The resulting disk-shaped tissue fragments are nearly 100% sterilized, and after
one to two months calluses will form on them. Figure 6.2 shows the tissue culturing
method used for the brown alga Ecklonia stolonifera (from the kelp order).
While medium preparation is the most important part of tissue culturing, recent
examination of physical conditions such as light, osmotic pressure, and agar concentration has resulted in callus induction being accelerated in the red algae
Chrondia crassicaulus, Laurencia intermedia and Grateloupia filicina when agar
concentrations approach 0.3–1.5%. Temperature and light intensity were first found
to have a large influence in the late 1990s (Table 6.5).
Table 6.2 (continued)
ASP 1
ASP 2 ASP 6
ASP 7 ASP 12 KDX
ASS 1
Glycylglycine
–
–
–
–
–
75 mg –
HEPES
j
–
–
–
–
–
–
100 mg
pH
7.6
7.8
7.6
7.8–
8.0
7.8–80 8.3–8.4 8.0
a Preparation for metal mixture P II (in 1 ml): 1 mg Na 2 ÁEDTA, 0.01 mg Fe (as Cl
− ), 0.2 mg B (as
H 3 BO 3 ), 0.04 mg Mn (as Cl
− ), 0.005 mg Zn (as Cl
− ), 0.001 mg Co (as Cl
−
)
b
Preparation for metal mixture S II (in 1 ml): 1 mg Br (as Na
+ ), 0.2 mg Sr (as Cl
−
), 0.02 mg Rb
(as Cl
−
), 0.02 mg Li (as Cl
−
),0.05 mg Mo (as Na
+ ), 0.001 mg I (as K
+ )
c Preparation for metal mixture P8 (in 1 ml): 3 mg Na 3 -versenol, 0.2 mg Fe (as Cl
−
), 0.2 mg B (as
H 3 BO 3 ), 0.1 mg Mn (as Cl
−
),0.05 mg Zn (as Cl
−
), 0.01 mg Co (as Cl
−
), 0.05 mg Mo (as Na
+ ),
0.002 mg Cu (as Cl
−
)
d
Preparation for metal mixture for ASS (in 1 ml): 100 lg Fe (as Fe-sequestrene), 100 lg B (as
H 3 BO 3 ), 100 lg Mn (as Cl
−
), 10 lgZn (as Cl
−
), 1 lg Co (as Cl
−
), 10 lg Mo (as Na 2 MoO 4 ), 1 lg
Cu (as Cl
−
), 1 mg Br (as K
+ ), 100 lg Sr (as Cl
−
), 10 lg Rb (as Cl
−
), 10 lg Li (as Cl
−
), 1 lg I (as
K
+ )
e Preparation for vitamin mixture S3 (in 1 ml): 0.05 mg thiamine hydrochloride, 0.01 mg niacin,
0.01 mg calcium pantothenate, 1 lg p-aminobenzoic acid, 0.1 lg biotin, 0.5 mg inositol, 0.2 lg
folate, 0.3 mg thymine
f
Preparation for vitamin mixture 8A (in 1 ml): 0.2 mg thiamine hydrochloride, 0.1 mg nicotinic
acid, 0.04 mg putresine-2HCl, 0.1 mg calcium pantothenate, 5 lg riboflavin, 0.04 mg pyridoxine
2-HCl, 0.02 mg pyridoxamine-2HCl, 0.01 mg p-aminobenzoic acid, 0.5 lg biotin, 0.5 mg choline
citrate, 1 mg inositol, 0.8 mg thymine, 0.26 mg orotic acid, 0.05 lg vitamin B 12 , 0.2 lg folinic
acid, 2.5 lg folate
g
Preparation for ASS vitamin mixture (in 1 ml): 100 lg thiamine hydrochloride, 100 µg nicotinic
acid, 10 lg putresine-2HCl, 100 lg calcium pantothenate, 10 lg riboflavin, 10 lg
pyridoxine-2HCl, 10 lg pyridoxamine-2Hcl, 10 lg p-aminobenzoic acid, 1 lg biotin, 1 mg
inositol, 100 lg choline citrate, 100 lg thymine, 100 lg orotic acid, 1 lg cyanocobalamin, 1 lg
folate, 0.1 lg folinic acid
h
Preparation for KDS solution (in 1 ml): 7.84 mg KBr, 54.2 mg KCl, 1.95 mg SrCl 2 Á 6H 2 O,
0.01 lg cyanocobalamin, 0.05 lg biotin, 10.0 lg thiamine hydrochloride
i
Preparation for KDTM solution (in 1 ml): 668.4 lg HÁEDTA, 1.14 mg H 3 BO 4 , 199.0 lg
FeSO 4 Á7H 2 O, 3.9 lg CuSO 4 Á5H 2 O, 12.6 lg Na 2 MoO 4 , 36.0 lg MnCl 2 Á4H 2 O, 44.0 lg
ZnSO 4 Á7H 2 O, 4.0 lg CoCl 2 Á6H 2 O, 2.3 lg NH 4 VO 3, 3.9 lg KI
j
HEPES buffer solution: 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid
152
6 Seaweed Biotechnology
one to two months calluses will form on them. Figure 6.2 shows the tissue culturing
method used for the brown alga Ecklonia stolonifera (from the kelp order).
