Source: TENNESSEE STATE UNIVERSITY submitted to
GENETIC TRANSFORMATION OF PHLOX PANICULATA (GARDEN PHLOX) AND OF MONARDA DYDIMA (BEEBALM) FOR POWDERY MILDEW RESISTANCE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0198948
Grant No.
(N/A)
Project No.
TENX-0402-MAFCT1
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Jan 1, 2004
Project End Date
Sep 30, 2006
Grant Year
(N/A)
Project Director
Sauve, R.
Recipient Organization
TENNESSEE STATE UNIVERSITY
3500 JOHN A. MERRITT BLVD
NASHVILLE,TN 37209
Performing Department
AGRI SCIENCES
Non Technical Summary
While powdery mildew does not necessarily kill plants that it attack, it absorbs plant nutrients causing defoliation and a decline in health and vigor and makes all infected plants unsightly. The development of resistant cultivars of garden phlox and beebalm will reduce production costs, increase landscape succes and avoid the dependency on the use of fungicides.
Animal Health Component
(N/A)
Research Effort Categories
Basic
50%
Applied
(N/A)
Developmental
50%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20121201040100%
Goals / Objectives
The overall objective of this project is to develop selections of bee-balm and garden phlox that are resistant to powdery mildew. The first objective is to develop a regeneration system for these plants, the second is to develop a transforamtion system and the third is to evaluate genetically transformed plants for resistance to powdery mildew.
Project Methods
A regeneration system for bee balm and garden phlox will be developed for cultivars that are important to the nursery and perennial industries. Hypocotyls and cotyledons harvested from new growth and fully expanded leaves and flower stalks will be surface disinfected by immersion in a 1% by volume sodium hypochloride and 0.1% Tween 20 for 10 sec followed by a 10 sec dip in 70% ethyl alcohol. After 3 rinses in sterile deionized water, cotyledons, hypocotyls and flower stalks will be aseptically sectioned into 0.8 cm segments and leaves into 1.5 x 1.5 cm sections. These explants will then be plated onto Murashige and Skoog (MS) medium supplemented with 3% sucrose and solidified with 0.8% agar in peri dishes. To induce shoot formation, explants from each plant tissue types will be grown with factorial combinations of naphthalene-acetic acid (NAA)ranging from 0,05 to 5.0 mg/l and 6-benzylaminopurine (IBA) ranging from 0.05 to 5.0 mg/l. All cultures will be maintained at 24C under cool white fluorescent lights and sub-cultured on fresh media at 2-week intervals. Treatment number will total 40, with 10 explants per replicate from each explant source. To compare the effects of different cytokinins on shoot regeneration from leaf explants, kinetin, zeatin or thidiazuron will be substituted for BA. MS media will consist of 0.1 mg/l NAA and 5.0 mg/l cytokinin. Shoots will be rooted on MS medium supplemented with 2% sucrose and 0.05 mg/l IBA. Rooted plants will be transplanted into 3-qts pots containing a mixture of pasteurized field soil, peat, and perlite (1:1:1) and grown to flowering in the greenhouse. Explants from tissue culture plants will be incubated in a solution containing Agrobacterium tumefaciens strain EHA 105 that harbored the ESF 39A antimicrobial peptide gene following an established procedure in our lab. Infected explants will be cultured on solid callus induction media that contained MS basic salts supplemented with 50 mg/l kanamycin and different combination of cytokinins and auxins. Calli that survive for six weeks on media supplementd with kanamycin will be considered putatively transformed. After two sucultures on this selection media, calli will be tansferred to an embryo induction medium to regenerate new plants. Regenerated plants will be planted in pots and maintained in a greenhouse along with infected susceptible plants. The development of powdery mildew will be monitored at regular intrvals and disease severity will be rated monthly using a 1 to 5 scale. The overall performance of each cultivar over the study will be determined. Highly resistant cultivars will be field evaluated at two different locations.

Progress 01/01/04 to 09/30/06

Outputs
Monarda didyma (beebalm) and Phlox paniculata (garden phlox) were used to determine if they are capable of being transformed using Sinorizobium melloti, a bacteria commonly used as the vector for transferring genes into plant cells. Beebalm and garden phlox shoots were regenerated from leaf tissues obtained from fully grown plants. Explants were plated on Gamborg's medium supplemented with vitamins, 0 .1mg/l NAA and 1.0 mg/l of BA for beebalm and on Gamborg's medium supplemented with vitamins, 0.1 mg/l NAA and 5 mg/l of BAA for the garden phlox explants. For genetic transformation, shoots and calli regenerated from the two plant genera were inoculated with S. melloti harboring the vector that contained the GUSPLUS gene. Transgenic shoots were regenerated on Gamborg's medium supplemented with kanamycin as the selection agent. Enzyme assays and PCR tests were used to confirm the transformation events.

