was cut into 2-3 mm pieces and then they were pre-cultured on shoot regeneration medium (SRM), which comprised of MS basal salts +3% sucrose + BAP (15 µM) + IAA (2.5 µM) for zero days, 1-2 days, 2-3 days and 4-5 days. Bacterial strain and binary vector The Agrobacterium tumefaciens strain PGV2260 was used for transforming embryogenic calli of P. drummondii , which carried GUS binary plasmid vector PPZP20KWT with a marker gene and Nos promoter NPT-II: OcspA cassette (conferring kanamycin resistance) and a reporter gene GUS (intron)- 35S promoter cassette (Fig. 2). Agrobacterium -mediated genetic transformation Agrobacterium strain PGV2260, carrying GUS binary vector, was cultured overnight in 100 ml conical flask containing 30 ml YEM (yeast extract mannitol medium) + antibiotics (carbenicithin, spectinomycin and refampicin) on a shaker, 250 rpm, at 28°C. OD (Bacterial density) of the culture was checked intermittently after the gap of few hours and cultures at different ranges of OD (0.1- 1.0) at A600 were tried for different sets of transformation experiments (Table 1). After adjusting the OD of the bacterial culture, it was transferred to autoclaved Oakridge tubes for centrifugation at 3000-4000 rpm for 10 min at 28°C. The supernatant was discarded and the pellet dissolved in MSO (MS liquid medium without hormones) + acetosyringone 100 µM. The callus pieces in this medium were then transferred to an autoclaved screw- capped wide – mouthed jam bottle and to each piece was added to Agrobacterium culture of different OD. The bottles were gently stirred manually so that the callus gets properly infected with bacterial suspension. Along with the OD, infection time of bacteria was also standardized ranging from 5-30 min (Table 2). After pouring off the Agrobacterium suspension, the callus pieces were placed on autoclaved blotting paper. Blotted dry calli were then transferred to co-culture medium comprising SRM + acetosyringone (100 µM) for 2-3 days. The co-cultivated calli were finally transferred to selection medium containing SRM + augmentin (300 mg/l) + kanamycin (20- 50 mg/l) (Table S1). Kanamycin assay to optimize the concentration for stringent selection for the recovery of the transformed callus in selection medium Subculturing was done on the same selection medium after every 15 days up to 3 cycles of selection (Total 1½ months on selection medium). Untransformed callus did not survive on selection medium (Fig. 3A) whereas transformed callus could only regenerate on the selection medium (Fig.3 B). After 1½ months, callus was transferred to SRM ll containing MS basal salts +3% sucrose +BAP 10 µM +augmentin 300 mg (Fig. 3C). After 1 month of subculturing on SRM ll, 1-2 cm long shoots excised from regenerating callus were transferred to same medium but without kanamycin (Fig. 3 D). Transformed shoot multiplication, rooting and transplantation For multiplication of transformed shoots from the various experiments two approaches were followed. The small shoots (Ca 1-2 cm long) were individually transferred to fresh medium (SRM II without kanamycin) for further proliferation. Second, the longer shoots that had grown up to 4-5 cm, were cut into single node segments and each node segment transferred to fresh medium (SRM II without kanamycin) for further growth and multiplication (Fig. 3E). The number of shoots obtained at the end of a multiplication cycle was regarded as the rate of shoot multiplication. After 2- weeks shoots in vitro underwent flowering (Fig. 3F). However, for rooting, shoots measuring Ca. 4 cm, with 3-4 nodes, from transformed calli before in vitro flowering were excised and transferred to rooting medium (MS+IAA 7.5 µM ). The rooted plants (Fig. 3G) were washed in tap water to remove agar and then transplanted to soil in small plastic pots containing autoclaved soil as per procedure followed by Razdan-Tiku et al. (2014). The soil composition was one part vermiculate and 3 parts of garden soil. The transformed plants in pots were then covered with polythene bags with small holes (2 mm diameter) to maintain high humidity at least for 20 days. These plants were kept in tissue culture room with temperature 25 to 27 °C, relative humidity 50 to 60%, and 16 h photo period. After one month of acclimatisation, these plants were transferred to bigger pots (Fig. 3H) and subsequently shifted to transgenic green-house. GUS Assay Analysis of GUS activity in transformed plant parts was performed by histochemical assay as described by Jefferson et. al. (1987). Transformed calli, leaves and roots were incubated in 500 μL of GUS assay buffer [10 mg of X- gluc (5 – bromo 4 – chloro – 3 indolyl – glucuronide ) in 2 ml dimethyl formamide ] , 2 ml 5 mM potassium ferrocyanide , and 20 ml 0.1 M sodium phosphate buffer , at 37°C overnight . The solution was removed next morning and incubated materials were rinsed in 70% ethanol and examined for detection of blue colour (Fig. 4 A-C) before being stored in 40 % glycerol. Molecular analysis of putative transformants of P. drummondii The transgenic plants raised were analysed by PCR for the integration of the transgene. Using Techne PCR machine (U.K.), DNA was isolated from leaves in liquid nitrogen by a CTAB method as described by Doyle and Doyle (Doyle and Doyle 1987). About 100 ng of genomic DNA from untransformed control and the transgenic lines was taken separately, and mixed with 100 mM forward and reverse primers (Suppmentary Table No 2) in 7.5 μl of PCR buffer (10 mM of Tris HCl , 50 mM KCl , 2 mM MgCl 2 , 100 μM dNTPS mix) and 0.1 Taq DNA polymerase (Biotools , Spain), pH 8.3. The volume of mixture was raised up to 25 μl with SDW. DNA amplification was carried out in the thermal cycler programmed for 40 cycles as follows: 1 cycle of 5 min at 94 °C, followed by 39 cycles, each of denaturation at 94°C for 1 min, annealing for 1 min at 53 °C, and synthesis at 72 °C for 2 min, and finally 1 cycle of 10 min at 72 °C. After completion of the PCR 2 μl of 10x loading buffer was added to each of the samples. The amplification product was separated by electrophoresis in 1.2 % agarose gel containing 0.05 μl/ml ethidium bromide (Roche Diagnostic Gmbh, Germany) in 0.5x TBE buffer (0.045 M Tris- borate and 1mM Na 2 EDTA). The Hind III digested λ DNA was loaded in one of the lanes to serve as molecular size markers. After agarose gel electrophoresis (AGE), the gel (Fig.5 A-B) was photographed in UV light (LKb Pharmalia, USA). PCR experiment was repeated with the same samples 2 -3 times for the confirmation of results. The details of the primers used for npt -II gene and GUS are given in Table S2. Southern analysis with NPT -II gene as a probe Southern blot hybridization was performed to detect the NPT -II gene integration. Genomic DNA (10 µg) from different PCR positive transgenic lines as well as the untransformed control was digested with Eco RI, which released the NPT -II gene from the T-DNA and blots were prepared (on Nylon membrane Sigma, USA) , hybridised and washed with high stringency as per the standard protocol (Sambrook et al. , 1989). The NPT -II gene probe was prepared by using the random priming kit (Takara, Japan) as per the manufacturer’s guidelines. The probe was denatured before adding to the pre-hybridisation buffer. After hybridisation, membrane was washed and then exposed to X-ray film (Kodak); (Fig. 5C). Statistical analysis Each experiment was repeated thrice, data subjected to analysis of variance (ANOVA), and means were compared using SAS computer software according to Anderson – Cook 2004. Results For genetic transformation of P. drummondii , an efficient protocol has been devised based on the Agrobacterium -mediated transformation method. The details of procedures followed according to protocol are described in Section on material and methods. Based on this protocol, the following parameters were determined that facilitated efficient gene transformation in P. drummondii . Optimum Density (OD) of Agrobacterium and infection period Agrobacterium tumefaciens GV2260 strain carrying the GUS gene construct pPZp200 (Fig. 2) cultured in YEM medium at different ODs (0.1 – 1.0; Table 1) demonstrated the most effective transformation occurred using this bacterial strain at the optimum OD (0.3-0.5), A600. Explants infected with bacteria at same OD for different infection periods (5 min – 30 min) demonstrated gene transformation occurred at best at infection time of 10 min (Table 2). On higher OD at same infection time though the number of GUS positive calli were more but the number of transgenic plants regenerated happened to be very less. At lower OD the percentage of GUS transformed calli decreased with reduced potential for plant regeneration (Table 1). Similarly, with the increase in infection time (>10 min) plants regenerated from transformed calli had negligible or very low regeneration response (Table 2). Preculture of untransformed callus Prior to transformation as already mentioned, the untransformed callus was pre-cultured on shoot regeneration medium (SRM) for zero days, 1 -2 days, 2 -3 days and 4 -5 days to inculcate its better transforming efficiency. Most suitable callus for most effective genetic transformation was the one pre-cultured on SRM for 2-3 days. It contained high levels of cytokinin and low levels of auxin. Pre-cultured callus pieces (2-3 mm) were subsequently transformed by immersing in MSO medium containing Argobacterium (carrying gene construct) at standardised OD + Acetosyringone (100 µM) in a wide mouthed -autoclaved bottle and manually shaken for standardised period of 10 min. The infected calli were subsequently cultured on co-cultivation medium (SRM + acetosyringone (100 µM) for 2 – 3 days. These co-cultivated calli were finally transferred to a selection medium containing SRM + augmentin (300 mg/l) + kanamycin (30 mg/l). Although selection media with different concentrations of kanamycin were tested, proper selection of transformed callus (Fig. 3 B) occurred at antibiotic concentration 30 mg/l which regenerated more transformed shoot buds. At kanamycin concentrations below 30 mg/l although percentage callus survival was high but plants regenerated were mostly untransformed. At kanamycin concentrations above 30 mg/l, most calli turned pale -yellow or necrotic and died without showing any signs of regeneration. Plant regeneration from transformed callus Transformed callus were taken out from the selection medium and then transferred to SRM I containing MS salts + BAP (15 µM) + IAA ( 2.5 µM) + augmentin ( 300 mg/l ) + kinetin ( 30 mg/l ) and sub-cultured on same medium , every 15 days for at least one and a half month till small shoots appeared on the callus ( Fig. 3 C) . Transformation frequency and regeneration frequency of P. drummondii transgenic plants were 76% and 62%, respectively (Table 3). GUS assay was done with 2-week-old calli growing on selection medium to test the incorporation of GUS gene (Fig.4 A) and percentage calli showing GUS activity calculated. Highest percentage of GUS activity was shown to be around 70% when infection time was kept at 10 min and bacterial density 0.3 – 0.5, A600 (Table 2). After shoot bud formation callus was transferred to shoot regeneration medium II (SRM II). This regeneration medium contained MS salts + 3% sucrose + BAP (10 µM) + augmentin (300 mg/l) + kinetin (30 mg/l). After one month of sub-culturing on SRM II, 2 cm long shoots were excised from the base of callus and transferred to the same medium but without adding augmentin and kanamycin. This resulted in further elongation and multiplication of shoots (Fig. 3 D-E). In vitro flowering of transformed shoots also occurred on SRM II (Fig. 3F). Therefore, it became essential to induce rooting in transformed shoots on the medium MS + IAA (7.5 µM) before in vitro flowering (Fig 3G). The plantlets were finally transplanted to soil in pots (Fig. 3H) as per procedure described in material and methods. Morphology of Transgenics It was observed that floral and other characteristics of transgenic P. drummondii plants were quite different from control (normal /untransformed) plants. Height of transgenic plants was shorter than the normal plants. Stem and leaves of transgenics were very thick and dark green in contrast to the thin and light