An Examination of the Effect

Soil-Set Aid and Impro-

Grain on Programmed Cell

Death Rates in Arabidopsis

thaliana

Ciarán Lyne

14203766

MSc Plant Biology and Biotechnology

Supervisor: Dr. Paul McCabe

Submission Date: 4th

September 2015

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Abstract

The effect of two products produced by Alltech, Soil-Set Aid and Impro-Grain, on

Arabidopsis programmed cell death (PCD) was examined using the root hair assay and cell

suspension cultures. The toxic effect of the products was established and a protective effect

of Impro-Grain against heat induced PCD was also observed. These products have been

developed by the crop science division of Alltech as agricultural additives to enhance crop

yields.

The root hair assay provided a reliable in vivo model for looking at PCD in Arabidopsis

seedlings. PCD can be observed microscopically by its distinctive morphology (condensed

protoplast and retraction of the protoplast away from the cell wall). Impro-Grain was found to

give a protective effect to root hairs against heat induced PCD. The rate of heat treatment

induced PCD in seedlings exposed to the product were only 43.1% of the PCD rates in the

control seedlings.

Both Soil-Set Aid and Impro-Grain were found toxic to root hairs even at relatively low

concentrations, with Soil-Set Aid presenting higher toxicity. A toxic effect to root hairs was

observed in dilutions up to 500,000 and 15,000 in Soil-Set Aid and Impro-Grain respectively.

The toxicity was examined using a root hair assay after 24 hours incubation time in each

product. FDA viability stain was used to distinguish live and dead cells.

Cell cultures were also utilised to examine the toxicity of the products and yielded similar

results to the root hair assay, although the toxic effect appears to ease at much higher

concentrations.

This project has given an important insight into the physiological effects of the two products

on plant cells that could be potentially useful to the plant science and agricultural community.

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Table of Contents

Abstract ...................................................................................................................................... 0

Abbreviations ............................................................................................................................. 3

1. Introduction ............................................................................................................................ 4

1.1 Background to Programmed Cell Death (PCD)............................................................... 4

1.2 Regulators of Plant PCD .................................................................................................. 6

1.3 Plant Programmed Cell Death in Action: ........................................................................ 8

1.3.1 Environmental Stress Response ................................................................................ 8

1.3.2 The Hypersensitive Defence Response ..................................................................... 9

1.3.3 Senescence ................................................................................................................ 9

1.3.4 Development ........................................................................................................... 10

1.4 The Root Hair Assay for Studying PCD Rates in-vivo .................................................. 10

1.5 The Challenge of Improving Crop Yields ..................................................................... 11

1.6 About Alltech and the Products ..................................................................................... 13

1.6.1 Soil-Set Aid ............................................................................................................. 13

1.6.2 Impro-Grain ............................................................................................................ 13

1.7 Aims and Objectives ...................................................................................................... 14

2. Materials and Methods:........................................................................................................ 15

2.1 Growth of Plant Material: .............................................................................................. 15

2.2 Heat Shock of Seedlings: ............................................................................................... 16

2.3 Root Hair Assay/ Scoring Rates of PCD: ...................................................................... 16

2.4 Testing the Alltech Products: ......................................................................................... 18

2.5 Toxicity Tests................................................................................................................. 19

2.6 Examining the Effect of Alltech Products on Heat Induced PCD ................................. 19

2.7 Examining the Effect of Zinc on PCD Rates ................................................................. 19

2.8 Examining the Products Using Cell Cultures ................................................................ 19

2.8.1 Incubation Dilution Preparations ............................................................................ 20

2.8.2 Scoring PCD of Cell Cultures ................................................................................. 21

2.8.3 Packed Cell Volume ............................................................................................... 22

3. Results .................................................................................................................................. 23

3.1 Growth of Arabidopsis Seedlings .................................................................................. 23

3.2 The Root Hair Assay ...................................................................................................... 23

3.3 Toxicity Test of the Alltech Products ............................................................................ 25

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3.3.1 Toxicity of Soil-Set Aid .......................................................................................... 25

3.3.2 Impro-Grain Toxicity Test: ..................................................................................... 26

3.4 The Effect of the Products on PCD................................................................................ 27

3.5 Examining the Effect of Zinc Levels on PCD ............................................................... 31

3.6 Using Cell Cultures to examine the Products ................................................................ 32

3.6.1 Soil-Set Aid Toxicity .............................................................................................. 32

3.6.2 Impro-Grain Toxicity .............................................................................................. 33

3.6.3 Packed Cell Volume Impro-Grain .......................................................................... 34

4. Discussion ............................................................................................................................ 35

4.1 The Toxicity of Soil-Set Aid.......................................................................................... 35

4.2 Optimizing the Experimental Design with the Products................................................ 36

4.3 Possible Benefits to Agriculture .................................................................................... 38

4.3.1 Agricultural Application ......................................................................................... 40

Concluding Remarks ................................................................................................................ 40

Acknowledgments.................................................................................................................... 41

References ................................................................................................................................ 41

Supplementary Data: ................................................................................................................ 47

Abbreviations

AIF Apoptosis-inducing factor

AL-PCD Apoptotic like programmed cell death

FDA Fluorescein diacetate

HR Hypersensitive response

NAA 1-Naphthaleneacetic acid

PCD Programmed Cell Death

M+S Murashige and Skoog

ROS Reactive oxygen species

UV Ultraviolet

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1. Introduction

1.1 Background to Programmed Cell Death (PCD)

Programmed Cell Death (PCD) is a highly regulated process of cellular destruction. It is an

active, genetically controlled process that leads to the elimination of cells (Gadjev et al.,

2008). It has been implicated in a number of crucial processes throughout the animal and

plant life cycle, playing a role in development, defence, and in responses to biotic and abiotic

stress in plants. Programmed cell death can be utilised to regulate cell numbers and to remove

abnormal cells or cells that have already performed their function (Yuan et al., 1993).

Repeated cell division and differentiation in animals allows a fertilized egg to produce

billions of cells to create a body. This process produces many surplus or harmful cells that

need to removed or killed (Nagata, 1997). PCD encompasses multiple, possibly overlapping,

forms of controlled cell death pathways operating in plants (Kacprzyk et al., 2011).

A loss of the ability to regulate PCD can lead to serious problems within an organism. One

example of this is helper T-cells dying at too great a rate in AIDS, or, brain neurons dying

during Alzheimer’s (Pennell and Lamb, 1997). The importance of the ability to control cell

death in developmental processes was seen in early work with the nematode C. elegans.

Sulston and Horvitz, (1977) described the highly repetitive fate of cells and the effect on

development if any changes were made to the system that disrupted cells from undergoing

their intended fate. 1,091 somatic cells are formed during development and of these cells 131

undergo PCD in normal development. Development is hampered when genes controlling

developmental death are altered, ced-3 for example.

