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The advantageous provided by chloroplast DNA manipulation has seen growing interest into this field of research and development, particularly in agricultural and pharmaceutical applications.<ref name=":1" /> However, there are some disadvantageous in chloroplast DNA manipulation, such as the inability to manipulate [[Cereal|cereal crops]] DNA material and poor expression of foreign DNA in non- green plastids as mentioned before.<ref name=":1" /> Nevertheless, much progress has been made into plant transplatomics, for example, the production of edible vaccines for [[Tetanus]] by using a transplastomic [[Nicotiana|tobacco]] plant.<ref>{{cite journal | vauthors = Tregoning J, Maliga P, Dougan G, Nixon PJ | title = New advances in the production of edible plant vaccines: chloroplast expression of a tetanus vaccine antigen, TetC | journal = Phytochemistry | volume = 65 | issue = 8 | pages = 989–94 | date = April 2004 | pmid = 15110679 | doi = 10.1016/j.phytochem.2004.03.004 }}</ref>   
The advantageous provided by chloroplast DNA manipulation has seen growing interest into this field of research and development, particularly in agricultural and pharmaceutical applications.<ref name=":1" /> However, there are some disadvantageous in chloroplast DNA manipulation, such as the inability to manipulate [[Cereal|cereal crops]] DNA material and poor expression of foreign DNA in non- green plastids as mentioned before.<ref name=":1" /> Nevertheless, much progress has been made into plant transplatomics, for example, the production of edible vaccines for [[Tetanus]] by using a transplastomic [[Nicotiana|tobacco]] plant.<ref>{{cite journal | vauthors = Tregoning J, Maliga P, Dougan G, Nixon PJ | title = New advances in the production of edible plant vaccines: chloroplast expression of a tetanus vaccine antigen, TetC | journal = Phytochemistry | volume = 65 | issue = 8 | pages = 989–94 | date = April 2004 | pmid = 15110679 | doi = 10.1016/j.phytochem.2004.03.004 }}</ref>   


== Transformation technology and selection ==
== Transformation and selection procedure ==


=== Gene construct ===
=== Gene construct ===
The first requirement for transplastomic plant generation is to have a suitable [[Gene cassette|gene construct]] that can be introduced into a plastid like a chloroplast. There are several key features of a suitable gene cassette including but not limited to ('''1''') [[selectable marker]] ('''2''') flanking sequences ('''3''') gene of interest ('''4''') [[Promoter (genetics)|promoter sequences]] ('''5''') [[5' UTR]] ('''6''') [[3'-UTR|3' UTR]] ('''7''') [[Intergenic region|intercistronic elements]].<ref>{{cite journal | vauthors = Verma D, Daniell H | title = Chloroplast vector systems for biotechnology applications | journal = Plant Physiology | volume = 145 | issue = 4 | pages = 1129–43 | date = December 2007 | pmid = 18056863 | pmc = 2151729 | doi = 10.1104/pp.107.106690 }}</ref> Flanking sequences are crucial for introduction of the gene construct at precised determined points of the plastid genome through [[homologous recombination]].<ref name=":2" /> The gene of interests introduced have many different applications and can range from pest resistance genes to vaccine antigen production.<ref name=":2" /> Intercistronic elements (IEE) are important for facilitating high levels of gene expression if multiple genes are introduced in the form of an [[operon]].<ref name=":2" /> Finally, the 5' UTR and 3' UTR enhances ribosomal binding and increases transcript stability respectively.<ref name=":2" />
The first requirement for transplastomic plant generation is to have a suitable [[Gene cassette|gene construct]] that can be introduced into a plastid like a chloroplast in the form of an ''[[Escherichia coli|E. coli]]'' [[plasmid]] vector.<ref name=":02">{{Cite web|url=http://www.eurekaselect.com/72509/article|title=Plant Plastid Engineering|last=Wani|first=Shabir H.|last2=Haider|first2=Nadia|date=2010-10-31|website=Current Genomics|language=en|doi=10.2174/138920210793175912|pmc=PMC3048312|pmid=21532834|access-date=2020-04-16|last3=Singh|first3=Hitesh Kumar and N. B.}}</ref> There are several key features of a suitable gene cassette including but not limited to ('''1''') [[selectable marker]] ('''2''') flanking sequences ('''3''') gene of interest ('''4''') [[Promoter (genetics)|promoter sequences]] ('''5''') [[5' UTR]] ('''6''') [[3'-UTR|3' UTR]] ('''7''') [[Intergenic region|intercistronic elements]].<ref>{{cite journal|vauthors=Verma D, Daniell H|date=December 2007|title=Chloroplast vector systems for biotechnology applications|journal=Plant Physiology|volume=145|issue=4|pages=1129–43|doi=10.1104/pp.107.106690|pmc=2151729|pmid=18056863}}</ref> Flanking sequences are crucial for introduction of the gene construct at precised determined points of the plastid genome through [[homologous recombination]].<ref name=":22">{{cite journal|vauthors=Adem M, Beyene D, Feyissa T|date=2017-04-01|title=Recent achievements obtained by chloroplast transformation|journal=Plant Methods|volume=13|issue=1|pages=30|doi=10.1186/s13007-017-0179-1|pmc=5395794|pmid=28428810}}</ref> The gene of interests introduced have many different applications and can range from pest resistance genes to vaccine antigen production.<ref name=":22" /> Intercistronic elements (IEE) are important for facilitating high levels of gene expression if multiple genes are introduced in the form of an [[operon]].<ref name=":22" /> Finally, the 5' UTR and 3' UTR enhances ribosomal binding and increases transcript stability respectively.<ref name=":22" />


