diff --git a/wiki/pages/results.html b/wiki/pages/results.html
index 3ecd80ebaf1f81c3eaeeb5752022627dc629b25f..15f20c8cb3b7fc6716884c31098a0388c29763e3 100644
--- a/wiki/pages/results.html
+++ b/wiki/pages/results.html
@@ -294,7 +294,7 @@
<figure>
<img
class="img-inline"
- src="https://static.igem.wiki/teams/4729/wiki/results/pt/at-arqua1-ruby-hyg.png"
+ src="Ersetzen mit Tabelle"
alt="Graph with the caption: A. thaliana transformation baseline - 35S:RUBY (HygR) in ARqua1."
/>
<figcaption>
@@ -303,7 +303,7 @@
</figure>
<p>
- Three days after transformation, 46% of the 177 seedlings developed the red pigment betalain. This shows the expression of the RUBY reporter, corresponding to a transient or stable transformation of the T-DNA. After 7 days, 75% of the plants had developed hairy new tissue, with 55% of the plants having formed the red reporter dye. Hairy tissue was formed as regular roots, as well as hairy callus. More than 20% of the seedlings died between the first and second evaluation. One explanation for the high lethality could be the heightened plant defense due to the challenge by the bacteria, another explanation could be the particular stress of the plants due to a rough handling by the experimenters. Especially by incautious handling, microscopy under a coverslip can lead to high stress for the plants, from which some do not recover.
+ Three days after transformation, 46% of the 179 seedlings developed the red pigment betalain. This shows the expression of the RUBY reporter, corresponding to a transient or stable transformation of the T-DNA. More than 20% of the seedlings died between the first and second evaluation. One explanation for the high lethality could be the heightened plant defense due to the challenge by the bacteria, another explanation could be the particular stress of the plants due to a rough handling by the experimenters. Especially by incautious handling, microscopy under a coverslip can lead to high stress for the plants, from which some do not recover.
</p>
<figure>
@@ -333,121 +333,43 @@
While working on our baseline, we came to consider whether it might be interesting at a later stage to select the transgenic tissue by the inserted hygromycin resistance. Since hygromycin is expensive, we thought of cloning another antibiotic resistance as a selection marker into the 35S:RUBY plasmid that would be more widely applicable by other labs and iGEM teams. After some literature survey we decided to use kanamycin resistance. Even though we did not use the kanamycin resistance in 35S:RUBY:KanR afterwards, we initially ran a second baseline to check if ARqua1 with the modified plasmid gave the same results as the first baseline.
</p>
- <figure>
- <img
- class="img-inline"
- src="https://static.igem.wiki/teams/4729/wiki/results/pt/atarqua1-ruby-kan.png"
- alt="Graph with the caption: A. thaliana transformation baseline - 35S:RUBY:KanR in ARqua1"
- />
- <figcaption>
- <strong>Figure 12</strong>: Distribution of different phenotypes 3 days post infection (dpi) with 35S:RUBY:KanR in ARqua1 and 10 dpi.
-
- </figcaption>
- </figure>
-
<p>
- As you can see in Figure 12, 3 days after transformation, there was a transformation efficiency of 47% according to the first run. Remarkably, after 10 days, 35% of the plants counted on day 3 died. In fact, during the baseline from Figure 9, several new team members were incorporated in the plant laboratory who were still untrained in appropriate lenient handling. This confirms the hypothesis that a significant proportion of the deceased plants were caused by improper handling and cannot be explained by the virulence of the bacterium alone.
+ As you can see in Figure 9, 3 days after transformation, we could observe a transformation event in 47% of the 284 <i>Arabidopsis thaliana</i> used. Remarkably, after 10 days, 35% of the plants counted on day 3 died. In fact, since the Hygromycin baseline (Figure 9), several new team members were incorporated in the plant laboratory who were still untrained in appropriate lenient handling. This confirms the hypothesis that a significant proportion of the deceased plants were caused by improper handling and cannot be explained by the virulence of the bacterium alone.
</p>
<h3>Test for enhancement of natural virulence of <i>Agrobacterium rhizogenes</i> ARqua1 in <i>Arabidopsis thaliana</i> by treatment with vanillin</h3>
<p>
- While part of our RhizoGene team focused on targeting Agrobacterium virulence genes using inducible promoters, we looked in parallel for other ways to increase the transformation efficiency in the newly developed protocols for different plant species. During our research, we came across a publication in which different molecules were used to increase transformation efficiency (Cha et al., 2011).