Early research on PCD was heavily focused on animal cells. As there is a possible

evolutionary conservation of certain features of PCD between plants and animal cells

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research in plants has often focussed on the similarities between the two processes (Kacprzyk

et al., 2011). The origin of the process may be ancient in origin, arising possibly from a

single shared ancestor (Reape and McCabe, 2008).

Animal cells often (but not always) undergo a specific type of death when they initiate their

cell death mechanisms. This death is associated with a specific cellular morphology and is

termed apoptosis. Initially defined by Kerr, (1972), apoptotic morphology is characterized by

cell shrinkage, condensation and fragmentation of the nucleus and finally a break-up of the

cell into apoptotic bodies. Apoptosis has been described and named to distinguish it from the

unorganised cell death process necrosis. However, recent studies have suggested that necrosis

can also occur in a highly structured way and is controlled by gene expression. Examples of

controlled necrosis such as parthantos and oxytosis have been described and have challenged

the idea of necrosis being uncontrolled, but instead, is simply another form of cell death

(Berghe et al., 2014).

All cells have the required machinery to undergo PCD and rely on signals from neighbouring

cells to suppress these pathways. In animals it is known that signal molecules such as the

platelet-derived growth factor can suppress PCD. In plant cells, McCabe et al., (1997), found

that cells relied on signals that were released by the cells themselves to survive. These signals

were absent when cells were cultured at a low density (100 cells ml-1

) and the cells were

unable to survive. When cells were cultured at the same low density, in media supplemented

with growth signals, the cells were able to survive. Cells grown in fresh media (media that

had not been supplemented with growth signals) at a higher density were able to survive also,

suggesting that the signals needed to survive came from the cells.

Understanding the process of PCD in plants is increasing but still lags behind animal PCD

research. The process of cell death is an extremely complex phenomenon and a full

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understanding of the process in plant or animals systems is likely to challenge researchers

well into the future.

1.2 Regulators of Plant PCD

PCD is a highly regulated gene-directed process (Gadjev et al., 2008). In plant cells, after

activation of this pathway, there is a distinctive morphology that differentiates programmed

cell death from necrotic cell death. This morphologically defined death has been named

apoptotic-like PCD and features of this cell death are similar to those observed in animal cells

undergoing programmed cell death. It is protoplast retraction away from the cell wall that

distinguishes this apoptotic like cell death from other forms of cell death in plants.

Additionally, DNA cleavage is a marker for AL-PCD in plants. PCD-activated nucleases

cleave DNA between nucleosomes at liker sites resulting in fragments of DNA,

approximately 180 bp that can be separated by electrophoresis in agarose gels resulting in a

formation of a ‘ladder pattern’ (Reape and McCabe, 2008).

Apoptotic morphology has been described in the literature. The first paper to use this term

was published by Kerr et al., 1977 in an attempt to distinguish it from necrosis. Apoptotic

cells have a relatively conserved morphology consisting of cell shrinkage, nuclear

condensation and fragmentation and the eventual destruction of the cell into small intact

fragments (‘apoptotic bodies)’. Necrosis often results from exposure to conditions where cells

are killed before they are able to initiate their controlled apoptotic pathways. Necrosis results

in morphological changes in the cells where osmotic control is lost and the cell swells before

bursting and losing its contents. This in turn causes damage to neighbouring cells (Danon et

al., 2000). The distinctive morphology of apoptotic like cells in plants is a vital feature and

the cellular condensation may be an essential element of the process. Controlled retraction of

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the plasma membrane is an active process and may be a driver of some constituents of the

AL-PCD process (Reape and McCabe, 2013).

Although PCD in plants and animals share some similarities the process is not entirely

analogous (Rhoads and Subbiah, 2007). Plant cells, similar to animal cells, appear to release

cytochrome c from the mitochondria into the cytoplasm, have proteases that function as

caspases do in animals, and, form a mitochondrial permeability transition pore to initiate

PCD. Although little is known about the genetic regulation of plant PCD, the important

cellular regulators of the PCD process in plants are emerging.

Cytochrome c has been found to be released from the mitochondria during plant PCD, but

unlike animal apoptosis, it does not appear to be a direct activator of protease activity. In

animal cells, release of cytochrome c into the cytoplasm promotes the assemblage of a

caspase-activating complex leading to the eventual activation of the cell-death caspase

cascade (Reape and McCabe, 2008). Balk et al., (2003), using a free-cell Arabidopsis system,

found that purified cytochrome c itself was not sufficient to induce PCD. Cytochrome c may

play a role in plant PCD in other ways, perhaps by disrupting the electron transport chain,

amplifying the cell death process through a build-up of reactive oxygen species (ROS)

(Reape and McCabe, 2010).

The chloroplast is likely to also be involved in the regulation of PCD in plants as it is the site

of a large proportion of ROS production in the cell (Kacprzyk et al., 2011). During cellular

stress the chloroplasts produce increased levels of ROS. Using suspension cell cultures grown

in the dark or in the light Doyle et al., (2010), showed that AL-PCD was less prevalent in

cultures grown in the dark. Their work implicated chloroplasts to regulation of PCD in plants.

Kim et al., (2012) found that PCD is triggered when levels of singlet O2 is increased in the

chloroplast.

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The mitochondria are also a common site for ROS production (Diamond and McCabe, 2007).

A key role of the mitochondria is in interpreting environmental signals (Lam, 2001). This cell

organelle likely houses a number of key cell death initiating proteins such as Smac/Diablo

and apoptosis-inducing factor (AIF) where they are kept away from the cell components by

the physical barriers of the mitochondria. Changes to the permeability potential of the

mitochondrial membranes can release these proteins triggering PCD. The importance of the

mitochondria across most, if not all, eukaryotes have led to it being termed the ‘cellular

executioner’. This organelle can house numerous proteins that lead to PCD in plant cells.

Various environmental signals can trigger the mitochondria to release these proteins,

initiating PCD (Diamond and McCabe, 2007).

While there has been significant progress in recent times, there remains much to be

discovered as to the specifics of how this cell death programmes is regulated.

1.3 Plant Programmed Cell Death in Action:

1.3.1 Environmental Stress Response

Due to their sessile nature plants have had to develop strategies to adapt to the ever changing

environmental conditions they find themselves in. Key to this adaptability is the plasticity of

plant tissues, of which PCD plays an important role (Wituszynska and Karpinski, 2013).

