Plants comprising a low copy number of Ri genes

Abstract

Described is a plant transformed with one or more genes originating from the Ri plasmid of Agrobacterium rhizogenes by infection with A. rhizogenes comprising the Ri plasmid, or being progeny of such a plant, said plant or progeny comprising, in the genome thereof, 1 to 5 copies said one or more genes originating from the Ri plasmid. Further, the use of such a plant or progeny thereof as ornamental plants, the use as field crop species, for food extracts, cosmetics, perfumes or as medicinal plants is disclosed. Further disclosed are methods for the preparation of such plants. Said plants display an intermediate height and/or with a higher content of metabolites without significant reduction of flower number or flowering time delay as compared to control plants void of Agrobacterium rhizogenes sequences.

Claims

1. A method for the preparation of a plant comprising, in the genome thereof, at least 1 but no more than 5 copies of all the genes present on the Ri plasmid, comprising the steps of: (a) transforming tissue of the wild type plant with the Ri plasmid of Agrobacterium rhizogenes, (b) allowing the transformed tissue to develop roots having a hairy phenotype, (c) selecting, among the roots with hairy phenotype of step (b), a root where the hairy phenotype shows a maximum root hair length of at most half of the maximum root hair length observed in the roots obtained in step (b); (d) growing the selected root on a regeneration medium and allowing a transformed rooted plantlet to generate from the said selected root; (e) growing said transformed rooted plantlet into a mature transformed mother plant having a height of 25-75% of that of the corresponding wild type plant of step (a) and having not less than 80% of the number of flowers of the corresponding wild type plant of step (a); and (f) optionally, generating progeny of said plant of (e).

2. The method of claim 1, wherein step (c) further comprises selecting, among the roots with hairy phenotype of step (b), a root where the hairy phenotype shows a number of branches per length that is at most half of the maximum number of branches per such length observed in the roots obtained in step (b).

3. The method of claim 1, wherein step (e) further comprises selecting for plants or progeny not having a delayed flowering time by more than 4 days as compared to the corresponding wild type plant of step (a).

4. The method of any of claim 1, the selection further comprising assaying the number of copies of the genes originating from the Ri plasmid of Agrobacterium rhizogenes.

5. A method for the preparation of a plant comprising, in the genome thereof, at least 1 but no more than 5 copies of all the genes present on the Ri plasmid, comprising the steps of: (a) transforming tissue of the wild type plant with the Ri plasmid of Agrobacterium rhizogenes, (b) allowing the transformed tissue to develop roots having a hairy phenotype, (c) selecting a putatively transformed root having a hairy root phenotype; (d) growing the selected root on a regeneration medium and allowing transformed rooted plantlet to generate from the said selected root; (e) growing said transformed rooted plantlet into a mature transformed mother plant; (f) generating progeny of the mature transformed mother plant of step (e) by crossing, backcrossing and selfing, while selecting for progeny having an increased height as compared to the mature transformed mother plant of step (e) and a reduced height as compared to that of the corresponding wild type plant of step (a), and (g) repeating step (f) until the progeny results in mature plants having a height of 25-75% of that of the corresponding wild type plant of step (a) and having not less than 80% of the number of flowers of the corresponding wild type plant of step (a), and having in the genome thereof, at least 1 but no more than 5 copies of the Ri plasmid.

6. The method of claim 5, wherein step (f) and/or (g) further comprises selecting for plants or progeny not having a delayed flowering time by more than 4 days as compared to the corresponding wild type plant of step (a).

7. The method of claim 5, the selection further comprising assaying the number of copies of the genes originating from the Ri plasmid of Agrobacterium rhizogenes.

8. The method according to claim 1, wherein said plant comprises, in the genome thereof, at least 1 but no more than 3 copies of the Psi plasmid.

9. The method according to claim 5, wherein said plant comprises, in the genome thereof, at least 1 but no more than 3 copies of the Ri plasmid.

Description

(1) FIG. 1 depicts an illustrative A. rhizogenes Ri-plasmid from an agropine strain. The T-DNA contains two segments, T.sub.L and T.sub.R, which are separated by a 15 Kb sequence that is not integrated. The T.sub.L-DNA contains 18 open reading frames (ORFs) where the four root loci-genes reside. The T.sub.R-DNA contains several genes, including aux1 and aux2.

(2) FIGS. 2, 3 and 4 show roots of leaf discs of Solanum tuberosum, Dipladenia and Kalanchoe interspecific hybrid 2006-0199 (K. laciniata×K. blossfeldiana hybrid) respectively that have been exposed to Agrobacterium rhizogenes. A: non transformed control, B: transformed, hairy, C: transformed, less hairy.

(3) FIG. 5A shows a Kalanchoe interspecific hybrid 2006-0199 wild type (left) and Kalanchoe interspecific hybrid 4006-0199S2, regenerated from less hairy roots of (FIG. 4C), transformed with Agrobacterium rhizogenes, fifteen weeks after start of vegetative propagation under long day conditions (light from 02:00 to 17:00). Three weeks after sticking the cuttings the plants were moved to short day conditions for flower induction (light from 07:00 to 17:00). FIG. 5B shows a Kalanchoe interspecific hybrid 2006-0199 wild type (left) and and Kalanchoe interspecific hybrid 4006-0199K20.1 transformed with Agrobacterium rhizogenes (3 plants to the right) of the hairy type, regenerated from roots of FIG. 4B, fifteen weeks after start of vegetative propagation under long day conditions (light from 02:00 to 17:00). Three weeks after sticking the cuttings the plants were moved to short day conditions for flower induction (light from 07:00 to 17:00).

