SELEKSI
DARI KALUS YANG MENGHASILKAN
ANTOSIANIN TINGGI PADA
POL YCONUM TINCHTORIUMAIT
SELECTION OF HIGH MELDINGANTHOC YANIN
CALLUS
L I N E OFPOL YGONUM TINCHTORIUM AIT
A. ~rnawati*,
S.h4ayarnaH and M.
yo-
Abstract
The effectsof kind and concentration of plant growth regulators on antho-
cyanin synthesis of six cultivars o f
Pol-iurn tinchtorfurnAit were studied. The
cultivar tested were
(1)Shoujyacko Shirobana,
(2)Matsuae Ai, (3)
Hyaklcnn, (4) ShatyoukoAkabana, (5) Miyagi Ai and
(6)Akaguki Kosenbon. Among those cul-
tivars, cultivar no.
l , 2and 3 did not synthesize anthocyanin, cultivar no.
4pro-
duced slightly anthocyanin while cultivar no. 5 and
6produced large amount of
anthocyanin. The best condition to induce
anthocyanin formation was kinetin
(KIN)-indob acetic acid (IAA)
(KIN 10ppm; IAA
0 ,5,1,3,5,10 ppm) under light
condition using petiole as an explant. The amount of anthocyanin produced
byse-
lected calks was four times higher than unscreened callus. Indican was a little
detected on MeOH extract from green callus.
R ingkasan
Pengaruh d a r i jenis dan konsentrasi zat pengatur tumbuh pada s i n t e s a a n t o - sianin d a r i 6 kultivar P. tinch-m Ait t e l a h dipelajari. Kultivar yang diuji a d a - lah (1) Shoujyouko Shirobana, (2) Matsuae Ai, (3) Hyakkan, (4) Shoujouko Akabana, (5) Miyagi Ai, d a n (6) Akaguki kosenbon. Diantara kultivar tersebut, kultivar no. 1, 2 d a n 3 tidak menghasilkan antosianin, kultivar no. 4 menghasilkan antosianin da- l a m jumlah relatif kecil, sedang kultivar no. 5 dan 6 menghasilkan antosianin da- l a m jumlah besar. Kondisi t e r b a i k untuk menginduksi pembentukan antosianin a d a - l a h kinetin (KIN)-Indole acetic a c e d (IAA) (KIN 10 ppm: IAA 0.5, 1, 3, 5, 1 0 ppm) d i bawah pencahayaan berkesinambungan dan menggunakan petiole sebagai eks- plan. Kalus hasil seleksi menghasilkan antosianin 4 kali lebih tinggi d a r i pada kalus t a n p a seleksi. Indican dalam jumlah kecil d a p a t dideteksi pada e k s t r a k MeOH d a r i kalus hi jau.
*
P r e s e n t address: Jurusan Budidaya Pertanian, F a p e r t a IPB**
Dept. of P l a n t Protection, Fac. of Agriculture, Kobe Univ., J a p a n***
Lab. of P l a n t Cell Physiology, Dept. of Bioresource ScienceFac. of Agriculture, Kagawa Univ., Japan.
INTRODUCTION
Yolygonium tinchtorium Ait which is called Ai plant in J a p a n h a s been known t o produce indican, a n indigo precursor. Indigo i s a typical J a p a n e s e s t a i n for traditional J a p a n e s e clothes. This compound is also believed t o have anti- in- secticidal and anti- fungal activities. Under n a t u r a l condition t h e r e s i s t a n c e of plant against diseases i s possitively c o r r e l a t e d with t h e p r e s e n c e of red pigmenta- tion of t h e plant. S o m e cultivars of this plant have red pigmentation on t h e i r s t e m and flower. However, w e t h e r o r not, t h e plant r e s i s t a n c e a g a i n s t disease is caused by indican o r red pigment h a s n o t been fully conclusive. Nonetheless, indican a n d red pigment (anthocyanin) a r e economically important.
Tissue c u l t u r e method has been e x ~ l o i t e d to study biosynthesis, biochemical and g e n e t i c a l regulation of secondary m e t a b o l i t e s (Rhodes e t al, 1989). In addi- tion, tissue c u l t u r e method h a s been incorporated in industry, f o r example, in t h e production of red naphtoquinone anti- inflammatory drug, shikonin by suspension c u l t u r e of Lithospermum erythrorhizon (Tabata a n d Fijita, 1985).
