Agrobacterium-mediated Transformation of Buddleia Species

in HortScience
View More View Less
  • 1 Department of Plant Sciences, North Dakota State University, Department #7670, P.O. Box 6050, Fargo, ND 58108

Two Buddleia cultivars, B. davidii ‘Potters Purple’ and Buddleia ‘Lochinch’, were transformed using Agrobacterium tumefaciens strain EHA105 harboring the binary vector pBI121 carrying the neomycin phosphotransferase gene and β-glucuronidase gene (uidA). Transgenic plants were recovered from the Agrobacterium-infected leaf tissues through organogenesis in the selection medium (woody plant medium containing 250 mg·L−1 cefotaxime plus 500 mg·L−1 carbenicillin plus 40 mg·L−1 kanamycin). The rate of shoot regeneration from transformed leaf tissues increased from 5.7% to 32% through extending cocultivation time from 3 to 9 days. Integration of marker genes was verified with polymerase chain reaction (PCR) and Southern blot analysis. Southern blot analysis confirmed that one to three copies of transgenes were integrated into the buddleia genome. This transformation system could be used for improvement of buddleia or other related species. Chemical names used: 6-benzyladenine (BA), naphthalene acetic acid (NAA), acetosyringone (AS), 5-Bromo-4-chloro-3-indoxyl-beta-D-glucuronide cyclohexylammonium (X-Glu), cefotaxime (Cef), carbenicillin (Carb), kanamycin (Km).

Abstract

Two Buddleia cultivars, B. davidii ‘Potters Purple’ and Buddleia ‘Lochinch’, were transformed using Agrobacterium tumefaciens strain EHA105 harboring the binary vector pBI121 carrying the neomycin phosphotransferase gene and β-glucuronidase gene (uidA). Transgenic plants were recovered from the Agrobacterium-infected leaf tissues through organogenesis in the selection medium (woody plant medium containing 250 mg·L−1 cefotaxime plus 500 mg·L−1 carbenicillin plus 40 mg·L−1 kanamycin). The rate of shoot regeneration from transformed leaf tissues increased from 5.7% to 32% through extending cocultivation time from 3 to 9 days. Integration of marker genes was verified with polymerase chain reaction (PCR) and Southern blot analysis. Southern blot analysis confirmed that one to three copies of transgenes were integrated into the buddleia genome. This transformation system could be used for improvement of buddleia or other related species. Chemical names used: 6-benzyladenine (BA), naphthalene acetic acid (NAA), acetosyringone (AS), 5-Bromo-4-chloro-3-indoxyl-beta-D-glucuronide cyclohexylammonium (X-Glu), cefotaxime (Cef), carbenicillin (Carb), kanamycin (Km).

The genus Buddleia consists of more than 100 species, of which many are woody shrubs providing colorful and fragrant flowers for landscaping and gardening (Dirr, 1998; Hogan, 2003). Buddleia plants have a wide adaptability to various environmental conditions such as high soil pH and cold hardiness and can be grown in most states of the United States (Dirr, 1998; Hogan, 2003). Buddleia davidii, known as butterfly bush, is a perennial landscape plant that can also be used as an annual bedding plant. Butterfly bush was imported to North America from China in the early 1900s. It is characterized as a fast-growing deciduous shrub with a long flowering period and is well received by many gardeners. However, this species has such concerns as limited flower color (most reds and blues are muddled), excessive growth (1 to 5 m tall, lanky, and wide-spreading), and is a natural virus host (e.g., cucumber mosaic virus, alfalfa mosaic or tomato ringspot virus) (Podaras, 2005). Recently, invasiveness of this species has become an issue because Buddleia davidii is a prolific seed producer and its seeds can be dispersed by wind and water and may remain dormant in the soil for many years (Sheppard et al., 2006).

A colorful, compact, and sterile Buddleia davidii cultivar would be highly desirable for landscape purposes. Attempts to improve Buddleia davidii using conventional breeding methods have resulted in limited success. Different ploidy levels among species in the genus Buddleia may impede interspecific crosses; therefore, elite traits cannot be efficiently integrated into newly developed plants (Rose et al., 2000; Tobutt, 1993).

Transgenic plants have been obtained in many woody species (Petri and Burgos, 2005; Poupin and Arce-Johnson, 2005). However, transformation of Buddleia species has not been documented to date. The objective of this study was to develop a gene transformation protocol for further improvement of Buddleia species.