(4) FIG. 6 shows Kalanchoe pinnata AAE, rol transformed (left) and a control wild type plant (right), The plant of intermediate height (middle, K.pinnata rol-2 Pemba) was obtained by crossing and back crossing of a rol transformed plant of the left.

(5) FIG. 7 shows Rosa, rol transformed (left and middle) and a control wild type plant (right). The plant with intermediate height (middle) originates from a selection for less hairy roots.

(6) FIG. 8 shows Aster, rol transformed (left and middle) and a control wild type plant (right). The plant with intermediate height (middle) originates from a selection for less hairy roots.

EXAMPLES

(7) Plant Material

(8) In vivo plants of Kalanchoe pinnata, Kalanchoe interspecific hybrid 2006-0199, (Knud Jepsen A/S, Hinnerup, Denmark and AgroTech a/s, Tåstrup, Denmark) were cultivated in a greenhouse with temperatures of 20° C. at day and night, 16 hour day length and a light intensity of 260 μmol photons m.sup.31 2 s.sup.−1. In vitro plants were cultivated in growth chamber with temperatures of 25° C. at day and 22° C. at night, 13 hour day length and a light intensity of 75 μmol photons m.sup.−2 s.sup.−1. Plants of other species were purched from different suppliers.

(9) Leaf explants for each species/hybrid were used for control experiment. Leaves derived from in vivo material were sterilised in 70% EtOH for 1 min. followed by 20 min. in 1% NaOCl (VWR, Copenhagen, Denmark) and 0,03% (v/v) Tween 20 (Merck, La Jolla, USA) and washed 3 times in sterile water and were stored until excision.

(10) Bacterial Strain

(11) Agrobacterium rhizogenes strain ATCC43057 (A4) (kindly provided by Dr. Margareta Welander, Swedish University of Agricultural Sciences, Sweden) was used for induction of hairy roots. The strain was cultured in liquid MYA medium (Tepfer and Cassedelbart (1987) Microbiol Sci. 4, pp. 24-28. 1 mL of the bacterial glycerol stock (kept at −80° C.) was diluted in 10 mL MYA in a 50 mL Falcon tube and incubated for 8 h at 27° C. and shaken at 260 rpm. The solution was further diluted with 100 mL MYA in a 250 mL flask and shaken at 260 rpm for 24 h in darkness at 27° C. The OD.sub.600=0,4-0,6 was measured on Nanodrop 1000 (Thermo Scientific, Wilmington, Del., USA).

(12) Transformation

(13) Sterilized leaves or in vitro plant were excised to pieces of min 1 cm×1 cm and stored in sterile water until all explants were ready. The water was discarded from the explants and A. rhizogenes-suspension was added to cover all explants for 30 min. After 30 min. the A. rhizogenes-suspension was discarded and the slices were transferred, with a thin layer of the A. rhizogenes suspension on the surface, to co-cultivation plates for 24 h in darkness without selection. The explants were cultivated in the lab at temperatures at 22° C. in darkness. After co-cultivation the explants were transferred to 0-media (selection media) by drying the explants with pieces of ripped sterile filter paper. The leaf surface was as dry as possible on both sides of the excised leaf. The explants stayed in darkness until roots were developed enough to be transferred to regeneration media. The material was transformed over three sessions. The transformation was conducted with Solanum tuberosum (see FIG. 2), Dipladenia (see FIG. 3), Kalanchoe interspecific hybrid 2006-0199 (K. laciniata×K. blossfeldiana hybrid) (see FIG. 4-5), Kalanchoe pinnata (see FIG. 6), Rosa hybrida (see FIG. 7) and Aster novo-belgii (see FIG. 8). For each species the controls and putative transformants was performed the same day.

(14) Basic Medium

(15) The basic medium used as background of all media used was ½× MS (Sigma M0404) (consisting of Murashige and Skoog macro- and microelements) (Murashige and Skoog, 1962) at a concentration of 2,2 g L.sup.−1, 30 g L.sup.−1 sucrose (table sugar), 7 g L.sup.−1 bacto agar and 0,50 g L.sup.−1 2-(N-morpholino)-ethanesulphonic acid (MES) (Duchefa). The pH was adjusted to 6.3 by 1 M KOH and the media was autoclaved at 121° C. and 103,5 kPa.

(16) Co-Cultivation Medium

(17) Co-cultivation medium used for co-cultivation between explant and A. rhizogenes consisted of basic medium with 30 μg mL.sup.−1 acetosyringone (Sigma-Aldrich, Steinheim, Germany).

(18) Selection Medium

(19) Selection medium was a hormone-free medium used for root formation of putatively transformed explants and controls. Filter-sterilized antibiotics were added after autoclaving to the selection media to the basic medium. Selection media consist of basic media ½× MS medium with timentin (TIM) in the concentration of 100 mg L.sup.−1. Preferably, the selection medium contains arginine, preferably 0.5 mM arginine.