-For Agrobacterium-mediated gene transfer into plant hosts, the substance acetosyringone is often favored, which is naturally secreted by wounded dicotyledonous plants and induces Agrobacterium virulence genes. However, according to this paper, as an alternative to acetosyringone, other structurally similar phenolic compounds can also induce the virulence genes. For this reason, we decided to test vanillin in Agrobacterium culture to investigate an increase in transformation efficiency. This also offers the advantage that vanillin is significantly cheaper than acetosyringone. To achieve a higher probability of transformation events, we also centrifuged half of the bacterial culture with an OD(600) of approximately 1 and resuspended the pellet in the remaining culture. We added 500 µM vanillin to the bacterial culture with increased titer one hour before transformation.
+ While part of our RhizoGene team focused on targeting <i>Agrobacterium</i> virulence genes using inducible promoters, we looked in parallel for other ways to increase the transformation efficiency in the newly developed protocols for different plant species. During our research, we came across a publication in which different molecules were used to increase transformation efficiency (Cha et al., 2011).
+For <i>Agrobacterium</i>-mediated gene transfer into plant hosts, the substance acetosyringone is often favored, which is naturally secreted by wounded dicotyledonous plants and induces <i>Agrobacterium</i> virulence genes. However, according to this paper, as an alternative to acetosyringone, other structurally similar phenolic compounds can also induce the virulence genes. For this reason, we decided to test vanillin in <i>Agrobacterium</i> culture to investigate an increase in transformation efficiency. This also offers the advantage that vanillin is significantly cheaper than acetosyringone. To achieve a higher probability of transformation events, we also centrifuged half of the bacterial culture with an OD(600) of approximately 1 and resuspended the pellet in the remaining culture. We added 500 µM vanillin to the bacterial culture with increased titer one hour before transformation.
</p>
- <figure>
- <img
- class="img-inline"
- src="https://static.igem.wiki/teams/4729/wiki/results/pt/xat-arqua1-ruby-centr-van.png"
- alt="Graph with the caption:A. thaliana transformation - 35S:RUBY in ARqua1 centrifuged and additional vanillin "
- />
- <figcaption>
- <strong>Figure 13</strong>: Distribution of different phenotypes 3 days post infection (dpi) with 35S:RUBY:KanR in ARqua1 and 10 dpi. Vanillin was added while cultivation of Agrobacterium and into culture plates of plants. Bacterial culture was spinned down before transformation to increase concentration.
- </figcaption>
- </figure>
-
<p>
- The results 3 days after transformation were outstandingly good: 86% of the plants showed RUBY expression whereas without vanillin only 47% of the plants were RUBY positive. The development of new hairy tissue after 10 days also followed this trend and with a transformation efficiency of 75%. In this transformation, there were no dead plants after transformation. This proves that higher transformation efficiency with careful treatment does not lead to increased plant lethality at all. The decisive factor is the method of transformation, since the fraction of transformed plants among all survivors was much higher than in the approaches before. These overwhelmingly good results gave hope that more plant species could be successfully transformed by this adaptation of the protocol. The amazing results with Bambara groundnut confirmed this assumption (see results below).
+ The results 3 days after transformation were outstandingly good: 87% of the plants showed RUBY expression whereas without vanillin only 47% of the plants were RUBY positive. In this transformation, there were no dead plants after transformation. This proves that higher transformation efficiency with careful treatment does not lead to increased plant lethality at all. These overwhelmingly good results gave hope that more plant species could be successfully transformed by this adaptation of the protocol. The amazing results with Bambara groundnut confirmed this assumption (see results below).
</p>
<h3>Test of enhancement of the transformation efficiency by using the strain K599 in <i>Arabidopsis thaliana</i></h3>
<p>
- In addition to increasing the virulence of Agrobacterium strain ARqua1, we also searched for bacterial strains that might perform better in the plant species we transformed. In a comparison paper between Agrobacterium strains ARqua1, K599 and R1000, K599 showed superior results in transformation experiments compared to ARqua1 with a higher virulence (Foti & Pavli, 2020) . Therefore, we tested <i>Agrobacterium rhizogenes</i> K599 in Arabidopsis thaliana to observe the transformation efficiency with this strain. In K599, we transformed our 35S:RUBY:KanR plasmid.
