Plant Tissue Culture: Micropropagation and regeneration of

							2006 BIG Grant Application

Name of Faculty Proposer: Dr. Emily R. Nekl, Assistant Professor of Biology

Co-Faculty Proposer(s): None

Department(s): Biology

Phone: 841-4656

Email: enekl@highpoint.edu

Project Title: Plant Tissue Culture: Micropropagation and regeneration of herbaceous perennials
native to the eastern United States

Project Start Date and Expected Date of Completion: February 1, 2007 – January 31, 2009

Amount Requested: $24998.00

Project Abstract: This grant proposes the development of a facility with the ability to perform
and study plant tissue culture (micropropagation). Micropropagation is a technology that is
based on obtaining a mass of cells and placing it in a sterile growth environment with the correct
nutrients and hormonal balance for the formation of shoot and root systems. A micropropagation
facility will serve to enhance the laboratory component of several courses and provide research
opportunities for both students and faculty. Developing laboratories in upper-level courses will
provide students with hands on experience in designing/conducting experiments based on plant
biotechnology through the utilization of micropropagation technology. The micropropagation
facility will enable the department to build a research program that focuses on the development
of micropropagation techniques for native wildflowers, including the endangered Shortia
galacifolia Torr. & Gray (Oconee bells) and two species of Hepatica - Hepatica acutiloba
(Sharp-lobed Hepatica) and Hepatica americana (Round-lobed Hepatica). This will allow for
the development of novel, publishable micropropagation techniques, and the ability to increase
the physical numbers of these plants which in the future can be placed into the environment for
the purpose of conservation.


Faculty Proposer: _____________________________ Date: ________________

Department Chair: ____________________________ Date: ________________
Introduction

         Tissue culture is the process of growing biologically active cells in a laboratory for the
purpose of either research or propagation. Plant tissue culture (micropropagation) is the growing
of a plant from cells rather than from a seed or a cutting. Micropropagation was first developed
in the 1950’s and has become the primary method for propagation of many types of plants. This
technique is utilized by the horticulture industry to propagate over 200 million plants a year
(Kyte and Kleyn, 1996). It is also used by plant biotechnologists to study the biological
signaling processes involved with obtaining a mass of cells (an explant), and growing it into a
fully formed and functional plant specimen. Plant growth and development requires an
enormously complex series of signaling networks that are being studied by basic plant scientists
today.
         I propose the development of a facility with the ability to perform and study plant tissue
culture (micropropagation). Micropropagation is a technology that is based on obtaining a mass
of cells and placing it in a sterile growth environment with the correct nutrients and hormonal
balance for the formation of shoot and root systems. A micropropagation facility will serve to
enhance the laboratory component of several courses, allow for the development of two new
courses (Plant Propagation and Plant Pathology) and provide research opportunities for students
and faculty. Developing laboratories in upper-level courses (e.g. Plant Physiology,
Biotechnology, Molecular Biology, Cellular Biology) will provide students with hands on
experience in designing/conducting experiments based on plant biotechnology through the
utilization of micropropagation technology. The micropropagation facility will enable the
department to build a research program that focuses on the development of micropropagation
techniques for native wildflowers, including the endangered Shortia galacifolia Torr. & Gray
(Oconee bells) and two species of Hepatica - Hepatica acutiloba (Sharp-lobed Hepatica) and
Hepatica americana (Round-lobed Hepatica).


Micropropagation Technology

         Micropropagation is the basis of obtaining a mass of totipotent cells and placing it in a
sterile growth environment with the correct nutrients and hormonal balance for the formation of
shoot and root systems. Most plant cells are referred to as totipotent, indicating that they have
the potential to re-specialize and form into any type of specialized plant cell (e.g. cells involved
in water movement, nutrient movement, photosynthesis). In general, an explant, a portion of the
plant used to initiate a culture, can be taken from a plants stem, leaf, or root, surface sterilized to
remove any infectious bacteria or fungi and placed in a sterile nutrient media where the cells can
multiply into a plantlet. In order for this to occur there are three stages of sterile culture growth:
Stage I – establishment of the explant in the culture medium; Stage II – multiplication and Stage
III – rooting. Typically there is a unique nutrient media used for each stage, however this is
determined empirically for each plant species. Explants in Stages I and II do not contain roots,
which allows for the tissue to focus its energy on promoting shoot growth and multiplication.
Once Stage III is complete and roots are formed, the plantlet is then transferred to Stage IV,
which does not require sterile conditions, where it is placed into soil and acclimatized to the



