ENHANCEMENT OF LEUCAENA LEUCOCEPHALA TISSUE REGENERATION AND AGROBACTERIUM-MEDIATED

TRANSFORMATION

A THESIS SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAWAI'I AT MĀNOA IN PARTIAL FULFILLMENT OF THE REQUIRMENTS

FOR THE DEGREE OF

MASTER OF SCIENCE IN

MOLECULAR BIOSCIENCES AND BIOENGINEERING

May 2014

By

Jadd Correia

Thesis Committee:

Dulal Borthakur, Chairperson Qing Li

Jon-Paul Bingham

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ACKNOWLEDGEMENTS

This research was supported by the National Science Foundation award

CBET 08-27057. I would like to express my utmost gratitude and respect for Dr.

Dulal Borthakur for being my mentor and guide during my M.S. degree at UH

Manoa. Many thanks have to be given to my fellow 418 lab-mates. Without their

aid and support this work could not have been possible. To Dung Pham, thank

you for teaching me how to tissue culture and perform safe lab practices. To

Michael Honda, thank you for the unwavering support during the research.

Thank you to the other researchers on the 4th floor of the Agricultural and

Science building at UH Manoa for the expert advice and guidance during my

educational experience. Thank you to Dr. Christopher and Dr. Li’s labs for their

critical support.

My thesis committee also deserves a very special thank you for all their

time and guidance during my master’s degree. Dr. Li and Dr. Bingham were

always responsive to my questions and concerns as my research progressed.

Lastly, I would like to thank my family and friends for the love and support

during my master’s degree. I feel honored to have such wonderful family and

friends.

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ABSTRACT

Leucaena leucocephala, (leucaena) is a fast-growing leguminous tree with many

positive applications, ranging from biofuel/biomass to animal fodder. This

research aims to improve digestibility of leucaena by reducing the concentration

of the toxic amino acid mimosine. The approach to accomplish this was

transformation of leucaena with isolated genes from Rhizobium sp. strain

TAL1145 that are involved in degradation of 3-hydroxy 4 pyridone, which is a

precursor of mimosine biosynthesis. The main focus of this work was to improve

the existing transformation protocols by overcoming problems with tissue

regeneration and DNA transfer, which have been the limiting factors in replicating

previous work. Our experimental trials indicated three key limiting factors: (i)

production of phenolic exudates by explants, which deter tissue growth, (ii)

accumulation of necrotic material at explant cut surface, and (iii) inefficient

rooting as a hindrance for regeneration. We hypothesized that overcoming these

three specific barriers would improve tissue regeneration and genetic

transformation significantly. Reduced production of phenolic exudates was

accomplished by introducing 0.8-1.0% activated charcoal to adsorb the phenolics

released into the growth media. Introduction of a cell recovery phase and

supplementation of the medium with activated charcoal prevented development

of necrotic cell material. Proper root induction was achieved through two means:

(i) elongation media development: which enabled explants to elongate shoots

prior to root induction; and (ii) activated charcoal was utilized as a darkening

media agent for improved root induction. Our results thus far have established

that prevention of necrotic cell death coupled with timely induction of a healthy

root system through darkened media, improves both tissue regeneration and

transformation frequency of leucaena. PCR analysis has shown the presence of

the transgenes in 16 individuals. These explants originated from four

independent Agrobacterium-mediated transformation experiments. This research

contributes towards the development of mimosine-free leucaena and also the

improvement of transformation protocols for woody legumes.

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TABLE OF CONTENTS

ABSTRACT ....................................................................................................................... 3

TABLE OF FIGURES ...................................................................................................... 5

TABLE OF TABLES ......................................................................................................... 6

Chapter 1 .......................................................................................................................... 7

INTRODUCTION AND LITERATURE REVIEW ......................................................... 7

1.1. Vision .................................................................................................................... 7 1.2. Background ........................................................................................................... 7 1.3. Growth conditions/characteristics ......................................................................... 8 1.4. Nutritional content ................................................................................................ 9 1.5. Toxicity ............................................................................................................... 10 1.6. Biological engineering for plant genetic improvement ...................................... 11 1.7. Recalcitrant nature of woody legume plants to transformation/ regeneration .... 12 1.8. Advances in legume transformation ................................................................... 13 1.9. Justification and significance: ............................................................................. 14

Chapter 2 ........................................................................................................................ 15

LITERATURE REVIEW ................................................................................................. 15

2.1. Legume and woody plant tissue culture factors .................................................. 15 2.2. Cowpea (Vigna unguiculata) .............................................................................. 15 2.3. Chickpea (Cicer arietinum) ................................................................................ 16 2.4. Peanut (Arachis hypogaea) ................................................................................. 17 2.5. Soybean (Glycine max) ....................................................................................... 18 2.6. Pea (Pisum sativum) ............................................................................................ 19 2.7. Field Bean (Lablab purpureus) ........................................................................... 20 2.8. Willow (Salix matsudana) ................................................................................... 21 2.9. Bahera (Terminalia bellerica) ............................................................................. 22 2.10. Almond (Prunus dulcis) ...................................................................................... 23 2.11. Plant growth regulators ....................................................................................... 24 2.12. Role of activated charcoal ................................................................................... 29 2.13. Phenolic oxidation and exudate .......................................................................... 29 2.14. Improved rooting conditions ............................................................................... 30 2.15. Hypothesis........................................................................................................... 31 2.16. Objectives ........................................................................................................... 31

Chapter 3 ........................................................................................................................ 32

MATERIALS AND METHODS ...................................................................................... 32

3.1. Seed selection..................................................................................................... 32 3.2. Seed sterilization ................................................................................................ 32 3.3. Explant starting material .................................................................................... 33 3.4. Callus induction/ pre-culture media (CIM)........................................................ 33 3.5. A. tumefaciens culture ........................................................................................ 34

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3.6. Co-culture liquid suspension.............................................................................. 35 3.7. Transformation ................................................................................................... 35 3.8. Recovery stage 1 ................................................................................................ 36 3.9. Selection stage ................................................................................................... 36 3.10. Recovery stage 2 ................................................................................................ 37 3.11. Elongation stage ................................................................................................. 38 3.12. Rooting stage (RIM) .......................................................................................... 38 3.13. Transfer to potted soil ........................................................................................ 39 3.14. Tissue culture induction .................................................................................... 40 3.15. Herbicide selection test ..................................................................................... 41 3.16. RNA & DNA extraction from leaf and stem tissue .......................................... 43 3.17. PCR amplification of putative transgenic extracted DNA ................................ 45 3.18. Reverse transcriptase PCR ................................................................................ 46

Chapter 4 ........................................................................................................................ 47

RESULTS ........................................................................................................................ 47

4.1. Reduction of phenolic exudate and necrotic cell accumulation in callus induction media (CIM) stage ........................................................................................................ 48 4.2 Reduction of phenolic exudate and necrotic cell accumulation in multiple shoot induction (SIM) stage ................................................................................................... 50 4.3 Improved rooting conditions from introduction of elongation (EL) stage .......... 52 4.4 Root system environment improvements............................................................. 54 4.5 Lateral root induction improvement .................................................................... 56 4.6 A. tumefaciens-mediated transformation ............................................................. 58 4.7 Phosphenothricin-resistance assay: ...................................................................... 62 4.8 PCR Results: ........................................................................................................ 67

Chapter 5 ........................................................................................................................ 73

DISCUSSION .................................................................................................................. 73

TABLE OF FIGURES

FIGURE 1: BREAKDOWN OF MIMOSINE BY RUMEN MICROORGANISMS TO DHP 11 FIGURE 2: PCAM 3201 PLASMID WITH FUSION PYDA-GLY-GLY-GLY-PYD B FUSION PROTEIN 12 FIGURE 3: 2,4-DICHLOROPHENOXYACETIC ACID. 26 FIGURE 4: 1- NAPHTHALENEACETIC ACID 26 FIGURE 5: INDOLE-3-BUTYRIC ACID 27 FIGURE 6: 6- BENZYLAMINOPURINE 28 FIGURE 7: KINETIN 28 FIGURE 8: TISSUE CULTURE STAGES OF LEUCAENA EXPLANT GROWTH. 41 FIGURE 9: HERBICIDE SELECTION TEST 42 FIGURE 10: REDUCTION OF PHENOLICS THROUGH ADDITION OF ACTIVATED CHARCOAL 50 FIGURE 11: REDUCTION OF PHENOLICS AND NECROTIC CELL ACCUMULATION THROUGH THE

INDUCTION OF 2 RECOVERY PHASES 51 FIGURE 12: INTRODUCTION OF ELONGATION MEDIA TO THE TISSUE REGENERATION PROTOCOL

53

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FIGURE 13: ROOT SYSTEM DEVELOPMENT COMPARISON BETWEEN ORIGINAL RIM MEDIA AND OPTIMIZED RIM MEDIA 56