=== Transformation technologies ===
=== Transformation technologies ===
The most common method for plastid tranformation is [[Gene gun|biolistics]]: Small gold or tungsten particles are coated with the plasmid vector and shot into young plant cells or plant embryos, penetrating multiple cell layers and into the plastid.<ref name=":02" /> There will then be homologous recombination between the shot plasmid vector and the [[Genome|plastid's genome]], hopefully resulting in a stable insertion of the gene cassette into the plastid.<ref name=":02" /> Whilst the [[Transformation (genetics)|transformation]] efficiency is lower than in [[Agrobacterium tumefaciens|agrobacterial]] mediated transformation, which is also common in plant genetic engineering, particle bombardment is especially suitable for chloroplast transformation. Other transformation methods include the use of [[polyethylene glycol]] (PEG)- mediated transformation, which involves the removal of the plant [[cell wall]] in order to expose the [[Protoplast|"naked" plant cell]] to the foreign genetic material for transformation.<ref name=":02" /> PEG- mediated transformation however, is notoriously time consuming, very technical and labor intensive as it requires the removal of the cell wall which is a key protective structural component of the plant cell.<ref name=":12">{{Cite journal|last=Gan|first=Qinhua|last2=Jiang|first2=Jiaoyun|last3=Han|first3=Xiao|last4=Wang|first4=Shifan|last5=Lu|first5=Yandu|date=2018|title=Engineering the Chloroplast Genome of Oleaginous Marine Microalga Nannochloropsis oceanica|url=https://www.frontiersin.org/articles/10.3389/fpls.2018.00439/full|journal=Frontiers in Plant Science|language=English|volume=9|doi=10.3389/fpls.2018.00439|issn=1664-462X|pmc=PMC5904192|pmid=29696028}}</ref> Interestingly, a paper released in 2018 has described a successful plastid transformation of the chloroplast from the microalgae species ''[[Nannochloropsis|N. oceanica]]'' and ''[[Chlamydomonas reinhardtii|C. reinhardtii]]'' through [[electroporation]].<ref name=":12" /> Whilst no study has been attempted yet for plastid transformation of [[higher plants]] using electroporation, this could be an interesting area of study for the future.
The most common method to transform plastids is particle bombardment: Small gold or tungsten particles are coated with DNA and shot into young plant cells or plant embryos. Some genetic material will stay in the cells and transform them. The [[Transformation (genetics)|transformation]] efficiency is lower than in [[Agrobacterium tumefaciens|agrobacterial]] mediated transformation, which is also common in plant genetic engineering, but particle bombardment is especially suitable for plastid transformation.


