A non-tissue culture dependent genetic transformation of Marchantia polymorphya L.

doi:10.21203/rs.3.rs-2556225/v1

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A non-tissue culture dependent genetic transformation of Marchantia polymorphya L.

https://doi.org/10.21203/rs.3.rs-2556225/v1

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Marchantia polymorphya is one of the earliest terrestrial plant. As a new type of model plant, there are many advantages in plant biological researching, such as short growth cycle, haploid gametophyte dominance, sexual and asexual propagation simultaneously, and so on. In this study, a non-tissue culture dependent genetic transformation was developed for M. polymorphya. This method consists of immersion of explants (thalli or mature gemmae collected in field conveniently, without pre-culture) in solution of Agrobacterium, inoculation on soil simply, screening by spaying of selection solution directly. Over-expression of MpCCAAT-NFYB5(Mp6g07410.1) and corresponding changes of phenotype were observed. Furthermore, non-chimera transformant could be obtained rapidly through four cycles of gemmae reproduction. This method will facilitate further molecular biological works in this model plant.

Marchantia polymorphya

Agrobacterium tumefaciens

Non-tissue culture dependent transformation

non-chimera transformant

MpCCAAT-NFYB5

Marchantia polymorpha L. (Marchantiaceae), which is a member of the earliest land plant species and was recognized long ago (Bowman 2016), could be used as model plant material in many plant science researching works, such as to explore the mechanism of sexual reproduction (Hisanaga et al. 2019; Yamaoka et al. 2021), to reveal the developmental characteristics in cell level (Naramoto et al. 2022), and so on. M. polymorpha possesses many advantages for experimental operation in comparison with other model plant, such as easily culture in field (with soil) and in laboratory (in vitro culture), short life history, minor size for haploid body, propagules formation from sexual and asexual pathway, and so forth. Furthermore, publishings of whole genome sequences (Oda et al. 1992; Bowman et al. 2017) for M. polymorpha and for M. inflexa (Marks et al. 2019) and establishment of two open datbases — the MarpolBase for M. polymorpha Tak-1 strain and the MarpoDB for M. polymorpha Cam-1 strain (Delmans et al. 2017) — have promoted researches enormously.

Production of transgenic individuals could be carried out to satisfy humankind's requirements in agriculture and biological engineering or to explore gene function in research works. Genetic modified organisms should be obtained through various strategies or methods (Stewart, et al. 2011; Sathishkumar, et al. 2019). Genetic transformations by particle bombardment (microprojectile bombardment, or biolistic bombardment) and Agrobacterium-mediated methods have been reported in M. polymorpha with stable or transient expressions. Different genetic transformation by particle bombardment method in M. polymorpha adopted various plant materials as receptors: suspension-cultured cells (Irifune, et al. 1996; Nagaya, et al. 2011), whole thalli that originated from gemmae culture after two weeks (Takenaka, et al. 2000) or from spore culture after seven days (Chiyoda, et al. 2008), or fragment of mature thalli (gemmae culture with 2.5–3 weeks, Westermann, et al. 2020). Protocols (maybe with minor modifications) from Takenaka et al. (2000) had been employed to explore fatty acid metabolism (Kajikawa, et al. 2003; 2008) and to make mutant (Yamaoka, et al. 2004) in M. polymorpha. Because of convenient operations and low requirement in equipments, more attentions were paid to the Agrobacterium-mediated method, which was firstly applied in M. polymorpha by Nasu et al. (1997). All protocols based on Agrobacterium should be divided into two groups: infect of plant materials by co-cultured Agrobacterium in liquid system (Nasu, et al. 1997; Ishizaki, et al. 2008; Kubota, et al. 2013; Iwakawa, et al. 2021; Seo, et al. 2022) or on the surface of solid medium in Petri dish (AgarTrap: Agar-utilized Transformation with Pouring solutions, Tsuboyama, et al. 2014; Tsuboyama-Tanaka and Kodama, 2015; Tsuboyama-Tanaka, et al. 2015; Tsuboyama and Kodama 2018). Various plant materials, which were suspension-cultured cells, whole thalli from spore (Sporelings) or gemmae (gemmalings), or piecies from immature or mature thallus, also could be acted as receptors in genetic transformation of M. polymorpha. Differential protocols of Agrobacterium-mediated genetic transformation were applied in various researching works, such as to reveal characterisitics of auxin transcriptional response system (Flores-Sandoval, et al. 2015) and to establish genome editing method (Sugano, et al. 2018a; 2018b) in M. polymorpha.