FIGURE 14: LATERAL ROOT INDUCTION 57 FIGURE 15: PCR AMPLIFICATION OF DNA FROM PUTATIVE TRANSGENIC PLANTS # 1,3,4 67 FIGURE 16: PCR AMPLIFICATION OF DNA FROM PUTATIVE TRANSGENIC PLANTS # 2,5-12,14, 16 68 FIGURE 17: PCR AMPLIFICATION OF DNA FROM PUTATIVE TRANSGENIC PLANTS # 13 AND 15 68 FIGURE 18: PCR AMPLIFICATION OF DNA FROM PUTATIVE TRANSGENIC PLANTS # 1,3-10 70 FIGURE 19: PCR AMPLIFICATION OF DNA FROM PUTATIVE TRANSGENIC PLANTS # 2,3 71

TABLE OF TABLES

TABLE 1: COMPARATIVE COMPOSITIONS OF LEUCAENA AND ALFALFA LEAVES 9 TABLE 2: SUCCESSFUL AGROBACTERIUM-MEDIATED TRANSFORMATION OF LEGUMES 21 TABLE 3: SUCCESSFUL AGROBACTERIUM-MEDIATED TRANSFORMATION OF WOODY PLANTS 24 TABLE 4: MEDIA COMPOSITION FOR CIM PRE-CULTURE 34 TABLE 5: MEDIA COMPOSITION FOR A. TUMEFACIENS OVERNIGHT PRE-CULTURE 35 TABLE 6: MEDIA COMPOSITION FOR LIQUID CO-CULTURE 35 TABLE 7: MEDIA COMPOSITION FOR RM1 (RECOVERY MEDIA 1) 36 TABLE 8: MEDIA COMPOSITION FOR SIM SELECTION 37 TABLE 9: MEDIA COMPOSITION FOR RM2 (RECOVERY MEDIA 2) 37 TABLE 10: MEDIA COMPOSITION FOR ELONGATION PREPARATION 38 TABLE 11: MEDIA COMPOSITION FOR RIM PREPARATION 39 TABLE 12: HERBICIDE LEVEL SELECTION TEST WITH CONTROL EXPLANTS 41 TABLE 13: COMPOSITIONS OF THE ORIGINAL AND OPTIMIZED RIM MEDIA 54 TABLE 14: COMPARISON OF ROOT SYSTEM INDUCTION BY ORIGINAL AND OPTIMIZED RIM MEDIA

57 TABLE 15: NON PRE-CULTURE TRANSFORMATION GROUPS 59 TABLE 16: PRE-CULTURE TRANSFORMATION GROUPS 61 TABLE 17: PPT LEAF RESISTANCE ASSAY PHOTOS TWO WEEKS POST APPLICATION 63 TABLE 18: VISIBLE RANKING SYSTEM FOR CONTROL LEAF 66 TABLE 19: VISIBLE RANKING SYSTEM FOR PUTATIVE TRANSGENIC LEAF 66 TABLE 20: PCR CYCLE TIMES AND TEMPERATURES FOR PRIMER SET G32.0 67 TABLE 21: PCR CYCLE TIMES AND TEMPERATURES FOR PRIMER SET PYDA 69

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Chapter 1

INTRODUCTION AND LITERATURE REVIEW

1.1. Vision

In recent years, significant research efforts have been made to enhance our

food production through greater yields per hectare, stronger varieties and

increased nutritional value while simultaneously trying to decrease the price of

food for the consumer. These goals are difficult to accomplish together, but with

the help of biological engineering the arduous task of feeding the world’s

population (close to 9 billion by 2050, National Geographic) is much for feasible.

The vision of this particular study is to improve digestibility of a woody legume

named Leucaena leucocephala by Agrobacterium-mediated genetic

transformation.

Transformation of certain plant species was thought to be extremely difficult

due to their recalcitrant nature, but with time, a wide range of these species

including legume and woody plants have been successfully transformed.

Expanding the range of transformable plants increases the knowledge base of

plant transformation and its application in the real world. The goal we hope to

achieve is to develop and understand a more reproducible transformation

protocol for a previously recalcitrant legume.

1.2. Background

Leucaena leucocephala is a fast growing woody legume native to

southern Mexico, but can be found throughout the tropics today. This plant is

used by humans for a variety of purposes ranging from firewood, fiber, biomass

production and livestock fodder (Brewbaker et al. 1990). It is the last use of

fodder that I am interested in because my work involves engineering leucaena to

enhance its digestibility by ruminant animals.

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Due to leucaena’s many uses some people have held the plant in high

regard and believe that it is a miracle tree. Leucaena is capable of producing a

large volume of a medium-light hardwood for fuel with low moisture and a high

heating value. It also makes excellent charcoal, producing little ash and smoke.

The foliage of leucaena is nutrient rich for ruminants having both high protein and

mineral content. The Spanish, who came to Mexico in the 16th century,

recognized Leucaena for its excellent forage capability. They eventually brought

the plant with them to the Philippines, which was the jumping off point for

Leucaena to spread around the world (Brewbaker et al. 1990).

Leucaena’s increased use as a beneficial ruminant fodder has been

particularly dramatic in Australia. The main factor that drives this legumes

increased use is the ability of leucaena pastures to meet grazers’ needs for a

profitable system that simultaneously produces high quality beef. This is an

important point to note that leucaena is inexpensive to grow, but still produces a

high quality fed product that supports superior beef. The increase in overall

animal production /ha is 4 fold with leucaena fodder (Shelton et al. 2007).

Leucaena is thought to play an ever-increasing role in dry-land farming.

With rising global temperatures and increases in drought frequencies, leucaena

is being planted in an effort to convert marginal dry-land cultivation to a more

productive system. Australia is not the only place on the planet that will face

these challenges in the years to come concerning global warming. Leucaena has

already shown that it can produce high quality feed in arid conditions.

Environmental benefits of leucaena include dry-land salinity mitigation,

alkaline buffering capacity, improved water quality and improved soil fertility

through biological nitrogen fixation (Shelton et al. 2007).

1.3. Growth conditions/characteristics

This plant has been successfully planted around the tropics and the

subtropics all over the world. Leucaena does not tolerate frost so higher

latitudes cannot support leucaena growth (Fasolo et al. 1989). Although,

leucaena does tolerate more arid, drought like conditions, which is why it can be

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found growing where most other equally nutritious crops can’t survive. This

beneficial characteristic of leucaena gives the farmer an opportunity to have a

well-balanced feed product that can be grown throughout the tropics. Although,

not everyone feels that leucaena is a great leguminous tree to have around.

For some, leucaena is nothing more than a fast growing weed that out

competes native species (Lowe et al. 2000). Leucaena’s ability to quickly develop

seed banks, year round flowering and fruiting and self-fertilization contribute to its

invasiveness. These growth characteristics can be a problem for local

biodiversity restoration, although, the same physical attributes make leucaena a

very powerful forage plant for livestock. Leucaena’s invasiveness needs to be

kept under control, but it should not be targeted for eradication, as it is a powerful

tool for farmers trying to reduce food production costs by decreasing animal feed

costs.

1.4. Nutritional content

Leucaena is known for its high nutritional value with both macro and

micronutrients. The protein it provides to livestock is highly sought after and it

also boasts a balanced mineral and amino acid content. It is high in β- carotene

while having moderate tannin content to enhance the bypass value of the protein

(Jube et al. 2009). Leucaena has more than a two-fold concentration of β-

carotene compared to alfalfa.

Table 1: Comparative compositions of Leucaena and Alfalfa leaves

Component Leucaena leaf Alfalfa leaf

Total ash (%) 11.0 16.6

Total N (%) 4.2 4.3

Crude protein (%) 25.9 26.9

Modified-acid-detergent fiber (%)

20.4 21.7

Calcium (%) 2.36 3.15

Phosphorus (%) 0.23 0.36

ß- carotene (mg/kg) 536.0 253.0

Gross energy (kJ/g) 20.1 18.5

Tannin (mg/g) 10.15 0.13

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The total protein content of leucaena (15-18%) is much higher than that of

other common grasses and cereal straws (3-10%). High protein diet is very

important for farmers who are trying to feed and grow their cattle at a fast rate.

Leucaena is also used as a supplement to improve low quality forage feeds

(Soedarjo et al. 1996).

Digestibility and intake values for leucaena range from 50 to 71% and

from 58 to 85 g/kg (Jones 1979). The lower digestibility values of leucaena are

caused by mimosine and DHP when the diet was purely leucaena. Because of

the toxic effects of mimosine and DHP, animals diet cannot exceed

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Mid

Genes

Figure 1: Breakdown of mimosine by rumen microorganisms to DHP

1.6. Biological engineering for plant genetic improvement

Our goal is to biologically engineer Leucaena leucocephala to grow with

reduced mimosine content thereby allowing farmers to reduce their feed costs.

By supplementing their livestock’s diet with more leucaena that will have reduced

mimosine and 3-hydroxy 4 pyridone, farmers can feed their animals with a highly

nutritional food that is inexpensive and easy to cultivate.