Materials and Methods

Plant material.

Two cultivars, Buddleia davidii ‘Potters Purple’ and Buddleia ‘Lochinch’, were used in this study. In vitro cultures of the cultivars were subcultured every 4 weeks in Murashige and Skoog (MS) medium (Murashige and Skoog, 1962) supplemented with 2.67 μM benzyladenine (BA), 2% sucrose, and 0.35% phytagel (Sigma Chemical Co., St. Louis, MO) and adjusted to pH 5.8 before autoclaving. These cultures were maintained at 25 °C under cool-white light at 36 μmol·m−2·s−1 with a 16-h photoperiod.

Plant transformation.

An efficient shoot regeneration system of these two Buddleia cultivars was developed by Dai and Castillo (2007). Previous experiments determined that kanamycin at 40 mg·L−1 completely inhibited shoot regeneration from leaf tissues. Therefore, a series of media used for the buddleia transformation were made based on the result of the previous experiments:

  1. Shoot regeneration medium (SRM): For ‘Potters Purple’: woody plant medium (WPM) (Lloyd and McCown, 1980) + 0.35% phytagel + 5 μM BA + 5 μM indole acetic acid (IBA); for ‘Lochinch’: WPM + 0.35% phytagel + 20 μM BA + 4 μM IBA.

  2. Cocultivation medium (CCM): SRM + 200 μM acetosyringone (AS).

  3. Selection medium (SLM): SRM + CCK40 [250 mg·L−1 cefotaxime (Cef) + 500 mg·L−1 carbenicillin (Carb) + 40 mg·L−1 kanamycin] (antibiotics were filter-sterilized and added into the autoclaved medium when the medium temperature cooled down to ≈50 °C).

Agrobacterium strain EHA105 (Hood et al., 1993), carrying pBI121 (Clontech, Palo Alto, CA) containing the nptII gene encoding neomycin phosphotransferase and the uidA gene coding for β-glucuronidase (GUS) (Fig. 1), was grown overnight in Luria-Bertani medium with 100 mg·L−1 kanamycin at 28 °C in a shaker at 150 rpm. Cells were collected by centrifugation at 6000 rpm for 15 min, resuspended to 1.0 o.d. at Abs600 in WPM medium supplemented with 20 μM AS without kanamycin, and incubated at 28 °C in a shaker at 150 rpm for 2 h. In vitro leaves were cut through the main vein once (≈0.5 × 0.5 cm) and submerged in a bacterial culture solution for 30 min at 28 °C. Leaf explants were then removed from the bacterial culture solution, blotted on sterilized paper towels, and transferred to the CCM medium in petri dishes (100 mm × 15 mm, 25 mL medium) for cocultivation in the dark at room temperature. The effect of cocultivation time on transformation efficiency was tested. After cocultivation for 3, 6, and 9 d, leaf explants were washed twice with sterile deionized and distilled water (ddH2O) and once with sterile ddH2O plus 250 mg·L−1 Cef and 500 mg·L−1 Carb, and then blotted with sterile paper towels. These Agrobacterium-infected leaf explants were inoculated on the SLM for selection in the dark. Cultures were subcultured in fresh SLM and moved to a culture room under the regular culture conditions previously described. Each treatment (cocultivation time) had at least three SLM plates with 10 explants per plate. The control plates were SLM without kanamycin. The experiment was repeated three times.

Fig. 1.
Fig. 1.

Schematic representation of the T-DNA portion of pBI121 plasmid (Clontech, Palo Alto, CA). RB and LB = T-DNA right and left borders; NP = nopaline synthase promoter; NT = nopaline synthase terminator; nptII = neomycin phosphotransferase gene; uidA = β-glucuronidase gene; 35S-P = CaMV 35S promoter from cauliflower mosaic virus. The vector was introduced into the disarmed Agrobacterium tumefaciens EHA 105.

Citation: HortScience horts 44, 2; 10.21273/HORTSCI.44.2.526

Shoots regenerated/recovered from SLM were proliferated in MS medium supplemented with 2.67 μM BA and CCK40. Proliferated shoots (greater than 2.0 cm) were rooted based on the method of Dai and Castillo (2007). Rooted plants were transferred to Sunshine Mix #1 (Fisons Western Corp., Vancouver, Canada) and grown in the greenhouse under a 16–8-h photoperiod with the temperature adjusted to 25 °C in the daytime and 15 °C at night.

Histochemical β-glucuronidase assay.

