comparison of the effects of transgenes on osmotic …

COMPARISON OF THE EFFECTS OF TRANSGENES ON OSMOTIC STRESS IN POTATO

By

Bader Alsubaie

A THESIS

Submitted to

Michigan State University

in partial fulfillment of the requirements

for the degree of

Crop and Soil Sciences – Master of Science

2016

ABSTRACT

COMPARISON OF THE EFFECTS OF TRANSGENES ON OSMOTIC STRESS IN POTATO

By

Bader Alsubaie

The potato (Solanum tuberosum L.) is considered the fourth most significant food security crop

globally. As with other crops, growth of the potato plant is affected by many environmental stresses

such as drought, salinity, and intense temperature all of which reduce its productivity. In the case of

developing tolerance to abiotic stresses through transgenic approaches, genes such as

isopentenyltransferase (IPT), XERICO, and mannose 6-phosphate reductase (M6PR) have been

cloned for the purpose of addressing drought tolerance in potato and other crops. In this study, we

transformed the three different genes XERICO, M6PR, and IPT into the tetraploid S. tuberosum a

cultivar (CV) Désirée, a red-skinned variety with high transformation efficiency and susceptibility to

drought stress, to evaluate the response of transgenic lines to drought conditions in both the laboratory

and greenhouse. Due to its ability to modify the osmotic potential in a controlled manner, in vitro

laboratory experiments were conducted using poly ethylene glycol (PEG) to induce plant water deficit

in tissue culture experiments. Based on the height of plant, number of leaves, number of roots, fresh

and dry weight, all genetically engineered (GE) lines exhibited hypersensitivity to 4.8 % PEG in in-

vitro cultures. When the transgenic (XERICO, M6PR, and IPT) Désirée lines were subjected to severe

drought treatments (withholding water for 14 days in the greenhouse experiments) some events

recovered after re-watering, whereas the non-transgenic Désirée did not. Also, experiments

conducted with detached leaves from transgenic Désirée lines showed lower transpirational water

loss than the control non GE counterparts. These results suggest that the overexpression of XERICO,

M6PR, and IPT plants can confer improved drought tolerance in cultivated potatoes.

iii

ACKNOWLEDGEMENTS

First of all, I would like to thank my God for honoring me with his grace and mercy, and leading

me to what I have achieved. I would like to thank my family for their precious patience and

infinite support. I will never forget the continued guidance and help from my advisor Dr. David

Douches. Also, I would like thank Daniel Zarka, Joseph Coombs, Norma Carpintero, Kelly

Zarka, Alicia Massa, Swathi Nadakuduti, Sylvia Morse, Greg Steere, Matt ZuehlkeDonna Kells,

Chen Zhang, Kate McGlew, Felix Enciso-Rodriguez,Michael Hardigan, Maher Alsahlany,

Shafiqul Islam, Natalie Kirkwyland, Susan Akinyi Otieno. Also, I would like to thank all my

friends who supported me through my journey. Finally, it was a blessing to be a graduate student

in one of the best agriculture universities in the world Michigan State University.

iv

TABLE OF CONTENTS

LIST OF TABLES .......................................................................................................................... v

LIST OF FIGURES ....................................................................................................................... vi

KEY TO ABBERVIATIONS………………………………………………………………........vii

Chapter 1: Literature Review ......................................................................................................... 1 History and Importance of Potatoes ............................................................................................. 1 Drought Stress in Potato .............................................................................................................. 2

Breeding for Drought Resistance ................................................................................................. 3 Genetically Engineered Potato ..................................................................................................... 4

Selected Genes for Abiotic Stresses Tolerance ........................................................................... 6

Chapter 2: Evaluation Of Transgenic Potatoes ............................................................................... 9 Research Objectives ..................................................................................................................... 9 Material and Methods ................................................................................................................ 10

Plant Material and Transformation ....................................................................................... 10 Gene Constructs .................................................................................................................... 10

PCR Detection and Expression of Transgenes ..................................................................... 12 In Vitro Osmotic Stress Experiment ..................................................................................... 14

Greenhouse Reduced Watering Experiment ......................................................................... 17

Greenhouse Terminal Drought Experiment .......................................................................... 17

Rate of Leaf Water Loss ....................................................................................................... 20 Results ........................................................................................................................................ 21

Identification of Transgenic Plants ....................................................................................... 21

Identification of Individual Plants Expressing the Transgene .............................................. 22 In Vitro Drought Assessment................................................................................................ 26

Greenhouse Terminal Drought Experiment .......................................................................... 37 Relative Leaf Water Content ................................................................................................ 40

Leaf Water Loss Over Time ................................................................................................. 41 Reduced Water Experiment .................................................................................................. 43

Discussion .................................................................................................................................. 46 In conclusion ......................................................................................................................... 49

Future work ........................................................................................................................... 50

REFERENCES ............................................................................................................................. 51

v

LIST OF TABLES

Table 1: Primer Sequences and expected product size ................................................................. 14

Table 2: PCR and RT PCR results for presence and expression of the transgene. ....................... 23

Table 3: Lines used in the experiments......................................................................................... 24

Table 4: Summary of the plant revival rates after re-watering ..................................................... 39

Table 5: Analysis of leaf water loss over time experiment. .......................................................... 42

vi

LIST OF FIGURES

Figure 1: Construct Design ........................................................................................................... 12

Figure 2: Summary of the in vitro PEG procedure ....................................................................... 16

Figure 3: Relative water content experiment procedure ............................................................... 19

Figure 4: Leaf water loss over time experiment procedure .......................................................... 21

Figure 5: PCR from transgenic lines were used in experiments ................................................... 25

Figure 6: RT-PCR from the transgenic lines used in experiments ............................................... 26

Figure 7A: Analysis of height of plants at 15 days ....................................................................... 29

Figure 7B: Analysis of number of leaves at 15 days .................................................................... 30

Figure 7C: Analysis of number of roots at 15 days ...................................................................... 31

Figure 8A: Analysis of plant height at 30 days............................................................................. 32

Figure 8B: Analysis of number of leaves at 30 days .................................................................... 33

Figure 8C: Analysis of root number at 30 days ............................................................................ 34

Figure 8D: Analysis of fresh weight at 30 days ............................................................................ 35

Figure 8E: Analysis of dry weight at 30 days ............................................................................... 36

Figure 9: The phenotypic transition through 14 days drought and re-watering ........................... 38

Figure 10: Relative water content of leaves of transgenic and non-transgenic plants .................. 40

Figure 11: Analysis of tuber number for reduced water experiment ............................................ 44

Figure 12: XERICO Appearance ................................................................................................... 45

Figure 13: Height of transgeneic and non-transgentic plants in reduced water ............................ 46

vii

KEY TO ABBERVIATIONS

ABA – abscisic acid

Bt – Bacillus thuringiensis

CBF – C repeat binding factor

CK – cytokinin

CPB – Colorado potato beetle

CV – cultivar

DW – dry weight

EPA – Environmental Protection Agency

FW – fresh weight

GE – genetically engineered

HP – height of plant

IPT – isopentenyltransferase

M6PR – mannose 6-phosphate reductase

NL – number of leaves

NR – number of roots

PCR – polymerase chain reaction

PEG – poly ethylene glycol

PLRV – Potato Leaf Roll Virus

PSARK – promoter senescence associated receptor protein kinase

PVY – Potato Virus Y

RT-PCR – reverse transcriptase polymerase chain reaction

viii

RWC – relative water content

TW – turgid weight

USDA – US Department of Agriculture

1

Chapter 1: Literature Review

History and Importance of Potatoes

The cultivated potato (Solanum tuberosum L.) is ranked fourth among the most significant

economical food crops following rice, wheat, and maize (Barnaby et al. 2015). Globally, more than

a billion people eat potatoes, and total potato production exceeds 300 million metric tons

(POTATOBUSINESS, 2016). More than 4,000 native potato varieties can be found with different

sizes, shapes, and colors and mostly originated in the Andes (Devaux et al. 2009). In addition, there

are more than 90 wild potato species with significant diversity for natural resistance to biotic and

abiotic stresses in spite of the fact that some of them are too bitter for consumption (Spooner, 2013).