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external environment. Theoretically, thousands of plants can be propagated using explants from a
single mother plant – the plant from which the explant was obtained. All these plantlets will
have the identical genetic makeup of the mother plant. This can be of extreme benefit based on
our ability to select parent plants that have beneficial traits such has increased insect or fungal
tolerance, heat or cold resistance, or particular morphological traits such as a particular flower
color or fragrance. Many plants have seeds that require specific treatment for germination such
as a cold period or scarification from the acidic nature of an animal’s stomach. Cuttings that are
rooted characteristically grow slowly, have a poor survival rate, and are vulnerable to disease.
Therefore, plant tissue culture is often the only practical way to produce large numbers of plants
in a relatively short amount of time for either introduction into the environment or to study a
particular aspect of plant growth and development at a molecular biological or biochemical scale.



Applications of Micropropagation Technology: propagation of native, threatened NC
wildflowers

        Native wildflower plants are of keen interest to plant scientists such as myself based on
the fact that they posses traits that are fit for the environment in which they are native. Native
plants do not require harsh chemicals such as fertilizers and pesticides that are harmful to the
natural environment. They require less water and prevent erosion as compared to non-natives
based on their ability to develop deep root systems that store large amounts of water.
Additionally, they provide shelter and food for wildlife and promote biodiversity in the natural
environment. Native plants are adapted to their environment and maintain unique traits as
compared to non-natives, including heat/humidity/cold tolerance, light intensity tolerance, and
insect and fungal resistance to those organisms prevalent in the native region. These adaptations
have a genetic basis, which as of yet have not been identified, and the potential benefit to human
culture is not known or understood. Unfortunately, with the advent of housing and commercial
development native wildflowers are not being considered when land is cleared. Luckily there are
two volunteer groups that I am aware of that are involved in ‘plant rescues’, the North Carolina
Native Plant Society and the Master Gardeners – both of which I am part of. These rescues are
extremely difficult to organize because land developers do not want a federally threatened plant
to be identified (we have many in North Carolina) which would result in the inability to develop
the land. Both of these organizations have done an excellent job in identifying land being
redeveloped and organizing rescues throughout the year. My involvement with these groups will
provide me with the opportunity to collect plant tissue, from native threatened species, and
propagate them using micropropagation technology. Additionally, there are a handful of small
plant nurseries that specialize in seed and cutting propagation of native wildflowers here in North
Carolina that would be a source of plant tissue. Additionally, I have a verbal collaboration
agreement with Craig Moretz, a PhD student at Wake Forest University and an expert in native
plants of North Carolina, for the collection of plant specimens of interest.




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Applications of Micropropagation Technology: students (classroom and research)

        High Point University students who are part of the biology department will have the
opportunity to be part of learning plant micropropagation either in the classroom or by
expressing an interest in conducting research alongside myself. Plant physiology would be the
first course to utilize this technology. In the course, students will learn how plant tissue culture
is performed, the basis of the nutrient solutions, and growing a plant from an explant. I am
particularly interested in instructing on the micropropagation of both lower plants, such as
Boston fern (Nephrolepis exalta ‘Bostoniensis’ - 3 months) and of higher plants, a flowering
plant such as Flaming Katy (Kalanoche blossfeldiana - 4-6 weeks). Interestingly, it has been
estimated that more Boston ferns are propagated via tissue culture than any other ornamental
crop (Kyte and Kleyn 1996). The protocols for both plants have been well developed and are
appropriate for junior/senior level biology students. In addition to learning micropropagation
techniques in Plant Physiology, it is important that students understand that a significant amount
of plant molecular biology/biochemistry research at the graduate level and in industry is done
utilizing techniques done within a sterile environment such as isolation of protoplasts - single
plant cells without a cell wall. Development of experimentation for studying protoplasts can be
achieved with the acceptance of this grant. It is critical for a student studying plant physiology to
understand how the business industry as well as graduate level research on plant tissue culture is
conducted. Additionally, the more opportunities we give our students to learn scientific
techniques, the more attractive they look to graduate schools as well as businesses who are
hiring.
        Outside of the classroom, there is potential for biology students to either aid in
conducting research concerning native plant propagation or developing semester or year long
projects in which they study another type of plant micropropagation for which they are
interested. Interested and eager students who have novel research ideas will be encouraged to
conduct projects. These students, either working on my research or their own will have the
opportunity to present their findings at national or region plant biologist and native plant
meetings, and if I see fit will be encouraged to publish their results in a peer reviewed journal.
Additionally, professors teaching classes such as Biotechnology Techniques, Molecular Biology
or Cellular Biology who have research interests of their own, or students with interest in research
projects will have the opportunity to utilize the equipment requested (see below).