(20) Regeneration Media

(21) Regeneration medium containing the hormone N-(2-chloro-4-pyridyl)-N-phenylurea (CPPU) was used for regeneration of nodules on the putatively transformed root clusters. Filter-sterilised hormones and antibiotics were added after autoclaving to the regeneration media. The CPPU-medium contained basic ½× MS medium with 1.5 μg L.sup.−1 CPPU together with TIM in the concentration 100 mg L.sup.−1.

(22) Co-Cultivation

(23) In all treatments the explants were co-cultivated for 24 hours. After co-cultivation, the explants were blotted onto sterile filter paper and thoroughly dried with ripped pieces of sterilised filter paper. Controls and putatively transformed explants were transferred to selection medium.

(24) Plant Selection

(25) After 24 hours of co-cultivation the explants were transferred to 0-media (selection medium) with 8 explants on each Petri dish. After several weeks the increasing number of roots and decreasing number of explants (due to vitrification—the leaf sections became glass like or because of infections) were monitored for the specific Petri dish in the treatment.

(26) Plant Regeneration

(27) When the roots of putatively transformed explants had developed to a length of 1.5-2 cm they were transferred in clusters, with a part of the explant to CPPU-medium. The transferred root clusters were placed in a climate chamber (Celltherm, United Kingdom) on shelves with 11 h daylight and day/night temperatures of 20/18° C. and an intensity of 45-70 μmol photons m.sup.−2s.sup.−1 (Philips, Amsterdam, The Netherlands). Only root clusters with A. rhizogenes treated explants was transferred. Here the number of root clusters was monitored as well as the number of nodules developing from the roots. Counting of nodule development was stopped when no positive development was observed after 30 days for any of the four species.

(28) Control Plants

(29) Control plants were treated like transformants but inoculated in MYA medium without bacteria and with a lower number of explants-25 per cultivar. The control experiment plants were conducted in parallel with the transformants.

(30) Plant Growth Conditions

(31) Plants described herein were grown in a greenhouse according to day length and temperatures as described in tables 1 and 2 below. The plants were produced in pots with a diameter of 10.5 cm or 13 cm. Cuttings were taken from vegetative (veg.) plants and grown and kept vegetative for the first 3-8 weeks following planting, depending on cultivar, species, genus and pot size. The plants described in table 1 were transferred to flower inducing conditions 4-9 weeks after planting. Between 13-19 weeks after planting, depending on cultivar, species genus, pot size, and time of year, the plants entered their generative (gen.) stage—were mature with flowers that were opening or about to open.

(32) The plants were grown under natural light conditions supplemented with 70 μmol photons m.sup.−2s.sup.−1 SON-T light when the natural light was less than 100 μmol/m.sup.2/s. All plants, except Phaelanopsis and Vanilla, were grown in a peat based soil mix and were watered with a solution containing 200 parts per million (ppm) nitrogen, 200 ppm potassium, 40 ppm phosphorous, 200 ppm calcium, 40 ppm magnesium, 60 ppm sulphate, 1 ppm iron, 0.6 ppm manganese, 0.1 ppm copper, 0.1 ppm zinc, 0.3 ppm borium, 0.03 ppm molybdenum. For all plants, except Phaelanopsis and Vanilla, shading with curtains was active when light intensity was higher than 450 μmol photons m.sup.−2 s.sup.−1 and humidity was kept in the range between 60-80% relative humidity.

(33) Phaelanopsis and Vanilla were grown in a bark based soil mix and were watered with a solution containing 50 parts per million (ppm) nitrogen, 50 ppm potassium, 10 ppm phosphorous, 50 ppm calcium, 10 ppm magnesium, 15 ppm sulphate, 0.2 ppm iron, 0.1 ppm manganese, 0.01 ppm copper, 0.01 ppm zinc, 0.05 ppm borium, 0.005 ppm molybdenum.

(34) Vanilla and Phaelanopsis plants were grown under natural light conditions. Shading with curtains was active when light intensity was higher than 250 μmol photons m.sup.−2 s.sup.−1 and humidity was keep in the range between 80-90% relative humidity.

(35) TABLE-US-00001 TABLE 1 Growth conditions Light period Light Night (Max Light) Dark period temp temp. Genus Short day 07:00-17:00 17:00-07:00 19° C. 21° C. Kalanchoe (gen.), Aster (gen.), Chrysanthemum (gen.), Euphorbia (gen.), Bouvardia (gen.), Rhodiola (veg.) Long day 02:00-17:00 17:00-02:00 19° C. 21° C. Kalanchoe (veg.), Aster (veg.), Chrysanthemum (veg.), Euphorbia (veg.), Bouvardia (veg.), Rhodiola (gen.), Strelitzia, Hibiscus, Mandevilla, Echinacea; Schisandra, Rosa, Ocimum, Capsicum, Ipomoea, Solanum, Nicotiana

(36) TABLE-US-00002 TABLE 2 Growth conditions for Vanilla and Phaelanopsis plants Light period Dark period Light temp Night temp. Vegetative growth before flowering 06:00-18:00 18:00-06:00 24° C. 24° C. Cooling for flower induction 06:00-18:00 18:00-06:00 14-17° C.   14-17° C.   Generative growth after flower 06:00-18:00 18:00-06:00 24° C. 24° C. induction
Molecular Analysis

(37) Six independent DNA isolations per sample have been done with Nucleomag 96 plant, (http:/www.mn-netcom/) on a KingFisher Flex (thermo scientific) according suppliers recommendations, with the modification that lysis buffer MC1 of the Nucleomag 96 plant kit has been supplemented with 1% PVP.