The potato is a vegetatively propagated plant with an ability to produce 5-20 new tubers per

plant that are clones of the mother plant. The potato can also produce flowers and berries that contain

up to 100-400 seeds. These true potato seeds can be planted to produce new tubers, but they each

have unique genetic combinations from both the maternal and paternal plants.

The cultivated potato belongs to the Solanaceae family, along with tomato, tobacco, and pepper.

Potatoes were introduced to Europe and North America (Spooner et al. 2005) from the Andes. Today,

potatoes are planted in over 100 countries around the world (International Potato Center, 2014).

Potatoes can grow under harsh conditions that other crops might not tolerate (Lutaladio and Castaldi,

2009). The geographic distribution of the cultivated potato shows that it can be grown in all regions

except Antarctica (Rowe and Powelson, 2002).

The potato is an important crop consumed by millions of people on a daily basis (Mullins et al.

2006). In 2013, the total world potato production was 376 million tons and the US ranked fifth, with

2

19 million tons (FAO, 2015). More than half of the total potato production globally is from

developing countries (Monneveux et al. 2013). Many developing countries depend on potatoes as a

staple food (Lutaladio and Castaldi, 2009), yet grow the crop in poor and undernourished soils.

Potatoes are sold in fresh market, and are used as raw material for chip processing. French fry

production contributes to an increase in farmer profit that helps to reduce poverty and improve

nutrition (Scott et al. 2000). The potato is also an important source of protein, vitamin C, and several

forms of vitamin B, carbohydrates, and minerals (Camire et al. 2009; White et al. 2009; Birch et al.

2012).

Drought Stress in Potato

When crops are subjected to abiotic stress, the resulting physiological and biochemical changes

can affect the growth and productivity of the plant (Pachauri, 2007). Growers lose billions of dollars

yearly because of abiotic stresses on crops (Senaratna et al. 2003). Drought is recognized as the most

prominent abiotic stress, reducing the productivity of agricultural crops globally with a negative effect

on the economy. The definition of drought is the shortage of water in the root zone, leading to reduced

crop productivity (Kramer et al. 1995).

Drought affects more than 9% of the world’s total arable land, and could lead to more than 50%

average yield loss of globally important crops (Bray et al. 2000). Drought stress can reduce plant

growth, stem height, leaf extension, and movement of stomata (Hsiao, 1973). Water deficit is a

serious problem that the potato crop suffers from, due to inconsistent rainfall or poor supplemental

irrigation system (Thiele et al. 2010). In the US, summer crops excluding the potato, suffered drought

and heat estimated to cause $40-88 billion in losses in 2011 and 2012 (NOAA, 2011, 2012).

3

The potato is susceptible to water deficit, which negatively affects tuber number (Eiasu et al.

2007), tuber size (Schafleitner et al. 2007) and tuber quality (Mackerron et al. 1988). Climate change

is expected to affect global potato yield with losses between 18-32% during the first three decades of

this century (Hijmans, 2003). Potato is susceptible to water stress because it has a shallow root system

compared to other crops (King et al. 2003). However, some potato species, such as Solanum acaule

and Solanum demissum showed high levels of drought tolerance in in vitro and greenhouse (Arvin

et al. 2008). Such species are being studied to determine the underlying genes and molecular

mechanisms for tolerance, with the intention of engineering these genes into susceptible potato

varieties so that they can tolerate drought. Search for genes that confer tolerance to drought will be

important to protect crops from the negative effects of climate change and thus improve crop

production (Lobell et al. 2011).

Breeding for Drought Resistance

Drought tolerance is a complex trait controlled by many genes (quantitative inheritance pattern).

These genes and their expressions are affected by various environmental elements, for example, heat

(Blum, 2011). Drought tolerant phenotypes can be rather complex. Drought stress can affect the

morphological and physiological traits in plants. In terms of physiological traits, researchers have

proven that there is a relationship between different physiological responses of crops, such as high

amounts of relative water and potential water, and their resistance functions under drought conditions

(Clarke et al. 1982; Ritchie, 1990). Drought tolerance can also be related to morphological responses,

such as early maturity, small plant size, and reduced leaf area (Rizza et al. 2004). Therefore, screening

for these traits can be difficult for conventional breeding. Also, since many genes control drought,

conventional breeding methods can be labor-intensive, expensive, and time consuming, as efforts are

4

required to separate undesirable traits from desirable traits (Nezhadahmadi et al. 2013). For this

reason, genetic engineering and molecular-marker strategies are used to increase the efficiency of

developing drought-tolerant germplasm (Gosal et al. 2009).

Genetically Engineered Potato

With respect to developing crops tolerant to abiotic stresses conventional breeding has only had

limited success, due to the quantitative nature (controlled by polygenes) of the traits. Since genetic

engineering technology has the ability to transfer specific genes that confer tolerance to abiotic

stresses, this method may provide more success. S. tuberosum L was one of the early crop plants that

used Agrobacterium mediated transformation technique (An, 1986) for genetic engineering. Tomato

was the first GE food crop that became commercially available in the US in 1994 (Kramer et al. 1994).

Many scientists use genetic engineering methods to improve the efficiency of breeding for abiotic and

biotic stresses tolerance. Many GE crops resistant to abiotic stresses are in the pipeline for

commercialization: drought-tolerant maize (Monsanto, 2013), rice (Stein et al. 2010), peanut (Qin et

al. 2011) ; salt tolerance cotton ( Liu et al. 2012), alfalfa ( Liu et al. 2011), tobacco (Jha et al. 2011);

salt and drought tolerant tomato (Goel et al. 2011). Several cultivars of GE potatoes were

commercialized and grown by farmers in the past, but were discontinued (except for the Innate potato

released in 2015) due to processing industry acceptance issues, although there were no safety concerns

with the products. For example, the Bacillus thuringiensis (Bt) potato resistant to the Colorado potato

beetle (CPB) was approved by the Environmental Protection Agency (EPA) in 1995 (EPA, 1999), and

sold commercially to US farmers. In addition, two other events combining the CPB resistance with

the Potato Leaf Roll Virus (PLRV), and Potato Virus Y (PVY) were also commercialized in 1998.

The Amflora potato, a GE potato that produces pure amylopectin starch and developed only for

industrial applications (in paper manufacturing and in the textile and adhesives industries) was

5

approved in the European Union market in 2010 (Abdallah, 2010). The Amflora was later discontinued

due to lack of public acceptance (Katarzyna, 2013).

Another example of a successful gene being introduced into a cultivated potato variety is the

introduction of the late blight resistance gene from the wild species S. bulbocastanum into S.

tuberosum. Potato late blight, caused by the fungal pathogen Phytophthora infestans, damaged the

potato crop in Ireland and caused the Irish potato famine (Haas et al. 2009). A late blight resistant gene

cloned from S. bulbocastanum and introduced into Katahdin, a susceptible cultivar, provided

resistance to late blight disease (Song et al. 2003).

In the MSU potato breeding program, genetic engineering technologies are being used to develop

tolerance to biotic and abiotic stresses as well as for improving quality traits. For example, resistance

to insect pests such as CPB (Cooper et al. 2007); tuber moth and CPB (Douches et al. 2002) as well

as resistance to the fungal pathogen P. infestans responsible for late blight disease (Kuhl et al. 2007)

have already been developed. In addition, tolerance to abiotic stresses such as cold (Nichol, 2011) has

also been developed. Furthermore, genetic engineering has been used to improve quality traits through

invertase silencing to lower reducing sugars, that produce dark color and bitter tasting in potato chips,

with acid invertase (K. Zarka, pers.com.), also gene editing (Butler and Douches, 2016).