Applications of Micropropagation Technology: faculty development

          While the intent to bring plant micropropagation to HPU is for the benefit of the students,
it is a technique that I highly value for the advancement of my own research interests. As
discussed earlier native plant micropropagation can be extremely beneficial for increasing the
numbers of native plant species, both endangered and potentially endangered, in endemic areas.
I am particularly interested in species of Shortia galacifolia and Hepatica acutiloba and
Hepatica americana.




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                                       S. galacifolia
              www.ncwildflower.com                      www.pdbase.com

        Shortia galacifolia Torr. & Grey, a member of the Diapensiaceae family is commonly
referred to as Oconee Bells. S. galacifolia is endemic to a small part of the southern Appalachian
Mountains, however small populations have been identified in Virginia, Tennessee, South
Carolina and Georgia. Loss of S. galacifolia can be attributed in part to horticultural collection
and damning construction projects. As a result, the numbers of S. galacifolia have dwindled and
this plant is classified as endangered in the state of North Carolina (North Carolina Department
of Agriculture and Consumer Services, Plant Industry Division, 2006). Interestingly, there are
large populations of its closest relatives, S. uniflora and S. exappendiculata in Eastern Asia
(Ronblom and Anderberg, 2002). It is a stemless perennial herb reaching approximately 6 inches
in height producing round waxy green leaves that persist through the year, turning reddish-
bronze in the winter. The flowers emerge in the early spring and are characterized as nodding,
pendulous, and bell shaped (Armitage, 2006). S. galacifolia is difficult to place in the
environment because they prefer moist peaty areas in the shade, typically in the shady woods
under Rhododendrons and Mountain Laurel or by stream banks (Jones and Augspurger, 1988). It
has poor seed dispersal, with the seeds often retained in the ovary and germinate in situ (Primack
and Wyatt, 1975). These plants require soil acidification, which naturally occurs in the soils of
North Carolina (Armitage, 2006). While the seeds germinate reasonably quickly, the seedlings
are very slow to set. Additionally, propagation through cuttings has been relatively unsuccessful,
and division is the best means of propagation at this point. S. galacifolia specimens are
extremely difficult to find for purchase; a Google search only gives three potential sources and
all sources are extremely expensive with the ability to only purchase one plant based on demand.
Therefore, development of micropropagation protocols for this plant would not only benefit the
conservation efforts to keep S. galacifolia in existence, but it would increase the availability of
plants to people interested in maintaining these plants in public and personal gardens. No
published work has been done on micropropagation techniques of this rare and sought after
specimen. I plan to obtain original specimens of S. galacifolia from a garden center in western
North Carolina – Gardens of the Blue Ridge, as well as collecting samples with Craig Moretz, a
PhD student at Wake Forest University who is an expert in native North Carolina wildflowers
and has access to a collection of S. galacifolia. Any success I have in culturing these plants
would be beneficial not only for the longevity of this plants existence, but would be a novel,
publishable finding. Previous tissue culture work has been done successfully on many plants
including part of the Ericales order (blueberry, azalea, rhododendron), from which the family
Diapensiaceae family is derived. I will begin my study of S. galacifolia utilizing previously
published methods from this order and fine tune the nutrient solutions to fit this particular plant’s


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micropropagation requirements. Eventually I would like to develop collaborations with
botanical gardens throughout North Carolina as well as native plant nurseries for distributing the
material to public and private gardens for the purpose of conservation and education.