In producing secondary m e t a b o l i t e through tissue c u l t u r e s o m e f a c t o r s have been identified to b e important. According to Zenk et a1 (1976) initialtissue which contains high a m o u n t of t h e secondary m e t a b o l i t e of i n t e r e s t must be chosen as t h e explant. P l a n t growth regulators a r e e f f e c t i v e t r i g g e r s of in v i t r o secondary metabolism, f o r e x a m p l e 5 x 1 0 - 5 ~ kinetin was e f f e c t i v e f o r maximum anthocya- nin synthesis in cell c u l t u r e s of Haplopappus gracilis (Constabel, Shyluk a n d Gam- borg, 1970). However in Daucus carota c e l l c u l t u r e induction of anthocyanin syn- thesis took p l a c e in t h e medium l a c k of 2,4-D (Ozeki and Komamine, 1985). Selec- tion of callus line was also proven t o b e a n e f f e c t i v e way of increasing secondary m e t a b o l i t e production.
In t h i s experiment, t h e e f f e c t s of explant, plant growth regulator on t h e a n - thocyanin production w e r e evaluated. An a t t e m p t of obtaining high yielding of an- thocyanin callus line by selection as well as analysis of indican c o n t e n t w e r e also reported.
MATERIAL AND METHOD
Six cultivars of Polygonum tinchtorium Ait w e r e studied in these, experiments. Those cultivars a r e 1. Shouyouko shirobana, 2.Matsuae Ai, 3. Hyakkan,4. Shouyou-
-
ko Akabana, 5. Miyagi Ai a n d 6. Akaguki kosenbon, which a r e numbered 1 to 6 res- pectively.Basal medium was of Murashige and Skoog (1962) consisted of NH4N03, KNO3, CaC12.2H20, MgS04-7H20, KH2P04, H3B03, m n S O 4 . 4 ~ 2 0 , ZhS04. 7H20, KI, N a ~ M o 0 4 . 2 H 2 0 , C u S 0 4 5 H 2 0 , CoC12.6H20, NazEDTA, F e S 0 4 7 H 2 0 , Thiamin HCI, Nicotinic acid, Pyridoxin HCI, Myo inositol, solidified with 8 gr agarll. Medium w a s adjusted at pH 5.6
-
5.8 with 1 N KOH o r 1 N NaOH prior t o autoclaving, a n d autoclaved at 121°C, f o r 15 min. The c u l t u r e s w e r e incubated t h e t e m p e r a t u r e of20° C under 16 hour light period unless it is specified.
a, Callus formation
G.5 c m of 1 week old etiolated seedling were used as t h e explants. Those explant w e r e inoculated in solid basal medium dispensed in plates. Each p l a t e contained
6 explants. T e n p l a t e s w e r e incubated in darkness while a n o t h e r 1 0 p l a t e s w e r e incubated under 16 hours light period. T h e growth regulators consisted of hours light period. The growth regulators consisted of 10-5M of kinetin, BA, IAA, NAA, 2,4-D and combination of IAAIKIN, 2. 4-DIKIN, NAA/KIN,IAA/BA, 2,4-D/BA and NAAIBA.
2. The e f f e c t of plant growth regulators on t h e growth of petiole explant.
L e a v e petioles measuring 0.5 c m of 2 - 4 months oldgreen house grown plants w e r e inoculated in basal medium incorporated with growth regulators. T e s t t u b e s with a r a t e of 1 e x p l a n t per t u b e r w e r e t o observe f o r e a c h t r e a t m e n t .