Commercialized GE crops have increased since 1994. A number of GE crops are commercially

planted globally. For example, GE crops were planted on 179.7 million hectares in 28 countries in

2015 (James, 2015). In the US, several GE crops have been evaluated to be safe for the environment,

and are now commercially available. Most of these crops have been approved for human consumption,

and animal feed. In 2015, approximately 93% of the cotton, soybean, and corn crops planted in the US

were GE varieties (USDA, 2015). As for potato; the US Department of Agriculture (USDA) approved

the Innate potato varieties for commercial planting. These potatoes were genetically engineered to

6

reduce bruising and browning, which lower the production of acrylamide upon fry processing (Waltz,

2015).

Selected Genes for Abiotic Stresses Tolerance

XERICO, mannose 6-phosphate reductase (M6PR), and isopentenyltransferase (IPT) have been

identified to produce abiotic stress tolerance. Abscisic acid (ABA) is a plant hormone that plays a

significant role in response to stress conditions in plants. Under drought conditions Ko et al. (2006)

characterized a RING-H2-type zinc-finger protein, also known as XERICO, that shows an increase

in cellular ABA levels. The authors used the Cauliflower Mosaic Virus (CaMV) 35S promoter to

drive constitutive expression of the XERICO gene in Arabidopsis, and observed a further increase in

cellular ABA, resulting in drought tolerance. Under drought treatment created by not watering the

plants for 10 days, CaMV35S XERICO up-regulated the transcription of AtNCED3, a gene that

produces a key enzyme in ABA biosynthesis, providing transgenic plants a greater tolerance to

drought stress (Ko et al. 2006). Also, fresh weight (FW) measurements indicated that water loss from

the leaves of transgenic plants was reduced to about 7.5%, whereas the leaves of non-transgenic plants

lost around 15% (Ko et al. 2006). In another study, Zeng et al. (2013) used the XERICO gene from

Arabidopsis under the control of CaMV35S promoter in rice. Detached rice leaves from the XERICO

expressing lines showed lower transpirational water loss in comparison to the control plants. Also,

transgenic rice lines showed a significant increase in ABA contents under drought and salt stress

conditions (Zeng et al. 2013).

Mannose 6-phosphate reductase (M6PR) is a key enzyme involved in mannitol biosynthesis, a

sugar alcohol that may help as a compatible solute to counter salt stress in celery (Zhifang and

Loescher, 2003). High compatible solute concentrations inhibit loss of water or balance salt

accumulation, however, this protection feature can occur at lower concentrations for significant

7

osmotic effects (Zhifang and Loescher, 2003). In this study, the authors used M6PR gene from celery

under the control of the CaMV35S promoter in Arabidopsis, a non-mannitol producing plant. Their

results showed that all the transgenic Arabidopsis lines contained mannitol throughout the plants.

Interestingly, M6PR-transgenic Arabidopsis plants were phenotypically similar in comparison with

non-transgenics in the absence of 300Mm NaCl. However, when the plants were subjected to salt

treatment in soil irrigated with 300Mm NaCl, mature M6PR-transgenic Arabidopsis showed a high

level of salt tolerance, growing, flowering and producing seeds whereas wild type (WT) showed

severe salt stress (Zhifang and Loescher, 2003).

It has been found that during drought, there is an increase in leaf senescence that reduces canopy

size, yield, and photosynthesis. For example, Rivero et al. (2007) showed that delaying leaf

senescence could enhance drought tolerance. Gan and Amasino (1995) showed that leaf senescence

can be delayed in transgenic plants by expressing the IPT gene as this gene produces an enzyme that

induces the production of cytokinin (CK), inhibiting leaf senescence. Rivero et al. (2007) used a

senescence associated receptor protein kinase promoter (PSARK) isolated from bean to express the

IPT gene in tobacco, producing drought tolerance by delayed leaf senescence. As expected in

transgenic tobacco plants, the expression of the IPT gene induced the synthesis of CK, which in turn

resulted in enhanced drought tolerance. Furthermore, under severe drought treatments, these

transgenic tobacco plants only partially wilted and did not display drought-induced senescence. Upon

re-watering the transgenic plants recovered and showed full turgor in leaves and resumed growth. In

addition, transgenic tobacco displayed greater root and shoot biomass and a 160% increase in seed

yield, compared to the non-transgenic plants. During drought, the water content of the transgenic

plants was only slightly reduced, from 86% - 92%. After re-watering, the water content of the

transgenic plants returned to control levels. Transgenic plants showed greater control of reactive

8

oxygen species and enhanced expression of stress-response transcripts. However, nitrogen

mobilization was not affected, no developmental changes were seen, and there was no delay in

flowering and seed set. Water use efficiency values in the transgenic plants were two to three times

higher than those before drought. When provided with only 30% of the amount of water used under

control conditions, minimal yield losses were observed in transgenic plants (Rivero et al. 2007).

In addition, Kuppu et al. (2013) used the IPT gene in cotton, under the control of an inducible

promoter, SARK. The transgenic cotton showed delayed senescence under water deficit treatment in

the greenhouse, and produced more root and shoot biomass compared to the non-transgenic line.

Moreover, the IPT plants showed a greater drought tolerance under growth chamber conditions

(Kuppu et al. 2013).

The goal of this research was to individually test several different transgenes and determine if

they could confer drought/osmotic stress tolerance in potato plants expressing each transgene and thus

reduce the water requirement, without significantly impacting the plant’s growth. There were three

main research objectives in this study. The first was to individually express the three different genes,

XERICO, IPT, and M6PR to confer abiotic stress tolerance in the potato cultivar (CV) Désirée, a

drought susceptible variety. The second objective was to use reverse transcriptase polymerase chain

reaction (RT-PCR) to select the events for gene expression. The third was to conduct laboratory and

greenhouse experiments to evaluate each event, and to characterize the value of these genes for

tolerance to drought and osmotic stress in potato.

9

Chapter 2: Evaluation of Transgenic Potatoes

Research Objectives

The purpose of this study was to individually examine a set of transgenes for their ability to

confer drought/osmotic stress tolerance in cultivated potato. The first objective was to transform the

potato CV Désirée, using each of the genes a) Arabidopsis XERICO, b) Celery mannose-6-

phosphate reductase (M6PR), and c) Agrobacterium isopentenyltransferase (IPT) gene. The first

two genes have been shown to confer drought and salt tolerance respectively, when overexpressed

in Arabidopsis (Zhifang and Loescher 2003; Ko et al. 2006). Meanwhile, the IPT gene, under the

control of water-deficit responsive and maturation specific PSARK, has been shown to result in

delayed leaf senescence and extreme drought tolerance in tobacco plants (Rivero et al. 2007), and

enhanced drought tolerance in cotton plants (Kuppu et al. 2013).

The second objective was to characterize the transgenic events using polymerase chain

reaction (PCR) with gene specific primers to confirm the presence of the transgenes, and RT-PCR

to observe the relative transcription of the transgenes in individual plants to identify which plants

could be advanced for further studies.

The third objective was to examine the growth characteristics of the GE plants in a water

restricted environment, which was created in vitro by modifying the osmotic potential in a

controlled manner using polyethylene glycol (PEG) supplemented growth medium (Barra et al.

2013). Additionally, plants were examined after growth in vivo in a greenhouse study using a

reduced watering protocol during the life of the plant, and a terminal drought protocol (Rivero et al.

2007). In this study, the effect of the three transgenes on conferring drought tolerance was observed

by comparing the transgenic plants with the non-transgenic counterparts of Désirée.

10

Material and Methods

Plant Material and Transformation

The potato CV Désirée, which is highly amenable to transformation, was independently

transformed with three vector constructs, each containing one of the osmotic stress genes using a

protocol as described by Cearley et al. (1997) (Figure 1). Cuttings for tissue culture were grown in

culture tubes in Murashige and Skoog basal medium with vitamins (MS) (PhytoTechnology

Laboratories, Overland Park KS); 4.3 g·L-1 MS, 30 g·L-1 sucrose, pH 6.0, 7 g·L-1 agar. The stock

cultures were maintained at 25°C, with a 16 h photoperiod. The construct pSPUD98 (Fig. 1A) is a

plant transformation vector with a T-DNA, which contains the XERICO gene (a gift from Dr.