In order to persist and be stably maintained in the cell, a plasmid DNA molecule must contain an [[origin of replication]], which allows it to be replicated in the cell independently of the [[chromosome]]. Because transformation usually produces a mixture of rare transformed cells and abundant non-transformed cells, a method is needed to identify the cells that have acquired the plasmid. Plasmids used in transformation experiments will usually also contain a gene giving resistance to an antibiotic (or, more recently developed resistance against a herbicide) that the intended recipient strain of bacteria is sensitive to. Selection for cells able to grow on media containing this antibiotic can then select the cells that have acquired the plasmid by transformation, as cells lacking the plasmid will be unable to grow.
In order to persist and be stably maintained in the cell, a plasmid DNA molecule must contain an [[origin of replication]], which allows it to be replicated in the cell independently of the [[chromosome]]. Because transformation usually produces a mixture of rare transformed cells and abundant non-transformed cells, a method is needed to identify the cells that have acquired the plasmid. Plasmids used in transformation experiments will usually also contain a gene giving resistance to an antibiotic (or, more recently developed resistance against a herbicide) that the intended recipient strain of bacteria is sensitive to. Selection for cells able to grow on media containing this antibiotic can then select the cells that have acquired the plasmid by transformation, as cells lacking the plasmid will be unable to grow.

Revision as of 10:18, 16 April 2020

A transplastomic plant is a genetically modified plant in which genes are inactivated, modified or new foreign genes are inserted into the DNA of plastids like the chloroplast instead of nuclear DNA.

Currently, the majority of transplastomic plants are as a result of chloroplast manipulation due to poor expression in other plastids.[1]

Chloroplasts in plants are thought to have originated from an engulfing event of a photosynthetic bacteria (cyanobacterial ancestor) by a eukaroyte.[2] There are many advantageous in chloroplast DNA manipulation because of its bacterial origin. For example, the ability to introduce multiple genes (operons) in a single step instead of many steps and the simultaneous expression of many genes with its bacterial gene expression system.[3] Other advantageous include, the ability to obtain organic products like proteins at a high concentrations and the fact that production of these products will not be affected by epigenetic regulation.[4] The reason for product synthesis at high concentrations is because a single plant cell can potentially carry up to a 100 chloroplast (like in Arabidopsis), which if all transformed can express the introduced foreign genes), compared to transformation of the nucleus which can only express one copy of the gene.[1]

The advantageous provided by chloroplast DNA manipulation has seen growing interest into this field of research and development, particularly in agricultural and pharmaceutical applications.[4] However, there are some disadvantageous in chloroplast DNA manipulation, such as the inability to manipulate cereal crops DNA material and poor expression of foreign DNA in non- green plastids as mentioned before.[4] Nevertheless, much progress has been made into plant transplatomics, for example, the production of edible vaccines for Tetanus by using a transplastomic tobacco plant.[5]   

Transformation and selection procedure

Gene construct

The first requirement for transplastomic plant generation is to have a suitable gene construct that can be introduced into a plastid like a chloroplast in the form of an E. coli plasmid vector.[6] There are several key features of a suitable gene cassette including but not limited to (1) selectable marker (2) flanking sequences (3) gene of interest (4) promoter sequences (5) 5' UTR (6) 3' UTR (7) intercistronic elements.[7] Flanking sequences are crucial for introduction of the gene construct at precised determined points of the plastid genome through homologous recombination.[8] The gene of interests introduced have many different applications and can range from pest resistance genes to vaccine antigen production.[8] Intercistronic elements (IEE) are important for facilitating high levels of gene expression if multiple genes are introduced in the form of an operon.[8] Finally, the 5' UTR and 3' UTR enhances ribosomal binding and increases transcript stability respectively.[8]

Transformation technologies

The most common method for plastid tranformation is biolistics: Small gold or tungsten particles are coated with the plasmid vector and shot into young plant cells or plant embryos, penetrating multiple cell layers and into the plastid.[6] There will then be homologous recombination between the shot plasmid vector and the plastid's genome, hopefully resulting in a stable insertion of the gene cassette into the plastid.[6] Whilst the transformation efficiency is lower than in agrobacterial mediated transformation, which is also common in plant genetic engineering, particle bombardment is especially suitable for chloroplast transformation. Other transformation methods include the use of polyethylene glycol (PEG)- mediated transformation, which involves the removal of the plant cell wall in order to expose the "naked" plant cell to the foreign genetic material for transformation.[6] PEG- mediated transformation however, is notoriously time consuming, very technical and labor intensive as it requires the removal of the cell wall which is a key protective structural component of the plant cell.[9] Interestingly, a paper released in 2018 has described a successful plastid transformation of the chloroplast from the microalgae species N. oceanica and C. reinhardtii through electroporation.[9] Whilst no study has been attempted yet for plastid transformation of higher plants using electroporation, this could be an interesting area of study for the future.