colour of stem and leaves of normal plants. Morphology of flowers in particular showed huge variations in different transgenic lines in comparison to normal untransformed plants (Fig. 6. A-E). In the genus Phlox (including P.drummondii ) as well as in family Polemoniaceae to which it belongs, flowers are radially symmetrical and pentamerous (each floral whorl consists of 5 sepals and 5petals fused into a cup, or tube-shaped structure (Fig. 6A). Stamens are 5, epipetalous, the length of all anthers and their filaments being almost same they are attached at the base of petals and reach up to the mouth of the corolla tube (Fig. 6C). However, in our investigations on GUS transformed P. drummondii plants the flower structure was found paradoxically different as it showed marked variations due to the occurrence of partitions in the corolla tube resulting in petals becoming free except at the base (more like a polypetalous condition; Fig. 6 B). Corolla tube length in the control or untransformed plants being Ca. 1cm, the length of corolla tube in transgenic plants on contrary was Ca. 2 cm. While in control plants, epipetalous anthers reach the same height (Fig. 6 C), the transgenic plants exhibited variation in their height. Two stamens being long are slightly exerted towards upper mouth of the corolla tube, another two stamens are of medium size and reach till the middle of the corolla tube, whereas the fifth stamen is short and appears at the base of the corolla tube (Fig 6 D). Different transgenic lines also showed variations in flower colour ranging from light pink to reddish pink (Fig. 6 E2 and E5). Further, shape of the petals varied as they are broader in control plants whereas transgenic flowers had narrow petals pointed at the top (club shaped; Fig. 6 E2, E4 and E6). Another striking feature being that few transgenic lines showed flowers with an increased number of petals (6-8; Fig. 6 E3, E5 and E6) thus defying symmetrical pentamerous condition of 5 petals in control plants (Fig. 6 E1). GUS assay was also performed in shoots and roots of both transgenics and control. Appearance of blue colour confirmed the transgenic nature of plants (Fig. 4 B, C). Molecular characterization of putative transgenics As mentioned earlier 70 % of the calli infected by Agrobacterium carrying vector T-DNA GUS and NPT -II genes construct at 0.3- 0.5 OD, A600, and infection time of 10 mins showed maximum GUS activity (Table. 2). The putative transgenics were characterised for the integration of vector T-DNA GUS and NPT -II genes into the host plant genome by doing PCR. The PCR was performed with the DNA isolated from untransformed (control) and transformed lines using NPT -II (marker) and GUS (reporter) gene specific primers. The amplified product of both the genes was 500 bp gene fragment which was found in 70% putative transformants (Fig.5 A, B), thus confirming their transgenic nature. PCR positive lines were further checked by Southern hybridization using NPT -II gene specific probe (Fig 5C). For Southern hybridization, genomic DNA isolated from transformed and untransformed (control) plants were digested with the restriction enzyme Eco RI, which cuts the T-DNA fragment of 2.3 kb containing GUS - NPT -II gene (Fig. 5C). The results obtained from molecular analysis correlated with morphology-based identification of putative transgenic lines regenerated from transformed embryogenic calli of P. drummondii . Discussion Development of ornamental varieties that have much improved floral characters is one of the important aspects of floriculture. Biotechnological approaches along with the classical breeding methods are being used to improve floral or other important traits in ornamentals to supplement their commercial value. Improvements desired in flowers generally relate to fragrance, morphological appearance of sepals and petals, their colour, diseases and stress resistance, enhanced vase life, etc. Transgenic strategies seem to have immense potential to produce novel flower phenotypes which otherwise are not found in nature or produced through traditional breeding procedures (Kim 2020 ). In this context the improvement of flower traits through gene transformation has been attempted in ornamental flowering plants like Gerbera (Chung et al., 2016 ), Petunia (Bashandy and Teeri 2017 ), rose (EL-Tarras et al., 2017 ), carnation (Yantcheva et al., 1997 ) and chrysanthemum (Shinoyama et al., 2012 ) with some success. Insertion of alien desired genes into a plant requires development of a reproducible protocol which was prime objective of this study for P. drummondii . Till date, no such studies have been carried out on any species of the genus Phlox . It required many efforts to devise and standardize each and every step of genetic transformation of P. drummondii. Certain very important parameters had to be standardized, such as choice of explant, selection of Agrobacterium strain with binary vector gene construct, infection time of this bacterium, optical density (OD) of the bacterial suspension culture, concentration of selection marker (kanamycin) and acetosyringone in the medium during co-cultivation, time period as well as temperature requirement for co-cultivation, and type of bacteriostatic agent used. Transformation protocol for P. drummondii required the development of an efficient tissue culture technique for effective regenerative response of explant and transformed tissues. Selection of explant was important as different explants (leaf discs, florets, petals, stem cuttings, roots, cotyledonary leaves, embryogenic calli etc.) showed difference in transformation and regeneration potential. Elomma and Holton (1994) also observed this phenomenon in other ornamental plants ( Gerbera , chrysanthemum, etc.). In P. drummondii zygotic embryo- derived callus was most suitable for Agrobacterium - mediated transformation using the protocol described and standardized in this paper. Preculture of calli proved an important parameter because most of the cells undergo rapid divisions and thus remain in an exponential growth phase which makes them more amenable to transformation. Such inferences were also reached based on the evidences of researches on other plant systems (Hiei et al., 1994 ; Vijayachandra et. al., 1995 ). According to Kumria et al. ( 2001 ) preculture of callus on high cytokinin (BAP 15 µM) or low auxin (IAA 2.5 µM) medium enhances the cell division as well as promotes Agrobacterium infection. OD of the bacterium suspension carrying gene construct was found equally critical. At lower OD (0.1–0.3) GUS activity was less pronounced depicting lower or negligible transformation frequency, while at higher OD (0.5-1.0) the extensive blackening of transformed calli proved detrimental for regeneration. Therefore, the agrobacterium strain PGV2260 suspension used in present study showed best GUS activity at O.D (0.3–0.5). The infection period of bacterium also varied and 10 min was found optimum for infection and transformation. Importantly, keeping the dry infected calli on SRM for at least 2 days was one more step required for successful transformation before shifting them to selection medium (SRM + kanamycin). Another interesting observation in our studies has been that the transformed callus showed no GUS activity in absence of acetosyringone in the medium. Therefore, acetosyringone (100 µM) in the medium was optimum and essential to promote both, GUS activity, and shoot regeneration. Acetosyringone, a phenolic compound, is emitted in wounded plant tissues which seems important for Agrobacterium to enter the host plant tissue-cell cytoplasm and transmit T-DNA along with associated proteins (T-complex) into the host pant genome for stable integration (Lacroix and Citovsky 2013 ). Cefotaxime and augmentin were tried as bacteriostatic in selection medium. Augmentin (300 mg/l) proved better as it restricted overgrowth of Agrobacterium and supported regeneration of transformed calli as well as shoot bud differentiation on selection medium SRMI (Fig. 3 B). Differentiation of shoot buds on the transformed callus also occurred on the same selection medium but for further shoot development (elongation and multiplication of shoots) a different shoot regeneration medium (SRMII) was standardized and used. While developing the genetic transformation protocol for P. drummondii some very interesting and remarkable results were achieved. Transformed plants showed different floristic characteristics from the normal plants. A major change was the trend towards polypetalous condition of the flower (Fig. 6 B) as against the floral architecture of gamopetalous tubular corolla in Phlox drummondii , and other members of the family Polemoniaceae. Another variation in floral architecture observed was that in some GUS transformed P. drummondii plants the number of petals increased (6–8) as against normal 5 petals found in normal plants (Fig. 6E3, E5, E6). Petal shape also changed along with its colour. The colour was more intensified and there was a paradigm shift in the position of anther filaments (Fig. 6E2 and E5). Reason behind these variations could be the impact of “insertional mutagenesis” in GUS transformed plants of P. drummondii leading to formation of new variant lines of this ornamental. Insertional mutagenesis is an alternative means of altering gene function caused by insertion of foreign gene into the genome of host plant. The foreign T-DNA carrying GUS : NPT-II by insertion changes the host plant gene expression with GUS acting as a marker for identification of variations in morphological characteristics of transgenic plants. Similar observations were reported in Arabidopsis due to the involvement of either transposable elements or T-DNA (Krysan et al., 2000 ; Bundock et al., 2012 ). Researchers have substantiated that T-DNA acts as an insertional mutagen and such mutations are chemically and physically stable through multiple generations (Wisman et al., 1998 ). T-DNA inserted mutants could be cloned easily as reported by Sangwan and Sangwan-Norreel ( 1989 ). Several large- scale T-DNA insertional mutagenesis projects are being undertaken for cloning mutant genes (Gelvin 2017 ; Jupe et al., 2019 and Pucker et al., 2021 ). In P. drummondii , the plausible explanation for noticeable variation in floral architecture of transgenic plants regenerated in present investigation could be that foreign T- DNA by insertion into host plant genome must have altered the functional genes responsible for expression of floral characteristics, thus almost warranting a rechristening of such transgenic taxa that show nearly polypetalous trend as against gamopetalous nature of normal plants in Phlox . Therefore, in present study a protocol devised successfully transformed P. drumondii resulting in transgenic plants that have striking variations in floral features hitherto not reported. Conclusion Genetic improvement of ornamentals is an ongoing process. Over the years new varieties of ornamental plants have been produced by conventional procedures, such as selection, cross hybridization, or mutation breeding. Success achieved in ornamentals following these traditional procedures has remained restricted. As a result, attempts are being made to apply non-conventional in vitro methods. GM technology could be one such tool useful for improvement of traits in original ornamental cultivars. Gene transformation techniques are known to modify target traits due to direct integration of bacterial T-DNA in to host genome. These techniques thus are used as a supplement to traditional breeding methods. In the present study, an efficient Agrobacterium - mediated genetic transformation protocol using reporter (GUS) and marker genes (NPTII) was developed for P. drummondii , an important ornamental, which impacted its floral structure. This transformation procedure is first attempt for any species in the genus Phlox and has potential for transfer of genes of interest in other Phlox species, e.g., polyamine biosynthesis genes for delaying senescence and increasing the vase life. The new transformed variant varieties could be propagated vegetatively or sexually for perpetuation of new traits. Abbreviations CRMM – Callus Regeneration and Multiplication medium SRM – Shoot regeneration medium MS – Murashige and Skoog medium YEM – Yeast Extract Mannitol medium SRM I – Shoot Regeneration medium I SRM II - Shoot Regeneration medium II MSO - Murashige and Skoog liquid medium GUS: – Glucuronidase NPT-II: – Neomycin Phosphotransferase II Declarations Conflict of interests Authors declare that they have no conflict of interest. Author’s contributions SNR and MKR conceived the idea and MVR designed the experiments. ART carried out the experiments, generated the data. ART and MVR analysed the data. ART wrote the manuscript. MVR, SNR and MKR edited, and finalized the manuscript. All the authors read and approved the final version of the manuscript. Acknowledgements I thank Prof. M.V. Rajam, Department of Genetics & Prof. S.M. Raina, Department of Botany, University of Delhi for providing facilities for providing facilities to conduct this study. 