Leaves from the recovered plants were subjected to GUS screening as described by Jefferson (1987). In brief, in vitro young and greenhouse-grown leaves were submerged in a GUS staining solution containing 200 μL ddH2O and 200 μL X-Glu solution (2 mg·mL−1; Gold Biotechnology, Inc., St. Louis, MO). After incubating at 37 °C overnight, stained leaves were gradually bleached with 70% to 100% ethanol. GUS staining was observed and photographed under a microscope.

Polymerase chain reactions.

Genomic DNA was extracted from young leaves of putative transformed and nontransformed buddleia plants based on the method of Lodhi et al. (1994). Reactions of polymerase chain reaction (PCR) were carried out in 25 μL volume containing 200 μM dNTPs, 1 μM of each oligonucleotide primer, 2.5 units Taq DNA Polymerase (Promega, Madison, WI), and 25 ng DNA. The reaction conditions were: 1 cycle at 94 °C for 5 min, 40 cycles of 94 °C for 1 min, 60 °C for 1 min, and 72 °C for 30 s and then one cycle at 72 °C for 7 min. Amplified DNA fragments (10 μL of reaction) were electrophoresed on a 1% agarose gel, stained with ethidium bromide, and visualized under ultraviolet light. The primers used for screening transgenes were: nptII reverse: 5′-GCAGGCATCGCCATGGGTCACGACGA-3′ and nptII forward: 5′-GCCCTGAATGAACTGCAGGACGAGGC-3′; and uidA reverse: 5′-CCCGGCAATAACATACGGCGTG-3′ and uidA forward: 5′-CCTGTAGAAACCCCAACCCGTG-3′; which produced 410-bp and 365-bp products, respectively.

Southern blot analysis.

Approximately 25 to 35 μg of genomic DNA was digested in a 50 μL reaction with 1 μL HindIII restriction enzyme at 37 °C for 2.5 h, electrophoresed on a 0.8% TAE (Tris-acetate ethylenediamine tetra-acetic acid) agarose gel, and blotted to a positively charged Hybond-N+ nylon membrane (Amersham Pharmacia Biotech, Little Chalfont Buckinghamshire, U.K.). Similarly, digested DNA from untransformed buddleia plants was used as a negative control, whereas 25 to 40 ng of pBI121 plasmid DNA was used as a positive control. The blot was probed with randomly primed 32P-labeled nptII PCR product (4 μL ddH2O, 1 μL 6-mer oligo primers, 1.5 μL 5 mm dNTPs, 1.5 μL 10× Klenow buffer, 1 μL Klenow polymerase, and 5 μL dCTP 32P). Prehybridization was at 65 °C for 6 h in the hybridization solution [1% bovine serum albumin Fraction V (Sigma Chemical Co.), 0.5 M NaH2PO4 (pH 7.0), 7% sodium dodecyl sulfate (SDS), 1 mm ethylenediamine tetra-acetic acid]. The denatured DNA probe was added directly to the blot in the prehybridization mixture, hybridized at 65 °C for 16 h, and then washed with 2× SSC (200 μM sodium chloride and 200 μM sodium citrate) for 35 min followed with 0.5× SSC + 0.1% SDS, and 0.1× SSC + 0.1% SDS for 10 min each at 65 °C on a shaker. The blot was exposed to x-ray film (Kodak, New York, NY) at –80 °C for 72 h and developed per the manufacturer's instructions.

Statistical analysis.

Data from all experiments were subject to analysis of variation and mean comparison using the GLM procedure of SAS software Version 9.1 (SAS Institute, 2004).

Results and Discussion

Effect of cocultivation time on shoot recovery.

In vitro shoots were regenerated from leaf explants infected with Agrobacterium EHA105 carrying pBI121 after 8 weeks' culture (two subcultures) in the selection medium (Table 1; Fig. 2A). No shoot was regenerated from noninfected leaf explants. Cocultivation time significantly affected regeneration rate. An average of 32% of leaf explants produced shoots from SLM medium containing 40 mg·L−1 kanamycin after 9 d cocultivation, whereas only 5.7% and 10.6% of explants developed shoots after 3 and 6 d cocultivation, respectively. However, leaf quality was crucial for a long (greater than 6 d) cocultivation. Several other experiments showed that cocultivation with EHA105 more than 6 d caused severe damage to leaf tissues, resulting in no shoot regeneration. However, mature leaves (3 to 4 weeks old in vitro) showed tolerance to an extended cocultivation time and were easier to recover from the infection of Agrobacterium than young leaves (1 to 2 weeks old), allowing the plant cells to initiate regeneration more easily. Regenerated shoots from the selection medium were continuously subcultured three to four times in medium containing CCK40 and then maintained in antibiotic-free medium. No Agrobacterium was detected from grinded tissues of transgenic plants inoculated on the antibiotic-free culture medium, indicating that all bacteria had been killed by antibiotics (cefotaxime and carbenicillin) after three to four consecutive subcultures. Confirmed transgenic plants were grown in the greenhouse (Fig. 2B).

