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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
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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
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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
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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.
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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
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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.
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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-
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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.
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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.
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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.
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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
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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
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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).
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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,
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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
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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
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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.
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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
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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
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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
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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
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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.
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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
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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.
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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
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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.
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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)
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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.
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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.
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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
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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.
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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.
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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.
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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.
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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