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Biol. Plant.58:153-158. Razdan A, Razdan M, Rajam MV, Raina SN (2008). Efficient protocol for in vitro production of androgenic haploids of Phlox drummondii . Plant Cell Tiss. Org. Cult. 95:245-250. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual (2nd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. Sangwan RS, Sangwan-Norreel BS (1989). Genetic transformation and plant improvement. The Impact of Biotechnology on Agriculture (Kluwer Academic Publishers), pp. 299-337. Sharma R, Messar Y (2017). Transgenics in ornamental crops: Creating novelties in economically important cut flowers. Curr. Sci. 113:43-52. Shibata M (2008) Importance of genetic transformation in ornamental plant breeding. Plant Biotechnol. 25:3-8. Shinoyama H, Aida R, Ichikawa H, Nomura Y, Mochizuki A (2012) Genetic engineering of chrysanthemum ( Chrysanthemum morifolium ): Current progress and perspectives. Plant Biotechnol. 29:323–337. Tanaka Y, Katsumoto Y, Brugliera F, Mason J (2005) Genetic engineering in floriculture. Plant Cell Tiss. Org. Cult. 80:1-24. Umemoto N, Toguri T (2012) Peptide transporting to chromoplasts in petals and method of constructing plant having yellowish petals by using the same. Patent US 8143478 B2. Underwood BA, Clarke DG (2011) Transgenic ornamental crops. In Transgenic Horticultural Crops: Challenges and Opportunities (B. Mou and R. Scorza, eds), Boca Raton, FL: CRC press. pp. 55-82. Vijayachandra K, Palanichelvam K, Veluthambi K (1995) Rice scutellum induces Agrobacterium tumefaciens vir genes and T-strand generation. Plant Mol Biol. 29:125-133. Wisman E, Hartmann U, Sagasser M, Baumann E, Palme K, Hahlbrock K, Saedler H, Weisshaar B (1998) Knock-out mutants from an En-1 mutagenized Arabidopsis thaliana population generate phenylpropanoid biosynthesis phenotypes. Proc. Natl. Acad. Sci. USA 95:12432-12437. Yantcheva A, Vlahova M, Atanassova B, Atanassov A (1997) Direct organogenesis and plant regeneration of Carnation ( Dianthus caryophyllus L.), Biotechnol. Biotechnol Equip.11:60-65. Tables Table 1. Influence of bacterial density (OD 600 ) genetic transformation in 6-weeks-old callus of Phlox drummondii on selection medium and subsequent shoot regeneration from transformed callus Bacterial density (OD 600 ) Percentage callus showing GUS activity Average number of shoots obtained from tranformed explants 0.1 - 0.3 50 ± 0.10 c 30 ± 2.10 d 0.3 - 0.5 60 ± 0.25 bc 53 ± 0.10 c 0.5 - 0.7 80 ± 0.50 ab 20 ± 0.35 de 0.7 - 1.0 80 ± 0.28 ad 0 ± 0 e Means ± SE, n=50; means in the same column followed by different letters are significantly different at P ≤ 0.05 and F = 96.56Anderson – Cook 2004). Table 2. Effect of the infection time on genetic transformation during cocultivation, and subsequent shoot regeneration in 6-week-old transformed callus of Phlox drummondii on selection medium Infection time (Min) Percentage callus showing GUS activity Average number of shoots obtained from responding explants 5 45 ± 0.50 c 30 ± 0.02 d 10 70 ± 0.35 b 53 ± 0.01 c 15 80 ± 0.00 ab 15 ± 0.30 e 20 80 ± 0.10 ab 10 ± 0.00 e 30 85 ± 0.15 a 0 ± 0 e Means ± SE, n=50; means in the same column followed by different letters are significantly different at P ≤ 0.05 and F = 96.56 (Anderson – Cook 2004). Table 3. Transformation of embryogenic callus of Phlox drummondii with binary vector (pPZP200) carrying GUS reporter gene and NPT -II marker gene Number of calli co-cultivated Number of calli surviving on selection medium (SRM I) Average number of transformed shoots obtained from responding calli Percentage transformation Percentage regeneration 85 53 ± 0.1 c 40 ± 0.5 cd 62 ± 0.10 b 47 ± 0.25 c 85 50 ± 0 c 38 ± 0.3 d 63 ± 0.11 b 48 ± 0.05 c 85 65 ± 0.5 b 53 ± 0.25 c 76 ± 0.5 b 62 ± 0.10 b 85 49 ± 0.12 c 39 ± 0 d 58 ± 0.18 c 45 ± 0.0 c 85 47 ± 0.11 c 37 ± 0.10 d 63 ± 0 b 49 ± 0.01 c Means ± SE, n=50; means in the same column followed by different letters are significantly different at P ≤ 0.05 and F = 96.56 (Anderson – Cook 2004). Supplementary Files TableS1.docx TableS2.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 29 Jul, 2025 Reviewers invited by journal 28 Jul, 2025 Editor assigned by journal 25 Jul, 2025 First submitted to journal 23 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7162932","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":492165183,"identity":"9297b3ff-709a-4799-8d31-39c3498a24fa","order_by":0,"name":"Anupama razdan tiku","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7UlEQVRIiWNgGAWjYDACZjYkzgcgZmMnoIMHWQvjDJAWZkJaGJC0MPOASQJa7NnZ0iQYau4kbmc/nfjZ5tc2eT5mBsYPH3PwOuyYBMOxZ4k7e3I3S+f23TZsY2Zglpy5DZ8W9jYJBrbDiRsO5G6Qzu25zQjUwsbMS1DLP6CW8283/7bsuW1PhBagwxjbgFpu5G6TZvhxO5GwlsNsyRaJfYeNd854u82yt+F2chszYzNev7D3HzO88eHbYdnt/Lmbb/z4c9t2fnvzwQ8f8WgBgwQgNgAxGNvAZAMB9VAA1sLwhzjFo2AUjIJRMLIAALM3TeRL3aNyAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-5249-2292","institution":"University of Delhi - Ramjas college","correspondingAuthor":true,"prefix":"","firstName":"Anupama","middleName":"razdan","lastName":"tiku","suffix":""},{"id":492165184,"identity":"e88d0971-368d-47da-b1b2-c88d4f2d6555","order_by":1,"name":"M. V. Rajam","email":"","orcid":"","institution":"University of Delhi - South Campus","correspondingAuthor":false,"prefix":"","firstName":"M.","middleName":"V.","lastName":"Rajam","suffix":""},{"id":492165185,"identity":"c3e83817-9321-461e-a359-7732c5a93839","order_by":2,"name":"M. K. Razdan","email":"","orcid":"","institution":"University of Delhi - North Campus: University of Delhi","correspondingAuthor":false,"prefix":"","firstName":"M.","middleName":"K.","lastName":"Razdan","suffix":""},{"id":492165186,"identity":"680091d0-0eb9-4f57-b0a1-445b60ee4ae1","order_by":3,"name":"S. N. Raina","email":"","orcid":"","institution":"University of Delhi - North Campus: University of Delhi","correspondingAuthor":false,"prefix":"","firstName":"S.","middleName":"N.","lastName":"Raina","suffix":""}],"badges":[],"createdAt":"2025-07-19 08:17:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7162932/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7162932/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87927954,"identity":"8e67d2e1-c4e6-4c1d-9b79-f21c42b4958a","added_by":"auto","created_at":"2025-07-30 12:56:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":89026,"visible":true,"origin":"","legend":"\u003cp\u003eThree-week-old callus regenerated \u003cem\u003ein vitro\u003c/em\u003e from zygotic embryo.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7162932/v1/eb7ac83e0ea303ad4953aaa6.png"},{"id":87927248,"identity":"b9eec2c2-5608-4ffc-8c5d-cb66aed55629","added_by":"auto","created_at":"2025-07-30 12:48:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":52121,"visible":true,"origin":"","legend":"\u003cp\u003eShowing construct of Binary plasmid vector PPZP200 KWT containing marker gene as Nos Pr nptii: OcspA cassette (conferring Kanamycin resistance) and a reporter gene as GUS (int)- 35S pA promoter cassette.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7162932/v1/ed90b296c1ccfad71454d695.png"},{"id":87927252,"identity":"708b19ef-36db-4629-8c43-e83ff9ee9090","added_by":"auto","created_at":"2025-07-30 12:48:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":636162,"visible":true,"origin":"","legend":"\u003cp\u003e(A-H). Showing different stages of regeneration in 3-week-old transformed embryogenic callus (obtained from zygotic embryo culture) of \u003cem\u003eP. drummondii\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA. Shoot bud formation on the callus (untransformed) on SRM (shoot regeneration medium) with no antibiotics.\u003c/p\u003e\n\u003cp\u003eB. Shoot bud formation on the transformed callus on selection medium (SRM + 30 mg/l Kanamycin).\u003c/p\u003e\n\u003cp\u003eC. Transfer of shoot bud containing transformed callus to shoot proliferation medium (SRM II without kanamycin).\u003c/p\u003e\n\u003cp\u003eD – E. Elongation and multiplication of transformed shoots on shoot proliferation medium (SRM II)\u003c/p\u003e\n\u003cp\u003eF. Well-developed shoot obtained from transformed callus showing in vitro flowering on µG. rooting in vitro of transformed shoots of \u003cem\u003eP. drummondii \u003c/em\u003eon the medium MS + IAA (7.5 M).\u003c/p\u003e\n\u003cp\u003eH. Transplantation of in vitro rooted transformed shoots to soil and vermiculite mixture.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7162932/v1/3ba28864300e893f4dc4b26e.png"},{"id":87927250,"identity":"1ba6a37a-a025-49c5-b7aa-dfa52017b356","added_by":"auto","created_at":"2025-07-30 12:48:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":384489,"visible":true,"origin":"","legend":"\u003cp\u003eA-C). GUS Assay in different parts of transformed plant\u003c/p\u003e\n\u003cp\u003eA. Conformation of GUS gene (blue colour) in transformed callus.\u003c/p\u003e\n\u003cp\u003eB. Conformation of GUS gene (blue colour) in shoot of transformed plant.\u003c/p\u003e\n\u003cp\u003eC. Conformation of GUS gene (blue colour) in roots of transformed plant.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7162932/v1/7aacb0942f947569a52eda6a.png"},{"id":87927257,"identity":"0bef8370-4988-40e4-86c1-e72e8ccb3336","added_by":"auto","created_at":"2025-07-30 12:48:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":272541,"visible":true,"origin":"","legend":"\u003cp\u003e(A-C). Molecular characterization of putative transgenic plants\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003ePCR with \u003cem\u003eNPT-\u003c/em\u003eII gene-specific primers: Lane 1- 1 kb ladder; Lane 2 – plasmid DNA: Lane 3- untransformed control; Lanes 4-16 putative transgenic lines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B) \u003c/strong\u003ePCR\u003cstrong\u003e \u003c/strong\u003ewith \u003cem\u003eGUS\u003c/em\u003e gene- specific primers: Lane1- 1 kb ladder; Lane 2 - plasmid DNA; Lane 3- untransformed control; Lane 4 -16 putative transgenic plants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C) \u003c/strong\u003eSouthern analysis with \u003cem\u003eNPT-\u003c/em\u003eII gene as probe: Lane 1 - 1 kb ladder; Lanes 2 -11PCR- positive transgenic lines.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7162932/v1/e5f6d6653c8d597e9eb3b8a5.png"},{"id":87927960,"identity":"3fe72282-fbe0-468d-914b-89db45b2a3d6","added_by":"auto","created_at":"2025-07-30 12:56:41","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":744536,"visible":true,"origin":"","legend":"\u003cp\u003e(A-E)\u003c/p\u003e\n\u003cp\u003eVariations in colour, shape, position of anthers and number of petals in the flowers of transformed (tra) plants in comparison to the normal (con).\u003c/p\u003e\n\u003cp\u003eA. Flower from control untransformed plant.\u003c/p\u003e\n\u003cp\u003eB. Flower from transformed plant showing nearly polypetalous condition.\u003c/p\u003e\n\u003cp\u003eC. L.S Flower from control plant showing epipetalous position of anthers (all the anthers have same position and same size of filament).\u003c/p\u003e\n\u003cp\u003eD. L.S Flower from transformed plant showing change in position of anthers (2+2+1) and variation in size of filaments of different anthers.\u003c/p\u003e\n\u003cp\u003eE. Flowers from different transgenic lines showing variation in colour: dark pink (E2-E4, E6), Red (E5); and shape: club shaped and nearly polypetalous (E2, E4 and E6) with increase in number of petals :6-8 (E3, E5 and E6) in comparison to flower of control (untransformed) plant, i.e. pink, gamopetalous with five petals (E1).