Functional Validation of BrARF3.1 and BrAXR1 in Brassica rapa and Arabidopsis thaliana

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Functional Validation of

BrARF3.1 and BrAXR1 in

Brassica rapa and Arabidopsis thaliana:

Role in Heading Traits in Chinese Cabbage

Simon Lansu

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Wageningen University and Research centre Plant Science Group

Wageningen UR Plant Breeding Bachelor thesis

Version 3 May 2016 Author Simon Lansu

Avans University of Applied Sciences School of Life Sciences (ATGM)

simon.lansu@wur.nl / s.lansu1@student.avans.nl Teacher supervisor

Dr. Lute-Harm Zwiers

Avans University of Applied Sciences School of Life Sciences (ATGM)

lh.zwiers@avans.nl

Project supervisors Dr. Guusje Bonnema

Wageningen University and Research centre Wageningen UR Breeding

Guusje.bonnema@wur.nl

Johan Bucher, BSc

Wageningen University and Research centre Wageningen UR Plant Breeding

Johan.bucher@wur.nl

Xiaoxue Sun, MSc

Wageningen University and Research centre Wageningen UR Plant Breeding

Xiaoxue.sun@wur.nl

Project institution

Wageningen UR (University and Research centre) Plant Science Group

Wageningen UR Plant Breeding

Droevendaalsesteeg 1 (building 107, Radix) 6708 PB Wageningen

Educational institution

Avans University of Applied Sciences School of Life Sciences

Lovensdijkstraat 61-63 4818 AJ Breda

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Nederlandse Samenvatting

Dit afstudeerproject is onderdeel van het promotietraject van Xiaoxue Sun (sandwich PhD Wageningen UR, Plant Breeding en Institute of vegetables and flowers, Chinese Academy of Agriculture sciences (CAAS)). Het belangrijkste doel van haar promotieonderzoek is om de achterliggende genetica te begrijpen van koolhoofd vorming in Chinese kool (B. rapa ssp. Pekinensis).

Transformatie is een nuttig hulpmiddel in de moleculaire biologie en kan worden gebruikt om gen functies te valideren door knock-out van genen, over-expressie van genen, promotor studies met reportergenen, etc. B. rapa is echter vrij recalcitrant voor Agrobacterium gemedieerde

transformatie.

Het doel van dit project kan worden opgesplitst in twee subdoelen. De ontwikkeling van een transformatie protocol voor B. rapa met behulp van A. tumefaciens. En validatie van de gen functie van de B. rapa ARF3-1 en AXR1 genen in relatie met hun rol in de koolhoofd vorming van Chinese kool door transformatie van A. thaliana.

Er zijn geen geverifieerde B. rapa transformanten geproduceerd tijdens de experimenten om een werkend transformatie protocol te ontwikkelen. De uitgevoerde experimenten omvatte het geleidelijk opvoeren van de selectieve druk, het gebruik van acetosyringone in de co-cultivatie medium en het gebruik van verschillende A. tumefaciens stammen. Allereerst werd vastgesteld dat het geleidelijk toevoegen van hygromycine nog steeds een dodend effect heeft op geregenereerde scheuten. Daarnaast hebben we vermoedden dat het continu toevoegen van 10 mg/l hygromycine dodelijk is zelfs voor getransformeerde scheuten.

In de literatuur wordt vermeld dat de toevoeging van acetosyringone in het co-cultivatiemedium de transformatie frequentie in B. rapa verhoogd. Dit effect werd echter niet waargenomen gedurende de experimenten van dit onderzoek.

Twee A. tumefaciens stammen werden gebruikt in de B. rapa transformatie experimenten, AGL1 en GV3101. De GV3101 stam groeide sneller en om overwoekering te voorkomen werd cefotaxime aan het dekolonisatie/ selectiemedium toegevoegd als extra antibioticum. Cefotaxime onderdrukt echter de regeneratie van B. rapa scheuten en zal moeten worden weggelaten in de toekomstige experimenten.

De transformatie van A. thaliana heeft 27 transformanten opgeleverd bestaand uit 11 35s ::

BrARF3-1cc, 8 35s :: BrARF3-1pc, 3 35s :: BrAXR1cc, 3 35s :: BrAXR1pc en 2 arf4-2 BrAXR1cc

overexpressie lijnen. Drie onafhankelijke BrARF3-1 overexpressie T1 transformanten met het pak choi allel (planten 1, 6 en 7) hadden smallere naar buiten gebogen bladeren, terwijl onafhankelijke 35s::BrARF3-1 Chinese kool allel T1 transformanten geen duidelijke fenotype vertoonden

vergeleken met de Col-0 wildtype. Verdere fenotypering van deze overexpressie lijnen moet worden uitgevoerd nadat homozygote T3 lijnen geproduceerd zijn doormiddel van zelfbestuiving.

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Abstract

This thesis is part of the PhD program of Xiaoxue Sun (sandwich PhD Wageningen UR, Plant Breeding and Institute of vegetables and flowers, Chinese Academy of Agriculture sciences (CAAS)). The main topic of her PhD project is to understand the genetics behind heading traits of Chinese cabbage (B. rapa ssp. pekinensis).

Transformation is a useful tool in molecular biology and can be used to validate gene functions of candidate genes within organisms by knocking-out genes or overexpressing genes, do promotor studies with reporter genes, etc. However, B. rapa is quite recalcitrant for Agrobacterium mediated transformation. The aim of this project can be split into two parts. The development of an A.

tumefaciens mediated transformation system in B. rapa. And the validation of ARF3-1 and AXR1’s

function in heading of Chinese cabbage by transformation of A. thaliana.

No verified B. rapa transformants have been established as of yet, however various experiments have been conducted in order to try and establish a working transformation protocol. This included progressively increasing the selective pressure, the use of acetosyringone in the co-cultivation medium and the use of different A. tumefaciens strains. Progressively adding hygromycin selection first of all showed that non transformed shoots could still be killed when hygromycin was applied later on in the in vitro culture process. In addition, we suspected that continuous application of 10mg/l hygromycin was also lethal to transformed shoots. It has been reported that the addition of acetosyringone to the co-cultivation medium increases the transformation rate in B. rapa

considerably.1_{However, this effect was not observed during the experiments of this thesis. Two A.}

tumefaciens strains were used in the B. rapa transformation experiments, AGL1 and GV3101. The

GV3101 strain grew much faster and cefotaxime was added to the decolonization/selection media as an additional antibiotic to prevent overgrowth of A. tumefaciens. However, cefotaxime

represses shoot regeneration in B. rapa and should be omitted in future experiment.

The transformation of A. thaliana resulted in 27 transformants consisting of 11 35s::BrARF3-1cc, 8 35s::BrARF3-1pc, 3 35s::BrAXR1cc, 3 35s::BrAXR1pc, and 2 arf4-2 BrAXR1cc overexpression lines. Three (plants 1, 6, and 7) independent non heading BrARF3-1 allele overexpression T1 transformants had narrower outward curving leaves, while independent 35s heading BrARF3-1 allele T1 transformants showed no clear phenotype compared to the Col-0 wild type. Further phenotyping of these overexpression lines should be conducted after homozygous T3 lines are established by self-pollination.

