Genetic transformation of moss plant

Bryophytes are among the simplest and oldest of the terrestrial plants. Due to the special living environment and characteristics, bryophytes have become attractive experimental tools for the elucidation of complex biological processes in plants. Mosses grow rapidly when cultured on simple salt media, thereby making them ideal materials for metabolic studies, particularly transformation and gene homologous recombination. Thus, in recent times mosses such as Physcomitrella patens, Funaria hygrometrica, Ceratodon purpureus, and Tortula ruralis are being developed for genetic engineering studies. Recently, the finding of efficient homologous recombination of P. patens and yeast and murine cells could be comparable. So, the moss, P. patens has become an extremely powerful organism for the functional analysis of plant genes. In this report, we reviewed recent research progress so far made on genetic transformation of the bryophytes with emphasis on P. patens. We also discussed the advantages and limitation of bryophytes transformation.


INTRODUCTION
Bryophytes, small green land plants, are among the oldest terrestrial organisms and appeared 4.5-5 million years ago.It comprises three groups: Mosses, hornworts, liverworts.Bryophytes are higher non-vascular plants which show an alternation of generations.The life cycle *Corresponding author.E-mail: zhq_he@163.com.Tel: +86-717-6397188.
With the strong regeneration and short life cycle, mosses are ideal model systems for the study of plant.Any part of moss can be regenerated into protonemal, and then grow into gametophyte.The regeneration process does not need to go through dedifferentiation, but directly differentiate into protonemal.The diploid gametophyte, which is predominant in the life cycle, is also useful for mutant screening and genetic analysis, making mosses attractive for genetic studies.The moss species Physcomitrella patens is considered suitable for studying plant model system due to: 1.The relative simplicity of the species, 2. The common characteristics with higher plants, example phytohormones (Ashton et al., 1985), hormone response mechanisms (Knight et al., 1995), phytochrome and phytochrome-controlled photomorphogenesis (Cove et al., 1978;Kolukisaoglu et al.,1993), 3. The simple axenic propagation under control conditions (Knight et al., 1988) and, 4. The amenableness to study using genetic (Ashton et al., 1988), physiological and cytological (Doonan and Duckett, 1988) methods (Ashton et al., 2000).

LIFE CYCLE
Mosses, like most plants, show an alternation of generations comprising of a free-living haploid gametophyte stage and a diploid Sporophyte stage, with the Sporophyte being parasitic on the gametophyte.The Sporophyte produces spores by meiosis that germinates to produce further gametophytes.In this cycle, spores germinate to form protonemal tissue, consisting of simple cell filaments.The protonema is formed by chloronemal filaments packed with large chloroplasts and extend by serial division of their apical cells, with a cell cycle of about 24 h while the sub-apical cells of chloronemal filaments undergo a limited number of divisions to form side branches.The apical cells can transform to divide to caulonemal filaments.This caulonemal filament is another protonemal, with fewer cells and less Chloroplasts like the chloronemal, caulonemal filaments extension also undergoes serial division of the apical cells, but has a short life cycle of only 6 h.Besides, the linear extension rate of the caulonemal cell is as rapid as 40 μm/h, but only 2 μm/h for chloronemal filaments, allowing for rapid growth in the soil and agar surface (Cove and Knight, 1993).The sub-apical cells of caulonemal filaments can produce side branches via limited division and may develop into chloronemal filaments under high light conditions, while others develop into caulonemal filaments or buds.Then, the buds produce leafy gametophytes with gametangia and gametes (Zhao et al., 2004).In the genera Funaria and Physcomitrella, female and male gametes are produced by the same plant whereas; in Ceratodon there are separate sexes.Figure 1 summarizes the life cycle of mosses, illustrating some of the main stages in Physcomitrella (Cove, 2000).

IMPORTANCE OF TRANSGENIC MOSS
The moss, especially Physcomitrella patens, has recently become a powerful genetically tractable model plant system.With its efficient gene targeting (Schaefer and Zryd, 1997;Strepp et al., 1998), the P. patens have been used to study the basic developmental mechanisms of land plants, particularly flowering plant (Prigge et al., 2010).It also applied to analyze gene function (Brucker et al., 2005;Schaefer et al., 1997;Strepp et al., 1998) and express many complex secreted eukaryotic proteins  (Decker and Reski, 2007).The transgenic plants have a low-cost and safe pathogen-free advantage of producing complex recombinant pharmaceutical proteins.However, the high plants specific protein N-glycosylation was reported to be immunogenic in biopharmaceutical production (Faye et al., 2005).The moss, with the strong regeneration and feasibility of targeted gene replacements, has non-immunogenic humanized glycan patterns for recombinant biopharmaceuticals (Decker et al., 2008).