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7162932/v1/806594252334321c21e816ed.png"},{"id":87929393,"identity":"eed33de9-2202-47ad-8d7b-f611519faae3","added_by":"auto","created_at":"2025-07-30 13:12:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3702499,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7162932/v1/06134dd2-294d-4d55-9b2c-7e84f3947c49.pdf"},{"id":87927246,"identity":"4dc5b3ea-1546-4116-8d29-27ca2ba715ad","added_by":"auto","created_at":"2025-07-30 12:48:41","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":13909,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7162932/v1/09943806b5984219008c1263.docx"},{"id":87927955,"identity":"18583198-c0f9-4d8a-877b-6ccf8ce4da89","added_by":"auto","created_at":"2025-07-30 12:56:41","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":13709,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-7162932/v1/29ebf99227d1f55e20c225d9.docx"}],"financialInterests":"","formattedTitle":"\u003cp\u003eDevelopment of an efficient Agrobacterium-mediated genetic transformation for an important ornamental plant, Phlox drummondii Hook\u003c/p\u003e","fulltext":[{"header":"Key Messages","content":"\u003cp\u003eThis study showed that a novel agrobacterium mediated genetic transformation protocol established in important ornamental plant \u003cem\u003ePhlox Drummondii\u0026nbsp;\u003c/em\u003ealong with mutagenic changes reported in transgenics.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eOrnamentals constitute an important component of horticulture industry with direct impact on human life because of their aesthetic and economic importance. Flower crop cultivation is considered as a lucrative and income generating venture. More importantly, ornamentals play an important role in economic strengthening of many countries particularly in Kenya, Ethiopia, Costa Rica, Colombia and Ecuador (Sharma and Messar \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). For economic purposes, floral industry mostly utilizes cut flowers, loose flowers, potted- flowering and foliage plants. Further ornamental grasses, trees, shrubs and annuals are also grown, which fulfil the aesthetic needs and also form an integral part of ecosystem. Cut flowers in ornamentals like phlox have more market value, followed by flowering pot plants, nursery crops and trees (Shibata \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). \u003cem\u003ePhlox drummondii\u003c/em\u003e Hook. is an important ornamental species and a mark of eternal beauty in the genus \u003cem\u003ePhlox\u003c/em\u003e, which belongs to family Polemoniaceae comprising Ca. 67 annual and perennial species. Plants of \u003cem\u003eP. drummondii\u003c/em\u003e are grown all over the world because of its beautiful and attractive flowers, which occur in shades of purple, pink, Crimson, red, scarlet, violet and many other intermediate shades. Flowers are fragrant, trumpet shaped, 5- lobed with short narrow corolla tube appearing in clusters (broad flat-topped cymes) at the stem ends. Additionally, central eye of the flower emerging from corolla tube differs in colour from the rest of petals making it more attractive. Stigma and style with ovary emerge from the base of five epipetalous anthers growing up to the upper wall of corolla tube (Grant and Grant \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1965\u003c/span\u003e). Leaves as well as stem are soft, hairy, and sticky. \u003cem\u003eP. drummondii\u003c/em\u003e adores border clumps, central flower beds of lawns, window boxes, flower vases, hanging baskets, and sold as excellent cut flowers (Bailey \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1950\u003c/span\u003e ; Hay and Synge \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1969\u003c/span\u003e; Herman \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1973\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eA winter annual \u003cem\u003eP\u003c/em\u003e. \u003cem\u003edrummondii\u003c/em\u003e is native to grasslands and open woods of Central and Eastern Texas. The genus name \u003cem\u003ePhlox\u003c/em\u003e is derived from the Greek word \u0026ldquo;phlox\u0026rdquo;, meaning flame, in context of its intense flower colours. In 1835, Thomas Drummond originally collected seeds of this plant in Texas and subsequently sent them to England from where they were distributed to nurserymen in different European and other countries world over (Levin \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1977\u003c/span\u003e). Considering the striking features of \u003cem\u003eP. drummondii\u003c/em\u003e, its ornamental potential has not been exploited to full potential owing to sensitivity towards water stress, temperature, and fast maturing habit thus imparting limitations in its multiplication and floriculture trade. Even conventional practices like selection, sexual crossing, back crossing and mutation breeding could overcome these impediments as well as widen its genetic base only to a certain extent. Attempts needed to be initiated for achieving the horticultural excellence in \u003cem\u003eP. drummondii\u003c/em\u003e using other non-conventional procedures (Razdan et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, Razdan-Tiku et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Application of genetic transformation procedures is one of the biotechnological approaches that could be utilized to supplement the efforts of existing conventional methods. Through transformation procedures the potential to incorporate novel characters in plants is possible which may otherwise appear recalcitrant using conventional breeding procedures (Ali et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, Sharma et al. 2017). Molecular-based gene transformation technology in ornamentals has brought changes in plant morphology (Chandler and Lu \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2005\u003c/span\u003e, Casanova et al.2005), flower shape (Casanova et al.2005), flower colour (Meyer et al.,1987, Tanaka et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2005\u003c/span\u003e, Debener and Winkelmann \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2010\u003c/span\u003e, Chandler and Brugliera \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, Umemoto and Toguri \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2012\u003c/span\u003e ), flower fragrance (Mori et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2004\u003c/span\u003e., Dudareva \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2002\u003c/span\u003e., Underwood and Clarke \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), pest as well as disease resistance (Milosevic et al., 2011a, 2011 b, Milosevic et al., 2015 ), stress tolerance ( Lojic at al., 2015), and increased vase or post-harvest life necessary for cut flowers (Chandler and Sanchez \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Transgenic breeding in major cut flower crops like roses (Kim et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2004\u003c/span\u003e, Katsumoto et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), gladiolus (Graves and Goldman \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1987\u003c/span\u003e, Kamo et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2009\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) carnations (Lu et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1991\u003c/span\u003e, Holton et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1993\u003c/span\u003ea, Meng et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) and chrysanthemums (Kazeroonian et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) was reported to have improved important traits in these ornamentals thus providing huge profits and avenues (Sharma et al. 2017).\u003c/p\u003e\u003cp\u003eIn the present study, \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e-mediated genetic transformation procedure has been developed with an objective of producing \u003cem\u003eP. drummondii\u003c/em\u003e plants with new improved floral traits since this technology acts as powerful tool for delivery of alien gene(s) of interest inside the host plant for integration with the host plant nucleus. Needless to state that a wide range of plant species have been transformed through application of this procedure for improvement of traits of agronomic, horticultural and ornamental value, including important traits in tree species. Moreover, an added advantage in this technology is that transfer of small copy number of T- DNA is good enough to result in stable integration of foreign DNA into host plant genome (Hwang et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Considering these aspects an efficient \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated genetic transformation protocol was developed in present study for first time in \u003cem\u003eP. drummondii\u003c/em\u003e or the genus \u003cem\u003ePhlox\u003c/em\u003e.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cp\u003e\u003cstrong\u003ePlant material found suitable for genetic transformation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor genetic transformation of \u003cem\u003eP. drummondii\u003c/em\u003e 3-week-old callus induced from zygotic embryo and multiplied by repeated subculturing on CRMM (callus regeneration and multiplication medium) was found suitable (Fig. 1) although calli derived from cotyledonary leaves were also tried. CRMM constituted of MS basal medium + 3% sucrose + BAP (5 \u0026micro;M) + NAA (10 \u0026micro;M). Prior to transformation the zygotic embryo induced callus was cut into 2-3 mm pieces and then they were pre-cultured on shoot regeneration medium (SRM), which comprised of MS basal salts +3% sucrose + BAP (15 \u0026micro;M) + IAA (2.5 \u0026micro;M) for zero days, 1-2 days, 2-3 days and 4-5 days.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBacterial strain and binary vector\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain PGV2260 was used for transforming \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;embryogenic calli of \u003cem\u003eP. drummondii\u003c/em\u003e, which carried \u003cem\u003eGUS\u003c/em\u003e binary plasmid vector PPZP20KWT with a marker gene and Nos promoter NPT-II:\u0026nbsp;OcspA cassette (conferring kanamycin resistance) and a reporter gene \u003cem\u003eGUS\u003c/em\u003e (intron)- 35S promoter cassette (Fig. 2).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003e\u003cem\u003eAgrobacterium\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e-mediated genetic transformation\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAgrobacterium\u003c/em\u003e strain PGV2260, carrying \u003cem\u003eGUS\u003c/em\u003e binary vector, was cultured overnight in 100 ml conical flask containing 30 ml YEM (yeast extract mannitol medium) + antibiotics (carbenicithin, spectinomycin and refampicin) on a shaker, 250 rpm, at 28\u0026deg;C. \u0026nbsp;OD (Bacterial density) of the culture was checked intermittently after the gap of few hours and cultures at different ranges of OD (0.1- 1.0) at A600 were tried for different sets of transformation experiments (Table 1). After adjusting the OD of the bacterial culture, it was transferred to autoclaved Oakridge tubes for centrifugation at 3000-4000 rpm for 10 min at 28\u0026deg;C. The supernatant was discarded and the pellet dissolved in MSO (MS liquid medium without hormones) + acetosyringone 100 \u0026micro;M. The callus pieces in this medium were then transferred to an autoclaved screw- capped wide \u0026ndash; mouthed jam bottle and to each piece was added to \u003cem\u003eAgrobacterium\u003c/em\u003e culture \u0026nbsp;of different OD. \u0026nbsp;The bottles were gently stirred manually so that the callus gets properly infected with bacterial suspension. Along with the OD, infection time of bacteria was also standardized ranging from 5-30 min (Table 2). After pouring off the \u003cem\u003eAgrobacterium\u003c/em\u003e suspension, the callus pieces were placed on autoclaved blotting paper. Blotted dry calli were then transferred to co-culture medium comprising SRM + acetosyringone (100 \u0026micro;M) for 2-3 days. The co-cultivated calli were finally transferred to selection medium containing SRM + augmentin (300 mg/l) + kanamycin (20- 50 mg/l) \u003cstrong\u003e(Table S1).