More efficiently transformation protocols independent on plant tissue culture were established, such as floral-dip transformation of Arabidopsis thaliana (Davis, et al. 2009), ovary-injection transferring of Paphiopedilum Maudiae (Luo, et al. 2020), in planta transformation of Notocactus scopa cv. Soonjung (Seol, et al. 2008), Kalanchoe pinnata (Jung, et al. 2009), and so on. Recently, Cao et al. (2022) developed the CDB (cut-dip-budding) genetic delivery system and transformed six kinds of plant species without involve sterile tissue cultures on Petri dish. However, all protocols for genetic transformation of M. polymorpha (based on particle bombardment or Agrobacterium mediation) also involve in vitro culture to this day. Therefore, a troublesome pollution that is followed co-culture with Agrobacterium also impedes works frequently.

To avoid the troublesome content (i.e. tissue culture sterilely on Petri dish) and to make genetic transformation more simply and effectively in M. polymorpha, in this study, we test the genetic delivery directly into thalli or gemmae that are collected from soil culture individuals. Our results show effective genetic modifications could be carried out without tissue culture on Petri dish. These findings should facilitate molecular researching works of M. polymorpha in future.

Culture under selection condition could facilitate identification of positive individuals in genetic transformation. To verify selection condition independent on tissue culture, test of kanamycin sensitivity for M. polymorpha was carried out on soil culture or solution culture firstly. Gemmae (i.e. nascent or developing thalli) under solution culture condition are more sensitive than individuals (i.e. rapid growth thalli) under soil culture condition: for solution culture, all gemmae died with 1.0 mg·ml^− 1 kanamycin; for soil culture, however, all thalli expressed normal growth with 1.0 mg·ml^− 1 kanamycin and died with 11.5 mg·ml^− 1 kanamycin (Table 1, Fig. 1).

Table 1

Survival of *M. polymorpha* under different selection conditions
Content of kanamycin (mg·ml^− 1)		0	0.25	0.5	0.75	1.0	2.5	5.0	7.5	9.0	10.0	11.5
Death rate (%)	Soil cult.	0	–	–	–	0.0	0.0	0.2	2.8	67.4	94.9	100
Death rate (%)	Solution cult.	0	24.1	69.1	87.9	100	–	–	–	–	–	–

– means no test for soil culture or solution culture; numbers involved in test for soil culture and solution culture were 175 and 106, respectively.

Explants (cutting pieces of thalli or mature gemmae) were transformation directly through immersion simply, or through negative-pressure infiltration with immersion in injector. Plant materials that were treated with Agrobaterium were cultured on soil for two weeks, and then were selected for next two weeks by spraying of selection buffer (11.5 mg·ml^− 1 kanamycin) once every two days. Effective transformation of M. polymorpha could be achieved through these simply protocols (Fig. 2, Table 2). For ratio of survival after selection, there was not significant differences among differential explants, between methods of treatment, or among various treatment times. However, for all kinds of explants, higher ratio of transformant were obtained by negative-pressure infiltration than by immersion simply; and more time of treatment shuold reslulted in higer ratio of transformant by each of these two kinds of treatment (Table 2). These transformants (T₀ generation) are chimeras that were determined by GUS staining (Fig. 2I and J).

Table 2

Genetic transformation of *M. polymorpha* by tissue culture independent*
Treatments Explants	Times of direct immersion			Times of infiltration with injector
Treatments Explants	7 min	10 min	15 min	7 min	10 min	15 min
Thalli (3 w)	66/0/0	84/26.2/31.8	81/19.8/37.5	120/33.3/25.0	120/31.7/31.2	120/26.7/53.1
Thalli (4 w)	72/40.3/6.9	72/63.8/15.2	72/37.5/22.2	120/20.8/24.0	120/49.2/32.2	120/60.8/58.9
Gemmae	240/11.3/11.1	240/23.7/15.8	240/23.7/19.3	394/22.1/24.1	411/25.1/30.1	407/29.9/41.8

*data in table are numbers of explants / ratio of survival after selection / ratio of transformant identified by PCR. Ratio of survival (%) = (No. of survival / No. of transformation) × 100. Ratio of transformant (%) = (No. of PCR positive / No. of survival) × 100.