Kyung-Hwan Han, Michigan State University, East Lansing, MI) under the control of CaMV35S

promoter. Shoots recovered from transformation experiments were placed on a rooting medium.

Shoots that rooted in the selection medium were tested via PCR to confirm the presence of the gene.

Positive lines selected for further research were designated either DES.98, 97, or 96 and were

maintained in tissue culture then transplanted into pots in the greenhouse for bioassays.

Gene Constructs

The 1.9 kb PSARK IPT fragment was cut from plasmid PARC593 using EcoR1 and inserted

into binary vector pBINPLUS. The XERICO gene (610 bp) was synthesized and designed to

contain BamHI and XbaI sites and inserted between a CaMV35S promoter and a nopaline

synthase terminator in pBINPLUS binary vector. The shoots recovered were designated as

DES.98. The construct pSPUD97 (Fig. 1B) containing the gene M6PR (Zhifang and Loescher,

11

2003) a gift from Dr. Wayne Loescher, Michigan State University, East Lansing, MI, was used

to introduce the M6PR gene under the control of a CaMV35SS promoter in pBINPLUS binary

vector. Shoots recovered from transformation experiments were designated DES.97 and were

treated as described above. The restriction sites were designed by Zhifang and Loescher (2003).

The construct pSPUD96 (Fig. 1C) was used to introduce the IPT gene under the control of the

inducible SARK promoter (Arcadia Biosciences, Davis CA). Shoots recovered from

transformation experiments were designated DES.96 and were treated as described above.

12

PCR Detection and Expression of Transgenes

A single shoot was taken from each explant to eliminate the possibility of duplicate events.

DNA from individual plants that had rooted in the kanamycin selective medium was then subjected

to PCR, using gene specific primers to confirm the presence of the transgene. Using PCR, 34, 36,

and 26, transgenic events were examined to test for the presence of IPT, M6PR, and XERICO genes,

respectively. The DNA from each of these plants was isolated using the Qiagen Dneasy Plant Kit

according to the manufacturer’s directions (Qiagen, Valencia, CA USA). For PCR reactions, 2 µL of

DNA from each sample was mixed with 10 µL of Go Taq® Master Mix 2x (Promega, Madison, WI,

USA), and 1 µL each (10µM) of reverse and forward primers for the respective genes (Table 1) in a

20µL reaction. PCR cycling conditions were: 95°C for 3min, 35 cycles of 95°C for 30s, 50°C

(XERICO and IPT) or 55°C (M6PR) for 30s, 72°C for 45s and a final extension at 72°C for 5 min.

Figure 1: Construct Design

Figure 2Figure 3

Figure 4

Figure 5Figure 6

Figure 7Figure 8

Figure 9

Figure 10

The T-DNA regions of A. pSPUD98, B. pSPUD97 and C. pSPUD96

constructs depicting the positions of the XERICO and M6PR genes

containing the constitutive CaMV35S promoter, or the IPT gene containing

the inducible PSARK promoter. The nptII gene for kanamycin resistance

was used as the selectable marker.

13

PCR products were separated and visualized on a 1% agarose gel stained with 10 mg/µL ethidium

bromide.

Frozen transformed potato leaf tissue stored at -80°C was milled to powder under liquid

nitrogen, and RNA was extracted using an RNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA)

according to the manufacturer’s guidelines. The RNA was eluted in a final volume of 30 µL. To

remove contaminating genomic DNA, samples were then subjected to a deoxyribonuclease treatment

using an Ambion TURBO DNA-free kit (Invitrogen, Carlsbad, CA, USA). A Super Script One-Step

RT-PCR with Platinum Taq kit (Invitrogen, Carlsbad, CA, USA) was then used to perform the RT-

PCR according to the manufacturer’s directions. For the RT-PCR, 4 µL of RNA and 1 µL of each

primer (Table 1) were mixed with 10µL of 2X reaction buffer and 1 µL of SuperScript III RT/

Platinum Taq Mix in a final volume of 20 µL. The RT-PCR cycling conditions were: 50°C for 30

min, 94°C for two min, 30 cycles of 94°C for 15 S, 55°C for 30 S, 72°C for 45 S, and a final

extension at 72°C for five min. The RT-PCR products were then separated and visualized as

described above for the PCR products. Also, the RNA was run in the gel to demonstrate the quality

of samples before doing RT-PCR.

14

In Vitro Osmotic Stress Experiment

Tissue culture grown plants were assessed for their ability to grow on MS medium

supplemented with 4.8% PEG (mol. wt. 8000, SIGMA, USA). The pH of the media was adjusted to

5.8 ±, and the media solidified with 5 gL-1 agar (Caisson Labs, Logan UT, USA). To conduct this

drought experiment, five IPT, six XERICO, and seven M6PR transgenic events were used,

respectively (Table 2). These lines were chosen for the presence of appropriate gene expression,

expected phenotypes, and availability. Plant nodal segments of similar age and quality (second and

third nodes) were selected from transgenic and control Désirée plants, transferred to 4.8% PEG

medium, and grown under standard conditions for tissue culture. Nodal cuttings were also

transferred at the same time into regular MS growth medium to use for growth comparisons. The

tubes were placed in racks using a completely randomized design (CRD) with five replications. The

Table 1: Primer Sequences and expected product size

Gene Primer sequence Expected Product Size (bp)

XERICO

Forward 5’-GACAACATCATTTCTACCGACA-3’

595

Reverse 5’-TAGCTGTACACAACAAACACACTC-3’

M6PR

Forward 5’-GGTGTTTGGGAGGTTGATGC-3’

510

Reverse 5’-GTTACTCCTTTGGTTGCGCTC-3’

IPT

Forward 5’-GGTATCATCGCAGCCAAGCA-3’

476

Reverse 5’-GCTGCGTTAACTTGGGGGAA-3’

15

plants were maintained at 22°C, with a 16 h photoperiod. Measurements were recorded for number

of leaves (NL), number of roots (NR), and height of plants (HP) at 15 days and 30 days. Fresh

weight (FW) and dry weight (DW) were also measured at 30 days (Figure 2) (Barra et al. 2013).

Two-way analysis of variance was used to evaluate the effect of the different genes and drought

conditions.

16

(A) Explants were placed in standard MS and PEG medium.

(B) Number of leaves (NL), number of roots (NR), height of plant (HP) were

measured at 15 and 30 days.

(C) Fresh weight (FW) measured at 30 days.

(D) Dry weight (DW) measured at 30 days.

Figure 2: Summary of the in vitro PEG procedure

17

Greenhouse Reduced Watering Experiment

Four-week old tissue culture grown transgenic plants, and control Désirée plants were

transferred to a greenhouse for in vivo studies. For the experiment, four events from XERICO,

M6PR, and IPT were used with four replications for each event (Table 3). The plants were grown in

10.16 cm diameter pots containing Greens Grade soil (Siteone Landscape Supply, Dimondale, MI)

at 25±3οC, and supplemental light with a 16h light/8h dark cycle. Cheesecloth was used to line the

pots to prevent soil loss. During the experiment, plants were fertilized twice a week using Peters

Professional 20-20-20 (BFG supply, Kalamazoo, MI). Plants were divided into two representative

sets. In the first set, plants were watered until the soil was fully soaked. These plants received full

watering twice a week for the entire length of the experiment. The second set of plants were

watered with only 30% (restricted water) of the volume of water used for the first set, at the same

intervals mentioned above, for the entire length of the experiment. The two sets of plants were

arranged in a randomized complete block design. The height of each plant was measured each week

and FW above the ground, DW, and the number and size of tubers were measured at the end of the

experiment. The plants were allowed to grow for 10 weeks. One-way analysis of variance was used

to evaluate the effect of the different genes and drought conditions.