There are two approaches to develop mimosine and DHP free leucaena

plants. 1) Silence the gene/genes responsible for mimosine biosynthesis. 2)

Introduce exogenous genes into the leucaena genome that will target 3-hydroxy

4-pyridone the precursor molecule to mimosine. The first approach is not

currently possible because there is very little information on the biosynthetic

pathway of mimosine (Jube et al. 2009).

We decided on the second approach to produce a transgenic plant with

reduced toxic concentrations by targeting the degradation of the precursor

molecule to mimosine, 3-hydroxy 4 pyridone. First we identified and isolated two

genes from the root colonizing bacteria Rhizobium sp. strain TAL1145. These

two genes are pydA (meta-cleavage dioxygenase) and pydB (pyruvate

dehydrogenase) (Soedarjo et al. 1998). A construct with pydA-G3-pydB was

Mimosine 3,4 dihydroxy-

pyridine

3-hydroxy-4-1

pyridone

Ruminal

Bacteria

12

transferred into a pCAM binary vector and then transformed into a disarmed

c58C1 Agrobacterium strain.

1.7. Recalcitrant nature of woody legume plants to transformation/ regeneration

Leucaena, a woody leguminous plant, is known to be very recalcitrant to

tissue regeneration and transformation. The low success of woody legume

transgenic production has been attributed to poor tissue regeneration during in

vitro culture and lack of compatible gene delivery methods (Chandra et al. 2003).

While growing leucaena in vitro, these problem areas are compounded by the

lack of information on this particular plant species concerning tissue culture and

transgenic plant production.

One major gap in the leucaena knowledge base is the unknown

endogenous hormone levels that take place inside the growing plant tissue

during regeneration. Without the endogenous hormone information the

researcher faces difficulty in designing growth medias with correct concentrations

of exogenous hormone levels. Getting correct auxin vs. cytokinin hormone ratios

pCambia 3201.

LB RB

cat gene

Ori Ec

Ori At

bar gene pydA-GGG-pydB

fusion gene

35 SP

Figure 2: pCAM 3201 plasmid with fusion PydA-Gly-Gly-Gly-Pyd B gene

13

is vital for proper tissue regeneration. Another problem faced by leucaena

researchers is the lack of reproducible transformation protocols. Leucaena has

been transformed in the past using Agrobacterium, but the efficiency has always

been low (1-2% Jube 1999). Repeated attempts to produce transgenic plants

with earlier protocols have proven to be unsuccessful as well.

With so many unknown variables in woody legume tissue culture, our

preliminary experiments lead us to focus on 3 problem areas that are thought to

be the limiting factors affecting successful production of a transgenic leucaena

plant. The first hindrance is the production of phenolic exudate from the excised

plant starting material. Second, is the accumulation of necrotic cells on the cut

surface site of the explant. Third is a poor root regeneration system. We

hypothesize that overcoming these 3 limiting factors will result in a more efficient

and reproducible transformation and regeneration protocol.

1.8. Advances In legume transformation

The first step in developing a successful legume transformant is finding

the correct cellular tissue to integrate the new DNA into its chromosome. These

cells should be young, totipotent, fast dividing while also having the capacity to

regenerate into a new plant (Somers 2003). Next is deciding what transformation

system will be employed to transfer the T-DNA into the target cells chromosome.

Part of the low success of legume transformation can be attributed to the lack of

compatible gene delivery methods. These approaches range from micro-

injection, particle bombardment, gene gun, electrophoresis and Agrobacterium-

mediated transformation (Chandra et al. 2003). Methods such as particle

bombardment have shown success with certain plant species, but legumes have

proven to be recalcitrant to physical means for transformation. An alternative

strategy is to use the genus Agrobacterium, which has the ability to conduct

interkingdom genetic exchange or transformation (Gelvin et al. 2012).

Agrobacterium-mediated transformation is the approach that has been

successful for a wide range of plant species especially those who have thick

outer cell walls (Somers 2003). The Agrobacterium will begin its transformation

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once it receives a chemical signal in the form of acetosyringone. The T-DNA will

pass through the plant cell wall and plasma membrane, cross through the

cytoplasm, enter the nucleus, while simultaneously integrating a single stranded

DNA sequence into the host’s chromosome (Gelvin et al. 2012). Once the T-DNA

has been successfully integrated into the plant’s chromosome, the next stage of

transgenic regeneration is a solid system for selecting or identifying which cells

are transformed.

A strong selection system that has been used to successfully transform

legumes is the multiple shoot induction while under herbicide selection. The

theory behind this method is once you establish a putative transgenic shoot,

tissue regeneration while under herbicide selection, you can continue to induce

more shoots while excising individual shoots and moving them to a rooting

media. The transgenic shoot is thought to have come from a single transformed

cell, which is then induced to divide rapidly and form multiple shoots that will also

be of the transformed variety (Somers 2003).

1.9. Justification and significance:

Leucaena research is valuable because as a plant species, it can be used

in a wide diversity of applications. Fast growing and nutritious, this legume plant

can grow in harsh conditions where alternative feed crops cannot. The main

reason why leucaena is not more widely used as a feed crop is due to the lack of

a low toxin variety.

Traditional breeding has been an invaluable tool for humans throughout

our development, but it does have limitations. In our case, there is no variety in

the whole family of leucaena that does not have high mimosine content (Jube

and Borthakur 2009). Biological engineering is the most effective strategy to

create a transgenic plant.

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Chapter 2

LITERATURE REVIEW

2.1. Legume and woody plant tissue culture factors

Considering leucaena to be a woody leguminous tree, we have focused

on other legume and woody plant methodology for tissue culture and genetic

transformation. For simplicity of discussion we will cover the following areas: 1)

legume and woody plant tissue transformation, 2) plant growth regulators, 3) cell

growth inhibitory factors and 4) improved rooting conditions.

In this literature review section we will cover related legumes as well as

other woody plant species, similar to leucaena, in order to gain a better

understanding of the challenges that need to be overcome in order to

successfully transform this recalcitrant plant species. The ideas gained from

successful transformation of other recalcitrant plant species will help improve the

tissue culture and regeneration protocol for leucaena. The reason why we have

focused on related legumes and other woody plant species transformation

protocols is due to the limited literature on successfully transformed woody

leguminous plants. Both legumes and woody plants are known to be recalcitrant

to tissue culture, but very little information is known on woody leguminous plants.

Literature review will be done with a particular emphasis to try and

improve the following areas: 1) phenolic exudate, 2) necrotic cell buildup and 3)

rooting inefficiencies.

2.2. Cowpea (Vigna unguiculata)

Cowpea has been successfully transformed through Agrobacterium-

mediated transformation. The starting explant material was the cotyledonary

nodes from mature seeds (Popelka et al. 2005). The strain used to transform this

legume species was EHA 105. The selective pressure that was used to screen

for transformants was 150 mg/l of kanamycin.

16

Pre-culture media contained MS salts + 10 μM thidiazuron (TDZ a

commonly used cytokinin in tissue culture). The strain maintenance media was

solid YEP with 10 mg/l rifampicin + 50 mg/l kanamycin. The co-culture media

contained MS salts with 1 μM 6- benzylaminopurine (BA a synthetic cytokinin

modeled after naturally occurring cytokinins) + 100 μM acetosyringone to aid in

transformation.

After transformation, the explant material was moved to multiple shoot

induction media with 5 μM BA + 0.5 μM kinetin + 150 mg/l kanamycin for

selection against non-transformants. The lower overall hormone concentrations

were noted and proved effective for inducing multiple shoots. During the

selection stage necrotic cells were not allowed to accumulate on the explant

material. The dying cells were continuously removed from the healthy explant

tissue. The technique of consistent removal of necrotic cells should be employed

for leucaena tissue culture regeneration for this was considered a major limiting

factor in healthy plantet regeneration. Additionally, the explants were constantly

being plated on fresh media (1-2 weeks), which lowered the negative impacts of

phenolic exudate and resulting necrotic cell death.

Cell recovery phase after transformation and selection stages was

deemed to be critical for healthy shoot and root tissue regeneration. The shoot

recovery media contained the same concentrations of hormones as the selection

media, except that kanamycin (selection agent) was dropped once the transgenic

shoots had been screened for. The absence of antibiotic selection helped the

plantlets recover prior to rooting. Next stage was root induction on MS salt media

+ 2.5 μM of Indole-3-butyric acid (IBA an auxin used to induce rooting).

The final transformation frequency they were able to achieve was 1-3

transgenic plants per 1000 explants tested.

2.3. Chickpea (Cicer arietinum)

This grain legume was successfully transformed with the explant source

being mature embryonic axes. The use of mature embryos grown from seeds, is

an interesting alternative to the use of immature embryos such as with past

17

successful transformations of leucaena (Jube 2009). The use of mature embryos

enables researchers to store collected seeds and begin germination at any time

of the year.

The strain of A. tumefaciens used was LBA 4404. Selection agent used to

screen for putative transformed tissue was 50 mg/l kanamycin. Researchers

induced mature embryonic axes to form new callus growth, which was the target

tissue for transformation. CIM media started with higher auxin concentration of 5

mg/l of 2,4 dichlorophenoxyacetic acid for 7 days (2,4 D is a synthetic auxin).