Transformants (T₀) that was transformed from differential explants (three of four weeks-old thalli or mature gemmae) show three kinds of phenotypic changes as follows. Type I: more than two of new thalli simultaneously develop from the edge of the same old thallus instead of dichotomous branching (Fig. 3A); these new thalli could grow to enough size and be transplanted on soil for vegetative reproduction. Type II: new thalli development are based on normal dichotomous branching, however, various number of swelling structure (amorphous tissue) develop on the upper surface of thalli; these swelling structure could grow to form curling flat structure and could not be transplanted (Fig. 3B). Type III: new thalli develop on the tip of a stalk that is originated from apical meristem; new disk-like thalli are peltate and grow in a polarity manner (Fig. 3C); these new thalli could also be transplanted. Gemmae development in gemmae cup occurs on upper surface for these three kinds of transformants. Expressions of MpCCAAT-NFYB5 gene for all T₀ transformants that possess phenotypic changes are higher than wild type: Type III > Type II > Type I (Fig. 3D ~ F).

To obtain non-chimera transformants, sub-cultures of gemmae continuously were carried out. For T_n+1 generation (n = 0 ~ 3), four times of sub-culture repeatedly were performed. Gemmae (80 or so), which were used to one repeat, were collected from one gemmae cup of a T_n (n = 0 ~ 3) generation individual. Gemmae for different repeat were collected from different mother plant. For T₅ generation, four gemmae cups that were used to collect gemmae for each of four sub-cuculture repeats were originated from the same of T₄ mother individual. These mother plants were produced by an individual of former generation, and developed gemmae cup and gemmae firstly among its sister individuals. For T_n (n = 2 ~ 4) generation sub-culture, a part of individuals of one repeat survived under selection condition, showed amplification band in PCR, and showed local staining of GUS detection (Fig. 4A ~ C, E ~ G). These results showed that all individuals (T_n, n = 1 ~ 3) are chimeras. For T₅ generation sub-culture, however, all individuals of four repeats survived under selection condition and showed amplification band in PCR (positive of PCR identification). Furthermore, the GUS staining showed whole painting (Fig. 4D, H). These results indicated that a non-chimera transformant was obtained in T₄ generation.

Screening by selection buffer should decrease amounts of works that are required for transformants identification, such as total DAN extraction, PCR, electrophoresis, and so on. In all of works reported previously, which were aimed to genetic transformation through method of Agrobacterium mediated transformation and particle bombardment, operations were based on tissue culture. Hygromycin B, which was added in solid media with 10µg·ml^− 1 or pouring on solid media (1 ml of solution with 100µg·ml^− 1 for each Petri dish), was usually used as selection antibiotic in genetic transformation of M. polymorpha (Takenaka, et al. 2000; Ishizaki, et al. 2008; Kubota, et al. 2013; Tsuboyama and Kodama 2018). Although hygromycin was 20 times more toxic than kanamycin and should be more effective in selection (Padilla and Burgos 2010), kanamycin also should be used in selection of M. polymorpha (Yao 2022). Our tests about sensitivity of M. polymorpha to kanamycin showed that the lethal concentration (LC) is 1.0 m g·ml^− 1 for solution culture with 1/2 B5 medium, and is 11.5 m g·ml^− 1 for soil culture (Table 1). The high LC of kanamycin for soil culture could resulted from natural degradation of antibiotic molecules, absorption of soil particles, high resistant of growing plant body, and so on. Although high concentration of kanamycin is needed for selection of transformants in our works, effective selection could be achieved by the simple operation without involving in sterile tissue culture (Fig. 1).

Pre-culture of plant materials should be carried out to satisfy genetic transformation for M. polymorpha (Nasu, et al. 1997; Ishizaki, et al. 2008; Tsuboyama and Kodama 2018). However, collections of thalli or gemmae could be treated with Agrobacterium directly. Co-culture of explants and Agrobacterium, which is one important step of Agrobacterium mediated transformation, could result in excessive growth of Agrobacterium and abolish genetic transformation frequently because of enough nutrients supplement with medium. Co-culture through solution culture (1/2 B5 medium) in our works is similar to co-culture through tissue culture based on support of artificial medium. Even though no sucrose supplement, algae could be propagated rapidly and make trouble for subsequent operations because of non-sterile culture environment. Otherwise, co-culture though soil cultivation in our works could be carried out without retardation from Agrobacterium and algae. This situation should be resulted from little nutrients supplement by soil than artificial medium, or resulted from sophisticated interplay between various organisms in soil. Normal growth in co-culture and selection steps without disturbance of Agrobacterium and algae facilitate subsequent operations enormously. Effective transformation of MpCCAAT-NFYB5 (Mp6g07410.1) was achieved under these simple conditions. Over-expression of MpCCAAT-NFYB5 resulted in changes of phenotype (Fig. 3D ~ F). Modifications of new thalli formation in MpCCAAT-NFYB5 transfomants (Fig. 3A and B) are similar to formation of multiple leafy cotyledons in LEC1 (Leaf Cotyledon1) transgenic plants in Arabidospsis thaliana (Lotan, et al. 1998). These results indicate MpCCAAT-NFYB5 regulates cell division in thalli formation of M. polymorpha.