(38) qPCR Amplification was done in a DNA thermal cycler (CFX96 Touch Real-Time PCR Detection System, Bio-Rad) according to the following program: 92° C. for 2 min, followed by 45 cycles of 92° C., for 20 s, 59° C. for 20 s, and 72° C. for 20 s. The reactions for rolC and Ref1, table 1, where individually labelled with LCgreen+by using SALSA Polymerase according to the supplier's instructions (BioFire Defense, http://biofiredefense.com/; MRC-Holland, www.mlpa.com).

(39) TABLE-US-00003 TABLE 3 Primers used primer # target sequence 2142 roIC F CAATAGAGGGCTCAGGCAAG SEQ ID NO 9 2143 roIC R CCTCACCAACTCACCAGGTT SEQ ID NO 10 1654 Ref1 AATGAGGGCTTGTTGGATGA SEQ ID NO 11 1655 Ref1 TTTGAGTGATGGCTCCTTCC SEQ ID NO 12

(40) The use of ΔC.sub.t assumes that the two reactions have similar efficiencies and that they proceed in an independent way since they were carried out in separate wells. A relative quantification has been done by
ΔC.sub.t=C.sub.t(rolC)-C.sub.t(Ref1)
and ratio was calculated by
2.sup.−ΔCt
and the copy number is calculated on the basis that the analysed plants where primary transformants of tetraploid plants and therefor the ratio is multiplied by 4 in order to get the copy number.

(41) In order to determine the copy number of a primary tetraploid transformant a qPCR on rolC of A. rhizogenes with Ref1 as an internal Kalanchoe reference has been done on an untransformed control plant 2006-0199 (an interspecific hybrid having K.laciniata and K.blossfeldiana as parental lines), 3 independent primary transformants of said control plant (i.e. obtained by selection of the transformants without further crossing based on root morphology as described herein), 4006-0199K20.1, 4006-0119S11 and 4006-0199S2, as well as on Ri line 331 as described by Christensen, 2008, supra. Said transformants differ in their growth habit, table 1. For 2006-0199 as expected no rolC could be detected and hence on ratio or copy number could be determined, in case of the 3 A. rhizogenes transformants there is a clear difference in copy number compared to the reference gene Ref1. The semi-compact transformants 4006-0199S11 and 4006-0199S2 having intermediate height, have one copy of rolC compared to Ref1, whereas the compact transformant 4006-0199K20.1 showing dwarf growth has 11 copies of rolC, whereas Ri line 331 showed intermediate height, see table 4. It was also observed that the transformants 4006-0199S11 and 4006-0199S2 showed a similar flower number as the control, and no leaf wrinkling, whereas both 4006-0199K20.1 and Ri line 331 showed wrinkled leaves and a flower number reduced by about 50% as compared to the control. K.pinnata AAE was a non-transformed wild-type control, K.pinnata AAE rol was a rol transformed K.pinnata AAE showing dwarfism, wrinkled leaves, delayed flowering and about 50% of the number offlowers a compared to wild-type, having 10 copies of rolC. K.pinnata AAE-rol was backcrossed with wild-type AAE resulting in several lines, such as K.pinnata Rol-2 Pemba, having intermediate height, but leaves were not wrinkled, and the number of flowers and inflorescences was about the same as of the untransformed K.pinnata AAE. The rolC copy number in K.pinnata Rol-2 Pemba was determined to be 2. In another experiment, K.pinnata AAE was transformed with rol and in the primary transformants, a selection was made on root morphology as described herein, i.e. the root hair length of the selected transformants being at most half of the maximum root hair length observed among the transformants. K.pinnata K2 AAE and K.pinnata PinS A15 AAE were obtained this way, having a phenotype similar of that of K.pinnata Rol-2 Pemba, and having 2 and 3 rolC copies, respectively.

(42) Similar results were obtained for the other plant genera tested, where selection was made also based on root morphology as described above, see table 4.