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1. Introduction

This research project is part of the PhD program of Xiaoxue Sun (sandwich PhD Wageningen UR, Plant Breeding and Institute of vegetables and flowers, Chinese Academy of Agriculture sciences (CAAS)). The main topic of her PhD thesis is to understand the genetics behind heading traits of Chinese cabbage (B. rapa ssp pekinensis), with special attention to the role of ARF genes

1.1. Brassica rapa

Brassica rapa is a diploid crop that belongs to the Brassicaceae. The Brassica genus contains a

number of economical important species for both food and industrial purposes. The six Brassica species of the highest agricultural importance are the diploid species B. rapa (AA, n=10), B. nigra (BB, n=8), B. oleracea (CC, n=9) and the amphidiploid species B. napus (AACC, n=19), B. carinata (BBCC, n=17) and B. juncea (AABB, n=18).2_{The amphidiploid species originated from}

spontaneous hybridization and chromosome doubling of two diploid Brassica species.

Another member of the Brassicaceae family is Arabidopsis thaliana which is a model organism. B.

rapa and A. thaliana have syntenic genomes, however Brassica’s underwent a whole genome

triplication event and B. rapa genes show high similarity levels on a sequence level with

orthologous genes in A. thaliana which means that knowledge of A. thaliana is highly relevant for gene isolation and characterization in Brassica species.3

There is enormous morphologic variation in B. rapa which can be divided in three main groups: turnip, oil and leafy types. Turnips have tuberous storage organs and are mainly used for

consumption as vegetables by humans and as fodders by animals. The oilseed types are rich in oil and protein. The oil is mainly used for consumption in cooking, salads and margarine and also for fuel. The meal with the protein is mainly used as feed for animals.

The leafy type like Chinese cabbage (B. rapa ssp pekinensis) and Pak choi (B. rapa ssp chinensis) are used as vegetable and are an important crop in eastern Asia (Figure 1). 4-8

Figure 1 Examples of the morphological crop variation in B. rapa: from the top left to right. pak choi, Chinese cabbage, turnip, oilseed, purple pak choi, caixin, mizuna, purple caitai and takucai. The third line shows additional morphotypes or varieties of the previous morphotypes.84

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1.1. Selection Signals for domestication traits

The huge morphological variation in B. rapa is mainly caused by selection by humans during the domestication of these crops. The underlying genetic cause of this phenotypic plasticity still is poorly understood. Joined efforts of WUR Plant Breeding and IVF CAAS resulted in resequencing of the genomes of both heading and non-heading B. rapa genotypes to address this question 9_. The heading trait of Chinese cabbage is characterized by the enlarged inward curving leaves with a larger abaxial identity which form the leafy head. When looking for signals for selection you should focus on regions with low genetic diversity and positive selection of alleles when comparing the population of interest (here Chinese cabbages) with the control group (all other morphotypes). These regions with low genetic diversity are defined as a selective sweep (see Figure 2). Another method of detecting selective sweeps is to measure the linkage disequilibrium (LD). The uncovered selective sweeps in heading B. rapa where annotated into GO-terms which represent gene and gene product attributes. Common phytohormone GO-categories of auxin, cytokinin, gibberellins and jasmonic acid are enriched in selective sweeps of heading types along with GO-terms involved in signal transduction or transport related activity.9_{Phytohormones and their downstream effects} play an important part in plant development and morphology. Auxin response factor 3 of the ARF-tasiRNA pathway and Auxin Resistant 1 are located in selective sweeps in heading B. rapa. Both genes are involved in the regular auxin response within plants. The tasiRNA-ARF3 pathway is involved in the adaxial/abaxial polarity in A. thaliana.10-13

Figure 2 schematic representation of a selective sweep in humans: a beneficial mutation will rise in frequency. The image shows polymorphisms on a

chromosome, including the selected allele, before and after selection. Ancestral alleles are shown in grey and derived alleles are shown in blue. Positively-selected alleles (red) rise to high frequency, nearby linked alleles on the chromosome ‘hitchhike’ along with it to high frequency, creating a ‘selective sweep.’86

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1.1.1. Auxin Response Factors

Auxin Response Factors (ARFs) are transcription factors that regulate gene expression of auxin response genes. These genes contain TGTCTC motifs in the promotor region which function as the auxin response element (AuxRE). ARFs consist of modular domains which in general are the N-terminal B3 DNA-binding domain, a variable activation/repression domain or Auxin-response super family domain and a dimerization domain (CTD) or AUX/IAA superfamily domain which is absent in ARF3 on the C-terminus. The CTD domain is highly similar in amino acid sequence with domains III and IV of AUX/IAA repressors, see Figure 3 and Figure 4.

The role of ARFs in plant development is well-

established in genomic studies on Arabidopsis thaliana.14_{The ARFs of interest for this study ARF3} and ARF4 are considered to regulate adaxial/abaxial identity of leaves. Resequencing Chinese cabbage and Pak choi accessions shows an allelic difference in BrARF3-1, this G/C Single nucleotide polymorphism (SNP) at cDNA position 2,264 causes a differential translation of glutamine in the heading B. rapa and a histidine in the non-heading types. Around 90% of the heading B. rapa genotypes carry the G allele translating to glutamine. In contrast, around 75% of the non-heading genotypes carry the C allele.9_{Mutations in AtARF3 or in AtARF4 have shown abnormal leaf and} flowering organ development, even though we couldn’t confirm that for the arf4 knockout mutant in the Col-0 genetic background that we ordered from Nottingham Arabidopsis Stock Centre (NASC) B. rapa has paralogues to some of the Arabidopsis ARFs. B. rapa has two ARF3s (BrARF3-1 and BrARF3-2), the duplicates are the result of the whole genome triplication event, that is shared by all B. rapa’s. These duplicate genes could be

redundant gene copies and there is a possibility that they are Pseudogenes without any function or have sub or new function. The heading

morphotype is composed of many enlarged leaves that fold inwards, likely due to a larger abaxial surface. The leaf polarity is regulated by various transcription factors and small RNAs. ARF3/4 is regarded as an abaxial determinant of leaf polarity and is regulated by trans-acting small interfering RNA (tasiRNA). The regulation of ARFs by tasiRNA’s is a form of post transcriptional regulation. The tasiRNA’s bind to the mRNA of their target after which the mRNA is cleaved by AGO7 (Figure 5).14-17_{ARF3 and ARF4 are} considered to be related in function and are classified as a sister pair. ARF sister pairs have somewhat redundant roles in Arabidopsis which means that single mutants could have a weak phenotype.

Figure 3 The general structures of both ARF and AUX/IAA repressors. ARF contains the N-terminus B3 domain, the Auxin response super family domain (MR) and the dimerization domains of the AUX/IAA super family (III and IV).81

Figure 4 The gene structure of ARF3/4 in both B.

rapa (Br) and A. thaliana (At). The intron/exon

pattern of both the paralogues differs quite extensively.14

Figure 5 Schematic representation of the Transcription factors regulating leaf polarity in A.

thaliana: the blunt arrows represent down regulation

and the pointed arrows represent up regulation. The ARF3/4 genes are positively regulated by auxin and positively regulate other factors in the abaxial side of the leaf while it’s repressed by tasiRNA of the adaxial side of the leaf.85

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1.1.1. Auxin Resistant 1

The Auxin resistant 1 gene or AXR1 is an important regular of auxin response. AXR1 mutants can have various phenotypic defects in root formation, floral development, root hair growth, apical dominance, fertility, leaf shape. BrAXR1 has been observed to have a differential

expression pattern between heading and non-heading B. rapa. 9

AXR1 encodes for the N-terminal subunit in

the heterodimeric RUB-activating enzyme E1 which has an effect in auxin response. RUB is ubiquitin like protein which modifies other proteins by conjugating to target proteins. The C-terminal of the E1 enzyme contains a cysteine that forms a thioester bond with RUB1 and activates RUB1 in an ATP dependent manner resulting in an AMP-RUB1 intermediate. Important targets for RUB modification are the cullin proteins. Cullins are subunits of E3 complexes called SCFs which facilitate the transfer of ubiquitin to target proteins. AUX/IAA repressors are targeted for ubiquitination which targets the AUX/IAA repressors for degradation in the 26s proteasome, enabling auxin induced transcription (Figure 6). 18-26

