Propagation and molecular barcoding of Diplazium proliferum andD sylvaticumfor preservation intents


This undertaking is aimed towards the constitution and proof of an efficient and working protocol towards the extension through in vitro micropropagation and DNA extraction of the nonflowering plantsDiplazium proliferumandDiplazium Sylvaticum.Following the constitution of the protocol, it will be used to propagate critically endangered nonflowering plants in Mauritius.


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  1. To map the geographical distribution of the wild species ofDiplazium proliferumandDiplazium sylvaticum.
  2. To measure the biological factors impacting the population kineticss in situ.
  3. To set up a extension protocol for micropropagation ofDiplazium proliferumandDiplazium sylvaticum.
  4. To optimise a Deoxyribonucleic acid extraction protocol for nonflowering plants.
  5. To transport out morphological word picture ofDiplazium proliferumandDiplazium sylvaticum.


Mauritius is known to be a biodiversity hot spot which means it has a part with a considerable pool of biodiversity that is under menace from worlds.

Diplazium sylvaticumandDiplazium proliferumare two species of theDiplaziumgenus that are under great danger following the changeless force per unit area from human colonisation and invasive foreigner species such asPsidium cattleianum.

Furthermore, presently in vitro extension and molecular analysis has been done for these ferns which leave a great loop hole in the procedure of repopulation of these rare autochthonal ferns. Hence, a working protocol has to be established priory which subsequently will be of important importance for the preservation of other rare near nonextant ferns that are subjected to similar menaces.

Pteridophytes are a really huge group that is non decently represented on the planetary Red List and its preservation is really boring and hard. 770 species of nonflowering plants worldwide are listed as in danger by the International Union for Conservation of Nature ( IUCN ) while in 2008 ; merely 211 were in the Red List due to jobs in finding species with the new demands. This is because of the considerable spreads in the apprehension of their distribution, preservation position and biological science ( Sara Magriniet al. ,2010 ) .

Literature Review

  1. Taxonomic group

The categorization of extant ferns into genera is to this day of the month still ill established and more research at a generic degree is required. This is the instance particularly on the relationship among the genera in the largest households. Families such as Aspleniaceae, Athyriaceae, Blechnaceae, Dryopteridaceae, Polypodiaceae, Pteridaceae, Tectariaceae and Thelypteridaceae are non to the full classified in genera within the different households into a phyletic tree.The bulk of the Athyriaceae genera were placed in Woodsiaceae by Smith et Al. ( 2006a, 2008 ) . In the context of monophyly of Athyrium and Diplazium, more in deepness survey is required for a generic categorization ( Maarten Christenhuszet Al. , 2011 ) ,

  1. Life rhythm of aHomosporous nonflowering plants

The four chief phases of a fern from a spore are:

  1. The sporophyte or fern works proper
  2. The asexual spores are produced as a consequence of decrease division.
  3. The gametophyte or thallus works
  4. The oospores are produced as the consequence of the junction of egg and sperm

When a spore germinates, it produces a little fibril ( protonema ) from which a bosom shaped thallus grows from its terminal. A two phase procedure is undergone for the development of the gametophyte. In the first phase, the works form is a additive sum and in the 2nd phase, it is a solid sum. ( Schaffner, John, 1906 )

The thallus is supported liberally by chlorophyll and is anchored to the land through legion cellular rhizoids. The thallus is intersexs and hence can develop several spermaries and ovaries on the lower part. These are non completed attached to the thallus tissues. They can get away so the thallus is covered with H2O through a breach in the outer part of the antheridial wall. ( Schaffner, John, 1906 )

Fertilization occurs when a antherozoid passes down the cervix to the egg to conjugate with it. The developing embryo is wholly parasitic to the parent works which provides it with foods. Finally the embryo interruptions free from the hypertrophied venter of the archegonium to turn roots in the land below while a first foliage develops towards the light switching from a parasitic phase to an independent 1. ( Schaffner, John, 1906 )

  1. In vitroextension

The in situ preservation of ferns and their Alliess in their natural home grounds should ever the chief purpose. However, this is non ever possible ; hence, extra methods are required to back up and finish the in situ methods which will increase the chance of endurance of the single species. ( Sara Magriniet al. ,2010 )

Several studies of in vitro extension of spores have shown success in their cultivation but have indicated a considerable loss of spores during the sterilisation procedure, sterile sowing or due to taint. These jobs have led to the development of different spore sterilisation and seeding methods ( Wu Huaet al. ,2009 ) .

The in vitro sprouting of spores and following prothallus development and subsequent sporophyte growing can short-circuit some of the troubles linked with conventional methods. These troubles include: low sprouting rates, taint with prothalli from invasive species and the growing of excessively few sporophytes. The methods were described by Ford and Fay in 1990. Some illustrations of successful in vitro sprouting of rare species areCibotium schiedeifrom Central America andAngiopteris boiviniifrom the Seychelles ( Michael Fay, 1992 ) .

  1. Spore Viability

Spores viability can change drastically among nonflowering plants ; from a few yearss to a few old ages. Recently, it has been understood that the familial make-up and a few physiological features of spores need to be reappraisals to understand the spores` viability fluctuations ( Marjana Camloh, 1999 ) .