(43) TABLE-US-00004 TABLE 4 Growth habit and their copy number of roIC compared controls Copy number Growth habit Average compared to Sample FIG. (height) ΔCt Ratio control Solanum tuberosum.sup.c 2a Normal/control ND ND ND Solanum tuberosum* 2b Dwarf −0.89 1.85 7 rol transformed Solanum tuberosum** 2c Intermediate 0.22 0.6 3 Dipladenia.sup.c 3a Normal/control ND ND ND Dipladenia* 3b Dwarf −1.22 2.33 9 Dipladenia** 3c Intermediate −0.25 1.18 5 Kalanchoe int. hyb. 4a, 5a, 5b Normal/control ND ND ND 2006-0199.sup.c Kalanchoe int. hyb. 4b, 5b Dwarf −1.49 2.82 11 4006-0199K20.1* Kalanchoe int. hyb — Intermediate 1.70 0.31 1 4006-0199S11** height Kalanchoe int. hyb 4c, 5a Intermediate 2.02 0.25 1 4006-0199S2** height Kalanchoe Ri line 1d of Intermediate −0.8 1.74 7 331* Christensen 2008, supra Kalanchoe pinnata 6 Normal/control ND ND ND AAE, wild type.sup.c Kalanchoe pinnata 6 Dwarf −1.31 2.48 10 AAE-rol* Kalanchoe pinnata 6 Intermediate 1.4 0.4 2 rol-2 Pemba*** height Kalanchoe pinnata — Intermediate 0.7 0.61 2 Pin5 A15-AAE** height Kalanchoe pinnata — Intermediate 1.1 0.47 2 K2 AAE** height Rosa hybrida.sup.c 7 Normal/control ND ND ND Rosa hybrida* 7 Dwarf −1.68 3.20 13 Rosa hybrida** 7 Intermediate −1.12 0.46 2 Aster novi-belgii.sup.c 8 Normal/control ND ND ND Aster novi-belgii* 8 Dwarf −1.98 3.94 16 Aster novi-belgii** 8 Intermediate 0.25 0.84 3 .sup.ccontrol wild type *rol transformed **rol transformed and selected for root morphology ***rol transformed and backcrossed with wt

(44) Statistical Analysis

(45) K. pinnata functioned as a reference of the transformation. Similarly, control explants had five replicates but 5 explants per species/hybrids with a total of 25 per species/hybrids. Since the explants may be taken out of the experiment because of infection, the number of explants changed over time. The total number of explants was therefore monitored to obtain a better ratio between number of explants and formation of roots. The number of roots was monitored as the number increased. The average of surviving explants per petri dish and the average of roots per petri dish were calculated. The two averages were used to calculate a ratio for each petri dish to describe the number of roots per explants.

(46) V.sub.max (root development/days) was modeled with a linear regression and using the slope. Standard deviations (SD) and students t-test (t-test) were calculated in Excel for each observation to verify variation within the individual species/hybrids. ANOVA test was performed with R (R is a free software environment for statistical computing).

(47) Results

(48) The experiments involved a natural transformation with Agrobacterium rhizogenes to study the transformation efficiency for different species and hybrids and for plain material from in vivo and in vitro. The plants belonging to the species Strelitzia reginae, Aster novi-belgii, Aster dumosus, Chrysantemum morifolium, Chrysantemum×morifolium (syn. C.×grandiflorum e.g.Dendranthema hybrids, or hybrids between Chrysantemum morifolium and other Chrysantemum species e.g. Chrysanthemum indicum, Euphorbia pulcherrima, Euphorbia milli, Bouvardia longiflora, Hibiscus rosa-sinensis, Hibiscus schizopetalus, Hibiscus sabdariffa, Hibiscus syriacus, Hibiscus trionum, Hibiscus cannabinus, Mandevilla×amabilis, Mandevilla sanderi, Mandevilla splendens, Nicotiana tabacum, Nicotiana sylvestris, Nicotiana×sanderrae Phalaenopsis amabilis, Phalaenopsis amboinensis, Phalaenopsis aphrodite, Phalaenopsis appendiculata, Vanilla planifolia, Ocimum basilicum, Capsicum annuum, Capsicum baccatum, Capsicum chinense, Capsicum frutescens, Capsicum pubescens, Ipomoea batatas, Solanum lycopersicum, Solanum tuberosum, Solanum nicotiana, Echinacea purpurea, Echinacea angustifolia, Echinacea pallida, Rosa hybrida, Rosa canina, Rosa spinosissima, Rosa damascena “trigintipetala’, Rosa centifolia, Schisandra chinensis, Schisandra glabra, Schisandra rubriflora, Rhodiola rosea, Kalanchoe pinnata, Kalanchoe marmorata, Kalanchoe gastonis-bonnieri, Kalanchoe dixoniana, Kalanchoe humilis, Kalanchoe laciniata, were transformed with the conditions that were found optimal for K. blossfeldiana ‘Molly’ by Christensen et al., (2008, supra) or slightly changed for optimalisation for each of the species. K. blossfeldiana ‘Molly’ was used as a control within the transformants since the cultivar formed background of the transformation system.

(49) Root induction and growth were monitored as a total number of roots per petri dish in each treatment. Since some explants were removed due to infection the total number of explants over time was also monitored. This was done to obtain a more unbiased assessment when calculating the number of roots per explant in each plant line.