Figure 6 Auxin regulation of gene expression in A. thaliana. (a) Under low auxin concentrations the Aux/IAA proteins heterodimerize through the III and IV domains with the ARF transcription factors, repressing auxin-inducible gene expression. (b) RUB1 activated by the AXR1-ECR1 dimer is transferred to CUL1 by RCE1. This frees CUL1 from CAND1 so it can form the SCF complex with ASK1 and TIR1. CSN can remove RUB from CUL1 making it possible for CAND1 to disassemble the SCF complex to bind with CUL1. (c) AUX/IAA is recruited to the SCF complex through auxin. Ubiquitin is activated by E1 after which E2 conjugates ubiquitin to AUX/IAA. The ubiquitinylated AUX/IAA is recruited by the 26s proteasome for degradation. AUX/IAA itself is auxin inducible forming a negative feedback loop. Abbreviations: DBD, DNA-binding domain; E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; U, ubiquitin; R, RUB. *AFB1, AFB2, or AFB3. 18

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1.2. Transformation

Breeding for a specific trait can be quite laborious and can take quite a lot of time to achieve and some traits can’t even be achieved through conventional breeding methods. Alternative strategies, like in the case of this research project transformation is used for validation of gene functions by for instance creating knockout mutants or over expression mutants or by transforming these genes into model organisms and observing the phenotype of multiple transformants. Transformation of plant material is a relatively fast method that can be used to incorporate exogenous genetic material in the nucleus of the cell to alter the genetic background of the plant. The exogenous genetic material can either be incorporated in the genome or can be expressed in the nucleus without being incorporated (transient expression). The introduced material can only be inherited by further generations if the exogenous material is incorporated in the genome and will do so in a Mendalian fashion. Transformation can be used for transferring both genes from the plant itself or from sexually compatible plants called cisgenes. Or even genes from completely different species or organisms called transgenes. Multiple transformation methods are available, Agrobacterium mediated transformation is the method used in this study.

Figure 7 schematic model of the T-DNA transfer from Agrobacterium into the plant genome: 1. The virulence factors are released as a wound response. 2. The virulence factors are recognized by A. tumefaciens. 3. A.

tumefaciens attaches to the wounded plant cell. 4 and 5. The activated virulence proteins process and form the

T-DNA complex. 6. The T-DNA is transferred to the host cell. 7. The T-DNA is imported into the nucleus of the host cell. 8. Random T-DNA integration into the host’s genome. 9. Expression of the integrated genes from the T-DNA by the Host cell.43

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Agrobacterium is a genus of soil bacteria that are commonly found in the rhizosphere. Some Agrobacterium species are plant pathogens like Agrobacterium tumefaciens. Crown gall disease is

caused by A. tumefaciens and is characterized by tumour formation in plants. The tumours are caused by a DNA sequence (T-DNA) that is transferred into the host’s genome by Agrobacterium. This T-DNA is located on a tumour inducing plasmid or Ti-plasmid. Agrobacterium targets wounded plants by sensing small phenolic compounds like acetosyringone and sugars that are considered to be virulence factors (Figure 7). The DNA has two cis-acting elements that are essential for the T-DNA transfer. These cis-acting elements are bordering sequences one for each side of the

oncogenes in the wild Agrobacterium strains. The DNA sequence in between the border sequences is the DNA that is transferred by Agrobacterium; replacing the oncogenes with a gene of interest makes it possible to transfer the gene of interest into a plant cell. Disarmed Agrobacterium strains lack the tumour inducing genes in the T-DNA but still have a Ti-plasmid containing virulence genes that are needed for T-DNA transfer and integration.

The binary T-DNA transfer system is used during the experiments of this project. The binary system uses a disarmed strain of Agrobacterium that contains a helper Ti-plasmid and a binary vector. The helper Ti-plasmid only contains the virulence genes and an origin of replication (ori) for

Agrobacterium. The disarmed strain of Agrobacterium is transformed with a binary vector so it will

contain two plasmids. The binary vector contains the T-DNA border sequences with the genes of interest and plant selectable marker genes in between, both an ori for Agrobacterium and

Escherichia coli and a selectable marker gene for bacteria.

Transformation of plant tissue will yield transformed cells which have to be cultured into complete plants in in vitro culture.

1.3. Plant Tissue Culture

Plants can both reproduce sexually and asexually. The asexual propagation of plants can be done by using tissue (leaf, stem, roots) which contains totipotent cells to form a whole plant in vitro. The most critical part of in vitro tissue culture is a sterile environment because cells and tissue

extracted from parent plants lack natural defences against pathogens and the sugar rich media harbour an ideal environment for microbial growth. Tissue culture is conducted in a controlled environment usually at a temperature of 25o_{C with a 16H/8H day/night period. During tissue} culture plants are grown on a culture medium which contains nutrients (organic/inorganic salts, amino acids, sugar, vitamins) and other growth factors (cytokinins, auxins). The composites of the medium can be used to control the development and growth. The hormonal balance can be used to promote shoot or root development in vitro. Shifting to a higher auxin: cytokinin ratio is used to promote root development while a lower auxin: cytokinin ratio generally promotes shoot formation. The tissue culture process consists of a few general steps to grow a complete plant from explant material.

The first step is to select the explant material for in vitro culture. Preferably material that is in good physiological and disease free condition from juvenile plants.

The explant material should be sterilized before initiating tissue culture and be put on medium. The second step, shoot proliferation should be done on medium containing a high cytokinin: auxin ratio. The forming of plant organs from explant material can occur through direct or indirect organogenesis. In indirect organogenesis, a mass of dedifferentiated cells, callus, is formed first. Plant organs are regenerated from that callus. In direct organogenesis the organs are formed by using the cells of the explant directly as a precursor and therefore bypassing the callus phase. The next step, rooting is done by culturing the excised shoots on medium with a low cytokinin: auxin ratio. The last step is the transfer of the plantlets to a soil in a growth chamber in a greenhouse. This step is called hardening off in which the plantlets are gradually moved from in vitro conditions with among others high humidity to more natural conditions (Figure 8).27

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B. rapa is considered to be rather recalcitrant for shoot regeneration from hypocotyls and

cotyledons. The shoot regeneration potential is under strong genotypic control, which means that some B. rapa genotypes are more recalcitrant then others. The use of different explant material, explant age, plant hormone type, hormone concentrations and ethylene inhibitors are also

examples of parameters affecting shoot regeneration. An efficient regeneration protocol is required for transformation with Agrobacterium tumefaciens as both the infection and selection agents in the media decreases shoot regeneration efficiency significantly.28-34

1.4. Gateway Cloning

The binary vectors used in the experiments are Gateway recombinational cloning vectors. The Gateway system is based on site specific recombination of bacteriophage DNA into and out of the bacterial genome. This system allows easy transfer of DNA fragments between vectors while maintaining the same reading frame by using att recombination sites and Clonase enzyme mixes. The ORF of interest has to be amplified by PCR with attB sites added to the 5’ side of the primers. The fragment containing the ORF is then transferred to an entry vector with

attP sites. BP clonase facilitates the attB-attP

recombination resulting in a donor vector with the target sequence in between attL sites. The donor vector can be used to transfer the fragment of interest to an appropriate

destination vector containing the right selectable marker, promotor/terminator, fusion tag, etc. for the experiment. The destination vector contains

attR sites and uses LR clonase to facilitate attL-attR recombination resulting into

expression/binary clones with the sequence of interest in-between attB sites (Figure 9).35-37