Next, the auxin level was dropped to (0.05 mg/l of 2,4 D) for 10 days, which

lowered the stress introduced into the growing tissue by slowing down the rapid

cell division.

Low hormone levels (0.05mg/l 2,4 D and 0.02 mg/l IAA) at specific times

during culture were used to accomplish the goal of healthy tissue regeneration.

(Mehrotra et al. 2010). The approach of lower total hormone induction levels

during various growth stages, which lessened the stress introduced into the

regenerating tissue, can be applied to leucaena tissue culture. Stress and

resulting necrotic cell accumulation, was mitigated by shortening the co-culture

period to only 48 hours. Even with the shortened co-culture period, a

transformation frequency of 3.6% was achieved. The shortened time period for

explant and Agrobacterium interaction should be employed during leucaena co-

culture for this is a stressful period of the experiment.

2.4. Peanut (Arachis hypogaea)

Peanut has been transformed with the aid of A. tumefaciens. The explant

starting material was the cotyledon embryonic nodes. The Agrobacterium strain

of choice was LBA 4404. Selective agent used was phosphenothricin at 3 mg/l

(Iqbal, et al. 2011).

Co-culture for the peanut began at the same time as the shoot induction

stage. There was no pre-culture or callus induction period. This is an interesting

approach going for direct organogenesis of putative transgenic shoots as

opposed to developing undifferentiated callus tissue. Direct organogenesis may

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have a place in leucaena transformation, since the explants do not respond well

to prolonged periods of high hormone induction. SIM media contained 5 mg/l of

BA cytokinin, which is a notably high concentration considering leucaena was

induced to produce on average 5-7 shoots while under 3 mg/l of BA (Jube et al.

2009). The researchers were able to obtain large putative shoots after only 2

weeks on SIM media. Next, the putative shoots were transferred to a shoot

elongation media. Leucaena shoots are often too short after 6-8 weeks on SIM

media, which limits their ability to survive excision and make the transition to form

a healthy root system. Shoot elongation is a strategy that aids in leucaena tissue

regeneration by enabling shoots to develop size and strength prior to the very

critical root induction period.

Root induction for peanut was accomplished on a low auxin RIM media.

0.3 mg/l NAA was the concentration used to induce a healthy root system (Iqbal,

et al. 2011). This is another example of tissue regeneration of a recalcitrant

species being accomplished with a low hormone induction level.

2.5. Soybean (Glycine Max)

Soybean is an extremely valued and widely grown crop around the world.

A lot of work has been done to improve the transformation efficiency of soybean

because of the monetary value tied to producing this crop. Mature seeds instead

of immature seeds have been used as starting material for successful soybean

transformation. Mature seed germination is an alternative that allows researchers

to perform transformations year round as opposed to waiting for the ideal growth

time to collect fresh or immature seeds. A mature seed transformation protocol

would be beneficial when working with leucaena, because the best immature

seeds are only available for 3-4 months in the late summer and early fall.

The starting material for A. tumefaciens-mediated transformation was

freshly excised embryonic tips from the germinated mature soybean seeds. The

Agrobacterium strain was EHA 105. Selective agent used to screen for

transformants was kanamycin (Liu et al. 2004). Embryonic tip regeneration was

used to accomplish the goal of stable soybean transformation.

19

The pre-culture period of explants was short (24 h) prior to a 5-day co-

culture period with a high cytokinin induction hormone level of 6 mg/l of BA. This

is a long co-culture period compared to 48 hours for chickpea (Mehrotra et al.

2010). It was not clearly stated how the hyper-virulent strain EHA 105 was

removed from the infected explant material after the 5 day co-culture. The

lengthy co-culture period would enable a very high number of A. tumefaciens

cells to develop and would then have to be removed following co-culture.

The next stage was the resting or recovery media. This is another

example of a leguminous plant regeneration protocol utilizing a recovery period

post co-culture and prior to selection stage. Recalcitrant plant species seem to

need this vital cell rest period in order to have healthy tissue regeneration later

on. The transformation efficiency of this particular plant species was 15.8% which

is an extremely high percentage of success for a previously recalcitrant plant

species.

2.6. Pea (Pisum sativum)

Researchers working with peas have focused on finding the most suitable

A. tumefaciens strain that will efficiently transform the host genotype. After

testing three different bacterial strains the most effective at transforming pea was

the hyper-virulent EHA 105 with an 8.2% transformation frequency (Orczyk A,

Orczyk W. 1999).

The starting material for this transformation experiment was immature

cotyledons, which were induced to have direct organogenesis. The immature

cotyledons were pre-cultured for 2 days prior to the transformation event. Ten

explants were put in 100 ml of liquid medium supplemented with 100 μl of EHA

105 inoculum with a density of 1 x 10^9 cells/ml. Inoculation mixture was shook

at 120 rpm at 22 °C. After two days the cotyledons were put on solidified growth

medium supplemented with 500 mg/l of carbenicillin + 100 mg/l hygromycin. The

use of carbenicillin versus cefotaxime was noted to be successful at removing

the virulent strain EHA 105 post co-culture. Removal of A. tumefaciens post co-

20

culture with immature embryos of leucaena has proven to be difficult and a

potential limiting factor in tissue regeneration.

2.7. Field Bean (Lablab purpureus)

Field bean was successfully transformed using mature seeds as the

starting material. Seeds were germinated and the embryonic axes were targeted

for co-culture. The A. tumefaciens strain used to perform the transformation was

EHA 105. It was noted that this particular strain has been used to successfully

transform a wide range of recalcitrant legume species.

The targeted region for transformation was the apical meristematic cells of

the embryo. A small wound was created in this particular cellular region providing

an entry site for the bacterium infection. Wounding of the target cell material was

noted to be critical for successful T-DNA insertion into the explant chromosome.

It was noted that the emerging embryos were not excised in order to

perform the transformation. The researchers referred to this technique as in-

planta (Keshamma et al. 2011). This is a very interesting alternative to excision

of the embryo because the only injury that needs to be performed is a small

puncture instead of multiple cuts to separate out the target tissue. This method

should be tested with leucaena because excision of the immature embryos prior

to transformation generates phenolic exudate and resulting necrotic cell death. If

the amount of injury issued to the starting material can be lessened, the amount

of damaging phenolics in the growth media will be reduced.

21

Table 2: Successful Agrobacterium-mediated transformation of legumes

Species/

Genotype

A.

tumefaciens

strain

Explant

starting

material

Selection agent Citation

Pakistani

peanut/

Arachis

hypogaea

LB 4404 Cotyledon

embryonic

nodes

Phosphenothricin Iqbal M, et al.

2011

Cowpea/ Vigna

unguiculata

EHA 105 Cotyledonary

nodes from

mature seeds

Kanamycin Popelka J, et

al. 2005

Chickpea/

Cicer arietinum

LB 4404 Mature

embryonic

axes

Kanamycin Mehrotra M,

Sanyal I, Amla

D 2010

Soybean/

Glycine Max

EHA 105 Embryonic tip

from mature

seeds

Kanamycin Liu Hai-Kun, et

al. 2004

Pea/ Pisum

sativum

EHA 105 Immature

cotyledons

Hygromycin Orczyk A,

Orczyk W.

1999

Barrel Clover/

Medicago

truncatula

Agrobacterium

rhizogenes

Seedling

radicals

Kanamycin Dernier A, et

al. 2001

Field Bean/

Lablab

purpureus

EHA 105 In Planta

embryonic

axes

Kanamycin Keshamma et

al. 2011.

2.8. Willow (Salix matsudana)

This woody plant species was successfully transformed using mature

seeds as the starting material. The seeds were germinated and the first apical

meristematic growth was then excised just prior to Agrobacterium co-culture. The

authors found that excising the very first meristematic growth would expose

rapidly dividing apical cells to the Agrobacterium, and resulted in a transformation

frequency of 7.2% (Yang et al. 2012). This is a reasonably high transformation

frequency percentage for a woody plant species that has been recalcitrant in the

past.

22

The authors state that some of the factors affecting their transformation

frequency include the strain of A. tumefaciens (LBA 4404), the density to which

the Agrobacterium cells were grown (OD 600 of 0.6), co-culture period (4 days),

and the length of time the mature seeds had been stored (no difference under 1

year storage was noted). Cell density growth and co-culture period are just a

couple of the factors that still need further trials to determine the optimal

conditions for leucaena transformation.

First, mature seeds were pre-cultured on media supplemented with 1.0

mg/l zeatin + 0.1 NAA for 4 days allowing the first apical bud growth to appear.

The buds were excised and immediately infected with A. tumefaciens grown to a

cell density of OD600 of 0.6. The authors found that 4 days of pre-culture with

excised apical bud had 76.4% frequency of multiple shoot induction. The infected

buds were co-cultured at 21 ˚C for 4 days prior to moving to selection.