+ In addition to increasing the virulence of </i>Agrobacterium</i> strain ARqua1, we also searched for bacterial strains that might perform better in the plant species we transformed. In a comparison paper between</i> Agrobacterium</i> strains ARqua1, K599 and R1000, K599 showed superior results in transformation experiments compared to ARqua1 with a higher virulence (Foti & Pavli, 2020). Therefore, we tested </i>Agrobacterium rhizogenes</i> K599 in Arabidopsis thaliana to observe the transformation efficiency with this strain. In K599, we transformed our 35S:RUBY:KanR plasmid.
</p>
-
- <figure>
- <img
- class="img-inline"
- src="https://static.igem.wiki/teams/4729/wiki/results/pt/at-k599-ruby-kan.png"
- alt="graph with the caption: A. thaliana transformation - 35S:RUBY:KanR in K599"
- />
- <figcaption>
- <strong>Figure 14</strong>: Distribution of different phenotypes 3 days post infection (dpi) and 10 dpi with 35S:RUBY:KanR in K599.
- </figcaption>
- </figure>
-
<p>
- Similar to the increase in virulence by vanillin, 3 days after transformation by K599, there is a marked increase in RUBY positive plants with 73% compared to baseline with ARqua1 with 47%. Interestingly, however, after 10 days there was no development of hairy tissue consistent with the previously recorded transformation events. The low number of dead plants and the fact that the plants were very healthy also suggests that the higher virulence of K599 did not have a negative impact on the plants. It can be deduced that the T-DNA was very successfully transformed with the reporter gene in 35S:RUBY:KanR, but root hair induction was weaker than for ARqua1.
+ Similar to the increase in virulence by vanillin, 3 days after transformation by K599, there is a increase in RUBY positive plants with 72% compared to baseline with ARqua1 with 47% (Figure 9). A low death rate of treated plants also suggests that the higher virulence of K599 did not have a negative impact on the plants.
</p>
<h3>Test of enhancement of the transformation efficiency by using our composite parts</h3>
<p>
- Our team also focussed on the construction of composite parts which should enhance <i>Agrobacterium rhizogenes</i>-mediated plant transformation. Each version of our composite part consists of an inducible promoter, a version of VirG and one of 2 different backbones. The general idea was to fine-tune the expression of the transcriptional activator VirG as the master switch of all other virulence genes. Therefore, we were looking for an inducible promoter with a high dynamic range, low leakiness and no cross reactivity. The promoters of our choice were Ptac and Ptau which can be induced by IPTG and taurine (see above). The two versions TiBo542 and super80 of VirG differ in the independence of the sensory kinase VirA of the latter. whereas the pABCa has a single copy for Agrobacterium. The pSRK entry vector (Khan et al 2008) carries the pBBR1 (broad host range) ori and was initially selected for our VirG overexpression constructs, due to its medium copy number in Alphaproteobacteria and compatibility with <i>E. coli</i> (Blázquez et al., 2023;Antoine & Locht, 1992). Additionally, we wanted to use the pABCa backbone with its single copy ori. We tested different constructs containing various combinations of the previously mentioned parts in plants.
- </p>
-
- <figure>
- <img
- class="img-inline"
- src="https://static.igem.wiki/teams/4729/wiki/results/constructs-in-plants/ptac-tibo542-psrk.png"
- alt="graph with the caption: A. thaliana transformation - 35S:RUBY:KanR + Ptac_TiBo542[VirG]_pSRK"
- />
- <figcaption>
- <strong>Figure 15</strong>: Distribution of different phenotypes 3 days post infection (dpi) and 10 dpi with 35S:RUBY:KanR and Ptac_TiBo542_pSRK in ARqua1.
- </figcaption>
- </figure>
-
- <p>
- A. thaliana transformation via ARqua1 containing Ptac_TiBo542[VirG]_pSRK was the first construct tested. After 3 days we saw a total amount of 32% of plants which expressed RUBY. Seven days later, the number of RUBY-positive plants had increased to 57%. Thus, transformation efficiency after 3 days is 32% lower compared to the baseline with ARqua1 35S:RUBY:KanR, increasing by 24% after day 10.
+ Our team also focussed on the construction of composite parts which should enhance <i>Agrobacterium rhizogenes</i>-mediated plant transformation. Each version of our composite part consists of an inducible promoter, a version of VirG and one of 2 different backbones. The general idea was to fine-tune the expression of the transcriptional activator VirG as the master switch of all other virulence genes. Therefore, we were looking for an inducible promoter with a high dynamic range, low leakiness and no cross reactivity. The promoters of our choice were Ptac and Ptau which can be induced by IPTG and taurine (see above). The two versions TiBo542 and super80 of VirG differ in the independence of the sensory kinase VirA of the latter. Whereas the pABCa has a single copy for </i>Agrobacterium</i>. The pSRK entry vector (Khan et al 2008) carries the pBBR1 (broad host range) ori and was initially selected for our VirG overexpression constructs, due to its medium copy number in Alphaproteobacteria and compatibility with <i>E. coli</i> (Blázquez et al., 2023;Antoine & Locht, 1992). Additionally, we wanted to use the pABCa backbone with its single copy ori. We tested different constructs containing various combinations of the previously mentioned parts in plants.