1.5. Functional Validation in Arabidopsis thaliana

The introduction of a gene into an organism to alter its phenotype is as mentioned earlier an important tool in molecular biology. However, transformation of higher eukaryotes including many crop species can be quite challenging, this is also the case in B. rapa which is transformation recalcitrant. The use of an easily transformable model organism can prove to be a useful alternative. A popular model organism in plant science is A. thaliana, a small plant in the Brassicaceae family. A. thaliana has a small 135mb diploid genome which is divided over five chromosomes (n=5), it was the first sequenced plant genome. A. thaliana is quite useful for genetic experiments because of its short generation time, predominant self-pollination, and

substantial seed production. The small size and short generation time of this plant makes it easy to handle in laboratory conditions. The transformation of A. thaliana with A. tumefaciens has become a routine task by using the floral dip protocol. 38-41

Figure 8 schematic representatation of the tissueculture procces. 80

Figure 9 Schematic representation of the BP and LR clonase reaction of the Gateway system: BP clonase facilitates the attB-attP recombination resulting in the integration of the PCR product into the entry clone. LR

clonase facilitates attL-attR recombination resulting in

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1.5.1. Floral Dip

Floral dipping is quite a simple and fast transformation technique and is mainly used in Arabidopsis and some other Brassicaceae, but is not very successful in other plant species. The floral dipping method is as simple as submerging the flowering stalks with closed floral buds into an

Agrobacterium suspension. This way the transformation events occurs in the gynoecium of the T0 generation primarily transforming the female gametophytes resulting in mostly hemizygous transformants. The T0 plants are kept in the greenhouse to ripen until the seeds are harvested. Roughly 1% of the T0 generation seeds are transgenic seeds. The seeds are then germinated on medium containing selective agents to identify the T1 seedlings.41-45

1.6. Aim of the Thesis

This thesis has two parts, development of a transformation protocol of B. rapa and the

transformation of A. thaliana with heading related genes. The aim of the B. rapa transformation experiments was to develop a working Agrobacterium mediated transformation system within B.

rapa for functional studies. Due to challenges in this front A. thaliana is used as a model for the

validation of BrARF3-1 and BrAXR1’s function in heading of Chinese cabbage. The second aim was to generate CaMV 35s overexpression mutants of the Chinese cabbage (cc) and Pak choi (pc) alleles of BrARF3.1 and BrAXR1 in A. thaliana. These mutants were phenotyped and genotyped in comparison with the Col-0 wildtype.

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2. Materials and Methods

2.1. Constructs

Transformation experiments in both B.

rapa and A. thaliana were performed

with the A. tumefaciens strain AGL1 or GV3101 harbouring various vectors. 46 Transformation of B. rapa was

performed with the pGWB505 vector.47 This vector contains BrARF3-1 with the Chinese cabbage allele without the stop codon fused to Green Fluorescent Protein (GFP) behind the cauliflower mosaic virus 35S promotor and the hygromycin B phosphotransferase gene (hpt) for hygromycin resistance (Figure 10).

The floral dip experiments in A. thaliana were conducted with A. tumefaciens AGL1 only. In this experiment multiple constructs were used with 2 different backbones. The pK7WG7 vector 48_was used to generate BrARF3-1(Bra005465) or BrAXR1 (Bra015396) overexpression mutants. In total 4 different inserts were used behind an CaMV 35s promotor in pK7WG2; BrARF3-1 Chinese cabbage allele, BrARF3-1 Pak choi allele, BrAXR1 Chinese cabbage allele, or BrAXR1 Pak choi allele. The backbone also contains the neomycin phosphotransferase II gene for kanamycin resistance (Figure 11).

The pGWB433 vector 47_{was also} used for A. thaliana transformation. In this vector the Chinese cabbage

BrARF3-1 promotor was cloned in

frame with GUS gene (uidA) which makes it possible to visualize promotor activity. This vector also carries the nptII gene for kanamycin resistance in plants.

Initially another construct cloned by Xiaoxue Sun was intended for this purpose. This vector however could not be transformed into A.

tumefaciens. However, the presence

of the BrARF3-1cc promotor could be verified by PCR with 1U Dreamtaq polymerase, 1x Dreamtaq buffer, 200 µM DNTP’s, 0,5 µM of the both the forward and reverse BrARF3-1cc promotor primer. (for primer sequences see, Appendix B )

in a final volume of 20 µl. with the following program; initial denaturation of 5 minutes at 94o_C followed by 30 cycles of denaturation of 1 minute at 94o_{C, annealing of 1 minute at 60}o_{C, and} extension of 1 minute at 72o_{C followed by a final extension of 7 minutes at 72}o_{C with a hold at} 10o_C.

Which made it possible to use the plasmid DNA as a template to clone a new vector.

Figure 10 The pGWB505 vector used in B. rapa transformation. This vector has the CaMV 35s promotor, BrARF3-1 cds fused to GFP, NOS terminator and hpt between NOS promotor and terminator within the T-DNA border sequences. 46

Figure 11 The pK7WG2 vector used in A. thaliana transformation. This vector has the CaMV 35s promotor, in this case the BrARF3-1 cds, NOS terminator and nptII between NOS promotor and terminator within the T-DNA border sequences. The Sm/SpR gene outside of the T-DNA confers streptomycin and spectinomycin resistance for bacterial selection.47

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2.2. Gateway Cloning of pGWB433

The BrARF3-1cc promotor was amplified with attB site extended primers. This PCR reaction was conducted with Phusion polymerase (Invitrogen) which is a high fidelity polymerase with proofreading to reduce the chances of mistakes in the final sequence. The PCR was conducted with 0,4U Phusion high fidelity DNA polymerase, 1× Phusion HF Buffer, 200 µM DNTP’s, 0,5 µM of the both the forward and reverse attB BrARF3-1cc promotor primers, in a final volume of 20 µl. with the following program; Initial denaturation at 98o_{C for 30 seconds} followed by 30 cycles of denaturation at 98o_{C for} 10 seconds, annealing at 60o_{C for 30 seconds,}

extension at 72o_{C for 30 seconds, followed by final extension step at 72}o_{C for 10 minutes and an} 10o_{C hold. The resulting amplicon was loaded on agarose gel with gel red loading dye for}

verification.

The resulting BP product of the BrARF3-1cc promotor was used in a BP reaction with pDONR221 after purification with the Qiaquick PCR Purification kit (Qiagen).

The BP reaction was conducted with 30ng BP-BrARF3-1cc promotor and 75ng pDONR221 vector in a total volume of 4µl. 1 µl of BP Clonase II enzyme mix (Invitrogen) was added and then incubated overnight at room temperature. After incubation 0,5 µl of Proteinase K was added to the samples and were incubated for 10 minutes at 37o_{C. One Shot Top10 E. coli cells (Invitrogen) were} transformed trough heat shock and incubated in SOC medium (Appendix A ) for one hour at 37o_C before plating on LB (Appendix A Medium Composition) agar plates containing 50mg/l kanamycin. The transformed colonies containing the entry vector pENTR221 with the BrARF3-1cc promotor were verified by PCR. This PCR was conducted with 1U Dreamtaq polymerase, 1x Dreamtaq buffer, 200 µM DNTP’s, 0,5 µM of the both the forward and reverse BrARF3-1cc promotor primer, in a final volume of 20 µl. with the following program; initial denaturation of 5 minutes at 94o_{C followed by} 30 cycles of denaturation of 1 minute at 94o_{C, annealing of 1 minute at 60}o_{C, and extension of 1} minute at 72o_{C followed by a final extension of 7 minutes at 72}o_{C with a hold at 10}o_{C. The resulting} product was loaded on agarose gel with gel red loading dye.