Globally more than 800 million hectares of land are salt affected. This figure is only going to

increase into the future with natural salinization from sea water along with manmade salinity

from irrigation systems (Munns and Tester, 2008). PCD may be a short term coping

mechanism against salt stress. Huh et al., (2002) found that Arabidopsis plants were capable

of surviving salt shock by using PCD to eliminate salt loaded primary roots. In mutant

varieties the PCD process involved in shedding these roots was inhibited and the mutants

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were unable to withstand the same levels of salt concentration. PCD of the primary root

allowed for the growth of secondary roots more adapted to the saline environment.

PCD has been shown to occur in a number of studies where plants have been subjected to

abiotic stresses. Treatment with H202, ethanol, staurosporine has led to cellular death

displaying characteristic PCD morphology. Apoptotic like morphology has been observed in

a number of species following heat shock. Tobacco, carrot and soybean have all been

reported to undergo PCD in response to abiotic stress induced by heat shock (Reape and

McCabe, 2008).

1.3.2 The Hypersensitive Defence Response

The hypersensitive response (HR) is a rapid killing of cells surrounding the site of pathogen

infection utilized to stunt a pathogens growth and spread throughout the plant after infection.

The success of many pathogens and viruses depends on their ability to inhibit programmed

cell death during the hypersensitive response. The importance of this early response

mechanism is evident in transgenic tomato plants where the p35 gene of baculovirus was

expressed and subsequently exposed to tobacco mosaic virus. In the transgenic plant cell

death was delayed and the virus was able to spread beyond the initial area of infection (Lam

et al., 2001).

1.3.3 Senescence

Senescence is an age-dependant process that results in death at the end of the life span of the

cells involved. It occurs at the cell, tissue, and organ level. The process is not the passive and

unregulated death of cells. During senescence cell undergo conserved changes in structure,

gene expression and metabolism. As senescence progresses nutrients such as nitrogen and

phosphorous are recycled to other parts of the plant in a controlled manner (Lim et al., 2007)

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Senescence is cell death under the control of age and various other exogenous environmental

factors (Lim et al., 2007). Under drought conditions for example the senescence process has

been shown to become accelerated (Rivero et al., 2007).

1.3.4 Development

PCD occurring at specific times and locations throughout the development of vegetative

tissue is essential for correct development. Occurrences of PCD in plant tissue development

include trichome differentiation, the final step of xylogenesis and during root cap sloughing

(Kacprzyk et al., 2011). In carrot cell cultures, suspensor-like cells have been shown to

exhibit condensed morphology when they die (McCabe et al., 1997). In Norway spruce cell

cultures, where the PCD of the suspensor has been interfered with, cells are unable to

undergo embryogenesis. Gene silencing in transformed cell lines prevents accumulation of

metacaspases and prevents the suspensor from dying (Suarez et al., 2004).

1.4 The Root Hair Assay for Studying PCD Rates in-vivo

Early work on PCD in plants utilized the cell culture system as it provided a method for

visualizing cell death at the single cell level. Cell cultures offered the benefits of uniformity

of cells, reduced complexity, and accessibility to each cell (McCabe and Leaver, 2000).

In whole plants, cells that are undergoing PCD are often buried within the tissue and cannot

be visualised. In the past, cell cultures have proven useful for investigating PCD in individual

cells. Establishing cell cultures for this research, however, can be labour intensive and time is

required to reach a mature stable cell line (Hogg et al., 2011).

The root hair assay is an innovative method for determining the rates of PCD and necrosis

induced by environmental stresses in root hair cells (Hogg et al., 2011). This novel technique

has many advantages over the conventional method of using cell culture systems. The root

hair assay allows for the rapid scoring of large numbers of cells in a relatively short period of

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time. This is an in-vivo method that can be carried out within 6 days (from seed to result)

eliminating the lengthy and expensive process of establishing cell cultures prior to carrying

out the experiments.

Using root hairs has offered an opportunity to study PCD in individual cells in an in-vivo

model. These are single cells that can be easily visualised by a light microscope. Corpse

morphology can be used as a visual indicator of PCD and this allows for rapid quantification

of cell death rates. PCD rates using this method have mirrored results obtained using cell

cultures with similar treatments, proving it to be a reliable indicator of cell death processes

(Hogg et al., 2011). Seedlings are stained with fluorescein diacetate (FDA), a viability

indicator. Only viable cells are capable of cleaving FDA to form fluorescein. Root hair

morphological differences are relatively easy to visualise using a light microscope.

Distinguishing between viable cells, necrotic cells and PCD cells visually allows for accurate

quantification of the rate and mechanism of cell death. Scoring cells in this manner gives a

simple, accurate and reproducible system for calculating the effect of proteins or foreign

compounds on the induction or suppression of PCD (Reape and McCabe, 2013).

1.5 The Challenge of Improving Crop Yields

Increasing crop yields in the immediate future is crucial to achieve global food security. The

population worldwide is expected to reach 9 billion people by the year 2050. It is estimated

that to adequately feed this number of people food production will have to increase by up to

70% compared to current levels. This increased production has to occur with reduced inputs

and with little to no increase in land area used for crop production (Tester and Langridge,

2010).

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Crop yields, particularly in Europe, have shown some evidence of reaching a threshold level

where yield has stagnated over a number of years (Fig. 1) after a period of steady increases in

the aftermath of the ‘Green Revolution’ in the 1970’s (Grassini et al., 2013).

Figure 1: The levelling off of wheat yields over the last 30 years (Grassini et al., 2013).

Crop losses can occur for a number of reasons. Globally the primary cause of crop losses is

abiotic stress. The occurrence of these stresses is likely to increase into the future with global

climate change bringing about higher occurrence of extreme weather events such as extended

periods of high temperatures, torrential rain and drought (Lloret et al., 2012). Drought and

salinity are the two most common stresses faced by plants leading to yield losses. It is

expected that by 2050, 50% of all arable lands will face these sub-optimal conditions

(Vincour and Altman, 2005). Plants respond to abiotic stresses in a variety of ways including

changes at the transcriptome, cellular and physiological levels. Abiotic stresses have been

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estimated to reduce average yields in many of the major crops by up to 50% annually from

their potential maximum yield (Atkinson and Urwin, 2012).

1.6 About Alltech and the Products

Alltech is a global biotechnology company founded in 1977 by Irish biochemist Dr. Pearse

Lyons. Alltech’s core business is animal feed. While animal feed is Alltech’s primary

business they have diversified to include a crop science division that complements its animal

nutrition sector (http://www.alltech.com/about/our-story).

Alltech have produced two products of interest to this study, Soil-Set Aid and Impro-Grain

1.6.1 Soil-Set Aid

Soil-Set Aid is applied to the soil as a spray or via an irrigation system onto crop residues left

in the field after harvest or around the time of planting. According to Alltech, Soil-Set Aid

promotes plant growth by improving mineral availability for plant nutrition, by contributing

to a healthy agribiome and by supporting root development. Soil-Set Aid is composed of a

yeast cell wall extract and contains zinc (3.2%), copper (2%), iron (1.6%), and manganese

(0.8%), (Supplementary data). This product is currently patented by Alltech (US patent

8053391 for abiotic stress in plants).