Greenhouse Terminal Drought Experiment

For the greenhouse terminal drought experiment, we used the same type of soil and pots as

described above. The tissue culture plants were transferred to the greenhouse as described for the

reduced watering experiment. Four different events each were used from XERICO, M6PR, and IPT,

with four replications for each line (Table 3). Plants were watered as needed until they were 31 days

18

old, after which water was completely withheld for 14 days. The plants were then re-watered for

three days, and were scored as either living or dead. Also, pots were weighed daily to estimate water

loss. Leaf water content was also measured at the end of terminal drought experiment at 14 days.

Four fresh detached leaves were weighed (FW) from each line, including the controls from the

terminal drought experiment. To determine the turgid weight (TW), the same detached leaves were

soaked in water contained in Petri dishes for 24h, and then they were weighed. The leaves were

dried for 24h using an oven at 100 °C and weighed again to obtain dry weight (DW) (Rivero et al.

2007) (Figure 3). The relative water content (RWC) of the leaves from the terminal drought

experiment was determined by using the formula:

RWC = (FW – DW) / (Turgid weight – DW)

One-way analysis of variance was used to evaluate the effect of the different genes and drought

conditions.

19

(A) The detached fresh leaves were weighed

(B) The same leaves were soaked in water for 24h under laboratory

conditions and then weighed

(C) The leaves were put in the dryer for 24h and then weighed.

Figure 3: Relative water content experiment procedure

Figure 81

Figure 82

Figure 83

Figure 84

20

Rate of Leaf Water Loss

To determine if the expression of the transgenes effect leaf transpiration, the rate of leaf water

content loss was measured. Three detached leaves from fully water-expended plants were used from

each transgenic line as well as a Désirée control plant from terminal drought and reduced water

experiments. Detached leaves were placed in Petri dishes under the same conditions as intact plants,

and were weighed immediately, at 30 min, 60 min, two hour, three hour, and four hour time

intervals. The percentage of water loss over time was then calculated (Figure 4). JMP statistical

software and R were used to analyze all experiments data.

21

Results

Identification of Transgenic Plants

A total of 26 XERICO shoots selected after rooting on kanamycin selection media were

identified as positive for containing the transgene. A total of 36 M6PR shoots were selected after

rooting on kanamycin, however, only 24 plants were confirmed positive for the presence of the

transgene. A total of 34 IPT shoots were selected after rooting on kanamycin with 33 confirmed

positive for the presence of the transgene (Table 2). However, some of the lines were chosen in

drought tolerance experiments (Figure 5).

Three detached leaves from Désirée and transgenic lines were weighed over

different times to identify the water loss from the leaves.

Figure 96 Leaf water loss over time experiment procedure.

Three detached leaves from Désirée and transgenic lines were weighed over

different times to identify the water loss from the leaves.

Figure 4: Leaf water loss over time experiment procedure

Figure 128

Figure 129

Figure 130

Figure 131

Figure 132

22

Identification of Individual Plants Expressing the Transgene

A subset of events was subjected to end-point RT-PCR. Ten XERICO plants, 15 M6PR plants,

and 9 IPT plants were positive for transgene expression (Table 2) and some of these events were

chosen for the presence of appropriate gene expression (Figure 6) and used for bioassays in vitro and

in the greenhouse (Table 3).

23

Table 2: PCR and RT PCR results for presence and expression of the transgene.

XERICO PCR

RT-

PCR

M6PR PCR

RT-

PCR

IPT PCR

RT-

PCR

DES.98.04 + NO DES.97.01 + + DES.96.02 + + -

DES.98.05 + + DES.97.02 + + DES.96.03 + -

DES.98.07 + NO DES.97.03 + + DES.96.04 + +

DES.98.08 + + DES.97.04 + + DES.96.05 + -

DES.98.09 + NO DES.97.05 + + DES.96.07 + -

DES.98.11 + NO DES.97.06 + + DES.96.08 + +

DES.98.12 + NO DES.97.07 + + DES.96.09 + -

DES.98.13 + - DES.97.08 + - DES.96.10 + +

DES.98.18 + + DES.97.09 + + DES.96.11 + NO

DES.98.20 + NO DES.97.10 + + DES.96.12 + -

DES.98.21 + + DES.97.11 + + DES.96.14 + -

DES.98.23 + + DES.97.12 + + DES.96.15 + -

DES.98.26 + + DES.97.13 + + DES.96.16 + NO

DES.98.27 + + DES.97.14 + NO DES.96.18 + NO

DES.98.28 + NO DES.97.15 + NO DES.96.19 + NO

DES.98.30 + + DES.97.16 + + DES.96.20 - -

DES.98.31 + + DES.97.17 - NO DES.96.21 + +

DES.98.32 + - DES.97.18 + NO DES.96.22 + +

DES.98.34 + - DES.97.19 - NO DES.96.24 + -

DES.98.37 + NO DES.97.20 + - DES.96.25 + NO

DES.98.38 + + DES.97.21 - NO DES.96.26 + NO

DES.98.39 + NO DES.97.22 - NO DES.96.27 + -

DES.98.40 + NO DES.97.23 + NO DES.96.28 + -

DES.98.41 + NO DES.97.24 - NO DES.96.29 + NO

DES.98.42 + NO DES.97.25 + NO DES.96.30 + -

DES.98.43 + NO DES.97.26 - NO DES.96.31 + NO

DES.97.27 - NO DES.96.32 + -

DES.97.28 + + DES.96.33 + NO

DES.97.29 - NO DES.96.34 + +

DES.97.30 + NO DES.96.35 + NO

DES.97.31 - NO DES.96.36 + +

DES.97.32 - NO DES.96.37 + -

DES.97.33 - NO DES.96.38 + NO

DES.97.34 + + DES.96.39 + NO

DES.97.35 + NO DES.96.02 + NO

DES.97.36 - NO

(+) = Presence of gene and expression

(-) = Absence of gene and expression

(NO) = The lines were not tested with RT-PCR.








24

Table 3: Lines used in the experiments.

1PEG experiments. 2Terminal drought. 3Reduced water. 4Relative water content. 5Leaf water loss

over time.

Line PE1 TD2 RW3 RWC4 LWLT5

DES.98.08 X X X X

DES.98.21 X X X X X

DES.98.27 X X X X X

DES.98.31 X X X

DES.98.38 X X

DES.98.23 X X

DES.96.04 X X

DES.96.08 X X X X X

DES.96.22 X X X

DES.96.36 X X X X

DES.96.38 X X X

DES.97.01 X X X X X

DES.97.12 X X X X

DES.97.13 X X X

DES.97.02 X X X X

DES.97.04 X X

DES.97.07 X X

DES.97.11 X

DES.97.34 X X

DES X X X X X

25

The presence of XERICO DES.98 (A), M6PR DES.97 (B), and IPT DES.96 (C) genes was

visualized by PCR using 100 bp ladder (L). Désirée DES and water used as a negative control

for XERICO, M6PR, and IPT . The expected product size for XERICO is 595 bp the XERICO

plasmid used as positive control.The product size for M6PR is 510 bp and the M6PR plasmid

used as positive control. The IPT product size is 476 bp and the IPT plasmid used as a positive

control.

DES = Control

DES.96 = IPT

DES.97 = M6PR

DES.98 = XERICO

L= Ladder 100 bp

A

A

A

A

A

A

A

A

B

B

B

B

B

C

C

C

C

C

Figure 5: PCR from transgenic lines were used in experiments

Figure 175

Figure 176

Figure 177

Figure 178

Figure 179

26

In Vitro Drought Assessment

A subset of lines were subjected to a series of drought experiments in order to test the

efficiency of IPT, XERICO, and M6PR events. For the in vitro drought assessment five, six and

seven IPT, XERICO, and M6PR events were used, respectively. Analysis of measurements taken

after 15 days showed significant reduction effect of the line (gene) and medium (non PEG or PEG)

The presence of appropriate gene expression of XERICO DES.98 and the RNA (A), M6PR

DES.97 and RNA (B), and IPT DES.96 and RNA (C) was visualized by RT-PCR. Water used

as a negative control for XERICO, M6PR, and IPT . Also, the RNA for the expected product

size for XERICO is 595 bp the XERICO. The product size for M6PR is 510 bp. The IPT

product size is 476 bp.