Selection was performed with kanamycin in the media for up to 8 weeks

until multiple shoots could be seen growing from the original explant. The authors

noted that 13 explant lines showed the most rapid growth and development,

where most others became chlorotic, grew slowly and then died. Screening for

the best individuals was very important in generating successful transformants

(Yang et al. 2012). This approach of individual selective screening needs to be

employed in transformation of leucaena.

2.9. Bahera (Terminalia bellerica)

An efficient in vitro transformation and plant regeneration protocol was

developed for this multipurpose tree species. The starting material was excised

cotyledonary nodes (Dangi et al. 2012). Explants were inoculated on growth

medium supplemented with a range of BA (2.2, 4.4, 8.8, 13.2 μM) and kinetin

(2.3, 4.6, 9.2 and 13.9 μM). TDZ was also tested but was found to be ineffective

at producing healthy putative shoots that could be rooted. After 4 weeks, shoot

buds were cut into smaller pieces and sub-cultured on medium containing 8.9 µM

of BA for proliferation. This concentration of BA in the growth media was found to

be the most effective at producing healthy putative transgenic shoots.

23

Once the putative shoots reached a length of 2-3 cm, they were placed on

medium supplemented with a range of IAA and IBA for rooting. After 4 weeks,

the cultures were evaluated and moved to potted soil (Dangi et al. 2012).

The A. tumefaciens strain that was used during this transformation

experiment was EHA 105. The authors used this particular strain due to its

hyper-virulence. No detail was given as to the methodology of techniques used to

remove the Agrobacterium post co-culture.

It was noted that higher regeneration and transformation efficiency was

achieved with pre-cultured cotyledonary nodes, rather than direct excision

followed by immediate infection. A past successful transformation protocol of

leucaena found that pre-culture of explants prior to transformation resulted in the

production of stable transformants. There were no stable leucaena transformants

produced without a pre-culture period (Jube et al. 2009).

2.10. Almond (Prunus dulcis)

Researchers were able to develop transgenic almond trees through

Agrobacterium-mediated transformation. The starting material used was leaf

segments taken from germinated almond seeds cultured on MS medium

containing 0.3 mg/l BA and 0.01 mg/l IBA (Miguel et al. 1999).

Explants used for transformation were young, fully expanded leaves of 3-

week-old micro-propagated shoots. The leaves were wounded to provide an

entry site for the DNA transfer to take place. The use of leaf segments is an

alternative starting material choice for transformation. No protocol or technique

was mentioned to mitigate the development of phenolic exudate resulting from

multiple cuts or excision points. Leucaena explant material has been shown to

produce heavy amounts of phenolic exudate and necrotic cell buildup as a result

of physical damage or cuts made to the tissue.

Co-cultivation was conducted for 3 days in darkness on MS media

supplemented with 1.5 mg/l TDZ + 0.5 mg/l IAA + 0.01 mg/l 2,4 D. The A.

tumefaciens strain that was used to conduct the transformation was EHA 105.

After 20 days on initial culture media, the explants were transferred to a shoot

24

elongation media supplemented with 1 mg/l BA + 50 mg/l kanamycin. Shoots

surviving after 3 weeks on selection were excised and cultured on a micro-

propagation media for 2 rounds.

The researchers working with this transgenic almond attributed a 7-fold

increase in transformation frequency to pre-culture time of explant material prior

to the transformation event. The second major factor effecting transformation

was the A. tumefaciens strain selected. EHA 105 was found to have a much

higher percentage of transformation success than the alternative strain LBA 4404

(Miguel C et al. 1999). It was noted that pre-culture and selection of EHA 105 as

the Agrobacterium strain were reoccurring factors that had shown success in

production of stable transformants from recalcitrant plant species.

Table 3: Successful Agrobacterium-mediated transformation of woody plants

Species/

Genotype

A.

tumefaciens

strain

Explant

starting

material

Selection agent Citation

Willow/ Salix

matsudana

EHA 105 Apical

meristemic

embryo

Kanamycin Yang J et al.

2012

Bahera/

Terminalia

bellerica

EHA 105 Cotyledonary

nodes

Kanamycin Dangi B et

al. 2012

Almond/

Prunus

dulcis

EHA 105 Excised leaf

segments

Kanamycin Miguel C et

al. 1999

2.11. Plant growth regulators

Plant growth regulators (PGR’s) or plant hormones are chemicals that

regulate the growth and development of plant tissue. PGRs are vital to many

biochemical and physiological aspects of plant tissue generation. During

transformation and regeneration, plant hormones are employed to accomplish

formation of callus, multiple shoot induction, shoot elongation and root system

25

development in the explant material. Since there is very little information known

on the hormone levels during leucaena early stage tissue development, more

work needs to be done to find the ideal ratio of cytokinin and auxin hormone

concentrations at all stages of development concerning tissue regeneration.

Plant tissue culture and transformation require hormones at specific times

to ensure proper regeneration and growth. Plantlets also need to be able to

disengage the effects of hormones when they are no longer needed (Davies

2010). This pattern of hormone induction followed by hormone concentration

decrease (recovery media phase) needs to be employed during leucaena tissue

culture to encourage fast and rigorous development. Hormones are critical in

providing the spark or jumpstart to tissue regeneration, but prolonged exposure

of explant cells to PGRs can induce stress and even cell death.

There are four main classes of plant growth regulators used in tissue

culture, namely auxins, cytokinins, gibberellins and abscisic acid. During this

project we focused on the use of auxins and cytokinins to accomplish our desired

phenotypic responses with leucaena. A balance of these two hormonal types

must be reached for each section of explant culture.

2.11 (a) Auxins

At the molecular level, auxins are essential for growth, affecting both cell

division and expansion. Auxin concentration levels together with local factors

contribute to cell differentiation and tissue regeneration. Depending on the type

of tissue, auxins may promote axial elongation and lateral expansion (Taiz 1998).

The auxins that were utilized in leucaena tissue media composition were

2,4 Dichlorophenoxyacetic acid (2,4 D), 1-Naphthaleneacetic acid (NAA) and

Indole-3-butyric acid (IBA).

26

Figure 3: 2,4-Dichlorophenoxyacetic acid.

2,4-D is a commonly used auxin hormone in plant tissue culture. It is a

synthetic auxin that is also used as an herbicide. Our use of this hormone in

media culture was to cause rapid division and growth of cells. 2,4 D is absorbed

by the cell tissue and translocated to the meristematic region of the plant (Suwa

1996). Rapid and unsustainable growth ensues. The uncontrollable cell division

and growth only continues while the plant tissue is under 2,4-D hormone

induction. 2,4-D was used only during the callus induction stage. The expected

phenotypic response of the explant material is rapid cell division and

multiplication of undifferentiated cells, which can then be induced to produce

apical buds.

Figure 4: 1- Naphthaleneacetic acid

NAA is another commonly used synthetic auxin hormone. This organic

molecule was used in both shoot and root production for leucaena tissue culture.

Additionally, NAA aided in vegetative propagation of multiple shoots and stems. It

is known to greatly increase cellulose fiber formation when paired with another

plant hormone gibberellin (Morikawa et al. 2004). During leucaena regeneration,

27

varying concentrations of NAA were employed depending on the desired tissue

growth.

Figure 5: Indole-3-butyric acid

The exact cellular mechanisms of IBA are still being worked out, but

genetic evidence has shown that IBA may be converted to a similar plant

hormone Indole-3-acetic acid (IAA) once taken up by the plant cells (Zolman et

al. 2008). IAA is an abundant plant hormone that is produced endogenously. IBA

is also naturally occurring, but only in small amounts.

IBA was employed during root system formation of leucaena. The

concentration of the auxin IBA (1.0 mg/l) needed to be higher than the cytokinin

kinetin (0.1 mg/l) in order to produce roots as opposed to shoots.

2.11 (b) Cytokinins

Cytokinins promote cell division in plant shoots and roots during tissue

culture and regeneration. Cytokinins are involved primarily in cell growth,

division, shoot/bud formation, lateral bud formation, and leaf expansion resulting

from cell enlargement (Aremu 2012).

The ratio of cytokinin to auxin needs to be worked out in order to obtain

the fastest growth, healthiest shoots, and stem elongation during leucaena

regeneration. Cytokinins alone have no effect on parenchyma cells, but paired

with auxins in equal concentrations, the parenchyma cells develop

undifferentiated callus. Once the callus has formed, the cytokinin concentrations

are increased while auxin concentrations are lowered. Shoot buds will begin to

emerge from the callus as the higher cytokinin concentrations induce shoot and

28

cell elongation (Aremu et al. 2012). This pattern of rapid cell division, callus

formation, followed by multiple shoot induction is utilized during leucaena explant

culture. The cytokinins used during this experiment were 6- Benzylaminopurine

(BA) and Kinetin.

Figure 6: 6- Benzylaminopurine

BA is a synthetic cytokinin that elicites plant growth and development

responses. During leucaena regeneration, BA was used to induce multiple

shoots from the original explant material, which is not natural tissue development

for leucaena.