In order to persist and be stably maintained in the cell, a plasmid DNA molecule must contain an origin of replication, which allows it to be replicated in the cell independently of the chromosome. Because transformation usually produces a mixture of rare transformed cells and abundant non-transformed cells, a method is needed to identify the cells that have acquired the plasmid. Plasmids used in transformation experiments will usually also contain a gene giving resistance to an antibiotic (or, more recently developed resistance against a herbicide) that the intended recipient strain of bacteria is sensitive to. Selection for cells able to grow on media containing this antibiotic can then select the cells that have acquired the plasmid by transformation, as cells lacking the plasmid will be unable to grow.

Biological containment and agricultural coexistence

Genetically modified plants must be safe for the environment and suitable for coexistence with conventional and organic crops. A major hurdle for traditional nuclear genetically modified crops is posed by the potential outcrossing of the transgene via pollen movement. Initially it was thought that, plastid transformation, which yields transplastomic plants in which the pollen does not contain the transgene, not only increases biosafety, but also facilitates the coexistence of genetically modified, conventional and organic agriculture. Therefore, developing such crops was a major goal of research projects such as Co-Extra and Transcontainer.

However, a study conducted on the tabacco plant in 2007 has disproved this theory. A transplatomic tabacco plant generated through chloroplast mediated transformation was bred with plants that were male sterile with an untouched chloroplast.[10] The transplastomic plants were engineered to have resistance to the antibiotic spectinomycin and engineered to produce a fluorescent molecule (GFP).[11] Therefore, it was hypothesized that any offspring produced by from these two lines of tabacco plant should not be able to grow on spectinomycin or be fluorescent, as the genetic material in the chloroplast should not be able to transfer via pollen.[11] However, it was found that some of the seeds were resistant to the antibiotic and could germinate on spectinomycin agar plates.[11] Calculations showed that 1 out of every million pollen grains contained plastid genetic material, which would be hugely significant in an agricultural farm setting.[11] Whilst this study showed that transplatomic plants do not have absolute gene containment, it does highlight that the level of containment is extremely high and would allow for coexistence of conventional and genetically modified agricultural crops.[11]

There are public concerns about having antibiotic resistant marker genes in the environment, regarding a possible transmission of antibiotic resistant genes to unwanted targets including bacteria and weeds.[12] As a result of this, technologies have been developed to remove the selectable antibiotic resistance gene marker. One such technology that has been implemented is the Cre/lox system, where the nuclear encoded Cre recombinase can be placed under control of an inducible promoter to remove the antibiotic resistant gene once homoplasmicity has been achieved from the transformation process.[13]

Transplastomic tobacco

However, plastid transformation is suitable only for certain crop species, and the reliability of this method has only been proven for tobacco. Led by Ralph Bock from the Max Planck Institute of Molecular Plant Physiology in Germany, researchers studied genetically modified tobacco in which the transgene was integrated in chloroplasts.[14] Since past literature reported contradicting figures on the reliability of this process, the Co-Extra researchers analysed more than two million seedlings and found that less than 20 in 1,000,000 inherited the transgene. In the pollen of adult plants, the rate was even lower, remaining below 3 in 1,000,000. This reduction is because some parts of the seedlings are lost during their development into mature plants.

Because tobacco has a strong tendency towards self-fertilisation, the reliability of transplastomic plants is assumed to be even higher under field conditions. Therefore, the researchers believe that only one in 100,000,000 GM tobacco plants actually would transmit the transgene via pollen. Such values are more than satisfactory to ensure coexistence. However, for GM crops used in the production of pharmaceuticals, or in other cases in which absolutely no outcrossing is permitted, the researchers recommend the combination of chloroplast transformation with other biological containment methods, such as cytoplasmic male sterility or transgene mitigation strategies.

