Introduction

Species of seaweed widely cultivated in madura is euchema cottonii that is appellation in the
world trade for species of seaweed kappaphycus alvarezii , one of a breed of seaweed most
important and largest volume production in indonesia .This type of containing extracts of
karaginan useful as a a thickener ( gelling agent ) , penetral and solidified agent , and as a
fertilizer ( suryaningrum 1998 ) .The main problems in an effort to increase production seaweed
cultivation of a kind of kappaphycus alvarezii namely the availability of seeds good .One of our
causes were seeds susceptible against infection disease . Attack by epiphytic causing the
quality of seeds be unfit planting, hence security grass the sea will disease is indicators success
seaweed cultivation.Technology seaweed cultivation simple and cheap currently widely used
cultivator of the not yet supported by the availability of seeds free of disease and epiphytic (
specific pathogen and epiphyte free / spef).
Tissue culture is one of technique to produce stock seeds quality .Research that has been
carried by parenrengi et al .In 2007; hurtado and biter in 2007; hurtado et al .( 2009 and yunque
et al .For 2010; have been known that seaweed ( k . Alvarezii to dikultur tissue , but in research
was not examined specifically about contamination occurring and identification to species
contaminants. One of the problems occurring in tissue culture is the contamination .The
condition of in vitro who favored eksplan that is, containing sucrose and hara in concentration
high , moisture high and a suitable temperature to be liked microorganisms that often grow and
is now developing very rapidly beat eksplan .A source of organisms contamination can come of
the neighborhood who do not sterile , or existing in the cell when eksplan kappaphycus alvarezii
will dikultur .This needs to be done efforts control .The first step business sector was
identification the types of microorganisms source of contamination and he knows symptoms
attacks on eksplan kappaphycus alvarezii.
Study literature
Kappaphycus alvarezii is seaweed rhodophyceae class.Based on the identification karaginan
produced by kappaphycus alvarezii is the type kappa karaginan, so this type in taxonomy
changed its name from eucheuma alvarezii be kappaphycus alvarezii ( patadjai 2007 ).The
name alvarezii given on kappaphycus alvarezii derived from the name of the deceased vicente (
vic ) alvarez.Vic is a pioner in method cultivation cottonii ( patadjai 2007 ).The seaweed trading
more familiar with the name of eucheuma cottonii or cottonii course.The seaweed defined as
disruption of the structure and function being normal as the occurrence of a change growth rate,
sightings ( colors and shapes ) can influence the levels of productivity.Tissue culture or
cultivation in vitro is a methods to isolate part of a plant grown in artificial media sterile, in a
bottle sterile culture and in the condition of being asep. So that the parts can be increase itself
and regenerate it into a complete plant .Tissue culture is a series of the activities carried out by
to make part of a plant ( roots , shoots , growing tissue of plants ) growing be a plant intact (
perfect ) in vitro (Indrianto 2002)..
The basic theories the tissue culture is a. cells of an organism multicellular anywhere it is
actually same with cells a zygote because it comes from one of the cell ( omne cellula ex cellula
) . B. the theory totipotensi cells namely the cell theory by schwann and schleiden ( 1898 )
stated that cells having the nature of totipotensi , that each cell a plant that lives furnished with
genetic information and devices physiological complete to grow and developing be a plant whole
if the condition in accordance .This theory believe that any part of plants can multiplied for all
part of a plant consists of jaringan- living tissue
According to thorpe of ( 1981 ) there are three key principles in tissue culture: a. isolation plant
parts of a plant whole ( an organ, roots, leaves, a stem ) b. guard part of a plant last in the
environment appropriate and the condition of the culture that right c. nurture in the condition
aseptic.