Reverse Transcriptase PCR from the transgenic lines used in experiments.

The presence of appropriate gene expression of XERICO DES.98 and the RNA (A), M6PR

DES.97 and RNA (B), and IPT DES.96 and RNA (C) was visualized by RT-PCR. Water used

as a negative control for XERICO, M6PR, and IPT . Also, the RNA for the expected product

size for XERICO is 595 bp the XERICO. The product size for M6PR is 510 bp. The IPT

product size is 476 bp.

A

A

A

A

A

A

A

A

A

A

B

B

B

B

B

B

B

B

C

C

C

C

C

C

C

Figure 6: RT-PCR from the transgenic lines used in experiments

Figure 198

Figure 199

Figure 200

Figure 201

Figure 202

27

on HP, NL, and NR. However, there was no significant line (gene) by medium interaction effect on

any of the outcome measures (Figure 7 A, B, C). For the measurements taken after 30 days, a

significant effect of the line (gene) was observed on HP, NL, NR, and FW, but not on DW. The

medium also had a significant reduction in all the measurements. At 30 days, we found the line by

medium interaction effect was significant for only the height of plant and number of roots. However,

the non PEG medium lines were greater than the PEG medium (Figure 8 A, B, C, D, E). The

summary of data on HP, NL, NR, FW, and DW was displayed using box plots. The observations of

the plant height at 15 days was similar to the 30 day, plant height was greater in the control medium

than the PEG medium. For the PEG medium at 15 days, we found the highest average plant height

under the drought condition occurred with the IPT line DES.96.38 (Figure 7A). For the PEG

medium at 30 days, we found the IPT line DES.96.36, the XERICO line DES 98.08, and Désirée to

have the highest variability in plant height (Figure 8A). Of these, the highest average plant height in

the drought condition occurred with the IPT line DES.96.36. Interestingly, unlike what we found

from the 15 day measurements, the patterns observed in the two treatment conditions were very

different, suggesting that time had an effect between the two treatments. As expected, leaf number is

greater in the control medium than the PEG medium at both 15 and 30 days. For the PEG medium at

15 days, the highest average leaf number in the drought condition occurred with IPT lines

DES.96.38 and DES.96.36 (Figure 7B). For the PEG medium at 30 days, the highest average leaf

number occurred with the IPT lines DES.96.38 and DES.96.36 just as it was at 15 days (Figure 8B).

The number of roots was found to be greater in the control medium than in the PEG medium at 15

and 30 days. For the PEG medium at 15 days, DES.96.36 had the highest variability and highest

values for the number of roots (Figure 7C). For the PEG medium at 30 days, we found that unlike at

15 days, the M6PR lines DES.97.04 and DES.97.34 had the highest variability for number of roots.

28

All other lines except for DES.96.36 were severely affected by the PEG treatment (Figure 8C). Fresh

weight was also greater in the control medium than the PEG medium, suggesting a treatment effect.

For the PEG medium, DES.96.36 had the highest variability and values for fresh weight (Figure 8D).

Similarly, the dry weight was greater in the control medium than the PEG medium, indicating a

treatment effect. For the PEG medium, DES.96.36 had the highest variability and highest values for

dry weight just as for fresh weight (Figure 8E). In summary, the PEG 4.8 % treatment had a negative

effect on the lines. The IPT lines DES.96.36 and DES.96.38 showed good performance in 4.8 %

PEG medium, although was a negative effect from the PEG.

29

Lines

Lines

Lines

Lines

Lines

Lines

Hei

gh

t of

pla

nts

(cm

)

Désirée = Control

DES.96 = IPT

DES.97 = M6PR

DES.98 = XERICO.

Analysis of height of plants at 15 days.

Désirée = Control

DES.96 = IPT

DES.97 = M6PR

DES.98 = XERICO.


Désirée = Control

DES.96 = IPT

DES.97 = M6PR

DES.98 = XERICO.


Désirée = Control

DES.96 = IPT

DES.97 = M6PR

DES.98 = XERICO.

Figure 7A: Analysis of height of plants at 15 days

Figure 213A

30

Lines

Lines

Lines

Lines

Lines

Désirée = Control

DES.96 = IPT

DES.97 = M6PR

DES.98 = XERICO.

Analysis of number of leaves at 15 days.

Désirée = Control

DES.96 = IPT

DES.97 = M6PR

DES.98 = XERICO.


Désirée = Control

DES.96 = IPT

DES.97 = M6PR

DES.98 = XERICO.


Désirée = Control

Lea

f n

um

ber

Figure 7B: Analysis of number of leaves at 15 days

Figure 7B

31

Root

nu

mb

er

Désirée = Control

DES.96 = IPT

DES.97 = M6PR

DES.98 = XERICO.

Figure 7 C. Analysis of number of roots at 15 days.

Désirée = Control

DES.96 = IPT

DES.97 = M6PR

DES.98 = XERICO.


Désirée = Control

DES.96 = IPT

DES.97 = M6PR

DES.98 = XERICO.


Désirée = Control

Lines

Lines

Lines

Lines

Lines

Figure 7C: Analysis of number of roots at 15 days

Figure 215AFigure 7C

Figure 7C

Figure 216AFigure 7C

32

Hei

gh

t of

pla

nts

(cm

)

Désirée = Control

DES.96 = IPT

DES.97 = M6PR

DES.98 = XERICO.

Figure 8 A. Analysis of plant height at 30 days.

Désirée = Control

DES.96 = IPT

DES.97 = M6PR

DES.98 = XERICO.


Désirée = Control

DES.96 = IPT

DES.97 = M6PR

DES.98 = XERICO.


Désirée = Control

DES.96 = IPT

DES.97 = M6PR

DES.98 = XERICO.

Analysis of plant height at 30 days.

Lines

Lines

Lines

Lines

Lines

Figure 8A: Analysis of plant height at 30 days

Figure 8A

33

Lea

f n

um

ber

Lines

Lines

Lines

Lines

Lines

Désirée = Control

DES.96 = IPT

DES.97 = M6PR

DES.98 = XERICO.

Figure 8 B. Analysis of number of leaves at 30 days.

Désirée = Control

DES.96 = IPT

DES.97 = M6PR

DES.98 = XERICO.


Désirée = Control

DES.96 = IPT

DES.97 = M6PR

DES.98 = XERICO.


Désirée = Control

DES.96 = IPT

DES.97 = M6PR

DES.98 = XERICO.

Figure 8B: Analysis of number of leaves at 30 days

Figure 8B

34

Root

nu

mb

er

Lines

Lines

Lines

Lines

Désirée = Control

DES.96 = IPT

DES.97 = M6PR

DES.98 = XERICO.

Figure 8 C. Analysis of root number at 30 days.

Désirée = Control

DES.96 = IPT

DES.97 = M6PR

DES.98 = XERICO.


Désirée = Control

DES.96 = IPT

DES.97 = M6PR

DES.98 = XERICO.


Désirée = Control

DES.96 = IPT

DES.97 = M6PR

DES.98 = XERICO.

Figure 8C: Analysis of root number at 30 days

Figure 8C

35

Fres

h w

eigh

t (m

g)

Désirée = Control

DES.96 = IPT

DES.97 = M6PR

DES.98 = XERICO.

Figure 8 D. Analysis of fresh weight at 30 days.

Désirée = Control

DES.96 = IPT

DES.97 = M6PR

DES.98 = XERICO.


Désirée = Control

DES.96 = IPT

DES.97 = M6PR

DES.98 = XERICO.


Désirée = Control

DES.96 = IPT

Lines

Lines

Lines

Lines

Lines

Figure 8D: Analysis of fresh weight at 30 days

Lines

Lines

Lines

36

Dry

wei

gh

t (m

g)

Désirée = Control

DES.96 = IPT

DES.97 = M6PR

DES.98 = XERICO.

Figure 8 E. Analyzing dry weight at 30 days.