While medium preparation is the most important part of tissue culturing, recent
examination of physical conditions such as light, osmotic pressure, and agar concentration has resulted in callus induction being accelerated in the red algae
Chrondia crassicaulus, Laurencia intermedia and Grateloupia filicina when agar
concentrations approach 0.3–1.5%. Temperature and light intensity were first found
to have a large influence in the late 1990s (Table 6.5).
Table 6.2 (continued)
ASP 1
ASP 2 ASP 6
ASP 7 ASP 12 KDX
ASS 1
Glycylglycine
–
–
–
–
–
75 mg –
HEPES
j
–
–
–
–
–
–
100 mg
pH
7.6
7.8
7.6
7.8–
8.0
7.8–80 8.3–8.4 8.0
a Preparation for metal mixture P II (in 1 ml): 1 mg Na 2 ÁEDTA, 0.01 mg Fe (as Cl
− ), 0.2 mg B (as
H 3 BO 3 ), 0.04 mg Mn (as Cl
− ), 0.005 mg Zn (as Cl
− ), 0.001 mg Co (as Cl
−
)
b
Preparation for metal mixture S II (in 1 ml): 1 mg Br (as Na
+ ), 0.2 mg Sr (as Cl
−
), 0.02 mg Rb
(as Cl
−
), 0.02 mg Li (as Cl
−
),0.05 mg Mo (as Na
+ ), 0.001 mg I (as K
+ )
c Preparation for metal mixture P8 (in 1 ml): 3 mg Na 3 -versenol, 0.2 mg Fe (as Cl
−
), 0.2 mg B (as
H 3 BO 3 ), 0.1 mg Mn (as Cl
−
),0.05 mg Zn (as Cl
−
), 0.01 mg Co (as Cl
−
), 0.05 mg Mo (as Na
+ ),
0.002 mg Cu (as Cl
−
)
d
Preparation for metal mixture for ASS (in 1 ml): 100 lg Fe (as Fe-sequestrene), 100 lg B (as
H 3 BO 3 ), 100 lg Mn (as Cl
−
), 10 lgZn (as Cl
−
), 1 lg Co (as Cl
−
), 10 lg Mo (as Na 2 MoO 4 ), 1 lg
Cu (as Cl
−
), 1 mg Br (as K
+ ), 100 lg Sr (as Cl
−
), 10 lg Rb (as Cl
−
), 10 lg Li (as Cl
−
), 1 lg I (as
K
+ )
e Preparation for vitamin mixture S3 (in 1 ml): 0.05 mg thiamine hydrochloride, 0.01 mg niacin,
0.01 mg calcium pantothenate, 1 lg p-aminobenzoic acid, 0.1 lg biotin, 0.5 mg inositol, 0.2 lg
folate, 0.3 mg thymine
f
Preparation for vitamin mixture 8A (in 1 ml): 0.2 mg thiamine hydrochloride, 0.1 mg nicotinic
acid, 0.04 mg putresine-2HCl, 0.1 mg calcium pantothenate, 5 lg riboflavin, 0.04 mg pyridoxine
2-HCl, 0.02 mg pyridoxamine-2HCl, 0.01 mg p-aminobenzoic acid, 0.5 lg biotin, 0.5 mg choline
citrate, 1 mg inositol, 0.8 mg thymine, 0.26 mg orotic acid, 0.05 lg vitamin B 12 , 0.2 lg folinic
acid, 2.5 lg folate
g
Preparation for ASS vitamin mixture (in 1 ml): 100 lg thiamine hydrochloride, 100 µg nicotinic
acid, 10 lg putresine-2HCl, 100 lg calcium pantothenate, 10 lg riboflavin, 10 lg
pyridoxine-2HCl, 10 lg pyridoxamine-2Hcl, 10 lg p-aminobenzoic acid, 1 lg biotin, 1 mg
inositol, 100 lg choline citrate, 100 lg thymine, 100 lg orotic acid, 1 lg cyanocobalamin, 1 lg
folate, 0.1 lg folinic acid
h
Preparation for KDS solution (in 1 ml): 7.84 mg KBr, 54.2 mg KCl, 1.95 mg SrCl 2 Á 6H 2 O,
0.01 lg cyanocobalamin, 0.05 lg biotin, 10.0 lg thiamine hydrochloride
i
Preparation for KDTM solution (in 1 ml): 668.4 lg HÁEDTA, 1.14 mg H 3 BO 4 , 199.0 lg
FeSO 4 Á7H 2 O, 3.9 lg CuSO 4 Á5H 2 O, 12.6 lg Na 2 MoO 4 , 36.0 lg MnCl 2 Á4H 2 O, 44.0 lg
ZnSO 4 Á7H 2 O, 4.0 lg CoCl 2 Á6H 2 O, 2.3 lg NH 4 VO 3, 3.9 lg KI
j
HEPES buffer solution: 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid
152
6 Seaweed Biotechnology