Figure 7: Kinetin

Kinetin is often used in plant tissue culture for inducing the formation of

callus in conjuction with auxins. During leucaena tissue culture, kinetin was used

in lower concentrations (0.1 mg/l) to balance the heavier IBA (1.0 mg/l) hormone

induction during root system regeneration. Even though root formation is driven

29

by auxin induction, cytokinins must be present in order to obtain healthy root

formation.

2.12. Role of activated charcoal

Activated charcoal is a form of carbon, riddled with many small, low

volume pores that increase surface area for adsorption of molecules. This form of

carbon has been cleaned of impurities and oxidized (Thomas 1998). The

physical characteristics of activated charcoal have many applications in leucaena

tissue culture including hormonal concentration regulation, and cell protective

characteristics.

Activated charcoal is used to improve cell growth and development. While

adsorbing the damaging molecules, activated charcoal helps with pH adjustment

and plant growth regulator concentrations. Activated charcoal is thought to

adsorb some of the PGRs and nutrients being added to the growth culture and

then release them into the media as the concentrations drop due to cell division

and growth. This controlled release of the PGRs is believed to be very beneficial

in healthy cell development and tissue regeneration (Sáenz et al. 2010).

Leucaena explants have shown strong responses to varying hormonal

levels as well as time exposed to the hormones. Activated charcoal should be

employed to control the effects of the added hormones by establishing a

controlled release of additional PGRs added to the growth media.

2.13. Phenolic oxidation and exudate

Phenolic oxidation and brown exudate accumulation are problems in a

closed environmental system such as sterile tissue culture. Activated charcoal

drastically reduces the accumulation of these damaging phenolic compounds

through absorption. With the reduced amount of damaging molecules in the

growth media, necrotic tissue development also slows down drastically. This is

extremely important when working with plants that have high phenolic content

like leucaena. Necrotic cell accumulation leads to high leucaena explant tissue

loss and must be mitigated in order to increase regeneration rates.

30

Phenolic compounds in the growth media are also toxic to Agrobacterium

cells. This presents a huge problem when the bacterium is the selected DNA

transfer agent. Studies have showed that activated charcoal added to co-culture

mediums has improved transformation rates (Rathore et al. 2013, Yao et al.

2013).

Studies have also shown that adding anti-oxidants such as citric acid can

reduce the negative impact of the phenolic exudates and slow down cell

browning and death. Media fortifications, which included both activated charcoal

and anti-oxidants, had explants with the least amount of browning or cell death

(Thomas 1998). These various media additives are able to work together to

combat the accumulation of damaging phenolic exudates better than if they were

being added individually.

2.14. Improved rooting conditions

Getting proper rooting conditions can be very difficult to obtain when

working with a plant species that does not have a lot of literature or research

done in the past. Plant growth regulator concentrations, along with other media

fortifications, dictate how fast a healthy root system can develop. Problem is, with

a closed system, such as a Magenta 7 box, the hormones and nutrients added to

the media will remain in the media, affecting the local cellular developmental

environment, until used by the explant. With the use of activated charcoal in the

growth medium, the PGRs and nutrients are absorbed by the carbon material

and are then released into the growth media only as the concentrations begin to

drop after plant cell division and growth. This is vital for a plant like leucaena

because very little is known of the endogenous hormone levels during early

tissue development.

Simulating soil conditions in tissue culture setting by darkening the media

has been shown to improve rooting conditions as well. Activated charcoal is often

used for these purposes because it is black in color and can be added and mixed

slowly until reaching the desired dark pitch. Activated charcoal has been used in

this way for a variety of plant species including woody plants. With the species

Pinus pinaster, a researcher was able to improve the overall rooting capacity of

31

mature explants to an average of 78% (Dumas E, Monteuuis O. 1995). With a

transgenic pineapple study, the author wrote that the addition of activated

charcoal to the growth media considerably enhanced the rooting ability of the

transgenic shoots (Firoozabady et al. 2006).

2.15. Hypothesis

Tissue regeneration and transformation frequency of Leucaena leucocephala can

be enhanced by overcoming key limiting growth factors that decrease healthy

tissue development.

2.16. Objectives

Enhancement of tissue regeneration with aims of improving transformation frequency by:

a) Prevention of the production/and or negative impact of phenolic exudates

in the growth media

b) Inhibition of the accumulation of necrotic cells on the explant cut surface

site

c) Overcoming in vivo root system development inefficiencies

32

Chapter 3

MATERIALS AND METHODS

3.1. Seed selection

With seed selection it was vital to screen for medium-large green, healthy

looking seeds with no visible exterior damage or insect infestation. A small

percentage of the germinating seeds showed the ideal growth characteristics

right from the start of embryo excision and plating on growth media. The thinning

of weak embryos with delayed growth from the more vigorous fast growing

embryos was crucial. If there was a pause in explant development during tissue

regeneration, plantlets began to brown and die. Energy and resources being

taken away from healthy, re-generable explants, will negatively impact

transformation efficiency. Our approach was to allow only the very best of the

seeds to begin germination on induction media.

Leucaena produces seeds throughout the year in tropical regions

(Brewbaker 1990), but its best and most desirable seeds begin to arrive in late

May and continue through September. Seed collection needs to be the focus

during this time of the year. Following collection, transformation with

Agrobacterium and selective tissue culture of many explant groups should be

employed. The more excised embryos that are exposed to Agrobacterium, the

higher the probability of producing a stable transgenic plant. Another limiting

factor with successful leucaena transformation is healthy, re-generable cells that

can integrate the T-DNA, and continue to divide and replicate. Healthy fast

growing seeds have the correct target cells for replication and tissue

regeneration.

3.2. Seed sterilization

Seeds were removed by hand from the green seedpods and collected in a

magenta 7 box. First stage was surface sterilization. 1% dish wash detergent and

10 % sodium hypoclorite were added to 200-400 ml of ddH2O. The box was then

33

placed on a magnetic stirrer and spun at low velocity for 10 minutes. Next the

seeds were rinsed 3X with ddH2O until all was solution was removed.

The seeds were brought over to the sterilized flow hood and dried on

autoclaved filter paper. Next the seed is cut in half and the embryo side is kept.

The seed coat is carefully removed exposing the embryo and attached

cotyledons segments. We decided not to cut off the remaining cotyledon pieces

that were still connected to the embryo because we wanted to limit the amount of

cuts and overall stress impacted to the young embryo.

To reduce browning of explant starting material, the embryos were soaked

for 30 minutes at RT in an antioxidant solution (50 mg/l ascorbic acid + 75 mg/l

citric acid). The embryos were then 1) immediately transformed and co-cultured

with A. tumefaciens (Explant groups A) or 2) directly plated on pre-culture media

(Explants groups B) for 4-14 days prior to A. tumefaciens-mediated

transformation.

3.3. Explant starting material

Immature embryos were selected as the starting material for

transformation and regeneration of Leucaena leucocephala K636. The young

embryonic cells provided the correct target material for Agrobacterium-mediated

transformation. Young cells were critical to obtain both fast growth and healthy

tissue development. Mature shoot tips and excised immature cotyledons were

also extensively tested but were found to have problems with tissue regeneration

capabilities as well as consistent outside contamination.

The immature embryos were excised from the young seeds and were

either transformed immediately (Group A), or pre cultured for 4-14 d (Group B).

3.4. Callus induction/ pre-culture media (CIM)

CIM media served as the initial pre-culture medium for group B explants

that had 4-14 days of culture prior to transformation. This media also served as

co-culture media. The 0.8% activated charcoal is the new supplemental material

that will absorb the phenolic exudates and will help reduce the accumulation of

34

necrotic cells at the cut surface site on the embryo. Secondly, activated charcoal

will aid in the protection of Agrobacterium cells during co-culture by absorbing the

damaging phenolic exudates produced after embryo excision, which are toxic to

bacterial cells.

The explants and A. tumefaciens were plated and observed closely during

the co-culture period. If the A. tumefaciens cell concentration built up too high the

explant was washed with 10% sodium hypochlorite followed by 3x rinses in

sterile H20 before being put back onto clean CIM media. Total time on CIM

media for non pre-culture explants (group A) was 5-7 days and for pre-culture

explants (group B) total time on CIM was 10-21 days.

Table 4: Media composition for CIM pre-culture

Media Component Concentration

Murashige & Skoog basal medium ½ X

2,4-Dichlorophenoxyacetic acid 1.0 mg/l

6- Benzylaminopurine 0.5 mg/l

Sucrose 30 g/l

Activated Charcoal 0.8%

pH 5.8

3.5. A. tumefaciens culture

The Agrobacterium strain C58C1 was engineered to contain and express

the pCAM binary plasmid containing the bar gene for herbicide resistance and

the fusion gene product pydA-GGG-pydB between the LB and RB segments on

the plasmid.