           H. acutiloba                   H. americana
       www.gardensoftheblueridge.com    www.leroysplants.com

        Hepatica is a genus of herbaceous perennial plants that belong to the buttercup family,
Ranunculaceae. It is native to eastern North America, central and northern Europe and Asia.
The native species of North America include H. acutiloba – Sharp-lobed Hepatica and H.
americana – Round-lobed Hepatica. Hepatica grows in a wide range of conditions, it can be
found in deeply shaded woodlands and in the grasslands in full sun. These plants will grow in
both sandy and clay based soils, but prefers somewhat alkaline soils. They reach approximately
4-6 inches and produce basal dark leathery green leaves, each with three lobes. The flowers
range from white, bluish purple or pink and are supported singly on hairy, leafless stems
(Armitage, 2006). H. acutiloba is threatened in Connecticut and endangered in Florida and
populations are dwindling in the southeast from development projects. H. americana is
endangered in Florida. It has been suggested by Craig Moretz, a PhD candidate at Wake Forest
University who is an expert of native plants in North Carolina that Hepatica will be classified as
threatened in the near future based on horticultural enthusiasts who collect them from the wild
and development projects. This makes these two Hepatica species ideal for tissue culture. Work
on one of the Asian Hepatica species H. nobilis has been successfully cultured through
micropropagation. This is a plant that is raised and marketed commercially as an ornamental
plant in Japan and demand necessitated development of these procedures (Nomizu et al., 2004). I
plan to utilize the work done on H. nobilis to develop tissue culture techniques for H. acutiloba
and H. americana. The two varieties will be obtained from the Gardens of the Blue Ridge as
well as through my collaboration with Craig Moretz.
        Eventually I would like to allow for these native plants to be introduced back into the
landscape, both in gardens as well as into the wild. This part of the project will be many years
from now, and I plan to work alongside the North Carolina Botanical Garden, Tanglewood
Arboretum in Clemmons NC, as well as through groups such as the North Carolina Wildflower
Society to aid in establishing populations in North Carolina.




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Time table for development of the micropropagation facility

        I expect that laboratory set up will take approximately one year which will include the
completion of development of tissue culture laboratories for Plant Physiology. Concerning
micropropagation of Shortia and Hepatica, I believe that I will have begun the experimentation
for development of the protocols, however I do not believe that these will be completed by that
time and have asked for a two year time frame for this grant to obtain new plant specimens from
the Gardens of the Blue Ridge and fuel to collect samples with Craig Moretz in the second year.
At the end of two years, I believe that I will have begun the collection of primary data and will
be able to apply for external funding from the National Science Foundation in the programs
Undergraduate Research and Mentoring in the Biological Sciences (NSF: 06-591) and
ADVANCE: Increasing the Participation and Advancement of Women in Academic Science and
Engineering Careers (NSF: 05-584).

Micropropagation facility: equipment needs

         The development of a micropropagation facility will require a number of specific pieces
of equipment. The first and foremost piece that is absolutely necessary for this to be successful
is a laminar flow hood. This equipment will provide the necessary sterile environment to clean
the utilized tissue, prepare the explants, and to transfer the explants into the correct nutrient
media. Many micropropagation techniques require a series of nutrient solutions for complete
plantlet formation. This results in the movement of explants from one nutrient media to another.
This must be done in a bacterial and fungal free environment. If not, the tissue will be infected
and consumed by contaminating organisms and not survive. This is the primary expense for this
grant; however, it is extremely important as it is the basis of all the experiments that will be
performed. A laminar flow hood can be utilized for many types of molecular biological
experiments that require a sterile environment. Therefore, students from Plant Physiology as well
those in Cellular Biology, Molecular Biology, and Biotechnology Techniques will have the
opportunity to utilize this equipment. Additionally, any student or faculty based research
projects that require the usage of a sterile working environment will be able to utilize the
equipment. As a result, this equipment will impact potentially 20-25 students per year.
         In order to begin working with micropropagation I need a number of small pieces of
equipment including a pH meter to measure pH of solutions, weigh balance, a specific heating
unit that will sterilize forceps before they are used to transfer sterile explants, glassware,
chemicals to make nutrient solutions, light meter for accurate reading of light quality and
quantity for growth, specific lights containing the correct wavelengths of light, plant stands to
hold the tissue as it grows into plantlets, and a variety of chemicals. All the proposed items (see
budget) are required for the development of a micropropagation facility.




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                                         Literature Cited

Armitage AM. (2006) Armitage’s Native Plants for North American Gardens. Timber Press,
      Portland OR. 212, 213, 348 pp.
Jones SM and MK Augspurger. (1988) Seed germination and phenology of Shortia galacifolia
       T. & G (Diapensiaceae). Castanea 53:140-148.
Justice WS, Bell CR, Lindsey AH. (2005) Wild Flowers of North Carolina. 2nd ed. University of
        North Carolina Press – Chapel Hill NC. 78, 190 pp.
Primack RB and R Wyatt, (1975) Variation and taxonomy of Pyxidanthera (Diapensiaceae).
      Brittonia 27:115-118.
Nomizu T, Niimi Y, Han DS. (2004) Haploid plant regeneration via embryogenesis from anther
      cultures of Hepatica nobilis. Plant Cell, Tissue and Organ Culture. 79:307-313.
North Carolina Department of Agriculture and Consumer Services, Plant Industry Division
       (2006): http://www.agr.state.nc.us/plantind.
Plants Database (2006): http://plants.usda.gov.
Ronblom K and Anderberg AA. (2002) Phylogeny of Diapensiaceae based on molecular data
      and morphology. Systematic Botany 27:383-395.