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKanamycin assay to optimize the concentration for stringent selection for the recovery of the transformed callus in selection medium\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSubculturing was done on the same selection medium after every 15 days up to 3 cycles of selection (Total 1\u0026frac12; months on selection medium). Untransformed callus did not survive on selection medium (Fig. 3A) whereas transformed callus could only regenerate on the selection medium (Fig.3 B). \u0026nbsp;After 1\u0026frac12; months, callus was transferred to SRM ll containing MS basal salts +3% sucrose +BAP 10 \u0026micro;M +augmentin 300 mg (Fig. 3C). After 1 month of subculturing on SRM ll, 1-2 cm long shoots excised from regenerating callus were transferred to same medium but without kanamycin (Fig. 3 D).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransformed shoot multiplication, rooting and transplantation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor multiplication of transformed shoots from the various experiments two approaches were followed. The small shoots (Ca 1-2 cm long) were individually transferred to fresh medium (SRM II without kanamycin) for further proliferation. Second, the longer shoots that had grown up to 4-5 cm, were cut into single node segments and each node segment transferred to fresh medium (SRM II without kanamycin) \u0026nbsp;for further growth and multiplication (Fig. 3E). The number of shoots obtained at the end of a multiplication cycle was regarded as the rate of shoot multiplication. After 2- weeks shoots \u003cem\u003ein vitro\u003c/em\u003e underwent flowering (Fig. 3F). However, for rooting, shoots measuring Ca. 4 cm, with 3-4 nodes, \u0026nbsp;from transformed calli before \u003cem\u003ein\u003c/em\u003e \u003cem\u003evitro\u003c/em\u003e flowering were excised and transferred to rooting medium (MS+IAA 7.5 \u0026micro;M ). The rooted plants (Fig. 3G) were washed in tap water to remove agar and then transplanted to soil in small plastic pots containing autoclaved soil as per procedure followed by Razdan-Tiku et al. (2014). The soil composition was one part vermiculate and 3 parts of garden soil. The transformed plants in pots were then covered with polythene bags with small holes (2 mm diameter) to maintain high humidity at least for 20 days. These plants were kept in tissue culture room with temperature 25 to 27 \u0026deg;C, relative humidity 50 to 60%, and 16 h photo period. After one month of acclimatisation, these plants were transferred to bigger pots (Fig. 3H) and subsequently shifted to transgenic green-house.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGUS Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnalysis of GUS activity in transformed plant parts was performed by histochemical assay as described by Jefferson et. al. (1987). Transformed calli, leaves and roots were incubated in 500 \u0026mu;L of GUS assay buffer [10 mg of X- gluc (5 \u0026ndash; bromo 4 \u0026ndash; chloro \u0026ndash; 3 indolyl \u0026ndash; glucuronide ) in 2 ml dimethyl formamide ] , \u0026nbsp; 2 ml 5 mM potassium ferrocyanide , and 20 ml 0.1 M sodium phosphate buffer , at 37\u0026deg;C overnight . The solution was removed next morning and incubated materials were rinsed in 70% ethanol and examined for detection of blue colour (Fig. 4 A-C) before being stored in 40 % glycerol. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular analysis of putative transformants of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eP.\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;drummondii\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003etransgenic plants raised were analysed by PCR for the integration of the transgene. Using Techne PCR machine (U.K.), DNA was isolated from leaves in liquid nitrogen by a CTAB method as described by Doyle and Doyle (Doyle and Doyle 1987). About 100 ng of genomic DNA from untransformed control and the transgenic lines was taken separately, and mixed with 100 mM forward and reverse primers (Suppmentary Table No 2) in 7.5 \u0026mu;l of PCR buffer (10 mM of Tris HCl , 50 mM \u0026nbsp;KCl , 2 mM MgCl\u003csub\u003e2\u0026nbsp;\u003c/sub\u003e, \u0026nbsp;100 \u0026mu;M dNTPS mix) and 0.1 Taq DNA polymerase (Biotools , Spain), pH 8.3. The volume of mixture was raised up to 25 \u0026mu;l with SDW. DNA amplification was carried out in the thermal cycler programmed for 40 cycles as follows: 1 cycle of 5 min at 94 \u0026deg;C, followed by 39 cycles, each of denaturation at 94\u0026deg;C for 1 min, annealing for 1 min at 53 \u0026deg;C, and synthesis at 72 \u0026deg;C for 2 min, and finally 1 cycle of 10 min at 72 \u0026deg;C. After completion of the PCR 2 \u0026mu;l of 10x loading buffer was added to each of the samples. The amplification product was separated by electrophoresis in 1.2 % agarose gel containing 0.05 \u0026mu;l/ml ethidium bromide (Roche Diagnostic Gmbh, Germany) in 0.5x TBE buffer (0.045 M Tris- borate and 1mM Na\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eEDTA). The \u003cem\u003eHind\u003c/em\u003eIII digested \u0026lambda; DNA was loaded in one of the lanes to serve as molecular size markers. After agarose gel electrophoresis (AGE), the gel (Fig.5 A-B) was photographed in UV light (LKb Pharmalia, USA). PCR experiment was repeated with the same samples 2 -3 times for the confirmation of results. The details of the primers used for \u003cem\u003enpt\u003c/em\u003e-II gene and \u003cem\u003eGUS\u003c/em\u003e are given in Table S2.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSouthern analysis with \u003cem\u003eNPT\u003c/em\u003e-II gene as a probe\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSouthern blot hybridization was performed to detect the \u003cem\u003eNPT\u003c/em\u003e-II gene integration. Genomic DNA (10 \u0026micro;g) from different PCR positive transgenic lines as well as the untransformed control was digested with \u003cem\u003eEco\u003c/em\u003eRI, \u0026nbsp;which released the \u003cem\u003eNPT\u003c/em\u003e-II gene from the T-DNA and blots were prepared (on Nylon membrane Sigma, USA) , hybridised and washed with high stringency as per the standard protocol (Sambrook et al. , 1989). The \u003cem\u003eNPT\u003c/em\u003e-II gene probe was prepared by using the random priming kit (Takara, Japan) as per the manufacturer\u0026rsquo;s guidelines. The probe was denatured before adding to the pre-hybridisation buffer. After hybridisation, membrane was washed and then exposed to X-ray film (Kodak); (Fig. 5C). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEach experiment was repeated thrice, data subjected to analysis of variance (ANOVA), and means were compared using SAS computer software according to Anderson \u0026ndash; Cook 2004.\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eFor genetic transformation of \u003cem\u003eP. drummondii\u003c/em\u003e, an efficient protocol has been devised based on the \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation method. The details of procedures followed according to protocol are described in Section on material and methods. Based on this protocol, the following parameters were determined that facilitated efficient gene transformation in \u003cem\u003e\u0026nbsp;P.\u003c/em\u003e\u003cem\u003e\u0026nbsp;drummondii\u003c/em\u003e. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOptimum Density (OD) of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eAgrobacterium\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eand infection period\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAgrobacterium\u003c/em\u003e \u003cem\u003etumefaciens\u003c/em\u003e GV2260 strain carrying the \u003cem\u003eGUS\u003c/em\u003e gene construct pPZp200 (Fig. 2) cultured in YEM medium at different ODs (0.1 \u0026ndash; 1.0; Table 1) demonstrated the most effective transformation occurred using this bacterial strain at the optimum OD (0.3-0.5), A600. \u0026nbsp;Explants infected with bacteria at same OD for different infection periods (5 min \u0026ndash; 30 min) demonstrated gene transformation occurred at best at infection time of 10 min (Table 2). On higher OD at same infection time though the number of GUS positive calli were more but the number of transgenic plants regenerated happened to be very less. At lower OD the percentage of GUS transformed calli decreased with reduced potential for plant regeneration (Table 1). Similarly, with the increase in infection time (\u0026gt;10 min) plants regenerated from transformed calli had negligible or very low regeneration response (Table 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreculture of untransformed callus\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrior to transformation as already mentioned, the untransformed callus was pre-cultured on shoot regeneration medium (SRM) for zero days, 1 -2 days, 2 -3 days and 4 -5 days to inculcate its better transforming efficiency. \u0026nbsp;Most suitable callus for most effective genetic transformation was the one pre-cultured on SRM for 2-3 days. \u0026nbsp;It contained high levels of cytokinin and low levels of auxin. \u0026nbsp;Pre-cultured callus pieces (2-3 mm) were subsequently transformed by immersing in MSO medium containing \u003cem\u003eArgobacterium\u0026nbsp;\u003c/em\u003e(carrying gene construct) at standardised OD + Acetosyringone (100 \u0026micro;M) in a wide mouthed -autoclaved bottle and manually shaken for standardised period of 10 min. \u0026nbsp;The infected calli were subsequently cultured on co-cultivation medium (SRM + acetosyringone (100 \u0026micro;M) for 2 \u0026ndash; 3 days. These co-cultivated calli were finally transferred\u0026nbsp;to a selection medium containing SRM + augmentin (300 mg/l) + kanamycin (30 mg/l). Although selection media with different concentrations of kanamycin were tested, proper selection of transformed callus (Fig. 3 B) occurred at antibiotic concentration 30 mg/l which regenerated more transformed shoot buds. At kanamycin concentrations below 30 mg/l although percentage callus survival was high but plants regenerated were mostly untransformed. At kanamycin concentrations above 30 mg/l, most calli turned pale -yellow or necrotic and died without showing any signs of regeneration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlant regeneration from transformed callus\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTransformed callus were taken out from the selection medium and then transferred to SRM I containing MS salts + BAP (15 \u0026micro;M) + IAA ( 2.5 \u0026micro;M) + augmentin ( 300 mg/l ) + kinetin ( 30 mg/l ) and sub-cultured on same medium , every 15 days for at least one and a half month till small shoots appeared on the callus ( Fig.\u0026nbsp;3 C) . Transformation frequency and regeneration frequency of\u003cem\u003e\u0026nbsp;P. drummondii\u003c/em\u003e transgenic plants were 76% and 62%, respectively (Table 3). GUS assay was done with 2-week-old calli growing on selection medium to test the incorporation of GUS gene (Fig.4 A) and percentage calli showing GUS activity calculated. Highest percentage of GUS activity was shown to be around 70% when infection time was kept at 10 min and bacterial density 0.3 \u0026ndash; 0.5, A600 (Table 2). After shoot bud formation callus was transferred to shoot regeneration medium II (SRM II). This regeneration medium contained MS salts + 3% sucrose + BAP (10 \u0026micro;M) + augmentin (300 mg/l) + kinetin (30 mg/l). After one month of sub-culturing on SRM II, 2 cm long shoots were excised from the base of callus and transferred to the same medium but without adding augmentin and kanamycin. This resulted in further elongation and multiplication of shoots (Fig. 3 D-E). \u003cem\u003eIn vitro\u003c/em\u003e flowering of transformed shoots also occurred on SRM II (Fig. 3F). Therefore, it became essential to induce rooting in transformed shoots on the medium MS + IAA (7.5 \u0026micro;M) before \u003cem\u003ein vitro\u003c/em\u003e flowering (Fig 3G). The plantlets were finally transplanted to soil in pots (Fig. 3H) as per procedure described in material and methods.