</p>
<p>
- The next construct we tested was the improved version Ptac_super80[VirG]_pSRK.
+ <i>A. thaliana</i> transformation via ARqua1 containing Ptac_TiBo542[VirG]_pSRK was the first construct tested (Figure9). After 3 days we saw a total amount of 32% of plants which expressed RUBY. Seven days later, the number of RUBY-positive plants had increased to 57%. Thus, transformation efficiency after 3 days is 15% lower compared to the baseline with ARqua1 35S:RUBY:KanR. After10 days the efficiency increased 25%, making the transformation 11% more efficient compared to the baseline.
</p>
-
- <figure>
- <img
- class="img-inline"
- src="https://static.igem.wiki/teams/4729/wiki/results/constructs-in-plants/ptac-super80-psrk.png"
- alt="graph with the caption: A. thaliana transformation - 35S:RUBY:KanR + Ptac_super80[VirG]_pSRK"
- />
- <figcaption>
- <strong>Figure 16</strong>: Distribution of different phenotypes 3 days post infection (dpi) and 10 dpi with 35S:RUBY:KanR and Ptac_super80_pSRK in ARqua1.
- </figcaption>
- </figure>
-
- <p>
- When analyzing the transformation efficiency after 3 days we saw a drop of RUBY positive plants to 29%.
- </p>
-
<p>
- Both results were against our previous expectations, however after several rounds of trouble shooting we came up with the idea that Ptac still shows relatively high leaky expression despite having a big difference in expression levels of induced to uninduced. To evade leakiness we chose the lower expressed Ptau.
+ The next construct we tested was the improved version Ptac_super80[VirG]_pSRK. When analyzing the transformation efficiency after 3 days we saw a drop of RUBY positive plants to 29% compared to the baseline (Figure 9). Both results were against our previous expectations, however after several rounds of trouble shooting we came up with the idea that Ptac still shows relatively high leaky expression despite having a big difference in expression levels of induced to uninduced. To evade leakiness we chose the lower expressed Ptau.
</p>
-
- <figure>
- <img
- class="img-inline"
- src="https://static.igem.wiki/teams/4729/wiki/results/ptau-super80-psrk.png"
- alt=""
- />
- <figcaption>
- <strong>Figure 17</strong>: Distribution of different phenotypes 3 days post infection (dpi) and 10 dpi with 35S:RUBY:KanR and Ptau_super80_pSRK in ARqua1.
- </figcaption>
- </figure>
-
<p>
- When testing Ptau_super80[VirG]_pSRK we observed a slight improvement to 37% RUBY positive plants. Nonetheless, it is important to note that the total percentage was still lower than during the RUBY baseline experiments without ARqua1 containing any additional constructs. Upon evaluating our most recent results 10 days post-transformation with constructs containing the pSRK backbone, we were surprised to witness an unexpected increase in the number of RUBY-positive plants compared to the 3-day post-transformation results. <b>We were especially surprised by the Ptac_TiBo542_pSRK transformation results which showed a transformation efficiency gain of 9% in comparison to the 35S:RUBY:KanR baseline</b>. The current efficiency levels now seem to be on par with the outcomes from the baseline experiments. With the assumption that pABCa behaves similarly, we anticipate obtaining comparable results ten days after transformation, and we anticipate these results to be available within the next week.
+ When testing Ptau_super80[VirG]_pSRK we observed a slight improvement to 37% RUBY positive plants. Nonetheless, it is important to note that the total percentage was still lower than during the RUBY baseline experiments without ARqua1 containing any additional constructs. Upon evaluating our most recent results 10 days post-transformation with constructs containing the pSRK backbone, we were surprised to witness an unexpected increase in the number of RUBY-positive plants compared to the 3-day post-transformation results. <b>We were especially surprised by the Ptac_TiBo542_pSRK transformation results which showed a transformation efficiency gain of 11% in comparison to the 35S:RUBY:KanR baseline</b>. The current efficiency levels now seem to be on par with the outcomes from the baseline experiments. With the assumption that pABCa behaves similarly, we anticipate obtaining comparable results ten days after transformation, and we anticipate these results to be available within the next week.