Table 1.

The effect of cocultivation time on shoot recovery from putative transformants of two Buddleia cultivars.

Table 1.
Fig. 2.
Fig. 2.

(A) Shoots were regenerated from leaf explants infected with Agrobacterium EHA105 in woody plant medium containing 5 μM 6-benzyladenine, 5 μM naphthalene acetic acid, and CCK40. (B) Transgenic Buddleia plants were grown in the greenhouse.

Citation: HortScience horts 44, 2; 10.21273/HORTSCI.44.2.526

Histochemical β-glucuronidase assay.

GUS staining showed blue in the leaves collected from both in vitro and greenhouse-grown plants (Fig. 3), indicating that the uidA gene expressed in the leaf tissues. It was observed that blue staining can only be detected in in vitro young leaves for some lines. However, in a few other recovered lines, greenhouse-grown leaves showed much darker blue staining than in vitro leaves, showing a deferential expression of uidA gene under different conditions.

Fig. 3.
Fig. 3.

β-glucuronidase staining of a leaf from transformed (A) and nontransformed (B) Buddleia plants. The blue color in A is the result of active beta-glucuronidase activity.

Citation: HortScience horts 44, 2; 10.21273/HORTSCI.44.2.526

Polymerase chain reactions.

GUS-positive regenerated lines were proliferated to have enough leaf tissues for genomic DNA extraction and subjected to PCR verification. The expected fragments of uidA (356 bp) and nptII (410 bp) genes were successfully amplified from these plants using the specific primers (Fig. 4).

Fig. 4.
Fig. 4.

Polymerase chain reaction amplification of nptII (A) and uidA (B) genes in transformed buddleia plants. Lanes M are DNA ladders. Lane P is positive control of plasmid pBI121. Lane N is negative control of untransformed buddleia. Lanes 1 to 4 are transformed lines.

Citation: HortScience horts 44, 2; 10.21273/HORTSCI.44.2.526

Southern blot analysis.

Genomic DNA was digested with HindIII. Gel electrophoresis showed that DNA was well digested and separated. The digested DNA was hybridized with the 32P-labeled fragment of the nptII gene probe prepared from PCR product. The result showed that the transgenic lines exhibited one to three distinctive restriction fragments (Fig. 5), confirming that the nptII gene was integrated into the Buddleia genome.

Fig. 5.
Fig. 5.

Confirmation of transgene (nptII) in transformed Buddleia. Lanes N, P, and 1 to 2 are digested DNA with HindIII from nontransgenic control (N), plasmid pBI121 (P), and transgenic lines (1 and 2), respectively. The digested DNA blot was probed with randomly primed 32P-labeled nptII polymerase chain reaction product.

Citation: HortScience horts 44, 2; 10.21273/HORTSCI.44.2.526

In this study, genetic transformation of two Buddleia cultivars has been confirmed. Many factors such as genotype, Agrobacterium strain, conditions of infection, and cocultivation affect transformation frequency. The Buddleia cultivars used in this study are very amenable to plant regeneration from leaf tissues (Dai and Castillo, 2007). Thus, higher transformation efficiency may be achieved if some improvements can be made during the Agrobacterium infection and transformant selection. For example, preconditioning explants, application of vacuum technique during the infection process, using different Agrobacterium strains, and extension of the cocultivation time may increase the T-DNA delivery efficiency. The large F value of cocultivation time in this study indicated that the time of cocultivation was one of the most important factors attributing to a significant increase of shoot recovery from the selection medium, which is in agreement with other research (Cardoza and Stewart, 2003; Forreiter et al., 1997).