The transformed and verified colonies were cultured overnight in LB broth containing 50mg/l kanamycin at 37o_{C. the pENTR221 BrARF3-1cc promotor vector was isolated form these overnight} cultures with the Qiaprep Spin Miniprep kit (Qiagen).

25ng of the pENTR221 BrARF3-1cc promotor and 50ng of pGWB433 were combined in a final volume of 4µl. 1µl of LR Clonase enzyme mix (Invitogen) was added and the samples were incubated overnight at room temperature. After incubation 0,5µl Proteinase K (Invitrogen) was added and incubated for 10 minutes at 37o_{C. One Shot Top10 E. coli cells (Invitrogen) were} transformed with the resulting expression clone pGWB433 BrARF3-1cc promotor (Figure 12) by heat shock and incubated in SOC medium for one hour at 37o_{C before plating on LB agar plates}

Figure 12 The resulting pGWB433 BrARF3cc promotor vector after Gateway cloning, used in A. thaliana transformation. This vector has the Brassica ARF3cc promotor cloned in frame with the GUS reporter gene, NOS terminator and nptII between NOS promotor and terminator within the T-DNA border sequences.46

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16 containing 50mg/l streptomycin and 50mg/l spectinomycin. The resulting colonies were verified in the same manner as the entry clone. The verified colonies were cultures overnight in LB medium containing 50mg/l streptomycin and 50mg/l spectinomycin at 37o_{C. The construct was isolated} from these overnight cultures with the Qiaprep Spin Miniprep kit (Qiagen).

2.3. Transformation of A. tumefaciens

Electro competent A. tumefaciens cells were produced by harvesting the cells of 50ml overnight cultures by centrifugation at 4600g, 4o_{C for 15 minutes. the harvested cells were washed with} 50ml ice cold sterile Milli-Q water and centrifuged again at 4600g, 4o_{C for 15 minutes. The cells} were washed again but with 25ml sterile ice cold Milli-Q water and centrifuged at 4600g, 4o_{C for 15} minutes. The cells were resuspended in 1ml sterile ice cold Milli-Q water with 10% glycerol and centrifuged at 4600g, 4o_{C for 15 minutes. The cells were resuspended a final time in 500µl sterile} ice cold Milli-Q water with 10% glycerol and were divided into 50µl aliquots. The various vectors were introduced into A. tumefaciens through electroporation with the Equibio easyjecT

electroporator on default settings (Pulse is not controllable). The transformed cells were grown in LB medium without antibiotics for 3 hours. After 3 hours the bacteria were spread on LB selection plates with 50mg/l carbenicillin, 50mg/l chloramphenicol, 50mg/l spectinomycin and 50mg/l streptomycin for the AGL1 strain, and 10mg/l rifampicin, 50mg/l spectinomycin and 50mg/l streptomycin for GV3101. Single colonies were picked and used for colony PCR and used as inoculum for liquid LB cultures.

The PCR was conducted with 1U Dreamtaq polymerase, 1x Dreamtaq buffer, 200 µM DNTP’s, 0,5 µM of both the forward and reverse primers of BrARF3-1, BrAXR1 or nptII in a final volume of 20 µl. with the following program; initial denaturation of 5 minutes at 94o_{C followed by 30 cycles of} denaturation of 1 minute at 94o_{C, annealing of 1 minute at 60}o_{C, and extension of 1 minute at} 72o_{C followed by a final extension of 7 minutes at 72}o_{C with a hold at 10}o_{C. Samples were loaded} on a 1% agarose gel with gel red loading dye for analysis.

2.4. A. tumefaciens Culture

For the B. rapa transformation experiments, A. tumefaciens AGL1 was grown in LB medium with 50mg/l streptomycin 50mg/l spectinomycin 50mg/l chloramphenicol and 50mg/l carbenicillin added after autoclaving. The A. tumefaciens culture is grown for 72 hours at 28o_{C before harvesting the} cells by centrifugation of 5ml culture at 4570g for 10 seconds. The harvested cells were re-suspended in Agrobacterium induction medium a day before the transformation of B. rapa explants. Agrobacterium induction medium consists of 111,0mM glucose, 30,2mM MES, 18,7mM NH4Cl, 1,2mM MgSO4.7H2O, 2,0mM KCl, 0,12mM CaCl2.H2O, 9,0 µM FeSO4.7H2O, 2,5mM NaH2PO4 and 2,1mM Na2HPO4. pH was adjusted to 5,7 and the medium was filter sterilized. After re-suspension of A. tumefaciens in the Agrobacterium induction medium 20mg/l acetosyringone was added after which A. tumefaciens was cultured at 22o_{C in the dark overnight.}

A. tumefaciens GV3101 was grown in LB medium containing 10mg/l rifampicin, 50mg/l

streptomycin, and 50mg/l spectinomycin. Initially GV3101 was also grown for 72 hours at 28oC. however, GV3101 grows faster than AGL1 and the culture time has been decreased to 48 hours. The cells were harvested and resuspended in Agrobacterium induction medium in the same way as AGL1 but on the same day as the transformation experiment shortening the induction time to 3 hours.

2.5. B. rapa Transformation

Two accessions of B. rapa were used for transformation experiments. The Chinese cabbage (B.

rapa ssp pekinensis) CC285v (BrDFS_B_034) and yellow sarson (B. rapa ssp trilocularis) R-o-18

(pv-Br020235). These accessions were chosen for their ability to regenerate well in in vitro culture. Before use in transformation experiments, the seeds were surface sterilized by first washing them in 70% ethanol for 1 minute after which they were washed in 2% NaOCl for 15 minutes followed by 5 sub sequential washes in sterile Milli-Q water for 1 minute each. Around 30 seeds were sown on agar plates containing full strength MS (Appendix A ), 30g/l sucrose and 8g/l Phytoblend agar. The cotyledonary explants were cut after 4 days and placed petiole down into co-cultivation medium. The co-cultivation medium consists of full strength MS medium, 3% sucrose, 500mg/l MES and 0,8% Phytoblend at pH 5,7. 4mg/l BAP and 0,15mg/l NAA were added after autoclaving.

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17 The plates holding the explants are transferred to a crossflow cabinet and dipped for a few seconds into the A. tumefaciens AGL1 culture diluted to OD600 0,3 from an approximate OD600 of 1,0 (log phase), with Agrobacterium induction medium, and returned to the co-cultivation medium for 72 hours under scattered light.