Metodology
A method of this research was begun dated 28 may to 17 june 2013.The implementation of the
tissue culture done in the laboratory tissue culture agroekoteknologi and research on organisms
contaminants done in the laboratory of marine science university trunojoyo madura.The tools
applied to the process of tissue culture among others bottles culture, paper strain, a funnel, a
beaker glass 250 ml dan500 ml, autoclave, laminar flow, a microscope, glass preparat + cover
glass and any great tool.Seaweed material used is seaweed kind of kappaphycus alvarezii the
results of cultivation groups of farmers in village aengdake, kecamatan bluto, kabupaten
sumenep
A medium for tissue culture climbing into his waders and the media conway and seawater that
sterilized .The composition of media such as that is listed in appendix 1 .A source of eksplan
that had been selected based having the following criteria: ( a ) having many branches ,
luxuriance and sharp pointed ( b ) you shall never be found spotting and peel off ( c ) the color of
specific bright ( ) ( d ) the age of the growth of 25-35 the day , ( e ) weighs between 50-100
grams a rumpon and not hard hit a disease ice-ice .The sterilization had been proven a tool use
of alcoholic 70 % on its fine appearance and an autoclave while the material used in the
sterilization had been proven eksplan is betadine 0,5 % , and the waters of the sea sterile 25 ppt
.The composition of substances the sterilization had been proven eksplan set out in appendix 2
.Phases of the implementation the culture of the tissues until observation eksplan uses the
method is like what is done hurtado and biter ( 2007 ) , of bond issuance will be using the waters
of the sea sterile and the media conway as that it was falling down the bottom of it: A. the
preparatory tools and material 1 .Prepared instrument wash instrument with flowing water .
sterilize instrument instruments which will be used use of alcoholic , sterilization wet use
autoclave with the temperature 1210c to pressure 1.5 atm for 20 minutes
2. Preparing Materials
Lift sea water into ± 100ml culture bottle
Save seawater that has been in autoclave in sterile room
Screen media conway and sterilize it into autoclave with
temperature of 1210C at a pressure of 1.5 atm for 15 minutes.
B. stage planting a bottle the culture that already contains media , as well as other planting tools
sprayed with use of alcoholic 70 % .Pieces of a thallus taken and admitted to in a disinfectant
solution i ( betadine solution 1 percent where konsentrasinya 1 ml per 100ml sea water ) for 10
minutes later am flushed with sterile akuades to be further included in a disinfectant solution ii (
solution betadine 0.5 % where 0.5 ml per 100ml sea water ) for 10 minutes later am flushed with
aquades sterile for 3 times , the aim of this so that there is no one who settles on the remains of
a disinfectant material eksplan and drained in a petri dish Planting is done in water eksplan
laminar flow eksplan planted in a bottle containing media culture , in every bottle of grown using
tweezers tweezers eksplan with 1 .These activities are done in laminar flow , placed in the oc
bersuhu 25 .25 ppt salinitas media , ph 7,5 eksplan dikultur 120 and that there are as many as
120 units culture in a bottle .Then a bottle closed moved and arranged on a rack culture in
accordance with the plans , given 10 watts neon lighting . The current positions of the lights in
the upper part of a shelf a distance of 30 cm culture from a bottle , lighting programmed for 24
hours has continuously. The stage of the subculture of every 5 the day eksplan that will be
planted in the subculture of to a bottle the culture of with the new one which contains culture
media tissues by the the concentration of and parameter that equal to culture media compared
to a previous low . D. left the care of his family at the first stage left the care of his were made to
three the same type of chambers the culture of with in maintaining the cleanliness and room
temperature .The culture that plastic bottle full of liquid already containing the media and
eksplan sprayed by with alcohol 70 % in every day and planlet as well as a media that is
contaminated be released soon from a room and observable . E. the stage of data over the
withdrawal of funds the observation is made every day to go to see that they can have
contamination bacteria or fungi .The media as well as eksplan that is contaminated in shall bring
forth for them from a room and observable of his cell as well as species pengkontaminasinya (
fishing et al .2008 ) .But as for the data were drawn its industrial activity among others: a. the
percentage of contamination the percentage of contamination was based on the old a formula to
tell worn by amiluddin ( 2007 ) as laid down in the following: a x 100 % c ( % )

Treatment and control

Observation with regard to the contamination wearing 4 control is an organism that
contaminating the seaweed hospital in the sea, sample sea water, organisms contaminants at