(50) Root Development on 0-Media

(51) Root formation was found to take place in the following species; Strelitzia reginae, Aster novi-belgii, Aster dumosus, Chrysantemum morifolium, Chrysantemum×morifolium (syn. C.×grandiflorum e.g.Dendranthema hybrids, or hybrids between Chrysantemum morifolium and other Chrysantemum species e.g. Chrysanthemum indicum, Euphorbia pulcherrima, Euphorbia milli, Bouvardia longiflora, Hibiscus rosa-sinensis, Hibiscus schizopetalus, Hibiscus sabdariffa, Hibiscus syriacus, Hibiscus trionum, Hibiscus cannabinus, Mandevilla×amabilis, Mandevilla sanderi, Mandevilla splendens, Nicotiana tabacum, Nicotiana sylvestris, Nicotiana×sanderrae, Phalaenopsis amabilis, Phalaenopsis amboinensis, Phalaenopsis aphrodite, Phalaenopsis appendiculata, Vanilla planifolia, Ocimum basilicum, Capsicum annuum, Capsicum baccatum, Capsicum chinense, Capsicum frutescens, Capsicum pubescens, Ipomoea batatas, Solanum lycopersicum, Solanum tuberosum, Solanum nicotiana, Echinacea purpurea, Echinacea angustifolia, Echinacea pallida, Rosa hybrida, Rosa canina, Rosa spinosissima, Rosa damascena “trigintipetala’, Rosa centifolia, Schisandra chinensis, Schisandra glabra, Schisandra rubriflora, Rhodiola rosea, Kalanchoe pinnata, Kalanchoe marmorata, Kalanchoe gastonis-bonnieri, Kalanchoe dixoniana, Kalanchoe humilis, Kalanchoe laciniata, and was transferred to regeneration medium no later than 100 days After transfer to 0-media. At the time of the first time of transfer to regeneration medium putative transformants from Strelitzia reginae, Aster novi-belgii, Aster dumosus, Chrysantemum morifolium, Chrysantemum×morifolium (syn. C.×grandiflorum e.g. Dendranthema hybrids, or hybrids between Chrysantemum morifolium and other Chrysantemum species e.g. Chrysanthemum indicum, Euphorbia pulcherrima, Euphorbia milli, Bouvardia longiflora, Hibiscus rosa-sinensis, Hibiscus schizopetalus, Hibiscus sabdariffa, Hibiscus syriacus, Hibiscus trionum, Hibiscus cannabinus, Mandevilla×amabilis, Mandevilla sanderi, Mandevilla splendens, Nicotiana tabacum, Nicotiana sylvestris, Nicotiana×sanderrae Phalaenopsis amabilis, Phalaenopsis amboinensis, Phalaenopsis aphrodite, Phalaenopsis appendiculata, Vanilla planifolia, Ocimum basilicum, Capsicum annuum, Capsicum baccatum, Capsicum chinense, Capsicum frutescens, Capsicum pubescens, Ipomoea batatas, Solanum lycopersicum, Solanum tuberosum, Solanum nicotiana, Echinacea purpurea, Echinacea angustifolia, Echinacea pallida, Rosa hybrida, Rosa canina, Rosa spinosissima, Rosa damascena “trigintipetala’, Rosa centifolia, Schisandra chinensis, Schisandra glabra, Schisandra rubriflora, Rhodiola rosea, Kalanchoe pinnata, Kalanchoe marmorata, Kalanchoe gastonis-bonnieri, Kalanchoe dixoniana, Kalanchoe humilis, Kalanchoe laciniata, were significantly different from control.

(52) Possible use of Transformed Plants According to the Invention

(53) Kalanchoe

Examples

(54) Kalanchoe pinnata Kalanchoe marmorata Kalanchoe gastonis-bonnieri interspefic hybrid ‘Tropical Parfait’ Kalanchoe dixoniana Kalanchoe humilis Kalanchoe laciniata interspefic hybrid ‘Amazing Pink’

(55) Current use primarily as an ornamental plant. A reduced plant height and increased branching was observed as compared to non-transformed plants. Also content of secondary metabolites e.g. bryophillin A, showing strong anti-tumor promoting activity does make this genus/species interesting as a medicinal plant. Kalanchoe, in particular K. pinnata also contains-coumaric acid, Ferulic acid, Syringic acid, Caffeic acid, citric acid, isocitric acid, malic acid, P-hydroxybenzoic acid, Flavonoids as quercetin, kaempferol, quercetin-3-diarabinoside, kaempferol-3-glucoside, quercetin-3-L-rhamnosido-L-arabino furanoside, η-hentricontane, η-tritriacontane, Sitosterol. Studies showed that several of these compounds exhibited higher concentrations in rol transformed plants.

(56) Any of these compounds can be isolated from Kalanchoe and used as or in a human or animal necessity such as a medicament. Plants, transformed with the Ri plasmid of A. rhizogenes comprising the rol genes exhibited higher concentrations compared to control.

(57) Rosa

Examples

(58) Rosa hybrida Rosa canina Rosa spinosissima Rosa damascena ‘Trigintipetala’ Rosa centifolia

(59) Current use primarily as an ornamental plant. A reduced plant height and increased branching is observed as compared to non-transformed controls, making it possible to have sufficient branching from fewer cuttings per pot. Rosa is also having significant use in the perfume industry. In Europe, Rosa damascena ‘Trigintipetala’ is particularly used, and Rosa centifolia in other parts of the world. The main constituents are the fragrant alcohols geraniol and 1-citronellol and rose camphor. β-Damascenone is also a significant contributor to the scent. Plants, transformed with the Ri plasmid of A. rhizogenes comprising the rol genes exhibited higher concentrations compared to control.

(60) Strelitzia

Example

Strelitzia reginae

(61) Mainly used as a cut flower because of its size. A significant reduction in plant height and also significantly in production time (time to flowering) is observed in plants, transformed with Agrobacterium rhizogenes comprising the Ri plasmid.