Désirée = Control

DES.96 = IPT

DES.97 = M6PR

DES.98 = XERICO.


Désirée = Control

DES.96 = IPT

DES.97 = M6PR

DES.98 = XERICO.


Désirée = Control

DES.96 = IPT

Lines

Lines

Lines

Lines

Lines

Figure 8E: Analysis of dry weight at 30 days Analyzing dry weight at 30 days.

Lines

Lines

Lines

37

Greenhouse Terminal Drought Experiment

To assess the tolerance of transgenic plants to drought stress, the plants were exposed to drought

(withholding water for 14 days). The transgenic plants did not display any morphological alterations

in comparison with non-transgenic Désirée when grown before the drought treatment (Figure 9 A).

After one week without water, all plants displayed some stress (Figure 9 B). After two weeks

without water, all plants displayed severe stress symptoms, with control plants severely wilted and

transgenic plants partially wilted (Figure 9 C). All plants were then returned to their normal pre-

drought watering schedule for three days (Figure 9 D). Désirée did not recover and died. However,

some transgenic lines recovered and the leaves looked healthy and resumed their growth, after which

foliage was visually scored for revival. Table 4 summarizes the revival rates of transgenic plants.

The rate of revival for XERICO lines ranged from 25-75%. The rate of revival for M6PR lines

ranged from 75-100%. The rate of revival for the IPT lines ranged from 25-75%. There was a 0%

revival rate for the Désirée control plants. Pots were weighed during the two weeks without water

and the results indicated that rate and extent of drying remained the same. One-way analysis of

variance was used to analyze the water loss from the pots, and the results showed no significance in

the percentage of water lost from pots.

38

(A) XERICO, IPT, and M6PR plants with Désirée control prior to drought treatment with

fully healthy growth.

(B) XERICO, IPT, and M6PR plants with Désirée control at one-week drought condition

exposed some stress.

(C) XERICO, IPT, and M6PR plants with Désirée control at two-week drought treatment.

(D) Revival of plants following three days re-watering with Désirée as control (no revival).

Figure 9: The phenotypic transition through 14 days drought and re-watering

Figure 9: The phenotypic transition through 14 days drought and re-watering.

39

1 Revival Rate. 2 Control

Table 4: Summary of the plant revival rates after re-watering

XERICO RR1 (%) M6PR RR (%) IPT RR (%) Désirée2 RR (%)

DES.98.08 50 DES.97.01 75 DES.96.38 25 DES 0

DES.98.27 75 DES.97.13 100 DES.96.08 75

DES.98.38 25 DES.97.02 75 DES.96.36 25

DES.98.21 75 DES.97.12 75 DES.96.22 25

40

Relative Leaf Water Content

Four fresh detached leaves (Table 3) were weighed from each line including Désirée as a

control from the terminal drought experiment to determine the water content in the leaves at two

weeks withholding water (Figure 10). DES.97.01 had the highest value among the lines (36.9%),

whereas DES.98.21 and DES.96.38 had the lowest values for RWC (23%, 21%), showing a

significant difference in comparison to the highest value of DES.97.01. The lines DES.97.12, DES,

DES.98.27, DES.96.08, DES.97.02, DES.98.08, DES.97.13, DES.96.38, DES.96.36, and DES.96.22

had similar values for RWC (Figure 10).

DES = Control

DES.96 = IPT

DES.97 = M6PR

DES.98 = XERICO

RWC results after two weeks withholding water. Means with the same

letter are not significantly different (α = 0.05).

Lines

Table

25:

Analysis

of leaf

water

loss over

time

experime

nt.Lines

Mea

n o

f R

WC

(%

)

Figure 10: Relative water content of leaves of transgenic and non-transgenic plants

Table 1: Analysis of leaf water loss over time experiment.Lines

Table 2: Analysis of leaf water loss over time experiment.


41

Leaf Water Loss Over Time

In this experiment, three detached leaves from the control and sub sample of XERICO lines

DES.98.31 and DES.98.21 showed less wilting in the reduced water experiment (Figure 11). In

addition, subsamples of M6PR lines DES.97.13, DES.97.12, DES.97.02, DES.97.01, and XERICO

lines DES.98.08, DES.98.27, and IPT line DES.96.08 displayed a higher revival rate among the

lines in the terminal drought experiment that followed by re-watering (Table 3). All leaves were

weighed at designated time intervals to identify water loss over time (Figure 4). Results showed

significant differences between the lines at different time intervals (Table 5). All M6PR, IPT, and

XERICO lines had less water loss over time in comparison to Désirée control. Among these lines,

DES.97.12, DES.97. 13, DES.98.08, and DES.96.08 belonging to Group B, showed less water loss

over the time interval. However, DES.97.01 and DES.98.21 also belonging to Group B, did not

consistent water loss across the time intervals. Group A (DES) is the group that lost the most

amount of water, and was consistent over time. Group B, which showed a significant difference in

comparison with Group B, lost the least amount of water. Group AB (DES.97.02, DES.98.31,

DES.98.31, DES.98.27) had similar results, but did not have significant variances of the percentage

of water lost over time (Table 5).

42

DES = Control

DES.96 = IPT

DES.97 = M6PR

DES.98 = XERICO

All transgenic lines had much less water loss in comparison with

Désirée. Means with the same letter are not significantly different

(α = 0.05).

DES = Control

DES.96 = IPT

DES.97 = M6PR

DES.98 = XERICO

All transgenic lines had much less water loss in comparison with

Désirée. Means with the same letter are not significantly different

(α = 0.05).











43

Reduced Water Experiment

For the measurements taken for full water, no significant effect of the line was observed for

plant height, number of tubers, fresh weight, and dry weight except tuber weight. The F statistic for

ANOVA test was significant for the tuber weight, but the Tukey test was not. The Tukey test

requires a larger sample size to detect group differences than the overall ANOVA test (Brooks and

Johanson, 2011). The second set of plants were watered with 30% (restricted water) of the volume

of water used for the first set at the same intervals for the entire length of the experiment. Also, one-

way analysis of variance was used for this experiment. Measurements taken on the lines for plant

height, fresh weight, and dry weight showed no significant effect of restricted water. However,

there was a significant effect on tuber number although significance was not consistent with two-

way analysis of variance (Figure 11). DES.97.04 showed the highest value for mean tuber number

(6) among the lines whereas DES.96.08 with the lowest mean tuber number (1.25) was significantly

different from DES.97.04. Although the mean for the number of tubers for DES.97.07, DES.98.27,

DES.96.04, DES.97.01, DES.98.23, DES.96.36, DES.96.38, DES.98.21, DES.97.34, DES.98.31

and DES ranged from 1.3 - 4.7, they were not significantly affected by the restriction of water.

Restriction of water did not show any significant differences among the plants, some of transgenic

lines showed less stress visually based on morphological traits on leaves in comparison to Désirée

(Figure 12) and height between the two treatments (Figure 13).

44

The tuber number in restriction water treatment was significant but it wasn’t significant with

two-way analysis of variance. The transgenic M6PR DES.97.04 had the highest tuber number.

Means with the same letter are not significantly different (α = 0.05).

Lines

Lines

Lines

Lines

Lines

Lines

Lines

Lines

Lines

Lines

Lines

Lines

Lines

Lines

Full water

Full water

Full water

Full water

Full water

Full water

Full water

Full water

Full water

Restriction water

water

Restriction water

water

Restriction water

water

Restriction water

water

Restriction water

water

Restriction water

water

Restriction water

Figure 11: Analysis of tuber number for reduced water experiment

Lines

Lines

Lines

Lines

Lines

Lines

45

The transgenic XERICO lines DES.98.31 and DES.98.21 showed

less stress phenotypically when water was restricted at 30% in

comparison with Désirée.

Figure 12. The transgenic XERICO lines DES.98.31 and

DES.98.21 showed less stress phenotypically when water was

restricted at 30% in comparison with Désirée.

Figure 12: XERICO Appearance

Table 72: Analysis of leaf water

loss over time experiment.Lines


loss over time experiment.






loss over time experiment.