the storage space culture and organisms contaminants in the planting eksplan ( laminar flow
).Observation to see the bacteria and fungi done by taking olesan sample on the wall thallus,
sample sea water, and sample culture media formerly ignored in the maintenance cultural and in
laminar flow and planted into media that and diinkubasi at room temperature 370c for 24 hours
The condition epiphytic on treatment control
Seaweed affected by epiphytic in the sea characterized by the presence of algae filaments
attached to part the epidermis thallus kappaphycus alvarezii as seen in figure 4.1.Based on
identification in type and characteristics of algae filaments in line with the research largo ( 2002
), species identified is polysiphonia sp that is algae competitors that is epiphytic
The existence of the influx of competitive algae could result in the disruption of a thallus in the
granting of that the idea of nutrition and the light of the .If needed components out in the
metabolism of grow at a slow pace so long time later seaweed kappaphycus alvarezii can be
into a long slender , pulpy , pale and can couse the death .Of the observation of shows that
there has been some of these intractable disorders on a network that characterized by the
presence of that bump at the focal point of the epidermis and makes residents and road , of the
observation of morphology through a microscope that it is obtainable that polysiponia has had
filaments that are like a sacred tree and makes residents and road and send the game into extra
part of a cell wall by kappaphycus alvarezii .According to darmayati et al .( 2001 ) polyshiponia
sp is mikroalga the people of the the rhodophyta and it is automatically become influx of
competitive algae to the grass the sea that can be meyebabkan disappearing of seaweed field
sluggishness in especially photosynthetic a result of anything the surface of the thallus by
polyshiponia reflected the bank sound assets . Observation about bacteria and fungi to bacteria
obtained bacteria colonies globular yellow creamy and white in samples of derived from roomy ,
through staining grams known morphology bacteria shaped coccus and a curved stem ,
indicated the bacteria is is pseudomonas sp and vibrio sp , darmayati et al .In 2001 states that
almost all example seaweed taken from cultivation kappaphycus alvarezii in pari island , both of
illness or a healthy found bacteria group vibrio of a kind of aeromonas sp
The same also found in seaweed infected from cultivation dipantai takalar south sulawesi found
the vibrio sp , aeromonas sp , and pseudomonas sp , in a healthy seaweed found pseudomonas
sp .This indicates that in general are likely bacteria type in the sea is pseudomonas sp ,
aeromonas , and vibrio sp .The testing the sterile tissue culture space , sea water and materials
eksplan indicates that there are bacteria all these places .As for the planting eksplan ( laminar
flow ) culture did not find any symptoms of contamination Morphology a bacterium that there is
for me the seaweeds that at the well of eksplan and the waters of the sea as was almost the
same either as to form as well as her colour may be .Morphology the bacterium so it is
presented in figure 4.2 in the following.
Fungus contaminating consisting of saprolegnia sp and phythopthora sp that is fungus of the
class oomycota the fungus can only growing on surroundings having moisture high or watery.
Seaweed contaminated saprolegnia sp characterized by memutihnya color thallus, slimy dogged
by shit like white flour, and the outer skin or epidermisnya flaky that look the network in / the
medulla in thallus and the hifa-hifa white on the surface media.Morphology saprolegnia sp as
there are in figure 4.3
The diagnosis was laboratories by means of taking mycelia, laid on the surface slide glas and
ditetesi some water to be examined under the microscope.Mycelia cause saprolegniasis having
branching with hypha aseptate structure
Saprolegnia sp having features features can grow in hose temperature 0-35 � c , with a hose
growth optimal 15-30 � c and is the fungi oomycota which is called also with aquatic fungi can
live in environmental conditions watery / having moisture high .Saprolegnia sp generally strike
the wounded , and then can also spread on healthy tissue other .According to wilfred et al .(
1965 in ningsih ( 2011 ) of a fungus the family saprolegniaceae can live in the water fresh and
saltwater .Zoöspores a group of fungi sought for a substrate fertile , then settled and started
producing hypha .Mycelia grown on tissue who cuts or the site of infection , then spread to the
normal tissues around the location infection .An enzyme pelisis issued fungi would undermine
surrounding tissues , delete cells and development of mycelia the progressive , very solid and
panhandle into the water that look as cotton .
Phytoptora
Thallus that contaminated are pale green and there are mottled black dots to us .Morphological
observation network shows that there has been colored areas dark and globular as seen in the
picture 4.4 under this.