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Proposed Budget : HPU Micropropagation Facility

YEAR 1

Equipment
     Laminar Flow Hood                                           $11320.00
     (VWR - $11320; Fisher - $11785)
     Weigh balance                                                $1620.00
     (Fisher - $1620 ; VWR - $1875)
     Flourescent Fixtures                                          $948.00
     (9 @118.50)
     Micropipetters (3 @ $250)                                     $750.00
     pH meter                                                      $780.00
     Bacticinerator III (plus replacement heater)                  $415.00
     Light meter                                                   $188.00
     Hot Plate/Stirrer                                             $360.00
     Automatic Pipetter                                            $325.00
     Stainless Steel Bookshelf                                     $130.00
     Gro-Lux Lights (12 @ $25)                                     $300.00
     Cool white Lights (30)                                         $87.00
                                                         Total   $17223.00

Plant Specimens/Plant Collection
       Hepatica acutiloba (5 @ $9.35)                               $46.75
       Hepatica americana (5 @ $4.75)                               $23.75
       Shortia galacifolia (2 @ $30)                                $60.00
       Gas for collecting specimens (250 miles @ 0.36)              $90.00
                                                         Total     $220.50

Glassware
      Tissue Culture Containers (200)                              $347.00
      Test Tubes (245)                                             $346.00
      Test Tube caps/racks                                         $210.00
      Corning Tubes (50mL)                                         $252.00
      Media Bottles (10/250mL)                                      $86.00
      Media Bottles (10/500mL)                                     $103.00
      Media Bottles (10/1000mL)                                    $125.00
      Serological Pipets (5mL/25mL)                                $235.00
      Erlenmeyer Flask (12/250mL)                                   $60.00
      Erlenmeyer Flask (6/500mL)                                    $37.00
      Erlenmeyer Flask (6/1000mL)                                   $60.00
      Beaker (12/250mL)                                             $35.00
      Beaker (6/500mL)                                              $27.00
      Beaker (6/1000mL)                                             $50.00


                                              9
       Graduated Cylinder (4/10mL)                                      $47.00
       Graduated Cylinder (4/100mL)                                    $116.00
       Graduated Cylinder (2/500mL)                                    $112.00
       Graduated Cylinder (2/1000mL)                                   $120.00
       Syringes (10cc/90)                                               $96.00
       Syringe Filters (100)                                           $175.00
                                                             Total    $2639.00

Miscellaneous
       Miscellaneous Laboratory equipment (forceps, tape,             $1500.00
       latex gloves – S,M,L -, weigh boats, spatulas, wash
       bottles, magnetic stirrers)

Inorganic Chemicals
      Inorganic Chemicals used in Tissue Culture                      $1750.00

Organic Chemicals
      Organic Chemicals used in Tissue Culture                        $1000.00

Resource Books
      Micropropagation: Technology and Application.                    $135.00
      (1991) P. Debergh and H. Zimmerman
      Plant Tissue Culture: Techniques and Experiments.                 $70.00
      (2000) H.A. Smith
      Plant Tissue Culture Concepts and Laboratory                     $100.00
      Experiments. (2000) R.N. Trigiano and D.J. Gray
      Experiments in Plant Tissue Culture. (2004) J.H.                  $40.00
      Dodds and H.W. Roberts
      In Vitro Plant Breeding (2001) T. Acram, P. P.                    $70.00
      Kumar, P. Lakshmanan
                                                             Total     $415.00
YEAR 2

Plant Specimens/Plant Collection
       Hepatica acutiloba (5 @ $9.35)                                   $46.75
       Hepatica americana (5 @ $4.75)                                   $23.75
       Shortia galacifolia (3 @ $30)                                    $90.00
       Gas for collecting specimens (250 miles @ 0.36)                  $90.00
                                                             Total     $250.50

Total expected expenses (Year 1)                                     $24747.50
Total expected expenses (Year 2)                                       $250.50


Total Budget (Years 1 and 2)                                         $24998.00


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