</p>
<h4>Plasmid stability assay</h4>
@@ -455,12 +377,12 @@ For Agrobacterium-mediated gene transfer into plant hosts, the substance acetosy
Plasmid incompatibility occurs when two plasmids cannot stably coexist in the same bacterial cell line over multiple generations. Typically, closely related plasmids are incompatible, while distantly related ones are compatible. Incompatibility often arises from shared replicons with identical Rep protein specificity or controlling elements. Competition can also stem from similar partitioning systems. This incompatibility may be reciprocal, leading to equal chances of loss, or unidirectional, favoring the plasmid with additional advantages, such as a second replicon. Although classification systems exist for certain hosts, many plasmids defy categorization due to the absence of systematic incompatibility tests on all known plasmids.
</p>
<p>
- Many of the most widely used origins of replication from <i>E. coli</i>, such as ColE1 do not work in Alphaproteobacteria. Hence, some of the plasmid backbones used by our team contained 2 origins of replication: one for viability in <i>E. coli</i> during cloning steps and one for replication in Agrobacterium, or alternatively, a broad host range ori. Additionally, our iterations of the best composite part were designed to co-exist in Agrobacterium with the 35S:RUBY:KanR plasmid carrying the plant selection marker and the pVS1 ori. Therefore, the stability of plasmids carrying different origins of replication was tested in strains also carrying the 35S:RUBY:KanR plasmid.
+ Many of the most widely used origins of replication from <i>E. coli</i>, such as ColE1 do not work in Alphaproteobacteria. Hence, some of the plasmid backbones used by our team contained 2 origins of replication: one for viability in <i>E. coli</i> during cloning steps and one for replication in <i>Agrobacterium</i>, or alternatively, a broad host range ori. Additionally, our iterations of the best composite part were designed to co-exist in <i>Agrobacterium</i> with the 35S:RUBY:KanR plasmid carrying the plant selection marker and the pVS1 ori. Therefore, the stability of plasmids carrying different origins of replication was tested in strains also carrying the 35S:RUBY:KanR plasmid.
</p>
<h5>pSRK backbone & 35S:RUBY:KanR plasmid</h5>
<p>
- The pSRK entry vector carries the pBBR1 (broad host range) ori and was initially selected for our VirG overexpression constructs, due to its medium copy number in Alphaproteobacteria and compatibility with <i>E. coli</i> (Antoine & Locht, 1992; Blázquez et al., 2023). However, after the observations from plant transformations, we noticed that strains carrying both plasmids displayed lower transformation efficiency when compared to strains solely carrying the 35S:RUBY plasmid. This led to the suspicion that the two plasmids might be unstable when co-existing in Agrobacterium, negatively affecting cell health and thus decreasing overall transformation efficiency.
+ The pSRK entry vector carries the pBBR1 (broad host range) ori and was initially selected for our VirG overexpression constructs, due to its medium copy number in Alphaproteobacteria and compatibility with <i>E. coli</i> (Antoine & Locht, 1992; Blázquez et al., 2023). However, after the observations from plant transformations, we noticed that strains carrying both plasmids displayed lower transformation efficiency when compared to strains solely carrying the 35S:RUBY plasmid. This led to the suspicion that the two plasmids might be unstable when co-existing in <i>Agrobacterium</i>, negatively affecting cell health and thus decreasing overall transformation efficiency.
</p>
<p>
In order to verify this hypothesis, we conducted a stability assay in <i>A. rhizogenes</i> ARqua1. <i>A. rhizogenes</i> ARqua1 competent cells carrying the 35S:RUBY:KanR plasmid were transformed with the pM4_1_002 VirG overexpression plasmid and plated on selection media (Gentamicin + Spectinomycin). Colonies were picked and screened via colony-PCR to verify the presence of both plasmids. A cryo stock from one colony was used to inoculate liquid LB medium without antibiotics, gentamicin + streptomycin, and gentamicin + spectinomycin, which were then incubated overnight. On the following day, a 20 µL sample was taken from the overnight culture and frozen away, 1 µL of the overnight culture was used to inoculate fresh medium for a new overnight culture. This process was repeated until samples from 5 days were collected.