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMorphology of Transgenics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIt was observed that floral and other characteristics of transgenic \u003cem\u003eP. \u0026nbsp;drummondii\u003c/em\u003e plants were quite different from control (normal /untransformed) plants. Height of transgenic plants was shorter than the normal plants. Stem and leaves of transgenics were very thick and dark green in contrast\u0026nbsp;to the thin and light colour of stem and leaves of normal plants. Morphology of flowers in particular showed huge variations in different transgenic lines in comparison to normal untransformed plants (Fig. 6. A-E). In the genus \u003cem\u003ePhlox\u003c/em\u003e (including \u003cem\u003eP.drummondii\u003c/em\u003e) as well as in family Polemoniaceae to which it belongs, flowers are radially symmetrical and pentamerous (each floral whorl consists of 5 sepals and 5petals fused into a cup, or tube-shaped structure (Fig. 6A). Stamens are 5, epipetalous, the length of all anthers and their filaments being almost same they are attached at the base of petals and reach up to the mouth of the corolla tube (Fig. 6C). However, in our investigations on GUS transformed \u003cem\u003eP. drummondii\u003c/em\u003e plants the flower structure was found paradoxically different as it showed marked variations due to the occurrence of partitions in the corolla tube resulting in petals becoming free except at the base (more like a polypetalous condition; Fig. 6 B). Corolla tube length in the control or untransformed plants being Ca. 1cm, the length of corolla tube in transgenic plants on contrary was Ca. 2 cm. While in control plants, epipetalous anthers reach the same height (Fig. 6 C), the transgenic plants exhibited variation in their height. Two stamens being long are slightly exerted towards upper mouth of the corolla tube, another two stamens are of medium size and reach till the middle of the corolla tube, whereas the fifth stamen is short and appears at the base of the corolla tube (Fig 6 D). Different transgenic lines also showed variations in flower colour ranging from light pink to reddish pink (Fig. 6 E2 and E5). Further, shape of the petals varied as they are broader in control plants whereas transgenic flowers had narrow petals pointed at the top (club shaped; Fig. 6 E2, E4 and E6). Another striking feature being that few transgenic lines showed flowers with an increased number of petals (6-8; Fig. 6 E3, E5 and E6) thus defying symmetrical pentamerous condition of 5 petals in control plants (Fig. 6 E1). GUS assay was also performed in shoots and roots of both transgenics and control. Appearance of blue colour confirmed the transgenic nature of plants (Fig. 4 B, C).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular characterization of putative transgenics\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs mentioned earlier 70 % of the calli infected by \u003cem\u003eAgrobacterium\u003c/em\u003e carrying vector T-DNA \u003cem\u003eGUS\u003c/em\u003e and \u003cem\u003eNPT\u003c/em\u003e-II genes construct at 0.3- 0.5 OD, A600, and infection time of 10 mins showed maximum GUS activity (Table. 2).\u003c/p\u003e\n\u003cp\u003eThe putative transgenics were characterised for the integration of vector T-DNA \u003cem\u003eGUS\u003c/em\u003e and \u003cem\u003eNPT\u003c/em\u003e-II genes\u0026nbsp;into the host plant genome by doing PCR. The PCR was performed with the DNA isolated from untransformed (control) and transformed lines using \u003cem\u003eNPT\u003c/em\u003e-II (marker) and \u003cem\u003eGUS\u003c/em\u003e (reporter) gene specific primers. The amplified product of both the genes was 500 bp gene fragment which was found in 70% putative transformants (Fig.5 A, B), thus confirming their transgenic nature. PCR positive lines were further checked by Southern hybridization using \u003cem\u003eNPT\u003c/em\u003e-II gene specific probe (Fig 5C). For Southern hybridization, genomic DNA isolated from transformed and untransformed (control) plants were digested with the restriction enzyme \u003cem\u003eEco\u003c/em\u003eRI, which cuts the T-DNA fragment of 2.3 kb containing \u003cem\u003eGUS\u003c/em\u003e- \u003cem\u003eNPT\u003c/em\u003e-II gene (Fig. 5C). The results obtained from molecular analysis correlated with morphology-based identification of putative transgenic lines regenerated from transformed embryogenic calli of \u003cem\u003eP. drummondii\u003c/em\u003e.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eDevelopment of ornamental varieties that have much improved floral characters is\u003c/p\u003e\u003cp\u003eone of the important aspects of floriculture. Biotechnological approaches along with the classical breeding methods are being used to improve floral or other important traits in ornamentals to supplement their commercial value. Improvements desired in flowers generally relate to fragrance, morphological appearance of sepals and petals, their colour, diseases and stress resistance, enhanced vase life, etc. Transgenic strategies seem to have immense potential to produce novel flower phenotypes which otherwise are not found in nature or produced through traditional breeding procedures (Kim \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In this context the improvement of flower traits through gene transformation has been attempted in ornamental flowering plants like \u003cem\u003eGerbera\u003c/em\u003e (Chung et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), \u003cem\u003ePetunia\u003c/em\u003e (Bashandy and Teeri \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), rose (EL-Tarras et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), carnation (Yantcheva et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e1997\u003c/span\u003e) and chrysanthemum (Shinoyama et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) with some success. Insertion of alien desired genes into a plant requires development of a reproducible protocol which was prime objective of this study for \u003cem\u003eP. drummondii\u003c/em\u003e. Till date, no such studies have been carried out on any species of the genus \u003cem\u003ePhlox\u003c/em\u003e. It required many efforts to devise and standardize each and every step of genetic transformation of \u003cem\u003eP. drummondii.\u003c/em\u003e Certain very important parameters had to be standardized, such as choice of explant, selection of \u003cem\u003eAgrobacterium\u003c/em\u003e strain with binary vector gene construct, infection time of this bacterium, optical density (OD) of the bacterial suspension culture, concentration of selection marker (kanamycin) and acetosyringone in the medium during co-cultivation, time period as well as temperature requirement for co-cultivation, and type of bacteriostatic agent used.\u003c/p\u003e\u003cp\u003eTransformation protocol for \u003cem\u003eP. drummondii\u003c/em\u003e required the development of an efficient tissue culture technique for effective regenerative response of explant and transformed tissues. Selection of explant was important as different explants (leaf discs, florets, petals, stem cuttings, roots, cotyledonary leaves, embryogenic calli etc.) showed difference in transformation and regeneration potential. Elomma and Holton (1994) also observed this phenomenon in other ornamental plants (\u003cem\u003eGerbera\u003c/em\u003e, chrysanthemum, etc.). In \u003cem\u003eP. drummondii\u003c/em\u003e zygotic embryo- derived callus was most suitable for \u003cem\u003eAgrobacterium\u003c/em\u003e- mediated transformation using the protocol described and standardized in this paper. Preculture of calli proved an important parameter because most of the cells undergo rapid divisions and thus remain in an exponential growth phase which makes them more amenable to transformation. Such inferences were also reached based on the evidences of researches on other plant systems (Hiei et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Vijayachandra et. al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). According to Kumria et al. (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) preculture of callus on high cytokinin (BAP 15 \u0026micro;M) or low auxin (IAA 2.5 \u0026micro;M) medium enhances the cell division as well as promotes \u003cem\u003eAgrobacterium\u003c/em\u003e infection. OD of the bacterium suspension carrying gene construct was found equally critical. At lower OD (0.1\u0026ndash;0.3) GUS activity was less pronounced depicting lower or negligible transformation frequency, while at higher OD (0.5-1.0) the extensive blackening of transformed calli proved detrimental for regeneration. Therefore, the agrobacterium strain PGV2260 suspension used in present study showed best GUS activity at O.D (0.3\u0026ndash;0.5). The infection period of bacterium also varied and 10 min was found optimum for infection and transformation. Importantly, keeping the dry infected calli on SRM for at least 2 days was one more step required for successful transformation before shifting them to selection medium (SRM\u0026thinsp;+\u0026thinsp;kanamycin). Another interesting observation in our studies has been that the transformed callus showed no GUS activity in absence of acetosyringone in the medium. Therefore, acetosyringone (100 \u0026micro;M) in the medium was optimum and essential to promote both, GUS activity, and shoot regeneration. Acetosyringone, a phenolic compound, is emitted in wounded plant tissues which seems important for \u003cem\u003eAgrobacterium\u003c/em\u003e to enter the host plant tissue-cell cytoplasm and transmit T-DNA along with associated proteins (T-complex) into the host pant genome for stable integration (Lacroix and Citovsky \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Cefotaxime and augmentin were tried as bacteriostatic in selection medium. Augmentin (300 mg/l) proved better as it restricted overgrowth of \u003cem\u003eAgrobacterium\u003c/em\u003e and supported regeneration of transformed calli as well as shoot bud differentiation on selection medium SRMI (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Differentiation of shoot buds on the transformed callus also occurred on the same selection medium but for further shoot development (elongation and multiplication of shoots) a different shoot regeneration medium (SRMII) was standardized and used. While developing the genetic transformation protocol for \u003cem\u003eP. drummondii\u003c/em\u003e some very interesting and remarkable results were achieved. Transformed plants showed different floristic characteristics from the normal plants. A major change was the trend towards polypetalous condition of the flower (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e6\u003c/span\u003eB) as against the floral architecture of gamopetalous tubular corolla in \u003cem\u003ePhlox drummondii\u003c/em\u003e, and other members of the family Polemoniaceae. Another variation in floral architecture observed was that in some GUS transformed \u003cem\u003eP. drummondii\u003c/em\u003e plants the number of petals increased (6\u0026ndash;8) as against normal 5 petals found in normal plants (Fig.\u0026nbsp;6E3, E5, E6). Petal shape also changed along with its colour. The colour was more intensified and there was a paradigm shift in the position of anther filaments (Fig.