Trials in a number of countries have found that soils treated with Soil-Set Aid provided plants

with better access to nutrients in the soil, promoting a healthy growth environment. Soil-Set

Aid has been recommended by Alltech for use with alfalfa, grass, maize, soybean, wheat,

barley and oats. This product, in accordance with EC 834/2007 is suitable for use in organic

farming.

1.6.2 Impro-Grain

Impro-Grain is a blend of ingredients that have been derived from fermentation processes. It

is a unique combination of micro-nutrients and a natural surfactant. Impro-Grain contains

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zinc (1.2%) and manganese (0.8%). Over 26 independent field trials in a number of countries

such as Ireland, Australia, Turkey and South Africa Impro-Grain were found to increase

yields by an average of 1 tonne/ hectare or 13% in wheat and barley crops.

According to Alltech, Impro-Grain works by streamlining plant metabolic processes.

Improving metabolism and photosynthesis provides the plants with more energy with which

to produce biomass. This product has been recommended by Alltech for use with alfalfa,

maize, soybean, wheat, barley and oats. In accordance with EC 834/2007 Impro-Grain is

suitable for use in organic farming.

1.7 Aims and Objectives

This project aims to examine the effect of two products developed by Alltech on the rate of

programmed cell death in Arabidopsis seedlings induced by heat shock.

To carry out this project Arabidopsis seedlings were grown and incubated in the products at

various concentrations for a period of time before they were observed to determine if there

was any change in PCD rates. To achieve this goal, the concentration where any toxic effect

of these products is too great needed to be determined. The temperature point best suited to

testing the products also needed to be determined.

Cell cultures were used to test whether any toxic effect of these products on root hairs

translated to the undifferentiated cells found in cell cultures. Cell cultures were also used to

determine whether cells growth in light dark conditions make any difference to the effect of

the products.

These products have been developed by Alltech as an additive to increase crop yields. The

goal of the project is to determine the action of these products on the PCD pathways of

Arabidopsis seedlings to understand how the products affect their targets. If the mode of

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action can be determined then the efficiency with which they are used and produced could be

optimized.

2. Materials and Methods:

2.1 Growth of Plant Material:

Arabidopsis thaliana Columbia-0 (Col-0) seedlings were used for the root hair assays in this

experiment. For the root hair assay seedlings were 5-6 days old. Seedlings were grown on

solid growth medium. Half-strength Murashige and Skoog medium (MS/2) was prepared as

follows; per 500 mls, 1.1g MS basal salt mixture, 5g sucrose, pH adjusted to 5.6-5.8. Agar

(6g) was added (final gelling agent, concentration 1.2%) and the medium was sterilized by

autoclaving.

Seeds were placed into the growth media under aseptic conditions in a laminar flow hood.

Prior to using the laminar flow hood the surfaces were wiped with 70% EtOH.

Seeds were surface sterilized before plating. Sterilization was carried out by submersion in

10% Milton Bleach solution, mixing by inversion occasionally. Seeds are subsequently

washed with deionised water 3-4 times.

Seeds were placed individually in 3-4 rows on solid media in square Petri plates (12x12 cm)

by suction using 200µl pipette tips. Plates were sealed with parafilm and placed in the dark at

4ºC for 24 hours to allow vernalization to occur for uniform germination of each of the seeds.

The seeds were moved to constant light at 25ºC after 24 hours where they were placed in a

vertical position allowing roots to grow on top of the media and not into the media. This is

done to allow for movement of seedlings with less mechanical damage to cells.

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After 5-6 days the seedlings are ready to be used for the root hair assay to determine PCD

occurrence.

2.2 Heat Shock of Seedlings:

Twenty-four-well cell culture plates were used for heat shock. These plates allow for the

simultaneous treatment of a large number of seedlings.

One ml of MS/20 (to prepare 500ml; 0.11g MS basal salt mixture, adjust the pH 5.6-5.8 and

autoclave) is added to each well. Arabidopsis seedlings are transferred to individual cells

using a forceps. One ml is added to all of the 24 wells to ensure that heat transfer is uniform

to all of the cells of the plate. Transfer of seedlings should be gentle to avoid mechanical

damage to the root hairs. The cultures plates can be sealed using Leucopore tape.

Water baths (Grant OLS 200) were set to the desired temperatures, with the shaking feature

set to ‘off’. The culture plates containing the Arabidopsis seedlings were placed on the

surface of the water for ten minutes to perform the heat shock.

Once the ten minutes elapsed the culture plates were removed and placed in the light at 22ºC

until scoring for the occurrence of cell death.

2.3 Root Hair Assay/ Scoring Rates of PCD:

Directly prior to performing the root hair assay a 0.001% w/v FDA solution is prepared. For

1ml FDA solution: add 10µl FDA stock solution to 990µl deionised water.

A drop of FDA solution was transferred directly to glass microscope slides and whole

Arabidopsis seedlings were stained directly on the slides. The seedlings were immediately

examined under white and fluorescent light.

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Seedlings were examined under white and fluorescent light. Starting at the tip of the roots

individual root hairs were examined for FDA staining and cellular morphology.

Individual root hairs are recorded as viable if they stain FDA-positive, necrotic if they do not

stain and do not exhibit PCD morphology. PCD root hairs do not stain with FDA and exhibit

the specific morphological traits associated with this type of cell death; a condensed

cytoplasm and retraction of the protoplast away from the cell wall (Fig. 2).

Using mechanical counters root hairs are scored until approximately 100 hairs per seedling

have been counted.

Figure 2: Apoptosis-like PCD and necrosis morphologies in Arabidopsis root hairs. Root hairs

were treated with the Alltech products for 8 and scored for morphology 16 h later, after FDA

staining. (A) Viable root hair cells cleave FDA and fluoresce under light at a wavelength of

490 nM. (B) Apoptotic-like PCD cells cannot cleave FDA, do not fluoresce and the

protoplast retracts from the cell wall. (C) Necrotic cells, cannot cleave FDA, do not fluoresce

and show no evidence of protoplast retraction (Reape and McCabe, 2013).

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2.4 Testing the Alltech Products:

Dilutions of Soil-Set Aid and Impro-Grain were prepared before use each day. The product

was stored in darkness in the fridge (4°C) (Fig. 3).

Figure 3: Soil-Set Aid (L) and Impro-Grain (R)

Prior to use each product was well shaken to ensure that the active ingredients are evenly

distributed within the product as a solid forms on the bottom of the tubes (Fig 4).