3. Selection f o r high yielding indican and anthocyanin callus line.
The r e d d e s t a n d t h e biggest calli grew on t h e medium containing KIN/IAA w e r e used as explants. These petiole derived callus w e r e subcultured t o basal me- dium containing : (a) I O - ~ M K I N / ~ O - ~ M NAA, (b) 106M I K I N / I O - ~ M NAA. A r a t e
of 6 calli p i e c e s w e r e inoculated in o n e plate. The callus c u l t u r e s w e r e incubated at 20°C, a n d 1 6 hours illumination. Two weeks a f t e r incubation half of e a c h callus, a b o u t 0.5 g w e r e analyzed f o r anthocyanin c o n t e n t while t h e o t h e r half w e r e c u t i n t o 4 pieces a n d t r a n s f e r e d t o a fresh medium of t h e s a m e formulation f o r a n - o t h e r 2 weeks. Callus which indicated t h e highest anthocyanin c o n t e n t w e r e t h e n maintained f o r f u r t h e r subculture for t h e callus line selection. The calli of low anthocyanin c o n t e n t w e r e discarded. As a control t r e a t m e n t initial calli grew on medium containing
KINIIAA
w e r e also subcultured a n d analyzed t h e i r anthocyanin c o n t e n t without subjected t o any selection.b. Extraction and estimation of anthocyanin content
Callus t o b e analyzed was t a k e n o u t f r o m p l a t e and cleaned f r o m a g a r with tissue paper and measured its fresh weight. As much as 0.1 g. f.w. callus was sus- pended in 2 ml of 0.1% HCI MeOH overnight, t h e n centrifuged at 2000 rpm for 10 minutes. The s u p e r n a t a n t was measured for i t s adsorbance in a c u v e t t e of 1 c m pathlength with a UV-VIS double b e a m spectrophotometer; Jasco
U
best- 30, at wavelengths ranging f r o m 200-
1100 nm. Absortion peak was reached at 525 nm.c. Extraction and Estimation of Indican
Fresh Calli of 2 g r a m w e r e homogenized and e x t r a c t e d in 1 5 m l cold a c e t o n at 4"C, overnight. The e x t r a c t s w e r e subsequently filtered. The f i l t r a t e s w e r e then, e v a p o r a t e d t o dry at 40°C under vaccuum. These dried e x t r a c t s w e r e sus- pended with 1 m l methanol. Then, t h e solutions w e r e centrifuged at 2000 rpm for 10 minutes. The s u p e r n a t a n t s were finally injected t o HPLC. Indican c o n t e n t was t o be d e t e c t e d at 254 nm with Hitachi L-6000 pump, Hitachi L-4200 UV-VIS de- t e c t o r and Hitachi D-2500 c h r o m a t o integrator. As t h e solvent was 5% CH3CN in 0.1% H3PO4. Indican c o n t e n t was determined by comparing t h e peak of s a m p l e t o t h e peak of indican standard.
Result and Discussion
a. Hypocotyl explant
under n a t u r a l condition, t h e s t e m colour of cultivars 1, 2, 3, 4 a r e g r e e n while of cultivars 5 a n d 6 show r e d pigmentation. Those d i f f e r e n c e In pigmentation was evident e i t h e r under light o r dark. However hypocotyls of cultivars 5 a n d 6 which were exposed t o light evidently showed m o r e pigmentation, as i t was observed vi- sually t h a n t h e ones which w e r e incubated in darkness. Light has been shown t o play a n i m p o r t a n t role in inducing anthocyanin synthesis (Nozue and Yasuda, 1985; Rengel and Kordan, 1987). eventhough in t h e case of vitis culture, light was n o t necessary f o r anthocyanin synthesis. (Yamakawa et al, 1983).
A f t e r o n e week of culture, calli w e r e f o r m e d on t h e hypocotyl explants in- cubated in t h e medium containing (1 - - 5 ~ ) : 2 weeks calli w e r e also formed f r o m t h e hypocotyl explants c u l t u r e d in t h e medium containing BA, IAAIKIN, IAAIBA.
Tabel 1. Pengaruh beberapa zat pengatur tumbuh pada pembentukan antosianin, kalus dan a k a r d a r i eksplan hipokotil d a r i 6 kultivar Yolygonum tinchto- rium Ait yang tumbuh di bawa pencahayaan, sesudah 2 minggu.
Table 1 . The e f f e c t s of various plant growth regulatorson red pigment formation, callus and root format ion of six-cultivars o f Yolygonum tinchtorium Ait hypocotil explants grown under light condition after 2 weeks.