46

Discussion

Drought is a serious abiotic stress factor that restricts the productivity of crops with

socioeconomic impacts (Salinger et al. 2005). Developing adaptive agricultural systems are

important for the changing environment and use of genetic engineering to develop stress tolerance

of crops has become an important tool to enhance the breeding process (Zhang et al. 2011). The

goal of this study was to compare the effect of osmotic stress responsive genes when expressed in

potato CV Désirée. We hypothesized that the three different genes the Arabidopsis XERICO gene,

(A) indicates restricted water and (B) indicates fully watered. The transgenic M6PR

DES.97.01 showed similar in the height results in A, and B treatments. The transgenic

XERICO DES.98.31 line was higher in B in comparison to A although it was not

significant. The transgenic IPT DES.96.08 showed similar results for height in A and B

treatments. The non-transgenic line Désirée was severely effected in A treatment in

comparison to B treatment.

Figure 217Figure 13. The height of the transgenic and non-transgenic plants in the reduced

water

experiment.

(A) indicates restricted water and (B) indicates fully watered. The transgenic M6PR DES.97.01

showed similar in the height results in A, and B treatments. The transgenic XERICO DES.98.31

line was higher in B in comparison to A although it was not significant. The transgenic IPT

DES.96.08 showed similar results for height in A and B treatments. The non-transgenic line

Figure 13: Height of transgenic and non-transgenic plants in reduced water

experiment.






47

the celery mannose-6-phosphate reductase (M6PR) gene, and Agrobacterium isopentenyltransferase

(IPT) will improve drought resistance in CV Désirée, a susceptible variety. For in vitro drought

assessment experiment, we used 4.8% PEG to induce osmotic stress. Based on the study by Barra et

al. (2013) where 149 INIA-Chile potato genotypes were evaluated using PEG with different

concentration in the beginning, 4.8 % PEG concentration was able to distinguish the genotype

responses to drought such as plant height, leaf and root number, fresh and dry weight. These results

classified the genotypes as high, intermediate, low, and very low tolerance to water stress.

However, we found that the 4.8% PEG medium has a significant effect on all the

measurements taken, irrespective of the time they were taken. All transgenic lines displayed

hypersensitivity to 4.8% PEG and the morphological traits such as NL, NR, HP, FW, and DW were

severely effected in comparison to control MS media without PEG.

In the Zeng et al. (2013) study, transgenic XERICO rice had higher revival rates in comparison

to non transgenic lines when the plants were exposed to drought by withholding water for 11 days.

The revival rate of XERICO rice plants was 49.2% whereas the control non-transgenic lines were

only 9.5%. They suggested that the enhanced drought tolerance of transgenic XERICO plants was

due to decreased water loss by transpiration. They supported this hypothesis by measuring fresh

leaf water loss over different time points as a signal of transpirational water loss. The leaves of the

XERICO line plants had much less water loss in comparison to the control. The conclusion of this

study was that overexpressing the XERICO gene in transgenic rice plants showed reduced

transpirational water loss conferring enhanced drought tolerance in rice by a significant increase in

ABA content. Many studies support that ABA is involved in improved drought tolerance by

increasing ABA content (Ko et al. 2006) resulting in stomatal closure to inhibit transpirational

water loss during drought conditions (Blatt, 2000).

48

The results of Rivero et al. (2007) showed that the WT tobacco plant did not recover from

withholding water experiment for 15 days whereas the transgenic IPT plants recovered and leaves

showed full turgor and the plants continued their growth after re-watering. Even though the ABA

increased the drought tolerance in plants, transgenic IPT plants were not associated with drought

tolerance in their experiment. Also, the transgenic IPT plants had higher water content in

comparison with WT. The conclusion of this study was that CK content was associated with the

drought tolerance in IPT tobacco by delaying leaf senescence.

In our data, we had similar results to the two previous studies in terms of the different revival

rates compared to control. We observed different revival rates among M6PR, XERICO and IPT

lines. However, the non transgenic Désirée could not recover upon re-watering and died. With the

revival of XERICO, IPT, and M6PR potato lines in the terminal drought experiment, the results

suggest that overexpression of M6PR, XERICO and IPT confer enhanced drought tolerance in

potato.We hypothesized that drought tolerance caused by transgenic plants may have a reduced

transpiration rate (Zeng et al. 2013) or the water content in the leaf was higher during the drought

period (Rivero et al. 2007). For this reason, we conducted two additional experiments to identify the

reason behind the drought tolerance. The first experiment was to determine RWC. The results of

RWC from Rivero et al. (2007) showed the WT had reduced water content (60%) compared to IPT

transgenic plants (92% to 86%) during the drought period when there was no significant difference

in the soil moisture level. In our data, while there were some minor differences in RWC of the lines,

the majority of plants showed similar amounts of water loss from leaves during the drought period

(Figure 10) as determined from dry pots. So, the genes were not causing the plant leaves to keep the

water content higher during the drought period rather they must be providing some other protective

components allowing the cells to recover when water becomes available.

49

The second experiment was to measure the leaf water loss over time. The purpose of this

experiment was to test whether a decreased transpiration rate might indicate increased water use

efficiency so the plant can survive a longer period of drought. Results of a similar study by Zeng et

al. (2013) on leaf water loss over time showed that transgenic leaves of XERICO plants had much

less water loss in comparison to non-transgenic counterpart. Our data confirmed these results as

transgenic lines had less water loss over time in comparison to the non-transgenic Désirée (Table 5).

These results suggest that transgenic plants confer improved drought tolerance by decreased

transpiration rate in leaves during drought conditions. In a study by Rivero et al. (2007), where

transgenic tobacco plants grown under two watering systems, full water (1 liter/day) and restricted

water (0.3/day) were tested for four months showed that seed yield and biomass of WT plants were

affected by the restricted water conditions showing a reduction of 57% in biomass and 60% seed

yield. However, the transgenic plants showed minimal reduction, although not statistically

significant in biomass and seed yield (8-14%). In our data, the statistical analysis of results did not

show a significant difference between the transgenic lines in restricted water compared to full water

system for fresh weight, dry weight, plant height, number of tubers and weight of tubers. However,

there was visually significant results for some transgenic lines. Some of the transgenic lines

displayed a drought tolerant phenotype. The leaves of these transgenic lines especially XERICO lines

showed less stress (Figure 12). Also, some of the transgenic lines showed differences in height

between the two treatments, albeit not statically significant (Figure 13).

In conclusion

The goal of this study was to determine if the transgenic lines from M6PR, IPT and XERICO

conferred drought resistance to the potato CV Désirée which is susceptible to drought. The results

50

indicate that the three genes can confer drought resistance to the potato CV Désirée. Some of the

transgenic lines showed promising results from the series of drought experiments such as the M6PR

lines DES.97.12, DES.97.13, DES.97.02, DES.97.01, XERICO lines DES.98.08, DES.98.31,

DES.98.27, DES.98.21, and the IPT line DES.96.08. These lines will be used in the future

bioassays.

Future work

Some results of the different experiments were promising and hence need to be repeated with a

larger number of the plants. For future molecular work, we will screen additional lines that might

show high expression. Also, we will re-design the XERICO gene construct by changing the

constitutive promoter to an inducible promoter. For the in vitro experiment, we will use different

PEG concentration because 4.8 % PEG seems too high for drought treatments on the lines. Also, we

will use different time measurements because the growth of plants increased with time. In RWC

experiment, we will measure the RWC at different time points before and during the drought

treatment to identify the RWC in the plants. It is hard to control the weather in Michigan, but a field

trial is essential to test the performance of transgenic plants for drought tolerance. The GE lines that

showed promising results in in vitro and in vivo will be evaluated in field trials with irrigation and

without irrigation as just the GE potato lines containing the CBF ( C repeat binding factor) gene

was treated at the Montcalm Research Center (Nichol et al. 2015). Finally, for the commercial

purposes, we will consider inserting combination of these three genes to provide more durable

drought resistance to the potato.

51

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52

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