Figure 4: An example of the solid forming at the bottom of the tubes. Shaking prior to use is

required to dissolve back into solution.

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Dilutions were prepared by serial dilution. Stock was created by diluting the product 1000X

initially. Ten ml stock was prepared by adding 10µl of each product to 9.99 ml MS/20

solution. A vortex was used to ensure that the entire product was diluted evenly in the

solution.

2.5 Toxicity Tests

Seedlings were incubated in the products at various dilutions for 24 hours before scoring for

cell death rates.

2.6 Examining the Effect of Alltech Products on Heat Induced PCD

Col 0 seedlings (5-6 days old) were incubated in the products before heat shock.

Heat shock was carried out as per section 2.2.

The time selected for incubation and the period of time after heat shock before scoring were

chosen based on levels where PCD was seen to be approximately 50%. This allowed for

small changes in the rates of PCD to be easily observed.

2.7 Examining the Effect of Zinc on PCD Rates

A stock solution of 3.2% zinc was made dissolving 1.4g zinc sulphate heptahydrate

(ZnSO4.7H2O) in 10 ml deionised water.

This stock was diluted as described in section 2.4. 5-6 day old Arabidopsis seedlings were

incubated in the zinc solutions for 24 hours before being scoring.

2.8 Examining the Products Using Cell Cultures

Cell culture media was prepared as follows: 4.3g/L M+S basal salt mixture, 30g/L sucrose,

0.05 mg/L kinetin, 0.5 mg/L NAA. The pH is adjusted to 5.8.

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Media (100 ml) was poured into 250ml conical flasks and the top of the flask was covered

with tin-foil. The media was then autoclaved. Cell cultures are stored in constant temperature

with constant shaking. Cells are grown in constant light or constant dark. If cells are required

with high chlorophyll levels then they are grown in the light where they produce functional

chloroplasts. Cells grown in constant dark have no functional chloroplasts (Doyle et al.,

2010).

Cell subcultures are kept fresh by adding 10 ml of culture into 100ml of fresh media every 7

days. Subcultures are prepared aseptically in the laminar flow hood. Transfer of cells is

carried out via pipette and mechanical pipette filler. To minimise the risk of contaminating

the cultures new pipettes were used for each subculture. Contact of the pipette tip to all

surfaces was avoided to maintain aseptic conditions.

2.8.1 Incubation Dilution Preparations

To incubate the cell cultures with the products 25ml conical flasks were used. Five mls of the

cell culture were transferred to the flasks containing the product at various dilutions. Cell

cultures grown in the dark are left in the dark as close as possible to being used to ensure that

chlorophyll levels do not increase beyond desired levels.

1000 times dilution was created by adding 5µl of the desired product to 5ml of the cell

culture.

Greater dilutions were created by first making up a stock dilute of the desired product. To

make a dilution of 10,000 times for incubation, a stock of 100X dilution was made and from

this 50µl was added to 5ml of the cell culture.

1 4 2 0 3 7 6 6 | 21

Cell cultures are incubated in the product for 24 hours before scoring under UV microscope.

Viable cells stain fluorescent after FDA staining. Necrotic and PCD cells have distinctive

morphology that allows for their discrimination from one another.

2.8.2 Scoring PCD of Cell Cultures

PCD was scored as per the root hair assay with viable cells fluorescing under UV light.

Necrotic and PCD cells are distinguishable by their different morphological appearance.

Approximately 50µl of FDA solution (10µl/1ml deionised water) is placed on a microscope

slide. An equal volume (50µl) of the cell culture is dropped into the FDA stain and covered

with a glass slide.

Scoring cell cultures follows the same principal as the root hair assay. Viable cells fluoresce,

PCD cells display distinct morphology (retraction of the protoplast away from the cell wall

leaving a visible gap) and necrotic cells did not fluoresce or display distinctive PCD

morphology (Fig. 5).

1 4 2 0 3 7 6 6 | 22

Figure 5: Apoptosis-like PCD and necrosis morphologies in Arabidopsis cell cultures. Cells

were incubated in the Alltech products for 24 hours before scoring for cell death rates. Prior

to scoring cell were stained with FDA as per the root hair assay. (A) Viable root hair cells

cleave FDA and fluoresce under light at a wavelength of 490 nM. (B) Apoptosis-like PCD

cells cannot cleave FDA, do not fluoresce and the protoplast retracts from the cell wall. (C)

Necrotic cells, cannot cleave FDA, do not fluoresce and show no evidence of protoplast

retraction.

2.8.3 Packed Cell Volume

Ten ml of the cell cultures is placed in 15 ml tube and centrifuged at a low speed (1400

revolutions). The tubes are then left for a set length of time (for this project 2 days and 5 days

were chosen) and the cells that have accumulated at the bottom of the tube are measured.

Measurement of growth is carried out by measuring the height of cells that have accumulated

on the bottom of the tube after they have been centrifuged. The total height of liquid in the

tube in measured and the lowest point where cells have attached to the side of the tube is

measured. Measurements are taken in millimetres. The packed cell volume is calculated as

follows;

1 4 2 0 3 7 6 6 | 23

The figure obtained is the volume percentage of plant cells in the tube.

3. Results

3.1 Growth of Arabidopsis Seedlings

Seedlings grown on MS/2 media germinated uniformly after a vernalization period of 24+

hours. After 5 days in the growth room at constant light and temperature the seedlings were

ready for PCD testing by the root hair assay.

3.2 The Root Hair Assay

A ten minute heat shock of Col 0 seedlings produced the following rates of cell death at each

temperature (Fig. 6)

1 4 2 0 3 7 6 6 | 24

Figure 6: Heat curve produced 24 hours after 10 minute heat shock. Heat shock induces PCD

up to 55°C. At higher temperatures necrotic cell death is most abundant. Cell death at 25°C

(control) is background cell death.

At room temperature, 25ºC, the majority of cells (>70%) remain viable after 24 hours and

any cell death is deemed to be background death. Morphology of the dead cells indicates that

necrosis is almost absent and any dead cells have activated PCD at this temperature.

PCD rates increase until 55ºC. The increase in PCD is mirrored by a decrease in viable cells.

Necrotic cell death remains at background levels despite the rise in temperature. At 55ºC the

rate of necrosis cell death increases. Viable root hairs are no longer observed after 55°C.

Necrotic death increases after 55°C and accounts for >80% of root hairs at 75ºC.

A heat curve produced 6 hours after ten minutes heat shock mirrored the heat curve after 24

hours (Fig. 7).

0

10

20

30

40

50

60

70

80

90

100

25 49 51 53 55 65 75

% C

ells

Temperature

PCD

Necrosis

Viable

1 4 2 0 3 7 6 6 | 25

Figure 7: Heat curve produced when root hairs were scored 6 hours after ten minute heat

shock.