Chimeras in plant genetic transformation occur in operations when multi-cellular explants act as receptors for exogenous gene. To avoid problems resulted from chimera, non-chimera transformants should be produced through two strategies: single cell (i. e. protoplast or pollen) transformation and continuous selection of off-spring that are reproduced sexually from chimera transfromants resulted form genetic transformation with multi-cellular explants (Weigel and Glazebrook 2002; Stewart, et al. 2011). For M. polymorpha, transformations of suspension-cultured cells were explored by particle bombardment (Irifune, et al. 1996; Nagaya, et al. 2011) or Agrobacterium mediated method (Nasu, et al. 1997). Transformation of spores for M. polymorpha also could produce non-chimera transgenic plants. However, developing spores (sporelings), which is a nascent thallus containing many cells in fact, were used as explants other than single cell (Ishizaki, et al. 2008; Chiyoda, et al. 2008; Tsuboyama and Kodama 2014). Our results show that non-chimera transformant could be obtained rapidly from transformation of multi-cellular explants (i.e. thalli or gemmae) and subsequent selection of asexual off-springs (gemmae, Fig. 4).

In summary, a simplified transformation method for M. polymopha based on soil culture was developed in our works. This method consists of immersion of explants in solution of Agrobacterium (without or with negative pressure treatment by hand), inoculation on soil, screening by spaying of selection buffer. No pre-culture of explants (thalli or mature gemmae) that are collected conveniently, non-tissue culture dependence, and acquisition of non-chimera rapidly should simplify operations of genetic transformation in M. polymopha. This method will facilitate further works on the model plant.

Plant material and growth conditions

M. polymorpha were collected from wild (Huaxi, Guiyang, China. 106.6592°E, 26.4496°N; Alt. 1050 m) and were cultivated in garden with moderate shedding. Thalli that are cultured three or four weeks and mature gemmae that could disperse from gemma cups spontaneously were excised and collected respectively to genetic transformation.

Sensitivity test of M. polymorpha to kanamycin

Tests of sensitivity to kanamycin were carried out on the basis of soil culture or solution culture. For soil culture strategy, individuals from sowed gemmae on soil that was sterilized to prove without M. polymorpha propagules containing were treated with selection buffer (contained cefotaxime and various contents of kanamycin) after two weeks culture. Various selection buffer possess cefotaxime (1.0 mg·ml^− 1) and various kannamycin (1.0, 2.5, 5.0, 7.5, 9.0, 10.0, or 11.5 mg·ml^− 1). Sprayings of screen solution on soil and plants surface were carried out once every two days. For solution culture strategy, gemmae were cultured in liquid 1/2 B5 medium supplement without sucrose and with antibiotics (1.0 mg•ml^− 1 cefotaxime and 0.25, 0.50, 0.75, or 1.00 mg·ml^− 1 kannamycin). Numbers of survival individuals for all treatments were counted after two weeks treatments with various selection buffers.

Plasmid and A. tumefaciens strains

The plasmid PBI121 that possess kanamycin resistant (selection marker) and GUS gene (expression label) was used as a binary vector for carrying MpCCAAT-NFYB5 (Mp6g07410.1), which is a transcriptional factor from nuclear factor Y family, under the control of a cauliflower mosaic virus 35S promoter (Yao 2022). Competent state cells of A. tumefaciens strain GV3101 were prepared (Tu, et al. 2005). The transformation of PBI121-MpCCAAT-NFYB5 into bacterial cells was carried oout through the liquid nitrogen freeze-thaw method (Wise, et al. 2006 ). The bacterium single colony was selected to liquid culture firstly in YEP liquid medium (5 ml system), and then verified transformation by vector through PCR. The verified positive bacterium (1 ml solution) was then transferred into 50 ml YEP liquid medium in flask, suspension culture for bacteria collection: 6 ~ 8 h at 28°C with 250 rpm to OD₆₀₀ = 0.5 ~ 0.6. Bacteria deposit after centrifugation (6000 rpm, 10 min, RT) was re-suspended in transformation buffer at OD₆₀₀ = 0.5.