Many transgenic plants such as most monocot species have been initiated using embryogenic tissues from seedlings such as immature embryos, hypocotyls, and cotyledons. This limits application of gene transformation for improving vegetatively propagated species, especially for elite cultivar improvement, because of the trait segregation of seed-propagated plants. This is the first article reporting genetic transformation of Buddleia species, which was achieved by using leaf tissues from a mature plant. Therefore, this method can be used to improve target trait(s) of an elite plant cultivar without changing any other genetic makeup, providing a useful tool for developing new cultivars of this and other species.

Literatures Cited

  • Cardoza, V. & Stewart, C.N. 2003 Increased Agrobacterium-mediated transformation and rooting efficiencies in canola (Brassica napus L.) from hypocotyl segment explants Plant Cell Rpt. 21 599 604

    • Search Google Scholar
    • Export Citation
  • Dai, W. & Castillo, C. 2007 Factors affecting plant regeneration from leaf tissues of Buddleia species HortScience 42 1670 1673

  • Dirr, M.A. 1998 Manual of woody landscape plants 5th Ed Stipes Publishing Champaign, IL

  • Forreiter, C., Kirschner, M. & Nover, L. 1997 Stable transformation of an Arabidopsis cell suspension culture with firefly luciferase providing a cellular system for analysis of chaperone activity in vivo Plant Cell 9 2171 2181

    • Search Google Scholar
    • Export Citation
  • Hogan, S. 2003 Flora: A gardener's encyclopedia Timber Press, Inc Portland, OR

  • Hood, E.E., Gelvin, S.B., Melchers, L.S. & Hoekema, A. 1993 New Agrobacterium helper plasmids for gene transfer to plants Transgenic Res. 2 208 218

  • Jefferson, R.A. 1987 Assaying chimeric genes in plant—The GUS gene fusion system Plant Mol. Biol. Rpt. 5 387 405

  • Lloyd, G. & McCown, B. 1980 Commercially-feasible micropropagation of mountain laurel, Kalmia latifolia, by use of shoot-tip culture Proc. Intl. Plant Prop. Soc. 30 421 427

    • Search Google Scholar
    • Export Citation
  • Lodhi, M.A., Ye, G.N., Weeden, N.F. & Reisch, B.I. 1994 A simple and efficient method for DNA extraction from grapevine cultivars, Vitis species and Ampelopsis Plant Mol. Biol. Rpt. 12 6 13

    • Search Google Scholar
    • Export Citation
  • Murashige, T. & Skoog, F. 1962 A revised medium for rapid growth and bioassays with tobacco tissue culture Physiol. Plant. 15 473 497

  • Petri, C. & Burgos, L. 2005 Transformation of fruit trees. Useful breeding tool or continued future prospect? Transgenic Res. 14 15 26

  • Podaras, P. 2005 Breeding a butterfly bush Landscape Plant News 16 16 7

  • Poupin, M.J. & Arce-Johnson, P. 2005 Transgenic trees for a new era In Vitro Cell. Dev. Biol. Plant 4 91 101

  • Rose, J.B., Kubba, J. & Tobutt, K.R. 2000 Induction of tetraploidy in Buddleia globosa Plant Cell Tissue Organ Cult. 63 121 125

  • SAS Institute 2004 SAS/STAT 9.1 user's guide SAS Institute Inc Cary, NC

  • Sheppard, A.W., Shaw, R.H. & Sforza, R. 2006 Top 20 environmental weeds for classical biological control in Europe: A review of opportunities, regulations, and other barriers to adoption Weed Res. 46 93 117

    • Search Google Scholar
    • Export Citation
  • Tobutt, K.R. 1993 Inheritance of white flower color and congested growth habit in certain Buddleia progenies Euphytica 67 231 235

Contributor Notes

This research was supported in part by McIntire-Stennis Project ND06212 and Landscape Plant Development Center.

We thank Dr. J. Ransom for SAS analysis and Drs. M. Christoffers, H. Hatterman-Valenti, and D. Herman for reviewing the manuscript.

Assistant Professor.

Graduate Student.

Research Specialist.

To whom reprint requests should be addressed; e-mail wenhao.dai@ndsu.edu.

  • View in gallery

    Schematic representation of the T-DNA portion of pBI121 plasmid (Clontech, Palo Alto, CA). RB and LB = T-DNA right and left borders; NP = nopaline synthase promoter; NT = nopaline synthase terminator; nptII = neomycin phosphotransferase gene; uidA = β-glucuronidase gene; 35S-P = CaMV 35S promoter from cauliflower mosaic virus. The vector was introduced into the disarmed Agrobacterium tumefaciens EHA 105.