3.3 Toxicity Test of the Alltech Products

Seedlings were incubated in each product at various dilutions for 24 hours before scoring of

root hairs for PCD rates.

3.3.1 Toxicity of Soil-Set Aid

Soil-Set Aid was toxic to seedlings at all dilutions up to 500,000 thousand times diluted. The

toxic effect of Soil-Set Aid was found to gradually decrease after 500,000 thousand times and

the toxicity had disappeared when dilution reached 750,000 times (Fig. 8).

0

10

20

30

40

50

60

70

80

90

100

25 49 51 53 55 65 75

% R

oo

t H

airs

Temperature (°C)

Viable

PCD

Necrosis

1 4 2 0 3 7 6 6 | 26

Figure 8: Percentage of viable root hairs after 24 hours incubation in Soil-Set Aid at various

dilutions. Seedlings were incubated in the product for 24 hours before scoring the root hairs

for viability.

In the control >90% of root hairs remain viable after 24 hours. Soil-Set Aid was found to

cause cell death to >95% of root hairs in dilutions up to 25,000 times. At 100,000 times

dilution 21% of root hairs were viable after 24 hours. At dilutions from 500,000-750,000

there remains a slightly toxic effect. The toxic effect is found to be gone at 750,000 times

dilution (Fig. 8).

3.3.2 Impro-Grain Toxicity Test:

Impro-Grain was less toxic to the seedling than Soil-Set Aid. Dilutions up to 15,000 had a

negative effect on the viability of root hairs (Fig. 9).

92 92 92 92 92 94 97

83

0 0 3

21

63

81 82 81

0

10

20

30

40

50

60

70

80

90

100

1000 10000 25000 100000 200000 500000 625000 750000

Pe

rce

nta

ge o

f V

iab

le C

ells

Soil-Set Aid toxicity at various dilutions. Control is MS/20 solution

Control

Soil-Set Aid

1 4 2 0 3 7 6 6 | 27

Figure 9: Percentage of root hairs that remained viable after exposure to Impro-Grain at

various dilutions verses exposure to MS/20 solution. Seedlings were incubated for 24 hours

in the product before scoring root hairs for viability.

3.4 The Effect of the Products on PCD

Seedlings were incubated in various dilutions of each product and examined for rates of PCD.

Soil-Set Aid was found to have little to no effect on PCD rates at any dilution.

Impro-Grain was examined and when diluted 5000 times this product was found to have a

protective effect for root hairs against heat induced PCD. At 5000 times dilution rates of PCD

in the control were higher than in seedlings incubated in the product after heat shock at 49ºC

for ten minutes (Table 1).

91

77

91 97 94 91 91

54

68 78

89 96

88 92

0

20

40

60

80

100

120

1000 5000 10000 15000 25000 50000 100000

Pe

rce

nta

ge o

f V

iab

le C

ells

Imro-Grain Dilution

Control

Imrpo-Grain

1 4 2 0 3 7 6 6 | 28

Table 1: Percentage root hairs undergoing PCD after 10 minute heat shock at 49ºC.

Repeat 1 2 3 4 5 Mean

Control 73 56 58 62 81 66

Impro-Grain 56 47 43 59 56 52.2

% Difference 77 84 83 95 69 79

After 10 minute heat shock at 49°C seedlings incubated in Impro-Grain had rates of PCD that

were 79% of those observed in the control.

The rate of PCD induced as a direct result of the heat shock was compared for each treatment.

This was calculated by subtracting the background levels of cell death. The level of PCD

induced as a result of the heat shock was calculated by subtracting the levels observed in the

control temperature from those seen at 49°C.

1 4 2 0 3 7 6 6 | 29

Figure 10: Mean PCD induced directly as a result of heat shocking. Figures have been

corrected for background levels of PCD. Necrosis levels were unaffected by heat shock,

remaining <10%. The reduction in root hair PCD was due to an increase in viable root hairs.

The means were from 5 independent biological repeats consisting of >4 seedlings per

treatment.

0

10

20

30

40

50

60

Control Impro-Grain

% r

oo

t h

airs

PCDp=0.003

1 4 2 0 3 7 6 6 | 30

Table 2: Observed difference in PCD rates induced by heat shock in control vs. Impro-Grain.

Figures have been corrected for PCD induced by heat only.

PCD (% root hairs) 1 2 3 4 5 Mean

Control 50 49 36 49 48 46.4

Impro-Grain 27 7 14 34 18 20

% Difference 54 14 39 69 38 43.1

When the levels of PCD are corrected for background levels of cell death the rate of PCD

was 43.1% that of the control over 5 independent biological repeats of the experiment (Table

2, Table 3). Each experiment repeat consisted of >4 seedlings per treatment.

Table 3: Two-sample T-test assuming unequal variances between PCD induced by heat

shocking in seedlings pre incubated in Impro-Grain 5000X or MS/20. Significance is taken as

P<0.05.

t-Test: Two-Sample Assuming Unequal Variances

Variable 1 Variable 2

Mean 46.4 20

Variance 34.3 113.5

Observations 5 5

Hypothesized Mean Difference 0

df 6

t Stat 4.855698492

P(T<=t) one-tail 0.001417599

t Critical one-tail 1.943180281

P(T<=t) two-tail 0.002835198

t Critical two-tail 2.446911851

1 4 2 0 3 7 6 6 | 31

3.5 Examining the Effect of Zinc Levels on PCD

Zinc was found to have a toxic effect on root hairs (Fig. 11).

Figure 11: Viable root hairs as a percentage of viable root hairs observed in the control.

Dilutions correspond to the zinc equivalent in Soil-Set Aid at the same dilutions. The figures

are means obtained over 3 independent biological repeats with >4 replicates.

A toxic effect was observed in root hairs in dilutions up to 100,000 times. The percentage of

root hairs remaining viable increased as the volume of zinc in solution decreased. The

percentage of root hairs that underwent necrosis did not change significantly in the presence

of zinc, remaining at background levels. The results were obtained over 3 independent

biological repeats with 4 replicates in each experiment (Fig. 11).

0

20

40

60

80

100

120

1k 10k 50k 100k Control

% ViableCells VsControl

1 4 2 0 3 7 6 6 | 32

3.6 Using Cell Cultures to examine the Products

3.6.1 Soil-Set Aid Toxicity

Soil-Set Aid was found to be toxic to cell cultures. The toxic effect was seen in cell cultures

grown in both the light and the dark (Fig 12).

Figure 12: Percentage of viable cells in cell cultures exposed to Soil-Set Aid. Figures are a

mean obtained from 3 independent biological repeats.