Impacts
Monarda dydima 'Marshal's Delight' and Phlox paniculata 'Franz Shubert' can be transformed with the GUSplus gene using Sinorizobium melloti as the mediating vector. Transformed plants were regenerated; thus, if these plants were transformed with a gene that confers disease resistance, disease resistant plants could be obtained.

Publications

  • Smith, S. 2006. Regeneration of Phlox paniculata and Monarda didyma and Procedures for Genetic Transformation using Sinorhizobium melloti. Tennessee State University Master's Theses, 60 pages.
  • Bhatti, S. M. and Sauve, R. 2005. In vitro culture of Phlox paniculata and Monarda didyma. Southern Nursery Association Research Conference.49:366-368.


Progress 01/01/05 to 12/31/05

Outputs
A regeneration system for bee balm and garden phlox has been developed for two cultivars and a transformation system is being evaluated. These regeneration system were initaited form hypocotyls and cotyledons harvested from new growth, fully expanded leaves and flower stalks. After surface-disinfection by immersion in a 1% by volume of sodium hypochloride and 0.1 Tween 20 for 10 sec followed by a 10 sec dip in 70% ethyl alcohol, they were sujected to 3 rinses in sterile deionized water. The cotyledons, hypocotyls and flower stalks were aseptically sectioned into 0.8 cm segments and leaves into 1.5 x 1.5 cm sections. These explants were plated onto Murashige and Skoog's (MS) medium supplemented with 3% sucrose (w/v) and solidified with 0.8% agar (w/v) in Petri dishes. To induce shoot formation, explants from each plant tissue type were grown with factorial combinations of naphthalene-acetic acid (NAA) ranging from 0.05 to 5.0 mg/l and 6-benzylaminopurine (BA) ranging from 0.05 to 5.0 mg/l. All cultures were maintained at 24C under cool white fluorescent lights and sub-culture on fresh media at 2-week-intervals. Treatment number totaled 40, with 10 explants per replicate from each explant source. The percentage of explants with shoots and number of shoots per explant were recorded after 2 months of incubation. To compare the effect of different cytokinins on shoot regeneration from leaf explants, kinetin, zeatin or thidiazuron (TDZ) were substituted for BA. MS media consisted of 0.1 mg/l of NAA and 5.0-mg/l cytokinin. Shoots were rooted on MS medium supplemented with 2% sucrose (w/v) and 0.05 mg/l IBA. Rooted plants were transplanted into #1 pots (3 qts) containing a mixture of canadian peat, perlite and sand (1:1:1). The second phase of the project is the development of a transformation system. Explants from tissue culture plants will be incubated in a solution containing Agrobacterium tumefaciens strain EHA 105 that harbored the Gus marker gene. Infected explants will be cultured on a solid callus induction media that will contain MS basic salts) supplemented with 50mg/l kanamycin and different combinations of cytokinins and auxins. Calli that survived for six weeks on media supplemented with kanamycin will be considered putatively transformed. After two subcultures on this selection media, calli will be transferred to an embryo induction medium to regenerate new plants.

Impacts
While powdery mildew does not necessarily kill plants that it attack, it absorbs plant nutrients causing defoliation and a decline in health and vigor and makes all infected plants unsightly. The development of resistant cultivars of garden phlox and beebalm will reduce production costs, increase landscape succes and avoid the dependency on the use of fungicides.

Publications

  • No publications reported this period


Progress 01/01/04 to 12/31/04

Outputs
We have devised a regeneration system for the garden phlox and bee balm. Comparative studies were done with three cultivars of Phlox and five cultivars of Monarda. There were genotypic differences in response to treatments in cultivars of both genera. Within three weeks of culture, shoots formed from leaf segments. The Phlox cultivar Franz Schubert developed shoots from leaf explant on a medium that contained 0.1 mg/L NAA and 1 or 5 mg/L BAP. When the concentration of NAA was increased to 0.5 mg/L, adventitious roots appeared on the cultures along with the calli. The highest frequency of shoots was achieved from leaf explants of the Monarda cultivar Marshall's Delight. It ranged from 46 to 90%. Highly embryogenic calli were observed on the media that contained 0.1 mg/L NAA with either 1 or 5 mg/L BAP. Other cultivars such as May Night and Prairie Night regenerated adventitious roots in addition to calli formation. From these results we will perform genetic manipulation studies with leaf explants from 'Franz Shubert' and 'Marshall's Delight'. Thus far, we have not been successful in the regeneration of transformed calli. Different transformation procedures such bollistic bombardment are now being evaluated.

Impacts
Development of disease resistant bee-balm and garden phlox cultivars.

Publications

  • Bhatti, S. M. and Sauve, R. 2005. In vitro culture of Pholx paniculata and Monarda didyma. Proceedings of the SNA Research Conference, Atlanta, Ga.