  • View in gallery

    (A) Shoots were regenerated from leaf explants infected with Agrobacterium EHA105 in woody plant medium containing 5 μM 6-benzyladenine, 5 μM naphthalene acetic acid, and CCK40. (B) Transgenic Buddleia plants were grown in the greenhouse.

  • View in gallery

    β-glucuronidase staining of a leaf from transformed (A) and nontransformed (B) Buddleia plants. The blue color in A is the result of active beta-glucuronidase activity.

  • View in gallery

    Polymerase chain reaction amplification of nptII (A) and uidA (B) genes in transformed buddleia plants. Lanes M are DNA ladders. Lane P is positive control of plasmid pBI121. Lane N is negative control of untransformed buddleia. Lanes 1 to 4 are transformed lines.

  • View in gallery

    Confirmation of transgene (nptII) in transformed Buddleia. Lanes N, P, and 1 to 2 are digested DNA with HindIII from nontransgenic control (N), plasmid pBI121 (P), and transgenic lines (1 and 2), respectively. The digested DNA blot was probed with randomly primed 32P-labeled nptII polymerase chain reaction product.

  • Cardoza, V. & Stewart, C.N. 2003 Increased Agrobacterium-mediated transformation and rooting efficiencies in canola (Brassica napus L.) from hypocotyl segment explants Plant Cell Rpt. 21 599 604

    • Search Google Scholar
    • Export Citation
  • Dai, W. & Castillo, C. 2007 Factors affecting plant regeneration from leaf tissues of Buddleia species HortScience 42 1670 1673

  • Dirr, M.A. 1998 Manual of woody landscape plants 5th Ed Stipes Publishing Champaign, IL

  • Forreiter, C., Kirschner, M. & Nover, L. 1997 Stable transformation of an Arabidopsis cell suspension culture with firefly luciferase providing a cellular system for analysis of chaperone activity in vivo Plant Cell 9 2171 2181

    • Search Google Scholar
    • Export Citation
  • Hogan, S. 2003 Flora: A gardener's encyclopedia Timber Press, Inc Portland, OR

  • Hood, E.E., Gelvin, S.B., Melchers, L.S. & Hoekema, A. 1993 New Agrobacterium helper plasmids for gene transfer to plants Transgenic Res. 2 208 218

  • Jefferson, R.A. 1987 Assaying chimeric genes in plant—The GUS gene fusion system Plant Mol. Biol. Rpt. 5 387 405

  • Lloyd, G. & McCown, B. 1980 Commercially-feasible micropropagation of mountain laurel, Kalmia latifolia, by use of shoot-tip culture Proc. Intl. Plant Prop. Soc. 30 421 427

    • Search Google Scholar
    • Export Citation
  • Lodhi, M.A., Ye, G.N., Weeden, N.F. & Reisch, B.I. 1994 A simple and efficient method for DNA extraction from grapevine cultivars, Vitis species and Ampelopsis Plant Mol. Biol. Rpt. 12 6 13

    • Search Google Scholar
    • Export Citation
  • Murashige, T. & Skoog, F. 1962 A revised medium for rapid growth and bioassays with tobacco tissue culture Physiol. Plant. 15 473 497

  • Petri, C. & Burgos, L. 2005 Transformation of fruit trees. Useful breeding tool or continued future prospect? Transgenic Res. 14 15 26

  • Podaras, P. 2005 Breeding a butterfly bush Landscape Plant News 16 16 7

  • Poupin, M.J. & Arce-Johnson, P. 2005 Transgenic trees for a new era In Vitro Cell. Dev. Biol. Plant 4 91 101

  • Rose, J.B., Kubba, J. & Tobutt, K.R. 2000 Induction of tetraploidy in Buddleia globosa Plant Cell Tissue Organ Cult. 63 121 125

  • SAS Institute 2004 SAS/STAT 9.1 user's guide SAS Institute Inc Cary, NC

  • Sheppard, A.W., Shaw, R.H. & Sforza, R. 2006 Top 20 environmental weeds for classical biological control in Europe: A review of opportunities, regulations, and other barriers to adoption Weed Res. 46 93 117

    • Search Google Scholar
    • Export Citation
  • Tobutt, K.R. 1993 Inheritance of white flower color and congested growth habit in certain Buddleia progenies Euphytica 67 231 235

All Time Past Year Past 30 Days
Abstract Views 0 0 0
Full Text Views 106 25 4
PDF Downloads 81 34 1