0

20

40

60

80

100

120

1000 2500 5000 10000 Control

% C

ells

Soil-Set Aid Dilution

Light

Dark

1 4 2 0 3 7 6 6 | 33

Soil-Set Aid is toxic to cell cultures. Both cultures grown in the light and cultures grown in

the dark are affected by this product. Cell cultures grown in darkness are more susceptible to

this product than those grown in the light (Fig. 12). Only 7% of cells remain viable in light

grown cells at 2500 times dilution, whereas 53% remain viable in dark grown at the

equivalent dilution of Soil-Set Aid.

3.6.2 Impro-Grain Toxicity

Impro-Grain was found to have little toxic effect on cell cultures. The small toxic effect of

this product disappeared in much lower concentrations of the product (Fig. 13).

Figure 13: Percentage of viable cells in cell cultures exposed to Impro-Grain. Figures are a

mean obtained from 3 independent biological repeats.

0

20

40

60

80

100

120

Control 500 1000 5000 10000 50000

% V

iab

le C

ells

Impro-Grain Dilutions

Light

Dark

1 4 2 0 3 7 6 6 | 34

There is little toxic effect observed on cells once dilution has reached 1000 times in either the

light or dark grown cells. At 500 times dilution the number of viable cells drops significantly,

with only 61% and 73% remaining viable in light and dark grown cells respectively. The

toxic effect is not as strong in Impro-Grain as it is in Soil-Set Aid.

3.6.3 Packed Cell Volume Impro-Grain

After 2 days the light grown cells showed no significance difference in growth rates between

cells incubated with Impro-Grain and the control. There was also no difference in cells grown

in light or in the dark (Table 4).

The experiment was repeated, allowing 5 days for cells to grow.

Table 4: Packed cell volume for Arabidopsis cells incubated in Impro-Grain various dilutions

grown in the light and in the dark for 5 days.

Light Total Height (mm) Height Cells (mm) Packed Cell Volume (%)

Control 74 37 50

5,000 times dilution 70 36 51

10,000 times dilution 70 36 51

Dark

Control 52 22 42

5,000 time dilution 69 30 43

10,000 times dilution 68 35 51

1 4 2 0 3 7 6 6 | 35

4. Discussion

The root hair assay proved to be a useful method for determining PCD rates in-vivo in

Arabidopsis seedlings. Within 7 days of plating seeds the rate of PCD could be determined by

FDA staining and distinguishing morphological characteristics. Using this method it was

determined that Impro-Grain has a protective effect on root hairs against heat shock induced

PCD. Incubation in Impro-Grain at 5000 times dilution prior to heat shock saw a reduction in

root hairs undergoing PCD and remaining viable. PCD rates were 43.1% those of the control

when incubated in Impro-Grain prior to heat shock.

The root hair assay was also found to be an effective method of determining the toxicity of

the products being examined. Both Soil-Set Aid and Impro-Grain were toxic to Arabidopsis

root hairs. Soil-Set Aid was the more toxic of the two and the negative effect on root hairs

observed only lessened when diluted to >500,000 times. Using cell cultures this toxicity was

less extreme but still present in cultures grown in the light and the dark.

4.1 The Toxicity of Soil-Set Aid

The toxicity of Soil-Set Aid may be caused by the high levels of zinc in this product. Zinc is

an essential micronutrient in plants, yet, at higher concentration it has been shown to be toxic

(Subba et al., 2014). Helmersson et al., 2008 suggested that zinc may play a role in PCD rates

due to its inhibitory effect on plant metacaspases. Zinc is present in Soil-Set Aid at 3.2%

derived from zinc heptahydrate (Supplementary literature). To examine if zinc contributed to

the high toxicity of this product, seedlings were incubated in a zinc solution equivalent to the

zinc content in Soil-Set Aid at various dilutions. Zinc was found to induce PCD at relatively

low concentrations. Soil-Set Aid was found to be toxic to the seedlings at dilutions as large as

500,000X. The zinc solution was also found to be toxic to Arabidopsis seedlings. However,

the number of viable cells was greater in the zinc treatments at the same dilutions, suggesting

that it may contribute to the toxicity of the Soil-Set Aid but is not solely responsible.

1 4 2 0 3 7 6 6 | 36

Copper is also present in Soil-Set Aid at a significant level (2%). Copper been shown to have

negative effect on plant growth despite being an essential micronutrient for normal plant

metabolism. High levels of copper can affect how plants metabolise nitrogen (Xiong and

Geng, 2006). A recent study looking at the toxic effect of both zinc and copper in barley

found that the EC50 of copper ranged from 0.39-1.21µM, compared to an EC50 of 109-364µM

in zinc (Versieren et al., 2014). The negative effect of both zinc and copper was also found to

increase when the two metals interacted with each other. At a dilution of 500,000 times there

would be approximately 0.8µM of copper present in Soil-Set Aid and this is likely to have

played a large role in its toxicity and would be an interesting place to look in any future

research into this product.

It may be that cells that survive the initial toxic effect of Impro-Grain at 5000 dilution prime

their defences to cope with the heat shock better. PCD is a gene regulated process induced by

different stimuli (Rantong and Gunawardena, 2015). Environmental factors can contribute to

natural variation in gene expression (Choi and Kim, 2007). At 5000X dilution Impro-Grain

appears to create conditions that lead to altering the PCD process.

4.2 Optimizing the Experimental Design with the Products

The toxicity test and early test of PCD rates were found to give variable results. Early

experiments, before optimization of the experimental design, pre-incubation dilutions of

750,000 and 25,000 were used for Soil-Set Aid and Impro-Grain respectively with a heat

shock at 51ºC. Exposed to these conditions, the rate of root hair PCD fluctuated greatly. It is

possible that these experimental conditions were extremely close to the ‘point of no return’ in

plant PCD. It is at this point that plant cells are irreversibly committed to dying (van Doorn,

2005). The root hairs exposed to the conditions may not have been completely committed to

die at the chosen concentrations and temperatures as they may remain useful as a source of

minerals. At higher concentrations of the product this point of no return is passed and the

1 4 2 0 3 7 6 6 | 37

toxic effect is clear at concentration of up to 200,000-300,000 times dilution where viable

cells are almost entirely absent.

During this study there was a difficulty in choosing the best temperature and time points with

which to test any possible effect of the products on PCD. The time left between heat shocking

seedlings and subsequent scoring was eventually chosen as greater than 16 hours. PCD

morphology can be observed after 6 hours (as reported by McCabe et al., 1997), however the

consistency of results was found to vary greatly at this time point. The importance of the time

point at which cells are observed has been reported in the literature (Doyle et al., 2010). It is

likely that after 6 hours the process had yet to come to completion and those individual

seedlings were closer to PCD completion than others. The difference in root hair death rates

can be seen in this project when comparing viable cells from the heat curves obtained in this

project (Figure 6 and Figure 7). After 24 hours heat shock at 51ºC gave almost no viability

(<10%). At the same temperature heat shock, when root hairs were scored after 6 hours, the

percentage of viable cells was much higher (>30%), suggesting that the death process may

not have come entirely to completion after 6 hours. After 16 hours the results were more

consistent and the cell death process had likely run its course at this time point.