References

  1. ^ a b Rigano MM, Scotti N, Cardi T (2012-11-24). "Unsolved problems in plastid transformation". Bioengineered. 3 (6): 329–33. doi:10.4161/bioe.21452. PMC 3489708. PMID 22892591.
  2. ^ Raven JA, Allen JF (2003). "Genomics and chloroplast evolution: what did cyanobacteria do for plants?". Genome Biology. 4 (3): 209. doi:10.1186/gb-2003-4-3-209. PMC 153454. PMID 12620099.{{cite journal}}: CS1 maint: unflagged free DOI (link)
  3. ^ Adem M, Beyene D, Feyissa T (2017-04-01). "Recent achievements obtained by chloroplast transformation". Plant Methods. 13 (1): 30. doi:10.1186/s13007-017-0179-1. PMC 5395794. PMID 28428810.{{cite journal}}: CS1 maint: unflagged free DOI (link)
  4. ^ a b c Ahmad N, Michoux F, Lössl AG, Nixon PJ (November 2016). "Challenges and perspectives in commercializing plastid transformation technology". Journal of Experimental Botany. 67 (21): 5945–5960. doi:10.1093/jxb/erw360. PMID 27697788.
  5. ^ Tregoning J, Maliga P, Dougan G, Nixon PJ (April 2004). "New advances in the production of edible plant vaccines: chloroplast expression of a tetanus vaccine antigen, TetC". Phytochemistry. 65 (8): 989–94. doi:10.1016/j.phytochem.2004.03.004. PMID 15110679.
  6. ^ a b c d Wani, Shabir H.; Haider, Nadia; Singh, Hitesh Kumar and N. B. (2010-10-31). "Plant Plastid Engineering". Current Genomics. doi:10.2174/138920210793175912. PMC 3048312. PMID 21532834. Retrieved 2020-04-16.{{cite web}}: CS1 maint: PMC format (link)
  7. ^ Verma D, Daniell H (December 2007). "Chloroplast vector systems for biotechnology applications". Plant Physiology. 145 (4): 1129–43. doi:10.1104/pp.107.106690. PMC 2151729. PMID 18056863.
  8. ^ a b c d Adem M, Beyene D, Feyissa T (2017-04-01). "Recent achievements obtained by chloroplast transformation". Plant Methods. 13 (1): 30. doi:10.1186/s13007-017-0179-1. PMC 5395794. PMID 28428810.{{cite journal}}: CS1 maint: unflagged free DOI (link)
  9. ^ a b Gan, Qinhua; Jiang, Jiaoyun; Han, Xiao; Wang, Shifan; Lu, Yandu (2018). "Engineering the Chloroplast Genome of Oleaginous Marine Microalga Nannochloropsis oceanica". Frontiers in Plant Science. 9. doi:10.3389/fpls.2018.00439. ISSN 1664-462X. PMC 5904192. PMID 29696028.{{cite journal}}: CS1 maint: PMC format (link) CS1 maint: unflagged free DOI (link)
  10. ^ Ruf S, Karcher D, Bock R (April 2007). "Determining the transgene containment level provided by chloroplast transformation". Proceedings of the National Academy of Sciences of the United States of America. 104 (17): 6998–7002. doi:10.1073/pnas.0700008104. PMC 1849964. PMID 17420459.
  11. ^ a b c d e Ruf S, Karcher D, Bock R (April 2007). "Determining the transgene containment level provided by chloroplast transformation". Proceedings of the National Academy of Sciences of the United States of America. 104 (17): 6998–7002. doi:10.1073/pnas.0700008104. PMC 1849964. PMID 17420459.
  12. ^ Puchta, Holger (2003-08-01). "Marker-free transgenic plants". Plant Cell, Tissue and Organ Culture. 74 (2): 123–134. doi:10.1023/A:1023934807184. {{cite journal}}: Unknown parameter |name-list-format= ignored (|name-list-style= suggested) (help)
  13. ^ Bala A, Roy A, Das A, Chakraborti D, Das S (October 2013). "Development of selectable marker free, insect resistant, transgenic mustard (Brassica juncea) plants using Cre/lox mediated recombination". BMC Biotechnology. 13 (1): 88. doi:10.1186/1472-6750-13-88. PMC 3819271. PMID 24144281.{{cite journal}}: CS1 maint: unflagged free DOI (link)
  14. ^ Bock, Ralph; Karcher, D; Bock, R (2007), "Determining the transgene containment level provided by chloroplast transformation", Proceedings of the National Academy of Sciences, 104 (17): 6998–7002, doi:10.1073/pnas.0700008104, PMC 1849964, PMID 17420459

External links

  • Co-Extra Research on the co-existence and traceability of genetically modified plants
  • Transcontainer Developing biological containment systems for genetically modified plants
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