Protoplasts used for the transformation
Plant protoplasts which had its cell walls removed offer a versatile cell-based experimental system (Yoo et al., 2007).The cell wall is degraded by digestion with different mixture of hydrolytic enzymes example cellulase, hemicellulase.Protoplasts are used to create hybrid cells via protoplast fusion.In some cases they are totipotent, and are thus capable of regenerating into plant on appropriate culture medium.Such protoplasts can be used in plant transformation.Mosses, unlike other plant protoplasts, do not need phytohormones for regeneration, nor do they form a callus during regeneration.Instead, they regenerate directly into the filamentous protonemal thus mimicking a germinating moss spore.

Polyethylene glycol (PEG)-mediated transformation
The first stable genetic transformation of plant cells was reported in tobacco by Krens et al. (1982), who used the method of polyethylene glycol-mediated Ti-plasmid DNA uptake of protoplasts.The first successful transformation of moss was achieved by the transfer of resistance gene by polyethylene glycol (PEG)-mediated DNA uptake of protoplasts of the moss P. patens (Schaefer et al., 1991) (Table 1), a technique which is widely used for the transformation of seed plants.With slight modification, this technique could also be used for genetic transformation of the moss Ceratodon purpureus.Furthermore, the technique has also been used for the generation of targeted knockout mutants.Thus, PEGmediated DNA transfer has been selected for the construction of a high-throughput transformation system for both higher plants and mosses (Schaefer et al., 1991;Potrykus and Spangenberg, 1995;Zeidler et al., 1999).However, the efficiency of transformation using this technique is low, about 0.5 -1 transformant per μg DNA.Besides this low efficiency, PEG-mediated transformation requires more extensive tissue and protoplast manipulation, complicated procedure and high efficiency regeneration of protoplasts which is obtained under scrupulously sterile conditions (Šmídková et al., 2010).Table 1 shows PEG-mediated transformation of some species of mosses.

Microprojectile bombardment transformation
An alternative method widely used for transformation of higher plants and mosses is particle bombardment.This technique has successfully being used for the transformation of P. patens (Sawahel et al., 1992) and Marchantia polymorpha cells (Irifune et al., 1996).Biolistic transformation has several advantages over PEG-mediated transformation mainly because it can shorten the procedure to a minimum thereby avoiding microbial contamination.The biolistic transformation efficiency depends to a large extent on the form of DNA.Šmídková et al. (2010) reported that particle bombardment of tissues with 1 μm gold particles generated about 20 to 40 stable transformants per 1 μg DNA.They further reported that transformation of circular plasmids yielded higher frequencies of random transgene integration, and linear plasmids are even more efficient in targeted transformation using particle bombardment.

Agrobacterium-mediated transformation
Although Agrobacterium-mediated transformation was the method of choice for the genetic transformation of seed plants, mosses are not often amenable to Agrobacterium infection.However, report on Agrobacterium-mediated transformation of the Marchantia polymorpha by -Nasue et al. (1997) did not obtain intact transgenic plants using Agrobacteriummediated transformation.Contraily, Ishizaki et al. (2008) Jing et al. 229 established an efficient and reproducible Agrobacteriummediated transformation protocol for M. polymorpha using immature thalli .In Ishizaki et al. (2008) infected immature thalli developed from spores directly by Agrobacterium, and achieved high transformation efficiency.The high transformation efficiency could be attributed to the direct infection of the immature thalli with the Agrobacterium without going through protoplast isolation and regeneration steps in the transformation process.They further reported that acetosyringone is essential for Agrobacterium-mediated T-DNA transfer into M. polymorpha cells.The duration of pre-culture and cocultivation also have significant effects on the transformation efficiency of M. polymorpha.

TYPES OF REGENERANTS
All the methods used for transformation of moss yielded three types of regenerants after selection for plasmidencoded antibiotic resistance.These are transient, weak, and strong resistance phenotypes.Some transformed cells lose resistance and were probably expressing resistance transiently.Although others continue to show resistance, the resistance is weak and are lost when the transgenic moss are transferred to nonselective medium after about 14 days of growth.These transgenics were therefore termed unstable and were likely to replicate plasmid DNA outside of the chromosome (Knight, 1994).
In spite of these negative results, few transgenic mosses grew strongly on selective medium and when transferred to nonselective medium, they maintained their resistant phenotype with Mendelian pattern of inheritance (Schaefer et al., 1991).These transgenics were termed stable and confirmed the integration of the plasmid at a genomic locus (Ashton et al., 2000).According to Cove (2000) multiple copies of the plasmid DNA integrated at the same locus and in the same orientation.