After 72 hours of co-cultivation the explants were transferred to selection medium. This selection medium consists of full strength MS medium, 3% sucrose, 500mg/l MES and 0,8% Phytoblend at pH 5,7, 4mg/l BAP and 0,15mg/l NAA, 2,5mg/l AgNO3, 160mg/l timentin and 15 mg/l hygromycin after autoclaving. Non-dipped explants were placed on selection medium, selection medium without hygromycin and selection medium without any antibiotics as controls for selection and shoot

regeneration. The selection plates holding the transformed explants were returned to the climate room under scattered light. 30-34,49-54

2.5.1. Delayed and Progressive Addition of Hygromycin Selection.

Another approach used during this project was the gradual increase of selective pressure. This approach lets the transformed cells regenerate for a bit before adding selective agents to increase the chances of survival under selective pressure. 55-58

The seedlings used as explant material in these experiments were grown, cut and inoculated in the same manner as the previous experiments. After co-cultivation the explants were transferred to decolonization medium which consists of full strength MS medium, 3% sucrose, 500mg/l MES and 0,8% Phytoblend at pH 5,7, 4mg/l BAP and 0,15mg/l NAA, 2,5mg/l AgNO3, and 160mg/l timentin after autoclaving to remove the A. tumefaciens growing on the explants. Two plates with non-dipped control explants were also transferred to this medium and one control plate was transferred to the same medium but without the timentin. After two weeks the explants were transferred to low selection medium which consists of full strength MS medium, 3% sucrose, 500mg/l MES and 0,8% Phytoblend at pH 5,7, 4mg/l BAP and 0,15mg/l NAA, 2,5mg/l AgNO3, 160mg/l timentin and 5 mg/l hygromycin were added after autoclaving. One of the control plates was transferred to the same medium to test whether selection still has an effect on partially regenerated explants, another control is transferred to the same medium decolonization medium, and the last control is again transferred to medium without antibiotics. after two weeks the explants were transferred to the high selection medium with an increased hygromycin concentration of 10mg/l. the control on selection medium is also transferred to this medium with increased hygromycin concentration. And the other controls were transferred in the same manner as previously mentioned.

Explants were transferred every two weeks to the same media as previously mentioned till shoots could be excised and transferred to rooting medium which consists of full strength Gamborgs B5 medium (Appendix A ) with 3% sucrose, 0,8% Phytoblend agar. After autoclaving 160mg/l timentin and 0,15mg/l NAA were added.

A variation of this experiment was the addition om 100µM acetosyringone to the co-cultivation medium. This phenolic compound induces the virulence genes in A. tumefaciens and is added to try to increase the transformation efficiency.

Table 1 Overview of the various B. rapa transformation experiments conducted during this thesis.

Genotype A. tumefaciens _strain Treatment Number of _explants Experiment 1 R-o-18 AGL1 Direct selection 260

Experiment 2 CC285v AGL1 Progressive selection 200 Experiment 3 R-o-18 AGL1 Progressive selection 110 Experiment 4 R-o-18 AGL1 Progressive selection + _{acetosyringone} 200 Experiment 5 R-o-18 AGL1 Progressive selection 200 Experiment 6 R-o-18 AGL1 Progressive selection + _{acetosyringone} 270 Experiment 7 R-o-18 GV3101 Progressive selection 140 Experiment 8 R-o-18 GV3101 Progressive selection +

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18 The previous experiment with the gradual selection was also conducted with a different A.

tumefaciens strain, GV3101. The experiments were conducted almost in the same way but with the

addition of 250mg/l Cefotaxime to the decolonization/selection plates. This was done to decrease the change of bacterial overgrowth because of the much faster growth of A. tumefaciens GV3101.46

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19

2.6. A. thaliana Floral Dip

Seeds of A. thaliana Col-0 were placed on moist filter paper and transferred into the cold room at 4o_{C for a minimum of 48 hours of stratification. After this cold period the seeds were sown on peat} pots and were grown under long day conditions 16h light/8h dark at around 22o_{C. The first bolts} were cut off to remove the apical dominance which results in the growth of multiple bolts. The plants were dipped in an early flowering stage in which the plants carry mostly unopened flowers and very few fertilized flowers. The bolts of A. thaliana Col-0 were dipped in the A. tumefaciens suspension and swirled around for 3-5 seconds. The dipped plants were placed in a parchment bag and returned to the greenhouse. The bag was removed after 24 hours and the plants were

returned to the initial growth conditions to ripen before harvest.

The transformed seeds were surface sterilized by adding 1ml of 70% ethanol to the seeds in 1,5ml Eppendorf tubes for 1 minute. The ethanol was removed and 1ml 4% NaOCl with 0,02% Triton X-100 was added for 15 minutes and were shaken regularly. The NaOCl solution was removed by pipetting and the seeds were rinsed with sterile MQ water. The MQ water was refreshed every minute for 5 times. The sterile seeds were re-suspended in 1ml sterile 0,1% agarose and pipetted on selection plates containing ½ MS medium, 0.8% Phytoblend and 50mg/l kanamycin. The seeds were scattered over the plate with a Drigalski spatula. The sown plates were stratified at 4o_{C for a} minimum of 48 hours. The plates were transferred to a climate room with a long day cycle of 16h light/8h dark at 24o_C.

putative transformants were transferred to peat pots in the greenhouse, about 2 weeks after sowing. The peat pots containing the putative transformants were placed into plastic domes with two hatches to maintain high humidity for 48 hours, after which one of the hatches is opened. After 24 hours the second hatch is opened, and after a further 24 hours the dome is removed and the putative transformed plants were grown normally.

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20 In order to obtain homozygous transformants, the hemizygous T1 generation are self-pollinated. This results in a seed batch containing the T2 generation in which the transgenes are distributed in a Mendalian fashion (25% homozygous, 50% hemizygous and 25% without transgenes). When sowing the T2 generation on selection medium only the plants with the transgene will survive of which 33,3% is homozygous and 66,7% is hemizygous. In order to identify the homozygous plants, they should be self-pollinated again. The resulting T3 generation was sown on selection medium and if the T2 parent was homozygous it’s offspring should be as well, resulting in a 100% survival rate on selection medium. If the T2 parent was hemizygous only 75% of the plants should have survived on the selection medium (Figure 13).

2.7. Molecular Validation of Putative Transformants

A single leaf of putative transformants were harvested and stored directly in liquid nitrogen. The frozen samples were ground with a pestle and mortar to a fine powder before extraction of DNA and RNA. 1ml of TRIzol reagent (Invitrogen) was added to the ground samples and vortexed vigorously. 0,2ml of chloroform was added and mixed by inversion and incubated for 2-3 minutes at room temperature. The samples were centrifuged at 2000g for 20 minutes at 4o_{C. The aqueous} phase was transferred to an RNase free Eppendorf tube for RNA extraction the interphase and organic phase were used for DNA extraction.

0,4ml isopropanol was added to the aqueous phase, mixed and incubated for 10 minutes at room temperature. The samples were centrifuged at 2000g for 15 minutes at 4o_{C. the resulting RNA} pellet was washed with 0,4ml of 75% ethanol and centrifuged at 2000g for 7 minutes at 4o_{C. the} RNA pellet was air dried for 10 minutes and dissolved in 50µl sterile MQ water.

0,3ml of absolute ethanol was added to the interphase and organic phase, mixed and incubated for 2-3 minutes at room temperature. The samples were centrifuged at 2000g for 5 minutes at 4o_C. the resulting pellet was washed twice with 1ml of 0,1M sodium citrate in 10% ethanol for 30 minutes while mixing regularly. The sodium citrate was removed by centrifugation at 2000g for 5 minutes at 4o_{C. The resulting pellet was washed in 1,5ml 75% ethanol for 20 minutes while mixing} regularly and then centrifuged at 2000g for 5 minutes at 4o_{C. The DNA was dissolved in 50µl MQ} water overnight. The DNA samples were centrifuged at full speed for one minute and the

supernatant was transferred to a clean Eppendorf tube to get rid of the remaining cell debris. The putative transformants were verified by PCR with 1U Dreamtaq polymerase, 1x Dreamtaq buffer, 200 µM DNTP’s, 0,5 µM of both the forward and reverse nptII primers for A. thaliana and hpt forward and reverse for B. rapa in a final volume of 20 µl. with the following program; initial denaturation of 5 minutes at 94o_{C followed by 30 cycles of denaturation of 1 minute at 94}o_C, annealing of 1 minute at 60o_{C, and extension of 1 minute at 72}o_{C followed by a final extension of 7} minutes at 72o_{C with a hold at 10}o_{C. The resulting product was loaded on agarose gel with gel red} loading dye.