T r e a t m e n t s ( ~ o - ~ M )
No hormon KIN B A I A NAA 294-D IAA-KIN NAA-KIN 294-D-KIN IAA-BA NAA-BA 2,4-D-BA green s t e m
1,2,3,4l (1 00%)2 1,2,3 (1 00%) 1,2,3 (1 00%) 1,2,3,4 (1 00%) 1,2,3,4 (1 00%) 1,2,3,4 (100%) 1,2,3,4 (100%) 1,2,3,4 (1 00%) 1,2,3,4
( 100%) 1,2,3,4 (1 00%) 1,2,3,4 (1 00%) 1,2,3,4 (1 00%)
r e d s t e m
pink callus root
It's obvious t h a t auxin was important f o r callus formation. Table 1 shows t h e res- ponse of t h e hypocotyl explant in culture a f t e r 2 weeks. Calli formed in this ex- periment were generally white. Only t h e one formed in t h e medium containing IAA/KIN showed a l i t t l e pigmentation. Callus formed on t h e medium containing NAA was friable and showed a good appearance while callus formed on medium containing 2,4-D was compact but turned brown (indicated senescense) a f t e r 2 weeks. It is possibly because t h e concentration of t h e 2,4-D in t h e medium was t o o high.
b. Callus formation from petiole explants
Callus for mation from petiole explants required t h e presence of auxin with or without cytokinin in t h e medium. In f a c t calli were not formed in t h e medium containing BA, kinetin o r 4PU, but they were formed in t h e medium containing IAA, NAA or 2,4-D as well as in t h e medium containing both auxin and cytokinin.
The e f f e c t s of plant growth regulators on t h e morphogenetic response of pe- tiole explants could be described as follows.
Tabel 2. Kombinasi tat pengatur tumbuh terbaik untuk menginduksi kalus pada 6 kultivar Yolygonum tinchtorium Ait.
Table 2. The best combination of plant growth regulators t o induce callus in six cultivar o f Yolygonum tinchtorium Ait.
Cultivars Combination of plant growth regulators
1. Shouyouko shirobana 3 ppm BA
-
0.5 ppm NAA (++++++) 2. Matsue-Ai 3 ppm BA-
0.5 ppm NAA (++++) 3. Hyakkan 2 ppm BA-
0.5 ppm NAA (++++++) 4. Shouyouko Akabana 1 ppm KIN - 3 ppm 2,4-D (++++++)5. Miyagi Ai 3 ppm BA
-
0.5; 1; 2; 3 ppm NAA 0.05 pprn KIN-
1 pprn 2,4-D (+++++)6. Akaguki kosenbon 1 ppm BA
-
0.5 ppm NAA (+++++)+
: showed t h e relative s i z e of callus + : menunjukkan ulauvln relatif kalusIn case of combination of KIN-2,4-D; at 2 pprn KIN, in various concentration of 2,4-D, for six cultivars, t h e callus size is t h e lowest. Callus formed at 2 pprn 2,4-D in various concentration of KIN is bigger than t h a t at 1 pprn 2,4-D o r 3 pprn 2,4-D, e x c e p t for cultivar no. 4 (Kojoko Akabana). It seems t h a t 3 pprn 2,4-D was too high t o promote callus growth and 2 pprn kinetin inhibited callus growth.
In t h e combination of KIN-IAA, t h e increased of KIN concentration f r o m 0.5 pprn t o 3 pprn also caused t h e increased in thecallus size. Callus grown on t h e media containing 5 and 10 pprn KIN has t h e s a m e size as the one grown in t h e presence of 3 pprn KIN. The increased of IAA concentration from 0.5 pprn to 10 pprn at various concentration of KIN did not give any differences in callus size. This indicates t h a t KIN played more important role than IAA t o promote callus
In t h e case of combination of BA-NAA, in t h e a b s e n c e of BA, in a l l concen- t r a t i o n of NAA (from 0.5 ppm t o 3 ppm) only small callus size formed. But when BA was added, t h e callus size increased, while t h e root formation decreased. T h e best combination of BA-NAA to induce callus was d e p e n t on t h e cultivar, as shown in t a b l e 2.
The increased c o n c e n t r a t i o n of 4PU in all c o n c e n t r a t i o n of NAA caused t h e increased of callus size. While t h e increased of NAA concentration f r o m 0.1 ppm t o 5 ppm did n o t c a u s e t h e increased in callus size. This tendency w a s similar to BA-NAA tendency.