TRANSFORMATION EFFICIENCIES
The transformation frequencies of moss as reported in literature varied according to the method employed.Both the PEG-mediated and particle bombardment transformation method yielded almost the same frequency of transgenic moss.It has been estimated that PEG-mediated DNA uptake procedure requires more DNA (routinely 20 μg per transformation) while the particle bombardment required only 2 shots per transformation and 1 μg per shot (Cove, 2000).Consequently, the transformation efficiencies, defined as transgenic per ug of DNA by biolistic bombardment are higher than PEG-mediated method (Cove, 2000).The frequency of transformation by Agrobacterium tumefaciens in the presence of acetosyringone was reported to be 10% in M. polymorpha (Nasue et al., 1997).A study by Ishizaki et al. (2008) reported that hundreds of hygromycin-resistant plants per sporangium were obtained by co-cultivation of immature thalli of liverwort Marchantia polymorpha with Agrobacterium carried a binary vector.

HOMOLOGOUS RECOMBINATION
One of the most important characteristic of P. patens is its ability to integrate foreign DNA into its genome by homologous recombination at a very high rate than other plants (Schaefer et al., 1997).This characteristic which could make gene knockout possible could be used to analyze gene functions.Schaefer and Zryd (1997) demonstrated that the efficiency of gene targeting evaluated in their transformation experiments which designed to target three single-copy genomic loci in P. patens was above 90%, an observation similar to that of yeast.They also reported that the transformation efficiency was higher than previous reports on gene targeting in plants.These observation suggests that haplobiontic moss is the first example from the plant kingdoms which could be used to gene knock-out; previously this method was only applied in lower eukaryotes such as protozoa, yeasts and filamentous fungi.Strepp et al. (1998) disrupted the ftsZ gene which was isolated from the eukaryote Physcomitrella patens.The ftsZ gene encoding a homolog of the bacterial cell division protein FtsZ, an ancestral tubulin.It controls the chloroplast division in the P. patens.Seven out of 51 transgenics obtained were knockout plants generated by homologous recombination; they were specifically impeded in plastid division with no detectable effect on mitochondrial division or plant morphology.So, this was proved the effect of the FtsZ gene in the chloroplast division.
The reduction of adenosine 5'-phosphosulfate (APS) to sulfite catalyzed by adenosine 5'-phosphosulfate reductase (APSase) is considered to be the key step to the understanding of sulfate assimilation in higher plants.However, analogous to enteric bacteria, an alternative pathway of sulfate reduction via phosphoadenosine 5'phosphosulfate (PAPS) had been proposed in higher plants.To date, the presence of the corresponding enzyme, PAPS reductase, could neither be confirmed nor rejected in plants.Koprivova et al. (2002) disrupted the adenosine 5'-phosphosulfate reductase (APSase) single copy gene in P. patens by homologous recombination resulting in complete loss of the correct transcript and enzymatic activity.
The moss P. patens is thus the first plant species in which PAPS reductase had been confirmed on the molecular level; it is also the first organism where both APS-and PAPS-dependent sulfate assimilation co-exist (Koprivova et al., 2002).

Jing et al. 231 THE EXTANT PROBLEMS AND PREDICTION
In general, the transformation frequencies of mosses have been reported to be higher than other plants, nevertheless they have not reached the levels achieved in eukaryotic microbes such as yeasts.Although, the frequency of transformants achieved is on the ascendancy, and the reliability of the transformation procedure is improving too, efforts should be made to continue to find perfect the conditions that will improve both frequency and reliability of the methods used to achieve transgenic moss (Cove, 2000).Now the research platform with mosses has not been completed.However, a genetic linkage map has been yielded by crossing a French ecotype to a British ecotype (Kamisugi et al., 2008).We need more time to establish the cDNA library and genome DNA, which could be identified by using next-generation sequencing technologies (Prigge et al., 2010).
The mechanism of homologous recombination rate with P. patens has not yet been researched enough.The P. patens genome can only represent the portion of the terrestrial plant, which has not been studied by gene knockout.Thus, the mechanism of homologous recombination rate should be investigated.Mosses, especially P. patens, are the most attractive to study due to their simplified molecular biology characteristics, compared to higher plants.The development of transgenic studies in moss could make them model plants for genetic studies and also play a catalytic role in molecular biology in plants similar to that of Arabdopsis.

Figure 1 .
Figure 1.The life cycle of P. patens.All stages are haploid except the zygote and the Sporophyte.