Pre-incubation times were also important in this study. The times needed to allow the

products enough time to have an effect on the PCD pathways while ensuring that the

seedlings were scored within the 5-6 day window that is optimal for this method of PCD

scoring. In seedlings left 7 days or more after vernalization, a higher instance of death is seen

in root hairs versus controls after 5-6 days.

Variability in the results may be explained by natural variation amongst the seedlings.

Variety in life is seen every day and this may have been the cause of the large differences

seen in relation to PCD rates of seedlings when exposed to the same treatment.

1 4 2 0 3 7 6 6 | 38

That Impro-Grain is less toxic to the root hairs is not surprising. Impro-Grain was produced

for foliar application to plants, whereas Soil-Set Aid is to be applied to the soil before

planting so that the toxic effect may have lessened by the time crops are planted.

Both light and dark grown cell cultures were utilized as these products are designed for soil

application in the case of Soil-Set Aid and foliar application in the case of Impro-Grain. Cell

cultures grown in constant light produce functional chloroplasts as evidenced by their green

colour but cell grown in the dark do not. Due to the sucrose medium the cells are able to

grown in the dark while not producing functional chloroplasts (Doyle et al., 2010).

4.3 Possible Benefits to Agriculture

The work carried out in this study used only the dicot species Arabidopsis thaliana. For

practical application in agriculture the effect of these products on monocots will need to be

studied also, as it is monocots that are responsible for a large proportion of biomass produced

in agriculture (Panis, 2008).

A small increase in the percentage of cells capable of withstanding abiotic stress could have

a significant effect on the final yield produced. There are examples, such as the re-greening

of yellow leaves where PCD appears to have been arrested and even reversed (van Doorn

(2005). Studies looking at maize have found that delaying senescence has prolonging the

growing season, allowing for an increase in biomass produced (Lee and Tollenaar, 2007).

The toxic effect of these products on Arabidopsis was clear throughout the study. Soil-Set

Aid was more toxic than Impro-Grain in both the root hair assay and the cell cultures. It

would be interesting to determine if this effect is mirrored in monocot species, as this group

is responsible for the majority of agricultural biomass produced globally (Panis, 2008). Many

agricultural weeds that put a burden on yield production are dicotyledonous. Control of these

weeds can be achieved by using dicotyledonous-specific herbicides (Marshall et al., 2003). If

1 4 2 0 3 7 6 6 | 39

the toxicity of these Alltech products were not to translate to monocots then perhaps they

could be modified for use as environmentally friendly alternatives to current herbicides. The

importance of developing novel methods for controlling weeds into the future is one that

should not be dismissed. Current practices are over reliant on chemical herbicides. Herbicide

resistant weeds are becoming more common. 220 weed species have evolved resistance to

one or more herbicides as of 2014. The agricultural chemistry companies have not brought

any new chemicals to the market with novel target sites in a number of years pressuring

farmers to use current herbicides in novel ways. In addition, tougher regulation of these

chemicals has led to a number of herbicides being removed from the market further limiting

choices for farmers (Heap, 2014). Without innovation in this area crop yields will be put

under threat.

The use of growth stimulants is a growing practice in global agriculture. By 2018 the global

market for biostimulants is projected to reach over $2,200 million, growing by 12% each

year. The largest market for these products in 2012 was Europe (Calvo et al., 2014). Impro-

Grain was examined as a possible growth stimulant using a packed cell volume test. This is a

method used for determining the total cell volume as an indicator of cell growth (Street,

1977). Both light and dark grown cell cultures were incubated with Impro-Grain at 5,000 and

10,000 times dilution. The results of the packed cell volume test suggested that this product

had little positive effect on cell growth. A 1% increase in packed cell volume was seen in

cells incubated in Impro-Grain verses the control in light grown cells at both 5,000 and

10,000 times dilution. In dark grown cells there was a 9% increase in packed cell volume in

cells incubated in Impro-Grain at 10,000 times dilution. However, due to time limitations in

this study the packed cell volume test was only carried out for one replication. Before any

conclusions about its potential in this area can be drawn it needs to be examined further.

1 4 2 0 3 7 6 6 | 40

4.3.1 Agricultural Application

Rain fall and soil composition will have an effect on the concentration of any product in soil.

This is an important factor in determining how either Soil-Set Aid or Impro-Grain is best

applied in the field. This project examined the effect of these products at various dilutions

and found that at high concentrations there was a significantly toxic effect on root hairs (Fig.

8, Fig. 9). When looking at the effect of the products on PCD the concentrations were

extremely important. Impro-Grain was found to have a protective effect at 5000 times

dilution but at any other concentration that was looked at this effect was not seen. The use of

‘slow (controlled) release pellets’ perhaps is one way to overcome the challenge of ensuring

concentrations in the field are as close to optimal as possible. These pellets are made to

release their contents gradually when conditions are suitable (Hanfi et al., 2000). Pellets are

created by coating the product with a membrane that serves as a barrier to its diffusion into

the soil. Membranes can be composed of various materials such as chitosan and in recent

times polymer coated pellets have been popular (Ahmad et al., 2015).

Concluding Remarks The products tested in this project have a remarkable effect on plant cells. Further work is

needed to understand how they exert this effect over plant cells. At high concentrations this

product was toxic to root hairs. In the case of Impro-Grain it was found to have a protective

affect over heat induced PCD. Knowing that these products affect the PCD process in plants

gives potential for beneficial use. Abiotic stresses, such as heat, are responsible for huge yield

losses annually worldwide. With a greater understanding of how the products work, they

could become very useful to researchers or in agriculture in the future, providing a tool in the

challenge of sustainable food production.

1 4 2 0 3 7 6 6 | 41

Acknowledgments I would like to thank my supervisor Paul firstly. His input and guidance over the summer and

he was always available when needed for anything.

To Joanna, I would like to show my gratitude for all of her help. I can safely say that I would

not have been able to complete this thesis without her help in the lab and her critique of my

report.

I would like to thank everybody who worked over the summer in lab 2.67, Niall, John,

Frances, Dave and Theresa who created an atmosphere that was a joy to work in for the last 3

months and never turned me away when I needed help. My classmates deserve thanks for

their input and their company over the summer.

I would also like to thank my family for their unwavering support over my lifetime.

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