Table 2. s u m m a r i z e s t h e best growth regulators f o r t h e biggest petiole de- rived callus size f o r m e d in t h e medium. In s h o r t callus growth is d e p e n t upon t h e genotypes and t h e level of growth regulators c o n c e n t r a t i o n included in t h e me- dium.
c. Selection f o r anthocyanin high yielding callus line
Those t w o previous e x p e r i m e n t s significantly indicated t h a t only c u l t i v a r no. 5 (Miyagi Ai) a n d cultivar no. 6 (Akaguki Kosenbon) are worth t o b e explored f o r anthocyanin production, s i n c e t h e y showed a c l e a r red pigment f o r m a t i o n in a l l combinations of growth regulators. Furthermore, t h e colour f o r m a t i o n of Mi- yagi Ai cultivar is controlable f o r s o m e e x t e n t s i n c e it is d e p e n t upon t h e r a t i o of auxin a n d cytokinin ( t a b l e 3). Therefore f o r t h e callus line selection e x p e r i m e n t only callus derived f r o m petiole explants w e r e s e l e c t e d through subculture. Every subculture indicated o n e passage of selection.
The anthocyanin c o n t e n t of s c r e e n e d callus line was evidently increased with t h e passage of selection, while t h e c o n t e n t of anthocyanin of unscreened callus line remained constant. T h e i n c r e a s e of t h e anthocyanin c o n t e n t s t a r t to b e c o n s t a n t a f t e r 12 passages (fig. 1 and fig. 2.). Miyagi Ai cultivar was of i n t e r e s t t o b e cho- s e n as a model f o r callus line screening s i n c e it has been widely known. This re- s u l t a r e t r u e f o r both callus lines established in bothmedium containing 1 0 - 5 ~ ki- n e t i n 1 1 0 ' ~ ~ NAA a n d ~ o - ~ M k i n e t i n I l 0 - 6 ~ NAA.
These t w o media w e r e s e l e c t e d s i n c e callus grown o n t h o s e media showed b e t t e r pigmentation. T h e anthocyanin c o n t e n t of t h e s c r e e n e d callus increased u p t o 4 t i m e s fold a f t e r screening of 14 passages. Another interesting thing t o n o t e is t h a t anthocyanin c o n t e n t of callus line grown in t h o s e t w o d i f f e r e n t media we- r e also d i f f e r e n t eventhough t h e i r sizes w e r e a l m o s t t h e same. However it is di- .
f f i c u l t to conclude whether t h e d i f f e r e n c e is simply hormon dependence o r callus line variant dependence o r both, inherents with t h e selection itself. Nonetheless t h e selection of callus line resulted in yielding anthocyanin callus line.
d. Indican c o n t e n t of s e l e c t e d callus line
During t h e c o u r s e of selection, a n intriguing phenomenon was observed. The s c r e e n e d callus, in addition t o forming callus of red pigmentation,also f o r m e d ca- llus of g r e e n colour in t h e s a m e culture.
t h e r e d callus. This result suggest t h a t indican and anthocyanin h a v e d i f f e r e n t biosynthetic pathway. Consequently, higher indican production m a y require a di- f f e r e n t biochemical trlgger f o r c e l l s t o d i f f e r e n t i a t e toward indican systhesis.
CONCLUDING REMARKS
T h e a b o v e e x p e r i m e n t shows t h a t a g r e a t variability o c c u r s on callus culture. This variation, in o n e hand, c a n b e d i r e c t e d f o r yield improvement of c e r t a i n se- condary m e t a b o l i t e s as well as f o r biosynthesis of novel compund, but in t h e o t h e r hand c a n c a u s e a serious problem if i t is applied for l a r g e s c a l e production of se- condary metabolite. However, callus line selection c a n b e a n a l t e r n a t i v e method of improving c e r t a i n secondary m e t a b o l i t e yield by amplyfying t h e t a r g e t cells t o g e t s t a b l e lines.
REFERENCES
Constabel, F., J.P. Shyluk a n d O.L. Gamborg. 1971. T h e e f f e c t s of hormones on anthocyanin accumulation in c e l l c u l t u r e s of Haplopappus gracilis. P l a n t a 9 6 : 317
-
325.Nozue, M a n d Yasuda, H. 1985. O c c u r e n c e of anthocyanoplast on cell suspension c u l t u r e s of s w e e t potato. P l a n t C e l l R e p o r t s 4: 252 - 255.