@@ -529,7 +451,7 @@ Of the 14 Bambara groundnuts used, no plant showed root growth after 2 or 4 week
"
/>
<figcaption>
- <strong>Figure 20</strong>: Development of a hairy root in bambara groundnut 2 weeks after transformation with Agrobacterium ARqua1. </figcaption>
+ <strong>Figure 20</strong>: Development of a hairy root in bambara groundnut 2 weeks after transformation with <i>Agrobacterium</i> ARqua1. </figcaption>
</figure>
<figure>
@@ -539,7 +461,7 @@ Of the 14 Bambara groundnuts used, no plant showed root growth after 2 or 4 week
alt="Two hands with gloves hold a bambara groundnut plant with a handful of root system."
/>
<figcaption>
- <strong>Figure 21</strong>: Strongly developed root system of bambara groundnut 8 weeks after transformation with Agrobacterium ARqua1.</figcaption>
+ <strong>Figure 21</strong>: Strongly developed root system of bambara groundnut 8 weeks after transformation with <i>Agrobacterium</i> ARqua1.</figcaption>
</figure>
<p>
@@ -553,7 +475,7 @@ Of the 14 Bambara groundnuts used, no plant showed root growth after 2 or 4 week
alt="Image through binoculars showing a root section with a red color stripe running through it."
/>
<figcaption>
- <strong>Figure 22</strong>: Red root section of bambara groundnut 8 weeks after transformation with Agrobacterium ARqua1.</figcaption>
+ <strong>Figure 22</strong>: Red root section of bambara groundnut 8 weeks after transformation with <i>Agrobacterium</i> ARqua1.</figcaption>
</figure>
<figure>
@@ -563,14 +485,14 @@ Of the 14 Bambara groundnuts used, no plant showed root growth after 2 or 4 week
alt="Image through binoculars showing a root section with a red color stripe running through it."
/>
<figcaption>
- <strong>Figure 23</strong>: Red root section of bambara groundnut 8 weeks after transformation with Agrobacterium ARqua1.</figcaption>
+ <strong>Figure 23</strong>: Red root section of bambara groundnut 8 weeks after transformation with <i>Agrobacterium</i> ARqua1.</figcaption>
</figure>
<h3>Implementing a new transformation method: The stone wool method</h3>
<p>
- During the implementation of the cut dip budding protocol, we came across a method by Sebastian Cocioba via social media by chance. In fact, while working with the CDB protocol, we had thought about a way to further reduce the stress of the plants, as the roots suffered even when being carefully removed from the vermiculite. In the new method, rockwool is soaked with Agrobacterium culture and placed in a centrifuge tube filled with tap water. The plant, stripped of its roots, is placed in this assembly with the cutting side down and co- cultured with the bacteria. If root growth occurs, the roots can be assessed through the transparent tube wall. Expecting to follow up on the positive results with Bambara groundnut, we transformed dandelion, strawberry, and Bambara groundnut with <i>Agrobacterium rhizogenes</i> ARqua1 using the rockwool method. This involved adding 500 µM vanillin to the culture as in our previous, very successful CDB run and centrifuging the culture to double titer.
+ During the implementation of the cut dip budding protocol, we came across a method by Sebastian Cocioba via social media by chance. In fact, while working with the CDB protocol, we had thought about a way to further reduce the stress of the plants, as the roots suffered even when being carefully removed from the vermiculite. In the new method, rockwool is soaked with <i>Agrobacterium</i> culture and placed in a centrifuge tube filled with tap water. The plant, stripped of its roots, is placed in this assembly with the cutting side down and co- cultured with the bacteria. If root growth occurs, the roots can be assessed through the transparent tube wall. Expecting to follow up on the positive results with Bambara groundnut, we transformed dandelion, strawberry, and Bambara groundnut with <i>Agrobacterium rhizogenes</i> ARqua1 using the rockwool method. This involved adding 500 µM vanillin to the culture as in our previous, very successful CDB run and centrifuging the culture to double titer.
</p>
<figure>
@@ -587,7 +509,7 @@ Of the 14 Bambara groundnuts used, no plant showed root growth after 2 or 4 week
<!-- NOT COMPLETE -->
<p>
- After the impressive transformation efficiency of Agrobacterium strain K599 in A. thaliana, we decided to transform our crop plants with this strain as well. We proceeded as described above. For now we are still waiting for upcoming results.
+ After the impressive transformation efficiency of <i>Agrobacterium</i> strain K599 in A. thaliana, we decided to transform our crop plants with this strain as well. We proceeded as described above. For now we are still waiting for upcoming results.