A. tumefaciens cells were collected off solid LB plates containing 50 mg/l

chloramphenicol and 10 mg/l rifampicin and grown in 40-50 ml liquid LB media,

supplemented with 50 mg/l chloramphenicol + 10 mg/l rifampicin, overnight on a

rotating shaker at 250 rpm at 28 °C.

35

Table 5: Media composition for A. tumefaciens overnight pre-culture

Media Component Concentration

Luria Broth 1X

Chloramphenicol 50 mg/l

Rifampicin 10 mg/l

3.6. Co-culture liquid suspension

The freshly cultured A. tumefaciens cells were centrifuged for 3 minutes at

5,000 rpm to form a pellet. The pellet was then re-suspended in 40-50 ml liquid

CIM media supplemented with 200 μM of acetosyringone. Acetosyringone is

critical during this stage of the experiment for it induces the virulence genes in A.

tumefaciens initiating the transformation process.

Table 6: Media composition for liquid co-culture

Media Component Concentration

Murashige & Skoog basal medium ½ X

2,4-Dichlorophenoxyacetic acid 1.0 mg/l

6- Benzylaminopurine 0.5 mg/l

Acetosyringone 200 μM

Sucrose 30 g/l

pH 5.8

3.7. Transformation

The immature embryos in Groups B were grown on their pre-culture media

for 4-14 days prior to transformation. The overnight culture of A. tumefaciens was

centrifuged at 5,000 rpm to form a pellet and then re-suspended in co-culture

medium.

The embryos and A. tumefaciens culture were put into the same

eppendorf tube and placed on a rotating shaker for 1 hr at 250 rpm prior to 1 hr

of vacuum infiltration. The explants and A. tumefaciens were then removed from

the liquid co-culture medium and quickly dried on sterile filter paper. The infected

explants were then placed radicle side down into the solid CIM media initiating

36

the co-culture period. Co-culture was run for 4-7 days depending on the visual

health and hormonal response of the explant tissue. If phenolic exudates began

to damage the young tissue causing cell necrosis, the explants were washed free

of bacteria and moved to recovery media 1.

3.8. Recovery stage 1

After co-culture the putative transformed explant cells needed time to

recover after the bacterial infection. Recovery time was 5-7 days depending on

how quickly the explants developed new green growth. The recovery media was

new to this experiment and allowed time for the explant to recover post

transformation. Recovery media 1 stopped the development of necrotic cells by

removing growth hormones, which gave the embryonic cells time to recover prior

to the selection stage. The explants were kept in the dark for the recovery period.

Table 7: Media composition for RM1 (recovery media 1)

Media Component Concentration

Murashige & Skoog basal medium ½ X

Cefotaxime 250 mg/l

Sucrose 30 g/l

Activated Charcoal 0.8%

pH 5.8

3.9. Selection stage

After recovery stage 1 the explants were taken out of the dark and placed

into the light for 16hrs light/8 hrs dark for the remainder of the experiment.

Explant time on this media ranged from (6-14 weeks). The wide range of time

during selection was determined by the regeneration response of the individual

explant. The selective pressure in this media was 3 mg/l phoshenothricin, which

was confirmed to be the correct concentration through control explant trials.

The purpose of SIM media is to select against non-transformed cells while

simultaneously inducing the explant to produce multiple putative shoots.

37

Leucaena naturally produces 1 shoot per embryo, but with a high cytokinin

concentration in the media (3 mg/l BA), multiple shoot induction occurs. The

development of more than one shoot from the original embryonic tissue

increased the likelihood of a transformed shoot emerging. The putative shoots

had to overcome the selection pressure and eventually form a healthy root

system.

Table 8: Media composition for SIM selection

Media Component Concentration

Murashige & Skoog basal medium 1X

1-Naphthaleneacetic acid 0.25 mg/l

6- Benzylaminopurine 3 mg/l

Sucrose 30 g/l

Phosphenothricin 3 mg/l

Cefotaxime 250 mg/l

3.10. Recovery stage 2

After multiple shoot induction under selection pressure the putative

transformed shoots needed a second recovery period. Recovery time was 5-7

days. The recovery media 2 was new to this experiment and allowed time for the

putative shoots to recover after selection. Recovery media 2 slowed the

development of phenolic exudates by removing growth hormones, which gave

the putative shoots time to recover prior to the elongation and eventual rooting.

Table 9: Media composition for RM2 (recovery media 2)

Media Component Concentration

Murashige & Skoog basal medium ½ X

Cefotaxime 250 mg/l

Sucrose 30 g/l

Activated Charcoal 0.8%

pH 5.8

38

3.11. Elongation stage

After the selection and recovery stages the putative transgenic shoots

were moved to elongation media. The explant material stopped production of

more shoots and elongated the shoots present after selection. The elongation

media (EL) is new to this experiment and allowed the explant time to stop

producing new shoots and divert the energy to elongation, which proved vital for

the following root induction stage.

Table 10: Media composition for Elongation preparation

Media Component Concentration

Murashige & Skoog basal medium

Full strength

1-Naphthaleneacetic acid 0.1 mg/l

6- Benzylaminopurine 0.1 mg/l

Indole-3-butyric acid 0.1 mg/l

Sucrose 30 g/l

Activated Charcoal 1.0%

Cefotaxime 250 mg/l

Phosphenothricin 3 mg/l

3.12. Rooting stage (RIM)

The rooting stage was very important because without a healthy root

system the explants never made the transition to soil effectively. Explants were

grown for 3-8 weeks on RIM media until a root system was displaying main roots

with lateral root formation. Rooted transgenic plantlets were then moved to soil

pots.

Two major changes had to be made to the original RIM media: 1) newly

optimized plant growth hormone concentrations, 2) 1.0% activated charcoal was

supplemented to the rooting media, which functioned as a media darkening

agent mimicking natural soil conditions.

39

Table 11: Media composition for RIM preparation

Media Component Concentration

Murashige & Skoog basal medium 2/3 strength

1-Naphthaleneacetic acid 0.2 mg/l

Indole-3-butyric acid 1.0 mg/l

Kinetin 0.1 mg/l

Activated Charcoal 1.0%

Phosphenothricin 3 mg/l

3.13. Transfer to potted soil

Transferring to potting soil was first tested on control explants that had

been grown under the same conditions as transgenic explants except no

A.tumefaciens exposure. The best method determined was 100% soil in small

clear plastic containers with drainage holes drilled with a glass clear beaker

turned upside to create a small greenhouse for the recently transferred explant. If

the glass beaker was not used, we lost high percentage of explants during soil

transfer.

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3.14. Tissue culture induction

(a)

(b)

(c)

(d)

(e)

(f)

41

Figure 8: Tissue culture stages of leucaena explant growth. a) Co-culture of excised immature embryos + A. tumefaciens grown for 4 days on CIM media. b) Explant post CIM and Recovery 1 (3 weeks of culture), but prior to selection on SIM media. Only one shoot has begun to emerge from the excised immature embryo. c) Explant for two weeks on SIM media. A total of 3 shoots have begun to emerge from the apical meristematic region of the original excised embryo. d) Explant for 8 weeks on SIM media but prior to elongation media. Note the many shoots that have developed from the original embryo. e) Excised putative shoot bundle 3 weeks on elongation media. f) Rooting of putative transgenic shoots producing a healthy root system after 1 week on optimized RIM media.

3.15. Herbicide selection test

The herbicide selection test was conducted to confirm the appropriate

concentration of the herbicide phosphenothricin for selection of explants. The

control explants were grown under the same conditions and media hormone

levels as the experimental explants except they were not exposed to

Agrobacterium. A total of 100 control individuals (25 per herbicide level) were

tested on 4 different levels of herbicide selection.

Table 12: Herbicide level selection test with control explants

Herbicide Selection

Concentration

Number of Control

Individuals

Length of Time on Media

Average Percentage of Explant Loss

2.0 mg/l PPT 25 3 weeks 60-65%

2.5 mg/l PPT 25 3 weeks 75-80%

3.5 mg/l PPT 25 3 weeks 90-95%

4.0 mg/l PPT 25 3 weeks 100%

This table shows the results for the herbicide level control test. As the

selection concentration increased, so did the overall damage and rate of loss for

the control explants. This was an important experiment because it gave a visual

confirmation of the ability for the herbicide PPT to kill and select against non-

transformed explants.

42

a) b)

c) d)

Figure 9: Herbicide selection test a) The control explants under only 2.0 PPT selection were not completely killed, but had lost 90-100% of their leaves and had completely stopped any new growth. The branch nodes had turned brown and some necrotic cells had begun to build up. b) Control Explant 3 weeks under 2.5 mg/l PPT selection. The explants under 2.5 mg/l PPT selection had more green tissue loss and necrotic cell buildup than the explants under only 2.0 mg/l PPT. Leaf loss was very heavy and all growth was stopped and many shoots were withered. c) Control explant 3 weeks under 3.5 mg/l PPT selection. Explants under 3.5 mg/l PPT selection had almost no green tissue left at the end of the 3 week experiment. Heavy leaf and shoot loss. Browning throughout the explant and extensive damage to the lower half of the explant that was direct contact with the media. Almost all the control explants on this media either died or were on their way out at the end of the 3 weeks. d) Control explant 3 weeks under 4.0 mg/l PPT selection. Explants under 4.0 mg/l PPT selection had the most extensive damage. Virtually no green tissue left and total leaf loss and browning. All the control individuals died under this selection pressure.