Ozeki, Y a n d A. Komamine. 1981. Inducton of anthocyanin synthesis in relation t o embryogenesis in a c a r r o t suspension culture: corelation of metabolic di- f f e r e n t i a t i o n with morphologica differentiation. Plantarum. 53 : 570
-
577.Rengel. Z. a n d H e b e r t A. Kordan. 1978. E f f e c t s of growth regulators o n light- dependent anthocyanin production in Z e a Mays Seedlirigs. Physiol. Plantarum 6 9 : 511
-
516.Tabata, M. a n d Y. Fijita. 1985. Biotechnology in plant science. Ed. by Zaitlin. M., Day. P. a n d Hollander, A. Academic Press. New York.
Yamakawa. T., S. Kato. K. Ishida, T. Kodama and Y. Minoda. 1983. Production of anthocyanins by Vitis c e l l s in suspension culture. Agric. Biol. Chem. 47 (10): 2185
-
2191.Number of passsages
Gambar 1. Perubahan kandungan antosianin (mg/g berat kering) selama seleksi kalus yang tumbuh pada media yang mengandung KIN 1 0 - 5 ~ NAA 1 0 - 6 ~ . Data adalah rata-rata dari 3 ulangan contoh yang masing- masing dianalisa secara triplo.
Figure 1 . Change in anthocyanin content (mg/g dry weight) durin selection o f
B
Number of passages
Gambar 2. Perubahan kandungan antosianin (mg/g berat kering) selama seleksi kalus yang tumbuh pada media yang mengandung
KIN
1 0 - 6 ~ NAA M. Data adalah rata-rata dari 3 ulangan contoh yang masing- masing dianalisa secara triplo.Figure 2. Change in anthocyanin content (mg/g dry weight) durin selection o f
B
callus grown on media containing KIN 1 O ' ~ M NAA 10'M.
The dataRetention time ( m i d
G a m b a r 3. Kromatografi c a i r kinerja tinggi fraksi yang l a r u t MeOH dari e k s t r a k a c e t o n d a r i kalus hijau Yolygonum tinchtorium Ait.
SELEKSI
DARI KALUS YANG MENGHASILKAN
ANTOSIANIN TINGGI PADA
POL YCONUM TINCHTORIUMAIT
SELECTION OF HIGH MELDINGANTHOC YANIN
CALLUS
L I N E OFPOL YGONUM TINCHTORIUM AIT
A. ~rnawati*,
S.h4ayarnaH and M.
yo-
Abstract
The effectsof kind and concentration of plant growth regulators on antho-
cyanin synthesis of six cultivars o f
Pol-iurn tinchtorfurnAit were studied. The
cultivar tested were
(1)Shoujyacko Shirobana,
(2)Matsuae Ai, (3)
Hyaklcnn, (4) ShatyoukoAkabana, (5) Miyagi Ai and
(6)Akaguki Kosenbon. Among those cul-
tivars, cultivar no.
l , 2and 3 did not synthesize anthocyanin, cultivar no.
4pro-
duced slightly anthocyanin while cultivar no. 5 and
6produced large amount of
anthocyanin. The best condition to induce
anthocyanin formation was kinetin
(KIN)-indob acetic acid (IAA)
(KIN 10ppm; IAA
0 ,5,1,3,5,10 ppm) under light
condition using petiole as an explant. The amount of anthocyanin produced
byse-
lected calks was four times higher than unscreened callus. Indican was a little
detected on MeOH extract from green callus.
R ingkasan
Pengaruh d a r i jenis dan konsentrasi zat pengatur tumbuh pada s i n t e s a a n t o - sianin d a r i 6 kultivar P. tinch-m Ait t e l a h dipelajari. Kultivar yang diuji a d a - lah (1) Shoujyouko Shirobana, (2) Matsuae Ai, (3) Hyakkan, (4) Shoujouko Akabana, (5) Miyagi Ai, d a n (6) Akaguki kosenbon. Diantara kultivar tersebut, kultivar no. 1, 2 d a n 3 tidak menghasilkan antosianin, kultivar no. 4 menghasilkan antosianin da- l a m jumlah relatif kecil, sedang kultivar no. 5 dan 6 menghasilkan antosianin da- l a m jumlah besar. Kondisi t e r b a i k untuk menginduksi pembentukan antosianin a d a - l a h kinetin (KIN)-Indole acetic a c e d (IAA) (KIN 10 ppm: IAA 0.5, 1, 3, 5, 1 0 ppm) d i bawah pencahayaan berkesinambungan dan menggunakan petiole sebagai eks- plan. Kalus hasil seleksi menghasilkan antosianin 4 kali lebih tinggi d a r i pada kalus t a n p a seleksi. Indican dalam jumlah kecil d a p a t dideteksi pada e k s t r a k MeOH d a r i kalus hi jau.
