2. 2
Index
1. Executive summary.............................................................................................3
2. Placement Project................................................................................................3
3. Introduction..........................................................................................................3
4. Purpose Of Agresearch.......................................................................................3
5. Endophytes...........................................................................................................4
6. Methods of determination...................................................................................5
Western Blotting................................................................................5
Slide preparation................................................................................5
Isolation..............................................................................................6
7. Inoculation............................................................................................................6
8. References............................................................................................................7
3. 3
Executive Summary
The document provides the brief descriptionof placement project.
Agresearchis a government crown research institute, whose CEO is
Tom Richardson.Its science capability is divided into six groups
including Animal Productivity, Forage Improvement,Food & Bio-based
Products,Animal Nutrition & Health, Innovative Farm Systems and Land
& Environment.
Placement Project
I started my placementin the Plant and Forage department at the
AgResearchgrasslands. The projectwas about the endophytes living in
grasses.Under the supervision of Christine Voisey and Milan Gagic, I
got to know about the beneficialeffects of the endophytes to plants and
grasses. Differenttypes of methods were used to identify the presence
of endophytes in plants which are discussed below.
Introduction
AgResearchis a Government-owned Crown ResearchInstitute (CRI)
focused onsupporting the pastoral sectorthrough scientific research
and development. With around 850 staff members spread around 4
campus locations (Grasslands, Ruakura, Lincoln and Invermay) it exists
to serve the agriculture and biotechnologysectors of New Zealand
industry.
Purpose of AgResearch
Its purpose is to drive the New Zealand economic engine through
innovation, and will achieve this through the provision of research and
transfer of technology and knowledge. Organisation also enhance the
value, productivity and profitability of New Zealand’s pastoral, agri-food
and agri-technology sectorvalue-chains to contribute to economic
growth and beneficialenvironmental and socialoutcomes for New
Zealand. It also improve animal health, milk quality, productivity and
profitability of sheep and beef farms.
4. 4
Endophytes
Endophytes are naturally occurring fungal microbes that live in the
tissues of varied groups of plants, including many grasses. They are
ubiquitous and are found in almost all plant species to date. There is a
mutual relationship between the plants and endophytes i.e. endophytes
grow in between the cells of host plant drawing nutrients from it but in
return conferresistance to insects, pests,providing some drought
tolerance, protectionfrom overgrazing and prevent pathogenic
organisms. These fungal strands grow between the plant cells and
transmit themselves to next grass generation by growing into the
developing seed head and then to the subsequentgrass seedling (Fig.
1).
Fig. 1 Transmission of endophytes form seeds to new plants
5. 5
Methods of Endophytes Determination
Western Blotting: - It is a widely used basic analytical technique. In this
method, antibodies are used to identify target endophytes.The antibody
used is isolated from rabbit serum.
Principle: - The highest concentration of the endophytes is in the basal
part of the tiller. Cut the tiller as close to the base as possible.Remove
any dead leaf sheath as it may contain any saprophytic fungi. Gently
press each freshly prepared tiller onto the membrane.Once the
Nitrocellulose Membrane (NCM) sheet has been prepared it is ready for
developing. The sheets are then immersed in the solution for 15 minutes
and are kept on the shaker. The sheets are now ready to examine. The
positive tillers are dark red in colour whereas negative ones are light red
in colour. Wetthe blot sheet to make it easier to distinguish positive from
negatives.
Slide Preparation:In this method the epidermallayer of the tillers is
taken and used to make a slide. It is stained in the Aniline blue. The
slide was pre-heated before examining. The slides are then looked
under microscope.
The endophyte is thick highly stained dark blue hyphae and run
longitudinally along the plant cell.
6. 6
Isolations of FungalEndophyte from Grass Tissue:
1. Cut the entire tillers from the selected plant,at about 0.5 cm
above roots.
2. Remove the leaves and upper parts of tiller with a slanted cut and
dispose inautoclave bag.
3. Place the remaining pseudo-stemin McCartney bottle and label.
4. Cover the tiller in 96%drum ethanol. Decant ethanol onto next
sample and continue till end.
5. Cover each tiller with 10% (v/v) janola, agitate and incubate for 2-
3 min.
6. Wash with sterile water and decant to waste
7. Place the tillers on sterile filter paper and leave them to dry
properly.
8. Using flamed sterile scapel, remove the bottom of the tiller to give
a freshclean cut.
9. Carefully manipulate the tillers segments to separate the inner
layers.
10. Place each separate layer on the ABPDA agar and leave
them for sub-culturing.
11. Check growth regularly from all tillers under
stereomicroscope after2 days.
Inoculationswith Endophyteusing wound method
1. Cut a bit of an endophyte from the edge of the colony and remove
as much agar as possible.
2. Place the endophyte on the seedling plate for ease.
3. Cut the endophyte into tiny pieces
4. Lightly nick (do not kill the plant) the seedling at the meristem site.
5. Carefully put the mycelium onto the wound site.
6. After inoculation, wrap plates individually.
7. Place the plates vertically and incubate at 15-25˚C