(62) Aster

Example

(63) Aster novi-belgii Aster dumosus

(64) Mainly used as potted plant and cut flower. A reduced plant height and increased branching is observed as compared to non-transformed controls, making it possible to have sufficient branching from fewer cuttings per pot.

(65) Chrysantemum

Examples

(66) Chrysanthemum morifolium Chrysanthemum indicum Chrysanthemum×morifolium (Dendranthema hybrids)

(67) Mainly used as potted plant and cut flower, but also as ingredient in Chrysanthemum tea (Chrysanthemum indicum). Reduced plant height and increased branching was observed in transformants making it possible to have sufficient branching from fewer cuttings per pot when using the plants as potted plants. Flavor of tea extracts made from Ri-transformed Chrysanthemum indicum and Chrysanthemum morifolium appeared to be more intense than that of not transformed counterparts.

(68) Euphorbia

Example

(69) Euphorbia milli Euphorbia pulcherrima (Poinsettia)

(70) Mainly used as an ornamental plant. A reduced plant height and increased branching was observed as compared to non-transformed plants, making it possible to have sufficient branching without pinching and from fewer cuttings per pot.

(71) Hibiscus

Examles

(72) Hibiscus rosa-sinensis Hibiscus schizopetalus Hibiscus sabdariffa Hibiscus syriacus Hibiscus trionum Hibiscus cannabinus

(73) Mainly used as potted plant or ornamental garden plants in temperate areas (Hibiscus syriacus) and in the tropics/subtropics (all species). Reduced plant height and increased branching is observed in transformants making it possible to have sufficient branching without pinching and from fewer cuttings per pot. Hibiscus flowers contain anthocyanins, and plants transformed with the Ri plasmid of A. rhizogenes comprising the rol genes exhibited higher concentrations as compared to control.

(74) Dipladenia/Mandevilla

Examples

(75) Mandevilla×amabilis Mandevilla sanderi Mandevilla splendens

(76) Mainly used as potted plant or ornamental garden plants in tropics/subtropics. In transformants, reduced plant height and increased branching was observed making it possible to have sufficient branching without pinching and from fewer cuttings per pot. Costly work attaching the plants to a physical fixture will not be needed in rol transformants.

(77) Nicotiana

Examples

(78) Nicotiana tabacum

(79) Mainly used as crop plants for the production of tabacco. Tobacco leaves normally contain 2 to 8% nicotine and plants, transformed with the Ri plasmid of A. rhizogenes comprising the rol genes exhibited higher concentrations compared to control. Nicotiana sylvestris Nicotiana×sanderrae,

(80) Mainly used as ornamental/bedding plants. A reduced plant height and increased branching was observed making it possible to have sufficient branching without pinching and from fewer cuttings per pot.

(81) Bouvardia

Example

(82) Bouvardia longiflora

(83) Mainly used as potted plant and cut flower. In trasformed plants, a reduced plant height and increased branching is observed making it possible to have sufficient branching from fewer cuttings per pot with less chemical growth regulation needed.

(84) Phaelanopsis

Example

(85) Phalaenopsis amabilis Phalaenopsis amboinensis Phalaenopsis Aphrodite Phalaenopsis appendiculata

(86) Used as potted plant and cut flower, the largest potted plant product (produced numbers and turnover) in Europe. A reduced plant height and increased branching was observed in transformed plants, making it possible to have more spikes per plant using less chemical growth regulation with expected higher prices on the market.

(87) Vanilla planifolia

(88) Vanilla is one of the primary sources for vanilla flavouring, due to its high vanillin content (4-hydroxy-3-methoxybenzaldehyde) and plants, transformed with the Ri plasmid of A. rhizogenes comprising the rol genes exhibited higher concentrations compared to control.

(89) Basil

Example

(90) Ocimum basilicum

(91) Mainly used as an edible plant. The leaves may taste somewhat like anise, with a strong, pungent, often sweet smell, due to its content of metylchavicol, kineol and linalool. Plants, transformed with the Ri plasmid of A. rhizogenes comprising the rol genes exhibited higher concentrations compared to control.

(92) Capsicum

Examples

(93) Capsicum annuum, Capsicum baccatum, Capsicum chinense, Capsicum frutescens, Capsicum pubescens.

(94) Mainly used as spices and food vegetables, but Capsicum containing capsaicin (methyl vanillyl nonenamide), has also found use in medicines to stimulate blood circulation or to relieve pain. Plants, transformed with the Ri plasmid of A. rhizogenes comprising the rol genes exhibited higher concentrations compared to control.

(95) Ipomoea

Example

(96) Ipomoea batatas

(97) Known as sweet potato. Its large, starchy, sweet-tasting, tuberous roots are a root vegetable. Besides starches, sweet potatoes are rich in complex carbohydrates, dietary fiber and in beta-carotene (a provitamin A carotenoid), while having moderate contents of other micronutrients, including vitamin B5, vitamin B6, manganese and potassium. Plants, transformed with the Ri plasmid of A. rhizogenes comprising the rol genes exhibited higher concentrations compared to control.