\u0026nbsp;6E2 and E5). Reason behind these variations could be the impact of \u0026ldquo;insertional mutagenesis\u0026rdquo; in GUS transformed plants of \u003cem\u003eP. drummondii\u003c/em\u003e leading to formation of new variant lines of this ornamental. Insertional mutagenesis is an alternative means of altering gene function caused by insertion of foreign gene into the genome of host plant. The foreign T-DNA carrying GUS : NPT-II by insertion changes the host plant gene expression with GUS acting as a marker for identification of variations in morphological characteristics of transgenic plants. Similar observations were reported in \u003cem\u003eArabidopsis\u003c/em\u003e due to the involvement of either transposable elements or T-DNA (Krysan et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Bundock et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Researchers have substantiated that T-DNA acts as an insertional mutagen and such mutations are chemically and physically stable through multiple generations (Wisman et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). T-DNA inserted mutants could be cloned easily as reported by Sangwan and Sangwan-Norreel (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e1989\u003c/span\u003e). Several large- scale T-DNA insertional mutagenesis projects are being undertaken for cloning mutant genes (Gelvin \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Jupe et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2019\u003c/span\u003e and Pucker et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn \u003cem\u003eP. drummondii\u003c/em\u003e, the plausible explanation for noticeable variation in floral architecture of transgenic plants regenerated in present investigation could be that foreign T- DNA by insertion into host plant genome must have altered the functional genes responsible for expression of floral characteristics, thus almost warranting a rechristening of such transgenic taxa that show nearly polypetalous trend as against gamopetalous nature of normal plants in \u003cem\u003ePhlox\u003c/em\u003e. Therefore, in present study a protocol devised successfully transformed \u003cem\u003eP. drumondii\u003c/em\u003e resulting in transgenic plants that have striking variations in floral features hitherto not reported.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eGenetic improvement of ornamentals is an ongoing process. Over the years new varieties of ornamental plants have been produced by conventional procedures, such as selection, cross hybridization, or mutation breeding. Success achieved in ornamentals following these traditional procedures has remained restricted. As a result, attempts are being made to apply non-conventional \u003cem\u003ein vitro\u003c/em\u003e methods. GM technology could be one such tool useful for improvement of traits in original ornamental cultivars. Gene transformation techniques are known to modify target traits due to direct integration of bacterial T-DNA in to host genome. These techniques thus are used as a supplement to traditional breeding methods. In the present study, an efficient \u003cem\u003eAgrobacterium\u003c/em\u003e- mediated genetic transformation protocol using reporter (GUS) and marker genes (NPTII) was developed for \u003cem\u003eP. drummondii\u003c/em\u003e, an important ornamental, which impacted its floral structure. This transformation procedure is first attempt for any species in the genus \u003cem\u003ePhlox\u003c/em\u003e and has potential for transfer of genes of interest in other \u003cem\u003ePhlox\u003c/em\u003e species, e.g., polyamine biosynthesis genes for delaying senescence and increasing the vase life. The new transformed variant varieties could be propagated vegetatively or sexually for perpetuation of new traits.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCRMM \u0026ndash; Callus Regeneration and Multiplication medium\u003c/p\u003e\n\u003cp\u003eSRM \u0026ndash; Shoot regeneration medium\u003c/p\u003e\n\u003cp\u003eMS \u0026ndash; Murashige and Skoog medium\u003c/p\u003e\n\u003cp\u003eYEM \u0026ndash; Yeast Extract Mannitol medium\u003c/p\u003e\n\u003cp\u003eSRM I \u0026ndash; Shoot Regeneration medium I\u003c/p\u003e\n\u003cp\u003eSRM II - Shoot Regeneration medium II\u003c/p\u003e\n\u003cp\u003eMSO - Murashige and Skoog liquid medium\u003c/p\u003e\n\u003cp\u003eGUS: \u0026ndash; Glucuronidase\u003c/p\u003e\n\u003cp\u003eNPT-II: \u0026ndash; Neomycin Phosphotransferase II\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eConflict of interests\u003c/h2\u003e\u003cp\u003eAuthors declare that they have no conflict of interest.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor\u0026rsquo;s contributions\u003c/h2\u003e\u003cp\u003eSNR and MKR conceived the idea and MVR designed the experiments. ART carried out the experiments, generated the data. ART and MVR analysed the data. ART wrote the manuscript. MVR, SNR and MKR edited, and finalized the manuscript. All the authors read and approved the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e\u003cp\u003eI thank Prof. M.V. Rajam, Department of Genetics \u0026amp; Prof. S.M. Raina, Department of Botany, University of Delhi for providing facilities for providing facilities to conduct this study.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e\u003cp\u003eData will be available on request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAli N, Muhammad A, Jianming D, Noreen K, Tayyaba S, He S (2017) \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Biotechnological advancements for improving floral attributes in ornamental plants. Front. 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Cult. 78:107\u0026ndash;111.\u003c/li\u003e\n \u003cli\u003eKim JB (2020). Current status on applications of conventional breeding techniques and biotechnological system in ornamentals. J. Plant Biotechnol. 47: 107-117.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eKrysan PY, Young JC, Sussman M (2000). T-DNA as an insertional mutagen in Arabidopsis. Plant Cell 11: 2283-2290.\u003c/li\u003e\n \u003cli\u003eKumria R, Waie B, Rajam MV (2001). Plant regeneration from transformed embryogenic callus of an elite indica rice via \u003cem\u003eAgrobacterium\u003c/em\u003e. Plant Cell Tiss. Org. Cult.67: 63-71.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eLacroix B, Citovsky V (2013) The roles of bacterial and host plant factors in \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated genetic transformation. Intern. J. Dev. Biol. 57:467-481.\u003c/li\u003e\n \u003cli\u003eLevin DA (1977) The organization of genetic variability in \u003cem\u003ePhlox drummondii\u003c/em\u003e.\u0026nbsp;Evolution 31: 477\u0026ndash;494.\u003c/li\u003e\n \u003cli\u003eLojić M, Vinterhalter B, Subotić A and Vinterhalter D (2015). Differences in regenerative capacity of Oriental lily (\u003cem\u003eLilium\u003c/em\u003e sp.) cultivars.\u0026nbsp;Botanica Serbica 39:159-167.\u003c/li\u003e\n \u003cli\u003eLu CY, Nugent G, Wardley-Richardson T , Chandler SF , Young R, Dalling MJ (1991)\u003cem\u003e.\u003c/em\u003e \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation of carnation (\u003cem\u003eDianthus caryophyllus\u003c/em\u003e L.). Nat. Biotechnol. 9:864\u0026ndash;868.\u003c/li\u003e\n \u003cli\u003eMeng L, \u0026nbsp;Song J , \u0026nbsp;Sun S , Wang C (2009). The ectopic expression of \u003cem\u003ePttKN1\u003c/em\u003e gene causes pLeiotropic alternation of morphology in transgenic carnation (\u003cem\u003eDianthus caryophyllus\u003c/em\u003e L.). Acta Physiolog. Plant. 31:1155-1164.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eMeyer P, Heidmann I, Forkmann G, Saedler H\u0026nbsp;(1987). A new petunia flower colour generated by transformation of a mutant with a maize gene. Nature 330:677-678.\u003c/li\u003e\n \u003cli\u003eMilo\u0026scaron;ević S, Cingel A, Ninković S, Simonović A, Nikolić D, Jevremović S, Subotić, A. (2011b): Genetiĉka transformacija Impatiens hawkerii Bull. Pomoću \u003cem\u003eAgrobacterium\u003c/em\u003e \u003cem\u003etumefaciens\u003c/em\u003e c58c1pac1. XVI savetovanje o biotehnologiji, sa meċunarodnim uĉe\u0026scaron;ćem, Zbornik radova, Ĉaĉak. 16:471-476.\u003c/li\u003e\n \u003cli\u003eMilo\u0026scaron;ević S, Lojić M, Antonić D, Cingel A, Subotić A (2015): Changes of antioxidative enzymes in \u003cem\u003eImpatiens walleriana\u003c/em\u003e L. shoots in response to genetic transformation. Genetika 47:71- 84.\u003c/li\u003e\n \u003cli\u003eMilo\u0026scaron;ević S, Subotić A, Bulajić A, Đekić I, Jevremović S, Vuĉurović A (2011): Elimination of TSWV from \u003cem\u003eImpatiens hawkerii\u003c/em\u003e Bull. and regeneration of virus-free plant. Electr. J. Biotechnol. 14:1-10.\u003c/li\u003e\n \u003cli\u003eMori S, Kobayashi H, Hoshi Y, Kondo M, Nakano M (2004) Heterologous expression of the flavonoid 3\u0026apos;,5\u0026apos;-hydroxylase gene of \u003cem\u003eVinca major\u003c/em\u003e alters flower color in transgenic \u003cem\u003ePetunia hybrida\u003c/em\u003e. Plant Cell Rep. 22:415-421.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003ePucker B, Kleinb\u0026ouml;lting N, Weisshaar B (2021) Large scale genomic rearrangements in selected \u003cem\u003eArabidopsis thaliana\u003c/em\u003e T-DNA lines are caused by T-DNA insertion mutagenesis. BMC Genomics 22:599.\u003c/li\u003e\n \u003cli\u003eRazdan-Tiku A, Razdan M, Raina SN (2014) Production of triploid plants from endosperm cultures of \u003cem\u003ePhlox drummondii\u003c/em\u003e. Biol. Plant.58:153-158.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eRazdan A, Razdan M, Rajam MV, Raina SN (2008). Efficient protocol for in vitro production of androgenic haploids of \u003cem\u003ePhlox drummondii\u003c/em\u003e. Plant Cell Tiss. Org. Cult. 95:245-250.\u003c/li\u003e\n \u003cli\u003eSambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual (2nd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.\u003c/li\u003e\n \u003cli\u003eSangwan RS, Sangwan-Norreel BS (1989). Genetic transformation and plant improvement. \u0026nbsp;The Impact of Biotechnology on Agriculture\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(Kluwer Academic Publishers), pp. 299-337.\u003c/li\u003e\n \u003cli\u003eSharma R, Messar Y (2017). Transgenics in ornamental crops: Creating novelties in economically important cut flowers. Curr. Sci. 113:43-52.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eShibata M (2008) Importance of genetic transformation in ornamental plant breeding. Plant Biotechnol. 25:3-8.\u003c/li\u003e\n \u003cli\u003eShinoyama H, Aida R, Ichikawa H, Nomura Y, Mochizuki A (2012) Genetic engineering of chrysanthemum (\u003cem\u003eChrysanthemum morifolium\u003c/em\u003e): Current progress and perspectives. Plant Biotechnol. 29:323\u0026ndash;337.\u003c/li\u003e\n \u003cli\u003eTanaka Y, Katsumoto Y, Brugliera F, Mason J (2005) Genetic engineering in floriculture. \u0026nbsp;Plant Cell Tiss. Org. Cult. 80:1-24.\u003c/li\u003e\n \u003cli\u003eUmemoto N, Toguri T (2012) Peptide transporting to chromoplasts in petals and method of constructing plant having yellowish petals by using the same. Patent US 8143478 B2.\u003c/li\u003e\n \u003cli\u003eUnderwood BA, Clarke DG (2011) Transgenic ornamental crops. In Transgenic Horticultural Crops: Challenges and Opportunities (B. Mou and R. Scorza, eds), Boca Raton, FL: CRC press. pp. 55-82.\u003c/li\u003e\n \u003cli\u003eVijayachandra K, Palanichelvam K, Veluthambi K (1995) Rice scutellum induces \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e vir genes and T-strand generation. Plant Mol Biol. 29:125-133.\u003c/li\u003e\n \u003cli\u003eWisman E, Hartmann U, Sagasser M, Baumann E, Palme K, Hahlbrock K, Saedler H, Weisshaar B (1998) Knock-out mutants from an En-1 mutagenized \u003cem\u003eArabidopsis\u003c/em\u003e \u003cem\u003ethaliana\u003c/em\u003e population generate phenylpropanoid biosynthesis phenotypes. Proc. Natl. Acad. Sci. USA 95:12432-12437.\u003c/li\u003e\n \u003cli\u003eYantcheva\u0026nbsp;A, Vlahova M, Atanassova B, Atanassov A (1997) Direct organogenesis and plant regeneration of Carnation (\u003cem\u003eDianthus\u003c/em\u003e \u003cem\u003ecaryophyllus\u003c/em\u003e L.), Biotechnol. Biotechnol Equip.11:60-65.