</p>
<!-- NOT COMPLETE -->
@@ -598,7 +520,7 @@ Of the 14 Bambara groundnuts used, no plant showed root growth after 2 or 4 week
In a great collaboration with Lars Opgenoorth from the PhytOakmeter project at the University of Marburg, we got the opportunity to work with the oak clone DF159. Since the clone is axenically propagated, it was necessary to develop a sterile transformation protocol specifically adapted to this propagation procedurel. If you want to read more about our self-developed protocol, you can <a class="igem" href="https://2023.igem.wiki/marburg/oak-diary" >click here</a> for our "Oak diary".
</p>
<p>
- In the first round of experiments, we used the Agrobacterium strain ARqua1 to transform 53 oak plantlets. After the transformation day, we transferred the oaks to a medium containing 500 mg/L of cefotaxime and activated charcoal two days later. We then conducted an evaluation once or twice a week for three months, documenting health status and new, potentially transformed tissue.
+ In the first round of experiments, we used the <i>Agrobacterium</i> strain ARqua1 to transform 53 oak plantlets. After the transformation day, we transferred the oaks to a medium containing 500 mg/L of cefotaxime and activated charcoal two days later. We then conducted an evaluation once or twice a week for three months, documenting health status and new, potentially transformed tissue.
</p>
<p>
In the first week after transformation, almost all oaks lost their leaves due to the enormous transpiration stress the plants had suffered under the sterile bench. Only after about 3 weeks, lateral buds sprouted and new leaves formed. At 4 weeks after transformation, we were pleased to observe root formation in two of the oaks, but no hairy roots. In fact, the culture in activated charcoal medium for evaluation turned out to be very impractical, as the formation of new tissue could only be observed inaccurately. At 12 weeks after transformation, we removed the oaks from the medium, evaluated them by visual inspection, and microscopically examined cross-sections of the roots and shoots in hopes of detecting ruby red tissue. At that time 27 of the 53 plantlets had been lost due to contaminations. Of the 26 plants that were not contaminated, 4 oaks had formed new roots. Two of these oaks had also developed hairy roots. Unfortunately, we were unable to detect RUBY in the oaks, indicating the transfer of one of the endogenous T-DNAs of the ARqua1 strain. Nonetheless, using a completely self-created transformation protocol, we were able to detect transformation events in two Quercus robur, which is tremendous success, since we were not aware of any report on an existing oak transformation protocol. This gave us hope that further improvement of the protocol might provide even better results.
@@ -611,7 +533,7 @@ Of the 14 Bambara groundnuts used, no plant showed root growth after 2 or 4 week
alt="in the picture is a part of a large test tube with black medium in it. In the medium sits a small oak with a white root visible."
/>
<figcaption>
- <strong>Figure 25</strong>: Quercus robur with a visible root 5 weeks after transformation with agrobacterium ARqua1.</figcaption>
+ <strong>Figure 25</strong>: Quercus robur with a visible root 5 weeks after transformation with <i>Agrobacterium</i> ARqua1.</figcaption>
</figure>
<figure>
<img
@@ -624,7 +546,7 @@ Of the 14 Bambara groundnuts used, no plant showed root growth after 2 or 4 week
</figure>
<p>
- After the promising results of the first experiment, we repeated the transformation again with 51 oak plantlets. This time we made some adaptations to reduce the drought stress during handling under the laminar flow hood. The detailed description of the performed method can be found on the <a class="igem" href="https://2023.igem.wiki/marburg/experiments">experiment page</a>. As a bacterial strain we used Agrobacterium K599 andinduced the virulence according to the promising results in Bambara groundnut with 500 µM vanillin. In addition, the bacterial culture was spun down and subsequently resuspended to increase the bacterial titer of the suspension twofold.
+ After the promising results of the first experiment, we repeated the transformation again with 51 oak plantlets. This time we made some adaptations to reduce the drought stress during handling under the laminar flow hood. The detailed description of the performed method can be found on the <a class="igem" href="https://2023.igem.wiki/marburg/experiments">experiment page</a>. As a bacterial strain we used <i>Agrobacterium</i> K599 andinduced the virulence according to the promising results in Bambara groundnut with 500 µM vanillin. In addition, the bacterial culture was spun down and subsequently resuspended to increase the bacterial titer of the suspension twofold.
At the time of wiki publication, the transformation was 19 days ago, therefore no statement about the transformation events can be made yet. Nevertheless, it can already be seen that the further developed method has had a positive influence on the oaks. On the one hand, only one oak shed its leaves after the transformation, and new buds also formed within the first week. After 2 weeks 21 oaks have already built calluses. As an innovation, we have the oak's cutting edges on the outer wall of the new test tube.