43

3.16. RNA & DNA extraction from leaf and stem tissue

Total RNA and DNA extraction was performed using the TRI reagent

protocol. First stage is homogenization. The tissue is freshly excised from the

putative transgenic plants and weighed out to 80-100 mg. The tissue is placed in

a pre-cooled mortar and ground to a fine powder with the aid of liquid nitrogen.

The tissue must be ground to a fine powder containing no clumps. While the

tissue is still frozen powder, 1 ml of TRI Reagent is added to the mortar and more

liquid nitrogen is added and that mixture is ground further until all clumps are

removed and only powder remains. The frozen powder is quickly added to

nuclease free 2 ml tubes and allowed to come to RT for approximately 10

minutes. During this time the tubes are vortexed vigorously until the solution is

thoroughly mixed. The tubes are placed in a 4°C centrifuge and spun at full

speed for 10 minutes. After the spin down, the supernatant containing RNA is

pipetted to a new 1.5 ml centrifuge tube. The remaining material in the original 2

ml tube containing the DNA and protein is stored at – 80°C for further isolation.

RNA isolation:

The 1.5 ml tube containing the RNA supernatant is further separated with

the aid of chloroform. 0.2 - 0.4 ml of chloroform is added to each tube of RNA

supernatant and then vigorously vortexted for 15 seconds before being stored at

RT for 15 minutes. The tubes are then centrifuged at full speed for 15 minutes.

The supernatant should be clear of visible cellular material. The RNA is

transferred to another clean 1.5 ml tube.

The next stage is RNA precipitation. 0.25 ml of 100% isopropanol + 0.25

ml of (0.8 M sodium citrate + 1.2 M NaCl) is added to the RNA solution. The

mixture is vortexed for 1 minute and stored at RT for 10 minutes. Next, the

solution is centrifuged for 10 minutes at full speed, which forms a gel-like white

pellet on the bottom of the 1.5 ml tube. The supernatant is removed and the RNA

pellet is then washed with 1 ml of 75% ETOH. The pellet should be broken up

with vortexing or by physical disruption with a pipet tip. After 10 minutes at RT

the solution is centrifuged at 12,000 rpm for 5 minutes. The ETOH is removed

44

and the pellet is then dried for 10-15 minutes. Make sure that all the ETOH has

dried from the 1.5 ml tube prior to adding 50-100 μl of ddH2O.

DNA isolation:

The -80 °C stored organic material containing a mixture of DNA and

protein is allowed to come to RT. After the solution has become liquefied, 0.4 ml

of chloroform is added to the solution and vigorously vortexed for 30 seconds.

The mixture is stored at RT for 15 minutes prior to centrifugation at full speed for

15 minutes. The solution separates into 3 phases and the supernatant is carefully

removed. The remaining two lower phases contain a mixture of DNA and protein.

The next stage is DNA precipitation. 0.5 ml of 100% ETOH is added to the

mixture and vortexed for 30 seconds. The mixture is stored at RT for 10 minutes

prior to centrifuge at 4,000 rpm for 5 minutes. The supernatant is removed from

the pelleted material.

Following precipitation the pelleted DNA is washed 2x in a solution of 0.1

M trisodium citrate in 10% ethanol. Use 1 ml of wash solution for each of the two

pelleted DNA washes. At each wash store the DNA at RT for 30 minutes with

mixing prior to centrifugation at 4,000 rpm for 5 minutes. Next stage is to

suspend the DNA pellet in 1-2 ml of 75% ETOH at RT for 20 minutes with

consistent mixing and breaking up of the pellet by pipet tip if necessary. The

ethanol wash removes pinkish color from the DNA pellet.

DNA solubilization is the next stage in the DNA isolation. The 75% ETOH

solution is centrifuged at 4,000 rpm for 5 minutes, and the supernatant is

removed and the pelleted DNA is allowed to completely dry. There needs to be

no visible ethanol solution left in the tube. Once the DNA is completely dry, re-

suspend the pellet in either 100% nuclease free H20 or TE buffer solution. Place

the solution in 65 °C for 20 minutes to degrade any remaining nucleases. After

the heating, allow the DNA solution to come to RT and then quantify the DNA to

determine quality and quantity.

45

3.17. PCR amplification of putative transgenic extracted DNA

50 μl PCR reactions:

10ul of 5x Green flexi buffer

5ul of 25mM MgCl2 = 2.5mM

1ul each dNTP’s (A, G, C, T) 10mM = 0.2mM each

2ul upstream/downstream primers 10mM = 0.4mM each

0.2ul Go Taq polymerase (5U/ul) = 1.25U

Extracted explant DNA (150 - 400ng)

dd H2O fill up to 50ul

The individual components excluding the extracted DNA were added one

by one to a main master mix. The master mix is continuously mixed and

centrifuged down during the combination of the individual ingredients. After the

master mix has all the components added 46-49 μl of the mix is added to each

PCR amplification tube. The amount depends on the added template DNA (1-4

μl). The total volume of solution in each PCR tube is 50 μl.

The PCR tubes are then loaded into the Gene Amp machine and the

cycles were entered into the machine. The initial denaturation stage was set at

95 °C for 5 minutes. The 35 cycles were made up of a denaturation stage of 95

°C for 1 minute + annealing stage of 55 °C for 1 minute + and extension stage of

72 °C for 1:30 minutes. The annealing temperature/time and extension

temperature/time were optimized for primer set G3 2.0. The expected base-pair

amplification for primer set G3 2.0 was 848 bp. The second primer set used to

amplify DNA was pyd-A. The initial denaturation and final extension stages were

identical for both primer sets. The 35 cycles for primer set pyd-A were 95 °C for 1

minute + 58 °C for 1 minute + 72 °C for 1:15 minutes. The expected bp

amplification for primer set pyd-A is 469 bp.

After the 35 cycles a final extension stage of 72 °C for 10:00 minutes was

employed. After PCR amplification, the amplified DNA was run on 1% agarose

gel with 70 volts for 3-4 hours. The amplified DNA was run next to a known DNA

ladder to show the location of the amplified segments.

46

3.18. Reverse transcriptase PCR

Quality cDNA was synthesized post treatment with a Turbo Dnase

inactivation enzyme.

A primer set named Dioxygenase was used to amplify the newly

synthesized cDNA. The expected fragment size is 396 bp and will indicate the

expression of pydA-G3-pydB gene insert.

The PCR reaction was a 25 μl reaction:

5ul of 5x Green flexi buffer

2.5ul of 25mM MgCl2 = 2.5mM

0.5ul each dNTP’s (A, G, C, T) 10mM = 0.2mM each

0.5ul upstream/downstream primers 10mM = 0.4mM each

0.1ul Go Taq polymerase (5U/ul) = 1.00U

Extracted explant DNA and cDNA (75 - 150ng)

dd H2O fill up to 25ul

30 cycles for cDNA PCR were employed. The initial denaturation was 95

°C for 2:00 minutes. The cycle stages were denaturation at 95 °C for 45 seconds

+ annealing at 63 °C for 45 seconds + extension 72 °C for 1 minute. The final

extension was 72 °C for 5 minutes.

47

Chapter 4

RESULTS

In this results section I will cover the improvements made in tissue

regeneration concerning phenolic exudate reduction, necrotic cell accumulation,

and rooting inefficiencies. Results were reduction in phenolic output and cell

death. Root system improvements were achieved with faster and healthier root

development (lateral root induction).

Leaf herbicide application assay was conducted to determine the

phenotypic response of wild-type (control) versus putative transgenic leaves.

Results indicate a stronger resistance to the applied herbicide in the putative

transgenic plants, versus the more damaged wild-type plants.

T-DNA presence and expression was tested through PCR and reverse

transcriptase-PCR amplification of extracted DNA and RNA from the putative

transgenic plants.

48

4.1. Reduction of phenolic exudate and necrotic cell accumulation in callus induction media (CIM) stage

Reduction in phenolic exudate and in resulting necrotic cell death during

pre-culture and co-culture periods was achieved by supplementing a range of

activated charcoal (0.7-1.0% w/w) to the original Callus Induction Media CIM.

Earlier explant groups grown on original CIM media experienced phenolic

accumulation and necrotic cell development. We observed when explants

remained on the CIM media without activated charcoal for longer than 5-7 days

they would quickly turn brown and lose vitality. We attributed this slowing of

growth to phenolic exudate that accumulates in a closed growth system. When

phenolics build up in the media, necrotic cell death quickly follows, which greatly

reduces tissue regeneration capabilities. An antioxidant soak (75 mg/l citric acid

+ 50 mg/l ascorbic acid) was introduced during the excision stage in the original

protocol (Jube and Borthakur 2010) to help reduce pheno