Agrobacterium -mediated direct transformation

Cutting pieces of thalli (three or four weeks culture from mature gemmae on soil) or mature gemmae were used as explants for directly through immersion simply in transformation butter containing bacteria, or through negative-pressure infiltration treatment with injector after immersion immediately by hand (Fig. 6). Immerged plant materials were transferred on filter paper to suck up liquid on surface. Treated plant materials were transferred and inoculated on the sterilized soil in cups. After three days of dark culture and a week (for thalli pieces) or two weeks (for gemmae) of light culture in garden, growing individuals were screened by two-weeks spraying of selection buffer that contains cefotaxime (1.0 mg·ml^− 1) and kannamycin 11.5 mg·ml^− 1) once every two days. Surviving plants were transplanted with new soil to grow 4 ~ 6 weeks continuously.

Identification of transgenic plant by PCR and GUS staining

Surviving plants grown enough large were used to total DNA extraction by CTAB method (Rogers and Bendich 1998). PCR amplification for GUS gene was carried out in 25 µL reaction system using Es Taq MasterMix (Cat^# CW0690M, Biopike co.) supplement with template (0.25 µg), sense primer (0.4 µM, 5′-TCTGCGACGCTCACACCGAT-3′), and antisense primer (0.4 µM, 5′-GCCAACGCGCAATATGCCTT-3′). Amplification conditions was listed as follows: 94°C for 4'; 34 cycles of 94°C for 30", 59°C for 30", and 72°C for 2'; final extension at 72°C for 10'. PCR products were detection through electrophoresis with agarose gel (1%). Positive individuals in PCR identification were selected to GUS staining (X-Gluc, Cat^# G3060, Solarbio Co., Jefferson et al. 1987). Transformed individuals (T₀ generation) that are positive in PCR identification and GUS staining were designated specific material numbers and were cultured to characterize phenotype and to produce new gemmae for further tests.

Continuously Culture To Produce Non-chimera Individuals

Transformed individuals of T₀ generation are generally chimeras. Culture of M. polymorpha continuously based on asexual life cycle (from gemmae to thalli repeatedly) was used to produce non-chimera individuals. Thalli (T_n+1), which were developed from gemmae that produced by thalli (T_n) and grow under selection conditions (as above), were supplied to extract of total DNA and to identify transformed status. For one thallus/individual (T_n), determination of its transformed status was based on next generation gemmae/thalli (T_n+1) from four gemma cups (at least twelve gemmae for each cup). If one thallus/individual is transformed without chimera, its all offspring should be survival under selection conditions, and be positive in PCR identification, and should be whole stained instead of local staining in GUS detection.

Quantification of MpCCAAT-NFYB5

Thalli of individuals that are grown from gemmae on soil about four weeks were used to total RNA extraction by MolPure® Plant RNA kit (Cat^# 9109, TakaRa). Then, cDNA synthesized by Hifair® 1st Strand cDNA Synthesis Kit (gDNA digester plus) reagent Kit from YEASEN (Cat^# 11139ES60) was used to quantify expression of MpCCAAT-NFYB5. qRT-PCR was performed on Bio-Rad CFX96 instrument with ChamQ Universal SYBR qPCR Master Mix reagent (Cat^# Q711-02, Vazyme) according to manufacturer's instructions in 20 µL reaction system. MpAPT (adenine phosphoribosyl transferase, Mp3g25140) is selected as internal reference for quantification of MpCCAAT-NFYB5. Primers for qRT-PCR are listed as follows: sense primer (0.2 µM, 5′- CGAAAGCCCAAGAAGCTACC-3') and antisense primer (0.2 µM, 5′- GTACCCCCGGTTGCAATAAG-3′) for MpAPT (product length: 150 bp); sense primer (0.2 µM, 5′- ATTCTCCCAAAGCAGGTGGG-3′) and antisense primer (0.2 µM, 5′-GTACGGAGGATGCAGATACACC-3′) for MpCCAAT-NFYB5 (product length: 150 bp). Each analysis includes three biological repeats and three technical replicates. Amplification condition was listed as follows: 95℃ for 30'; 40 cycles of 95℃ for 5', 60℃ for 30'.

Supplementary Data

Supplementary data are available at Plant Molecular Biology online.

Data Availability

The data underlying this article are available in the article and in its online supplementary material.

Funding

This work was supported by the National Natural Science Foundation of China (grant No. 31960067 and 31560081).

Disclosures

The authors have no conflicts of interest to declare.

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A non-tissue culture dependent genetic transformation of Marchantia polymorphya L.

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Abstract

Figures

Introduction

Results

Discussion

Materials And Method

Plant material and growth conditions

Continuously Culture To Produce Non-chimera Individuals

Declarations

References

Status:

Version 1