(98) Tomato

Example

(99) Solanum lycopersicum

(100) The tomato is the edible, often red/orange/yellow/greenish fruit/berry. The fruit contains lycopene, a powerful antioxidant, has been linked with reduced risks of colorectal, gastric, lung, prostate, and pancreas cancer. We observe reduced plant height and increased branching making it possible to use the plant also as a houseplant with edible fruits or in gardens with limited space available. Furthermore we noticed that content of lycopene increased in fruits of plants, transformed with the Ri plasmid of A. rhizogenes comprising the rol genes.

(101) Potato

Example

(102) Solanum tuberosum

(103) The potato is and edible plant, containing vitamins and minerals, as well as an assortment of phytochemicals, such as carotenoids and natural phenols. Reduced plant height and increased branching was observed making it possible to use the plant also as a garden plant with edible tubers in gardens with limited space available Furthermore it was noticed that content of vitamin and mineral content increased in tuber of rol transformed potato plants. Also, a more intense taste was observed.

(104) Solanum nicotianum

(105) Solanum nicotianum is an intergeneric graft chimera of Nicotiana tabacum L. and Solanum laciniatum (Kaddoura, R. L. and Mantell, S. H., (1991) Ann Bot 68 (6): 547-556, and used as an ornamental plant. A reduced plant height and increased branching is observed as compared to non-transformed controls, making it possible to have sufficient branching from fewer cuttings per pot.

(106) Echinacea

Examples

(107) Echinacea purpurea Echinacea angustifolia, Echinacea pallida

(108) Echinacea is mainly used for herbal medicines—containing phenyl propanoid, echinacoside. The constituent base for Echinacea is complex, consisting of a wide variety of chemicals of variable effect and potency. The range of active substances have antimicrobial, stimulating or modulating effects on different parts of the immune system. All species contain phenols, phenyl propanoid constituents such as cichoric acid and caftaric acid are present in E. purpurea, other phenols include echinacoside. Other chemical constituents that may be important in echinacea health effects include alkylamides and polysaccharides. The immunomodulatory effects of echinacea preparations are likely caused by fat-soluble alkylamides (alkamides), Alkylamides have similar potency to that of THC at the CB2 receptor, with THC being around 1.5 times stronger (˜40 nm vs ˜60 nm affinities). However, potency is dramatically less than that of THC at the psychoactive CB1 receptor (˜40 nm vs ˜>1500 nm affinities). It was now observed that concentration of a range of chemical content increased in parts of plants, transformed with the Ri plasmid of A. rhizogenes comprising the rol genes. Furthermore plant height and increased branching was observed making it possible to use the plant also as a house/garden plant with ornamental value.

(109) Schisandra

Examples

(110) Schisandra chinensis Schisandra glabra Schisandra rubriflora (ornamental use)

(111) The berries of Schisandra are used in traditional Chinese medicine. Chemical constituents include the lignans schisandrin, deoxyschisandrin, gomisins, and pregomisin, which are found in the seeds of the fruit. It was observed that concentration of a range of chemical content increased plant parts of plants, transformed with the Ri plasmid of A. rhizogenes comprising the rol genes. Furthermore plant height and increased branching was observed, making it possible to use the plant also as a house/garden plant with ornamental value.

(112) Rhodiola

Example

(113) Rhodiola rosea

(114) Rhodiola has been used in herbal medicine in China, Russia and Scandinavia to better cope with the cold Siberian weather. The aerial portion is consumed as food in some parts of the world, sometimes added to salads. The root and other plant parts contain rosavin, rosarin, rosin and salidroside (and sometimes p-tyrosol, rhodioniside, rhodiolin and rosiridin. It was observed that the concentration of a range of chemical contents increased in plant parts of rol transformed plants. The plant is quite slow growing and the ornamental value of the plant is limited.

(115) Elevated Flavonoid Levels in Kalanchoe

(116) Wild type Kalanchoe pinnate and the transformed counterpart having 5 Ri gene copies as described in table 4 (i.e. obtained by backcrossing a primary Ri transformed K.pinnata with wild type untransformed K.pinnata) were taken and extracts were prepared by freeze drying 50 g leaves until the water loss was 85 w/w %, followed by incubation in methanol (5 weight parts methanol perweight part dried leaves) overnight in the dark at 5° C. The supernatant was collected and evaporated under pressure to complete dryness. Extract samples were prepared with a concentration of 100 mg/ml methanol and filtered (Sartorius, 0.20 μm). HPLC-MS/MS analysis (a combination of of high-performance liquid chromatography (HPLC) with mass spectrometry (MS), MS/MS being the combination of two mass analyzers in one mass spec instrument) was used to identify the different derivatives of the flavonoids according to their characteristic masses of the deprotonated molecular ions. [M-HT were used for identification, based on mass-to charge ratio (m/z; Quercetin=301; Isorhamnetin=315; Kaempferol=285). HPLC-MS/MS was performed in accordance with Tsimogiannis et al. (Molecules (2007) 12, 593-606.

(117) It was found that in wild type K. pinnata, the levels of kaempferol and isohamnetin were 2,12 and 4,74 μg per g plant material, respectively, whereas in the K.pinnata determined to have 5 copies of Ri genes, the said level were 2,41 and 6,03 μg per g plant material, respectively.