Morphology phytophthora sp having the sporangium shaped tapering until somewhat round,
pear-shaped, spores have feathers whip ( flagella ) so that can move in water, and pathogens
can form klamidospora round ( by the directorate of protection holtikultura 2011 )
Bactery contaminated
Contamination the bacteria characterized by eksplan shows symptoms wither and media smells
pretty nasty.Our observations tissue thallus showed cell walls that broke out the possibility of
because of bacterial infection.Observation morphology the bacteria done by first bred bacteria
up the wall thallus in the media and staining to be gram and identified through electron
microscope, after the culture of bacteria in the media agar and identify under a microscope
obtained that morphology bacteria is coccus single, pairs and and gram chain of negative so
that indicated that bacteria are streptococcuss sp.Morphology bacteria is as trlihat in figure 4.5
following:
Based on a picture 4.5 that it is obtainable that morphology and the color of a bacterium that
originated from the culture of to the control of the is relatively the same as the identification of
contaminants there is for me the eksplan that is contaminated so that an organism of
contaminants is indicated quite has been built in a source of eksplan
Change morphology cell
Tissue cells observed in a thallus healthy out more clearly with indicated spread evenly reach
other staining ( safranin ) keseluruh the surface tissue seaweed, safranin who spread equally
show that of a liquid cells absorbed flattened every the spaces in in the pokey for the state of the
cell walls it still complete and no the cytoplasm that broke while tissue thallus that contaminated
having the form of a cell that does not intact and appeared not evenly reach other color will be
employed due to the damage components components cells / rupture cytoplasm
Lakitan 2011 in 2011 arisandi ( claim that cell walls of plants have the major function as a
protector of the order of cells so that when the walls of cells damaged by disease so it can have
caused the changes the shape and size of cell .The damage to the cell walls can interfere with
the absorption of nutrients into the cell so that it can be interferes with the metabolism of and
impeded cell division .Getting in worse damage to cause the cell walls of rupture , so that fluids
out and resulting in the shape of the cell to not irregular and contracting bellows plasmolisis ( me
and start experienced the death of moses and wei 2008 in arisandi 2011 ) .The difference a cell
that healthy with a cell that contaminated is presented in figure 4.6
The percentage contamination
The percentage contamination is the ratio thallus seaweed infected with seaweed total counted
in percent.After the calculation on the percentage contamination the work of the tissue culture
this 11.7 % while the seaweed health is the 88,3 % with the number of contamination per day is
as been noted in figure 4.7 follows
In the first week of the culture that contaminated seaweed is a range of 8 a bottle where the
bottle there are the characteristics of the issuance of the empi features characterized by
memutihnya thallus , that foreigners by the excrement of slimy as a fine white flour , as well as
the outer skin or peel off epidermisnya and in the 2 a bottle there are the characteristics of the
existence of other black spots and a tanned lines on the wall of a thallus .Contamination on on
the first week occasion in day to 3.4 and five days into with the densest populations took place
on the day to 4 .On the second week after the number of contamination is a range of 6 a bottle
with 3 a bottle there are features features characterized by whiten thallus and 3 a bottle there
are the characteristics of the existence of other black spots and a tanned lines on the wall of a
thallus .Semangun in 2001 said that between infection and apparently symptoms sometimes if
they are the distance a long time but usually a symptom of diseases will be looks after the
occurrence of infection .
Survival rate
In the tissue culture done obtained that the survival rate was in 62,5 %, with total seaweed die
about 45 bottles and seaweed live 75 bottles as stated in the picture 4.8.It indicates that the
level of success of tissue culture seaweed included in the category of work because the number
of seaweed die less than the number of seaweed life.Based on mighty ( 2011 ) survival rate it is
one factor success in the tissue culture, if the number of living at harvest many and the dead just
a little this value kelulushidupan will high, instead if the number of dead much so that the was
still alive when done harvesting live a little of value kelulushidupan it will low
Conclusion
Based on the results obtained conclusion as follows: 1 .There are 3 species contaminants on
tissue culture seaweed kappaphycus alvarezii of fungi consisting of the saprolegnia sp ,
phythophthora sp and bacteria of streptococcus sp .2 .Contamination fungi indicated they have
the to the apparent eksplan seaweeds that will dikultur for the species of mushroom an aquatic
fungus or oomycota that can only be growing on the environment to the high moisture content or
watery .3 .A cell that contaminated are smaller and appear to be any pengkerutan compared to
a cell on a network thallus that healthy which appeared to be are much larger in size and it looks
the cell walls of stronger in bearing the shape of the cell
Suggestion
For the tissue culture conditions aseptic must be kept to prevent contamination, in addition the
research in the field of antibiotics and antifungal needs to be done to avoid any contamination
bacteria and fungi when done cultivation tissue culture seaweed k.alvarezii so that it can
produce the availability of seeds free of disease and epiphytic ( specific pathogen and epiphyte
free / spef )