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1. \u0026nbsp;Influence of bacterial density (OD\u003csub\u003e600\u003c/sub\u003e) genetic transformation in \u0026nbsp;6-weeks-old callus of \u003cem\u003ePhlox drummondii\u003c/em\u003e on selection medium\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;and subsequent shoot regeneration from transformed callus\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 195px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eBacterial density (OD\u003csub\u003e600\u003c/sub\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 195px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ePercentage callus \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;showing GUS \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eactivity\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 195px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eAverage number of shoots obtained from tranformed explants\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 195px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.1 - 0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 195px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e50 \u0026plusmn; 0.10 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 195px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e30 \u0026plusmn; 2.10 d\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 195px;\"\u003e\n \u003cp\u003e0.3 - 0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 195px;\"\u003e\n \u003cp\u003e60 \u0026plusmn; 0.25 bc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 195px;\"\u003e\n \u003cp\u003e53 \u0026plusmn; 0.10 c\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 195px;\"\u003e\n \u003cp\u003e0.5 - 0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 195px;\"\u003e\n \u003cp\u003e80 \u0026plusmn; 0.50 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 195px;\"\u003e\n \u003cp\u003e20 \u0026plusmn; 0.35 de\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 195px;\"\u003e\n \u003cp\u003e0.7 - 1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 195px;\"\u003e\n \u003cp\u003e80 \u0026plusmn; 0.28 ad\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 195px;\"\u003e\n \u003cp\u003e\u0026nbsp; 0 \u0026plusmn; 0 e\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eMeans \u0026plusmn; SE, n=50; means in the same column followed by different letters are significantly different at P \u0026le; 0.05 and F = 96.56Anderson \u0026ndash; Cook 2004).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2. \u0026nbsp;Effect of the infection time on genetic transformation during \u0026nbsp;cocultivation, and subsequent shoot regeneration\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ein 6-week-old transformed callus of \u003cem\u003ePhlox drummondii\u003c/em\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eon selection medium\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 196px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eInfection time\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(Min)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 196px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ePercentage callus\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eshowing GUS \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eactivity\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 196px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eAverage number of\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eshoots obtained from\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eresponding explants\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 196px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 196px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e45 \u0026plusmn; 0.50 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 196px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e30 \u0026plusmn; 0.02 d\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 196px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 196px;\"\u003e\n \u003cp\u003e70 \u0026plusmn; 0.35 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 196px;\"\u003e\n \u003cp\u003e53 \u0026plusmn; 0.01 c\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 196px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 196px;\"\u003e\n \u003cp\u003e80 \u0026nbsp; \u0026plusmn; 0.00 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 196px;\"\u003e\n \u003cp\u003e15 \u0026plusmn; 0.30 e\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 196px;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 196px;\"\u003e\n \u003cp\u003e80 \u0026nbsp; \u0026plusmn; 0.10 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 196px;\"\u003e\n \u003cp\u003e10 \u0026plusmn; 0.00 e\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 196px;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 196px;\"\u003e\n \u003cp\u003e85 \u0026nbsp; \u0026plusmn; 0.15 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 196px;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp;0 \u0026plusmn; 0 e\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eMeans \u0026plusmn; SE, n=50; means in the same column followed by different letters are significantly different at P \u0026le; 0.05 and F = 96.56 (Anderson \u0026ndash; Cook 2004).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3. \u0026nbsp;Transformation of embryogenic callus of \u003cem\u003ePhlox drummondii\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;with binary vector (pPZP200) carrying \u003cem\u003eGUS\u003c/em\u003e reporter gene\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;and \u003cem\u003eNPT\u003c/em\u003e-II marker gene\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eNumber of calli co-cultivated\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eNumber of calli surviving on selection medium (SRM I)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eAverage number of transformed shoots obtained from responding calli\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ePercentage\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003etransformation\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ePercentage\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eregeneration\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e53 \u0026plusmn; 0.1 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e40 \u0026plusmn; 0.5 cd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e62 \u0026plusmn; 0.10 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e47 \u0026plusmn; 0.25 c\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e50 \u0026plusmn; 0 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e38 \u0026plusmn; 0.3 d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e63 \u0026plusmn; 0.11 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e48 \u0026plusmn; 0.05 c\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e65 \u0026plusmn; 0.5 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e53 \u0026plusmn; 0.25 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e76 \u0026plusmn; 0.5 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e62 \u0026plusmn; 0.10 b\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e49 \u0026plusmn; 0.12 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e39 \u0026plusmn; 0 d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e58 \u0026plusmn; 0.18 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e45 \u0026plusmn; 0.0 c\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e47 \u0026plusmn; 0.11 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e37 \u0026plusmn; 0.10 d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e63 \u0026plusmn; 0 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e49 \u0026plusmn; 0.01 c\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eMeans \u0026plusmn; SE, n=50; means in the same column followed by different letters are significantly different at P \u0026le; 0.05 and F = 96.56 (Anderson \u0026ndash; Cook 2004).\u0026nbsp;\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-cell-tissue-and-organ-culture-pctoc","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcto","sideBox":"Learn more about [Plant Cell, Tissue and Organ Culture (PCTOC)](https://www.springer.com/journal/11240)","snPcode":"11240","submissionUrl":"https://submission.nature.com/new-submission/11240/3","title":"Plant Cell, Tissue and Organ Culture (PCTOC)","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Ornamental plants, Phlox drummondii, Genetic transformation, Agrobacterium, Floral variants","lastPublishedDoi":"10.21203/rs.3.rs-7162932/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7162932/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn ornamental plants, continuous attempts are being made to introduce new quality traits that have market value. Of various modes of plant propagation that have been followed for production and propagation new traits, the genetic transformation technology has reportedly played an important role in creating novel varieties of ornamentals of rose, dahlia, carnation, lily, tulips, gerbera, etc. Many of transformed varieties of these ornamentals reportedly produce plants that have improved aesthetic traits like flower colour, fragrance, variant floral architecture, and vase life. \u003cem\u003ePhlox drummondii\u003c/em\u003e Hook., also known as \u0026ldquo;Drummond\u0026rdquo; Phlox, is a prized ornamental among 67 species of the genus \u003cem\u003ePhlox\u003c/em\u003e. In the present investigation, an efficient \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated genetic transformation protocol was developed to study its impact on floral morphology, an important commercial trait, of \u003cem\u003eP. drummondii\u003c/em\u003e. About 3-4-week-old embryogenic calli derived from zygotic embryo on callus regeneration and multiplication medium (CRMM) comprising MS basal medium\u0026thinsp;+\u0026thinsp;3% Sucrose\u0026thinsp;+\u0026thinsp;BAP (5 \u0026micro;M)\u0026thinsp;+\u0026thinsp;NAA (10 \u0026micro;M) were pre-cultured on shoot regeneration medium (SRM) comprising MS medium with 3% sucrose\u0026thinsp;+\u0026thinsp;BAP (15 \u0026micro;M)\u0026thinsp;+\u0026thinsp;IAA (2.5 m\u0026micro; M) for 2\u0026ndash;3 days. \u003cem\u003eA. tumefaciens\u003c/em\u003e strain GV2260 carrying the \u003cem\u003eGUS\u003c/em\u003e gene construct (PPZP 200) was grown overnight in YEM medium and calli were infected with bacteria (A\u003csub\u003e600\u003c/sub\u003e 0.3\u0026ndash;0.5) for 10 min. Infected calli were subsequently co-cultured on the medium SRM\u0026thinsp;+\u0026thinsp;acetosyringone (100 \u0026micro;M) for 2- days. Selection of transformed calli was achieved by transfer of these co-cultured calli on the SRM I (SRM supplemented with kanamycin (30 mg/l) and augmentin (300 mg/l), which allowed the production of 76% transformed shoots. For shoot elongation SRM II MS basal salts\u0026thinsp;+\u0026thinsp;3%sucrose\u0026thinsp;+\u0026thinsp;BAP 10 \u0026micro;M\u0026thinsp;+\u0026thinsp;augmentin 300 mg\u0026thinsp;+\u0026thinsp;kanamycin (30 mg/l) was used. Rooting of transformed shoots occurred on MS\u0026thinsp;+\u0026thinsp;IAA (7.5 \u0026micro;M). Transformation status of putative transgenics was confirmed by PCR using primers(Supplementary Table No 5) specific for \u003cem\u003eGUS\u003c/em\u003e and NPT-II genes, and by Southern hybridization using NPT-II gene as probe. Genetic transformation protocol has been standardized in this ornamental species for the first time and transformants were observed having unique changes in floral architecture hitherto unknown.\u003c/p\u003e","manuscriptTitle":"Development of an efficient Agrobacterium-mediated genetic transformation for an important ornamental plant, Phlox drummondii Hook","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-30 12:48:36","doi":"10.21203/rs.3.rs-7162932/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-07-29T08:07:58+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-28T18:10:44+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-25T04:38:03+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell, Tissue and Organ Culture (PCTOC)","date":"2025-07-23T09:28:27+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-cell-tissue-and-organ-culture-pctoc","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcto","sideBox":"Learn more about [Plant Cell, Tissue and Organ Culture (PCTOC)](https://www.springer.com/journal/11240)","snPcode":"11240","submissionUrl":"https://submission.nature.com/new-submission/11240/3","title":"Plant Cell, Tissue and Organ Culture (PCTOC)","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"70adaa74-2d88-45ce-848f-d347ea1af0a9","owner":[],"postedDate":"July 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-07-30T12:48:37+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-30 12:48:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7162932","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7162932","identity":"rs-7162932","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}