Factors such as spore type, chlorophyllose and non-chlorophyllous or systematic part can impact the viability of spores. Non green spores can lose their viability during storage. Some possible grounds for this doomed of viability are references as growing boosters, and chromosomal divergences, biochemical and metabolic factors, loss of membrane unity, inactivation of enzymes and exhaustion of respiratory substrate. The high rate of respiration or inability to retrieve photosynthetic belongingss after dehydration in green spores has been proposed as the cause of speedy lessening of viability. Since there is a narrow scope of H2O contents possible for storage at conventional temperatures, Ballesteros and Walters ( 2007a, B ) have suggested that the harm by dehydration or low temperature can be avoided by the minute control of H2O degree within the spores. Lloyd and Klekowski ( 1970 ) have compared the viability of green spores ( chlorophyllose ) and non-green spores ( non-chlorophyllous ) . They have noticed that green spores germinate faster than non-green spores ; nevertheless, this belongings to shoot is lost rapidly over clip. Furthermore, their informations has shown that under research lab conditions, green spores have short life span ( 48 yearss ) while the non-green spores live on to about 2.8 old ages. It was suggested by Lebkuecher ( 1997 ) that the surprisingly short viability of green spores ofEquisetum hyemaleL. may be due to the doomed of its photosynthetic ability when the spores are rehydrated undermentioned dehydration. While the factors act uponing viability of spores are known, small is known about those impacting the spores under herbarium conditions ( Sara Magriniet al. ,2010 ) .

Proposed methodological analysis ( Tentative )

  1. Collection of spores from wild assortments in native woods
  2. Light to no sterilisation of spores
  3. Use of different PGR ( TDZ, BAP ) and concentration
  4. Use of unfertile MS and coconut H2O as medium for spores to shoot
  5. Statistical analysis of sprouting rate for the different PGR used
  6. Culture kept in unreal visible radiation to initial sprouting
  7. Optimization of DNA extraction protocol for ferns
  8. Quantification of Deoxyribonucleic acid
  9. Amplification of DNA utilizing different primers ( trnL-F, matK and rbcL )
  10. Sequencing of cistrons amplified and running it through BLAST hunt.

List of demands



Approximate costs




Deoxyribonucleic acid extraction kit



  1. TDZ
  2. Bap

Murashige & A ; Skoog

  • Major salts ( macronutrients )
    1. Ammonium nitrate
    2. Calcium chloride
    3. Magnesium sulfate
    4. Potassium phosphate
    5. Potassium nitrate
  • Minor salts ( micronutrients )
    1. Boric acid
    2. Cobalt chloride
    3. Cupric sulfate
    4. Ferric sulfate
    5. Manganese sulfate
    6. Potassium iodide
    7. Sodium molybdate
    8. Zinc sulfate
    9. Na2EDTA
  • Vitamins and organics
    1. i-Inositol
    2. Niacin
    3. Vitamin b6
    4. Vitamin b1
    5. Kinetin
    6. Glycine
    7. Edamine
    8. Agar
    9. Indoleacetic acid


  1. trnL-F
  2. matK
  3. rbcL


Micropippette tips

Conic flask



Apparatus & A ; Instruments


PCR machime




Rs……… .

Gantt chart









Literature Review

Methodology & A ; Experiment

Results & A ; Discussion

First Draft

Undertaking entry

Expected result

  1. To hold established a micropropagation protocol for the extension of autochthonal ferns similar toDiplaziumgenera.
  2. To hold identified the biological factors forestalling autochthonal ferns to propagate.
  3. To hold optimized a protocol for the DNA extraction of nonflowering plants.
  4. To hold sequenced a venue of cistron and run it through a BLAST hunt.


  • Maarten J.M. Christenhusz, Xian-Chun Zhang and Harald Schneider. ( 2011 ) . A additive sequence of extant households and genera of lycophytes and ferns.Phytotaxa19, 7–54.
  • Schaffner, John H. ( 1906 ) . A Life Cycle of a Homosporous Pteridophyte.Ohio Journal of Science6( 5 ) , 485-488.
  • Sara Magrini, Cristina Olmati, Silvano Onofri and Anna Scoppola. ( 2010 ) . Recovery of Feasible Germplasm from Herbarium Specimens of Osmunda regalis L.American Fern Journal100 (3 ) , 159-160.
  • Marjana Camloh. ( 1999 ) . Spore Age and Sterilization Affects Germination and Early Gametophyte Development of Platycerium bifurcatum.American Fern Journal89( 2 ) , 124-132.
  • Wu Hua, Chen Ping-Ting, Yuan Li-Ping and Chen Long-Qing. ( 2009 ) . An Efficient Method for Surface Sterilization and Sowing Fern Spores in vitro.American Fern Journal99( 3 ) , 226-230
  • Michael F. Fay. ( 1992 ) . Conservation of Rare and Endangered Plants Using in vitro Methods.In Vitro Cellular & A ; Developmental Biology. Plant28P (1 ) , 1-4.

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