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@@ -642,7 +564,7 @@ At the time of wiki publication, the transformation was 19 days ago, therefore n
<h3> Establishing <i>Agrobacterium rhizogenes</i>-mediated transformation in <i>Setaria viridis</i> </h3>
<p>
- Agriculture relies mainly on a handful of monocotyl crops such as maize, wheat and rice. Despite their immense agricultural and nutritional significance, monocots, which represent the most crucial food crops in terms of both quantity and calories, have been explored by fewer than five iGEM teams throughout the history of the competition. When aiming to facilitate Agrobacterium-mediated plant transformation and broaden the spectrum of genetically accessible plants it is also important to think about testing in monocots. Since monocots don't release phenols like acetosyringone after wounding the virulence genes of Agrobacterium do not get activated and therefore the monocot does not get transformed (Cao et al., 2023). To circumvent this, phenolic additives like vanillin and acetosyringone are added to the bacterial culture and also the growing medium of the infected plants. In this context decoupling of VirG from VirA might result in an induction of the general virulence without the need of additives. When trying to work with the emerging model-organism for monocots Setaria viridis, commonly known as green millet, we encountered several challenges. At first we had to overcome the hurdle of seed sterilization. For this we had to come up with a way of completely removing the husks around the seeds so that our sterilization solution can reach every part of the seed. After successfully plating out several rounds of seeds which did not contaminate had virtually no seeds germinating so we looked for ways to break seed dormancy. After troubleshooting and literature research we found a way to break seed dormancy by adding a gibberellic acid (Sebastian et al., 2014). After our first trial of transforming the seedlings we surprisingly got 40% RUBY-positive plants after 3 days and even 53% after 10 days, slightly exceeding the baseline results for Arabidopsis. All following germinating trials were unsuccessful which forced us to ultimately change to a close relative of our chosen plant: Setaria italica, commonly known as foxtail millet. This plant is cheap, readily available and germinates even without the need of added gibberellic acid. However, after several trials with multiple constructs and conditions we couldn't detect the RUBY reporter. This could be due to the fact that the 35S:RUBY:KanR promoter is relatively weak in monocots (Jang et al., 2002). Sadly we couldn't explore the possibility to exchange the promoter and optimize RUBY expression in Setaria during the time of our iGEM project.
+ Agriculture relies mainly on a handful of monocotyl crops such as maize, wheat and rice. Despite their immense agricultural and nutritional significance, monocots, which represent the most crucial food crops in terms of both quantity and calories, have been explored by fewer than five iGEM teams throughout the history of the competition. When aiming to facilitate <i>Agrobacterium</i>-mediated plant transformation and broaden the spectrum of genetically accessible plants it is also important to think about testing in monocots. Since monocots don't release phenols like acetosyringone after wounding the virulence genes of <i>Agrobacterium</i> do not get activated and therefore the monocot does not get transformed (Cao et al., 2023). To circumvent this, phenolic additives like vanillin and acetosyringone are added to the bacterial culture and also the growing medium of the infected plants. In this context decoupling of VirG from VirA might result in an induction of the general virulence without the need of additives. When trying to work with the emerging model-organism for monocots Setaria viridis, commonly known as green millet, we encountered several challenges. At first we had to overcome the hurdle of seed sterilization. For this we had to come up with a way of completely removing the husks around the seeds so that our sterilization solution can reach every part of the seed. After successfully plating out several rounds of seeds which did not contaminate had virtually no seeds germinating so we looked for ways to break seed dormancy. After troubleshooting and literature research we found a way to break seed dormancy by adding a gibberellic acid (Sebastian et al., 2014). After our first trial of transforming the seedlings we surprisingly got 40% RUBY-positive plants after 3 days and even 53% after 10 days, slightly exceeding the baseline results for Arabidopsis. All following germinating trials were unsuccessful which forced us to ultimately change to a close relative of our chosen plant: Setaria italica, commonly known as foxtail millet. This plant is cheap, readily available and germinates even without the need of added gibberellic acid. However, after several trials with multiple constructs and conditions we couldn't detect the RUBY reporter. This could be due to the fact that the 35S:RUBY:KanR promoter is relatively weak in monocots (Jang et al., 2002). Sadly we couldn't explore the possibility to exchange the promoter and optimize RUBY expression in Setaria during the time of our iGEM project.
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