The RNA was purified and DNase I treated with the RNeasy Mini kit (Qiagen). The RNA

concentrations were determined via Nanodrop. 500ng of RNA was used for cDNA synthesis with the iScript cDNA synthesis kit (Bio-Rad).

The expression of both the A. thaliana AtARF3 or AtAXR1 and the B. rapa BrARF3-1 or BrAXR1 was examined with PCR of the A. thaliana overexpression mutant’s cDNA. The PCR was conducted with 1U Dreamtaq polymerase, 1x Dreamtaq buffer, 200 µM DNTP’s, 0,5 µM of both the forward and reverse ARF3 or AXR1 primers for either A. thaliana or B. rapa or Actin forward and reverse as a control. in a final volume of 20 µl. with the following program; initial denaturation of 5 minutes at 94o_{C followed by 30 cycles of denaturation of 1 minute at 94}o_{C, annealing of 1 minute at 60}o_{C, and} extension of 1 minute at 72o_{C followed by a final extension of 7 minutes at 72}o_{C with a hold at} 10o_C.

the same experiment was conducted with both the cDNA and genomic DNA of the A. thaliana Col-0 wild type and the cDNA and gDNA of B. rapa R-o-18 as a positive control and to verify the

specificity of the primers.

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21

2.8. A. thaliana Mutant Phenotyping

The hemizygous primary transformants were phenotyped non-destructively by taking pictures of the rosette from a top view and a side view. The plants were compared with the wild type and with the allelic counterpart on leaf morphology.

The homozygous mutant lines were sown both in the green house and in the time-lapse studio for phenotyping. However, the wild type controls didn’t grow in the time-lapse studio which makes it hard to compare the mutant phenotypes. The time-lapse experiment was conducted by Johan Bucher. The time-lapse studio contains Unicolour LED modules by Philips and contains three colours red blue and white (2 times). The A. thaliana plants in the time-lapse were grown in long day conditions with 16 hours of light and 8 hours of dark with red at 20% power and blue at 10% power during the day.

The mutant lines sown in the green house were grown until they were bolting. The leaves of these plants were excised with a scalpel from the top of the rosette to the bottom and placed on a background to make it easier to count the amount of leaves and see individual leaf shapes better for comparison with the wildtype.

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22

3. Results

A number of constructs used in the experiments were provided by Xiaoxue Sun, while the

BrARF3-1cc promotor with GUS construct needed to be constructed.

3.1. Gateway Cloning of pGWB433

The first step in in the cloning process was the amplification of the BrARF3-1cc promotor with the

attB extended primers. the resulting PCR product was analysed on gel (Figure 14) before post PCR

clean up and usage in a subsequent BP reaction.

The resulting entry clone was transformed into E. coli after which the colonies on the selection plate were verified via colony PCR before culturing (Figure 15). all four of the tested colonies amplified the expected fragments, and thus contained the entry clone. Cultures from positive colonies were used for plasmid isolation for the LR reaction.

The expression clone pGWB433 with BrARF3-1cc promotor was transformed into E. coli after which a colony PCR was conducted for verification of the plasmid (Figure 16). All eight of the tested colonies yielded amplification of the BrARF3-1cc promotor. The cultures of positive colonies were used for plasmid isolation. The isolated pGWB433 BrARF3-1cc promotor vector was sent to GATC for sequencing with both the forward and reverse M13 sequencing primers. Sequencing with the M13 forward primer yielded a sequence which corresponds with the covered 5’ part of the

BrARF3-1cc promotor in alignment with the in silico constructed pGWB433 BrARF3-BrARF3-1cc promotor vector

(Appendix C ). Sequencing with the M13 reverse primers however, didn’t yield a sequence.

Figure 14 The attB-BrARF3-1cc promotor PCR product (1846 bp) after amplification with the attB extended

BrARF3-1cc

Promotor primers.

Figure 15 Colony PCR to verify the transformation of

pENTR221(BrARF3-1cc promotor) into E. coli with the

BrARF3-1cc promotor (1768 bp)

primer set. The tested colonies are numbered.

Figure 16 The colony PCR to verify the transformation of pGWB433(BrARF3-1cc promotor) into E. coli with the BrARF3-1cc promotor (1768 bp) primer set. The tested colonies are numbered.

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3.2. A. tumefaciens Transformation

Transformation experiments of B. rapa were conducted with two A.

tumefaciens strains, AGL1 and GV3101.

The A. tumefaciens strain AGL1 was transformed with the pGWB505 (with 35s:: BrARF3-1cc, 35s::BrARF3-1pc,

35s::BrAXR1cc, or 35s::BrAXR1pc.)

vector. The transformation of AGL1 with the various pGWB505 vectors has been verified by colony PCR with the

BrARF3-1 2F and 3R or BrAXR1 2F and

3R primers (Figure 17). Three colonies for each construct were tested except for the pGWB505 (BrAXR1cc) vector which only yielded two colonies. All the tested colonies yielded amplification of the insert, which confirms the

transformation of A. tumefaciens.

A. tumefaciens GV3101 was only

transformed with the

pGWB505(BrARF3-1cc) vector. Which was verified by PCR

with the hpt primer set (Figure 18). Only colonies two out of the seven colonies tested yielded amplification of the hpt gene. Colony three was picked for use in the transformation

experiments.

The transformation of A. thaliana was only conducted with the A. tumefaciens strain AGL1. This strain was

transformed with two different construct backbones, pK7WG2 and pGWB433. The transformation of A. tumefaciens with the pGWB433(BrARF3-1cc

Promotor) vector was verified by colony PCR with the BrARF3-1cc promotor primer set. (Figure 19). This resulted in the amplification of the BrARF3-1cc promotor in six out of eight of the tested colonies which verifies the presence of the pGWB433 vector. The transformation of A. tumefaciens AGL1 with pK7WG2(with 35s::

BrARF3-1cc, 35s::BrARF3-1pc, 35s::BrAXR1cc, or 35s::BrAXR1pc.)

was initially verified by PCR with the with the BrARF3-1 2F and 3R or

BrAXR1 2F and 3R insert specific

primers. However, the transformation efficiency when used in the floral dip transformation of A. thaliana was quite low. The first 20 selection plates with transformed seeds didn’t yield any transformants. In order to verify whether the pK7WG2 constructs were still present. The used glycerol stocks

Figure 17 Colony PCR to validate the transformation of A.

tumefaciens AGL1 with the pGWB505 binary vectors containing

the various inserts. PCR was conducted with insert specific primers, either the BrARF3-1 2F and 3R or BrAXR1 2F and 3R primers. the tested colonies are numbered.

Figure 19 Colony PCR with the BrARF3-1cc promotor (1768 bp) primer set to verify the transformation of A. tumefaciens AGL1 with the pGWB433(BrARF3-1cc promotor) binary vector. The tested colonies are numbered.

Figure 18 Colony PCR with hpt (199 bp) primers to verify the transformation of A. tumefaciens GV3101 strain with the pGWB505 (BrARF3-1cc) binary vector. The tested colonies are numbered.

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24 of A. tumefaciens AGL1 were streaked

on selection plates and a colony PCR was conducted with nptII kanamycin resistance primers (Figure 20). All of the tested colonies yielded amplification of the nptII gene which verifies the presence of the pK7WG2 vector.

3.3. B. rapa Transformation

The transformation experiments were performed on both R-o-18 and CC-285v, only cotyledonary explants were used with non-inoculated explants as control. The non-dipped explants were placed on media with selection agents to test for possible escapes on selection medium. Other controls included media without antibiotics and media with Timentin added as regeneration controls.

3.3.1.Direct Selection of R-o-18

During this experiment with direct selection, the inoculated cotyledonary explants of R-o-18 were transferred to selection medium with 15mg/l of hygromycin after co-cultivation. There was callus formation on 77 out of 260 cotyledonary explants. but there was also callus formation on the

non-inoculated control on selection medium, which could mean that the other calli were escapes. The calli were put on fresh medium with antibiotics after 4 weeks and most of the calli turned brown with the exception of one which turned a lighter shade of green but didn’t turn brown. The green callus was kept on selection medium until two shoots emerged which were transferred to rooting medium without hygromycin but with the addition of timentin to prevent overgrowth of potentially lingering A. tumefaciens.

Single leaf samples were taken from the putative transformants upon

transferring to rooting medium for molecular validation. The molecular validation was conducted via PCR with hpt primers and with primers for the virulence factor G (virG) of A.

tumefaciens to exclude false positive

Figure 20 Verification of the presence of the various pK7WG2 constructs in A. tumefaciens AGL1 by PCR of nptII (722 bp) with the nptII primer set.

Figure 21 PCR result of the putative B. rapa R-o-18 transformants, transformed with A. tumefaciens AGL1 pGWB505(BrARF3-1cc). Plasmid DNA of A. tumefaciens AGL1 pGWB505(BrARF3-1cc) was used as a positive control for both primer sets. Primers used are indicated: hpt primers (199 bp) and VirG primers (606 bp). The two tested plants are

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25 results. PCR of both the putative transformants resulted

in the production of virG amplicons, which verifies the presence of lingering A. tumefaciens on the shoots but makes it hard to verify whether the shoot is true transformed shoot. (Figure 21).

The putative transformants were kept on rooting medium until they were rooted and could be transferred to the soil into the greenhouse. The second shoot was lagging a bit in development behind the other shoot which was transferred to greenhouse earlier. A single leaf sample was taken from the putative transformant after recovering in the greenhouse from the transfer. This leaf sample was used for molecular validation in the same manner as the previous experiment. There was no amplification of hpt in this experiment and some faint bands after amplification of virG with diverging sizes which implicates aspecific amplification. This result suggests that this putative transformant does not carry the hpt gene for hygromycin resistance and is in fact an escape (Figure 22). The second shoot was also

transferred to the green house but was not yet verified via PCR due to the limited time of this thesis.

3.3.2.Progressive selection of putative transformants of CC-285v

In the progressive selection experiments the transformed explants were transferred to medium only containing timentin to repress the growth of A. tumefaciens. This way the transformed cells should have some time to regenerate and produce the hygromycin B phosphotransferase protein before the actual hygromycin selection.

Two different regeneration controls were used during this experiment. The first regeneration control didn’t have any antibiotics in the medium. This didn’t result in an 100% regeneration rate, only four out of the 10 explants used regenerated on the regeneration medium. The other

regeneration control was with the addition of timentin to the regeneration medium which is used to repress the growth of A. tumefaciens in the transformation experiments. This control was used to determine whether timentin has a negative effect on shoot regeneration of B. rapa. The explants on this plate with timentin (decolonization medium) appear to regenerate better than their counterparts on the medium without antibiotics. Not all of the regenerated shoots of the

regeneration controls stayed viable during the experiment and were combined after 11 weeks in a single tub of rooting medium. One of the rooting shoots started to flower in vitro after 13 weeks. The third non transformed control underwent the same treatment as the inoculated explants. This was to test whether the addition of hygromycin to the regeneration medium was still proficient in eliminating the already regenerating shoots. The low selective medium (5mg/l hygromycin)

repressed the development of the regenerating shoots, which started to brown. The non-inoculated CC-285v explants bleached completely after a further 2 weeks on the high selective (10mg/l hygromycin) medium.

The CC-285v cotyledonary explants which were inoculated with A. tumefaciens AGL1 harbouring the pGWB505(BrARF3-1cc) vector have started to form shoots after 3 weeks of in vitro culture with only 160mg/l timentin as antibiotic in the media. The progressive hygromycin selection of the inoculated CC-285v cotyledonary explants resulted in nine explants with still green callus out of 200 after 2 weeks of high selection. These greens calli were excised from the browned/bleached tissue and transferred to high selective medium again which reduced the number of explants with green callus to three. 4 weeks of high selection reduced the amount of explants with green callus to one. This last piece of callus bleached after another 2 weeks of high selection. This indicates that

Figure 22 PCR results of virG (606 bp) and

hpt (199 bp) of the putative B. rapa R-o-18

transformants, transformed with A.

tumefaciens AGL1 pGWB505(BrARF3-1cc).

Plasmid DNA of A. tumefaciens AGL1 pGWB505(BrARF3-1cc) was used as a positive control for both primer sets. Primers used are indicated: hpt primers, virG primers.

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26 even if the plant tissue is able to resist the selection, regeneration into a full shoot is still repressed under high selective pressure (10mg/l hygromycin) (Figure 23).

Figure 23 Transformation experiment 2 progressive hygromycin selection of CC-285v transformed with A.

tumefaciens AGL1 harbouring pGWB505(BrARF3-1cc). The progression in time is depicted in weeks after

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3.3.3.Progressive Selection of Putative Transformants of R-o-18

Experiment 3 (Figure 24) was conducted in similar fashion as the experiment with CC-285v but with R-o-18. The regeneration control without antibiotics of this experiment was contaminated with a bacterium after 5 weeks so this control was removed. The regeneration control with timentin yielded regeneration of all explants, but as with progressive selection experiment with CC-285v, not all the shoots couldn’t be kept viable during the whole experiment only having only 3 out of 10 explants left after 11 weeks of in vitro culture. The selection media were also successful in killing the non-transformed R-o-18 cotyledonary explants of this experiment even after partial

regeneration. The inoculated explants of experiment 3 yielded in 9 green shoots out of 110 R-o-18 cotyledonary explants after the first two weeks of high selection. these 9 green shoots were reduced to 5 slightly lighter coloured shoots after another 2 weeks of high selection (10mg/l

Figure 24 progression of Experiment 3, progressive hygromycin selection of R-o-18 transformed with A.

tumefaciens AGL1 harbouring the pGWB505(BrARF3-1cc) vector. The progression in time is depicted in weeks

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28 hygromycin). All the shoots completely bleached after 6 weeks on high selection medium (10mg/l hygromycin).

Experiment 5 (Appendix D ) is a repeat of experiment 3. In experiment 5 both the regeneration controls didn’t regenerate very well and almost completely browned after 7 weeks of in vitro culture. The inoculated explants of experiment 5 resulted in 10 green shoots after 2 weeks on 10 mg/l hygromycin selection, these shoots were slightly lighter coloured than the shoots regenerated from the transformed cotyledonary R-o-18 explants in experiment 3. After a further two weeks on 10 mg/l hygromycin selection all the still green shoots were bleached completely. It’s suspected that there was something faulty at the beginning of the experiment with either the co-cultivation of

Figure 25 Experiment 6 progressive hygromycin selection of R-o-18 cotyledonary explants transformed with A. tumefaciens AGL1 harbouring the pGWB505(BrARF3-1cc) vector with the addition of acetosyringone to the co-cultivation medium. The progression in time is depicted in weeks after sowing.

Functional Validation of BrARF3.1 and BrAXR1 in Brassica rapa and Arabidopsis thaliana