*
P r e s e n t address: Jurusan Budidaya Pertanian, F a p e r t a IPB**
Dept. of P l a n t Protection, Fac. of Agriculture, Kobe Univ., J a p a n***
Lab. of P l a n t Cell Physiology, Dept. of Bioresource ScienceFac. of Agriculture, Kagawa Univ., Japan.
INTRODUCTION
Yolygonium tinchtorium Ait which is called Ai plant in J a p a n h a s been known t o produce indican, a n indigo precursor. Indigo i s a typical J a p a n e s e s t a i n for traditional J a p a n e s e clothes. This compound is also believed t o have anti- in- secticidal and anti- fungal activities. Under n a t u r a l condition t h e r e s i s t a n c e of plant against diseases i s possitively c o r r e l a t e d with t h e p r e s e n c e of red pigmenta- tion of t h e plant. S o m e cultivars of this plant have red pigmentation on t h e i r s t e m and flower. However, w e t h e r o r not, t h e plant r e s i s t a n c e a g a i n s t disease is caused by indican o r red pigment h a s n o t been fully conclusive. Nonetheless, indican a n d red pigment (anthocyanin) a r e economically important.
Tissue c u l t u r e method has been e x ~ l o i t e d to study biosynthesis, biochemical and g e n e t i c a l regulation of secondary m e t a b o l i t e s (Rhodes e t al, 1989). In addi- tion, tissue c u l t u r e method h a s been incorporated in industry, f o r example, in t h e production of red naphtoquinone anti- inflammatory drug, shikonin by suspension c u l t u r e of Lithospermum erythrorhizon (Tabata a n d Fijita, 1985).
In producing secondary m e t a b o l i t e through tissue c u l t u r e s o m e f a c t o r s have been identified to b e important. According to Zenk et a1 (1976) initialtissue which contains high a m o u n t of t h e secondary m e t a b o l i t e of i n t e r e s t must be chosen as t h e explant. P l a n t growth regulators a r e e f f e c t i v e t r i g g e r s of in v i t r o secondary metabolism, f o r e x a m p l e 5 x 1 0 - 5 ~ kinetin was e f f e c t i v e f o r maximum anthocya- nin synthesis in cell c u l t u r e s of Haplopappus gracilis (Constabel, Shyluk a n d Gam- borg, 1970). However in Daucus carota c e l l c u l t u r e induction of anthocyanin syn- thesis took p l a c e in t h e medium l a c k of 2,4-D (Ozeki and Komamine, 1985). Selec- tion of callus line was also proven t o b e a n e f f e c t i v e way of increasing secondary m e t a b o l i t e production.
In t h i s experiment, t h e e f f e c t s of explant, plant growth regulator on t h e a n - thocyanin production w e r e evaluated. An a t t e m p t of obtaining high yielding of an- thocyanin callus line by selection as well as analysis of indican c o n t e n t w e r e also reported.
MATERIAL AND METHOD
Six cultivars of Polygonum tinchtorium Ait w e r e studied in these, experiments. Those cultivars a r e 1. Shouyouko shirobana, 2.Matsuae Ai, 3. Hyakkan,4. Shouyou-
-
ko Akabana, 5. Miyagi Ai a n d 6. Akaguki kosenbon, which a r e numbered 1 to 6 res- pectively.Basal medium was of Murashige and Skoog (1962) consisted of NH4N03, KNO3, CaC12.2H20, MgS04-7H20, KH2P04, H3B03, m n S O 4 . 4 ~ 2 0 , ZhS04. 7H20, KI, N a ~ M o 0 4 . 2 H 2 0 , C u S 0 4 5 H 2 0 , CoC12.6H20, NazEDTA, F e S 0 4 7 H 2 0 , Thiamin HCI, Nicotinic acid, Pyridoxin HCI, Myo inositol, solidified with 8 gr agarll. Medium w a s adjusted at pH 5.6
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5.8 with 1 N KOH o r 1 N NaOH prior t o autoclaving, a n d autoclaved at 121°C, f o r 15 min. The c u l t u r e s w e r e incubated t h e t e m p e r a t u r e of20° C under 16 hour light period unless it is specified.
a, Callus formation
G.5 c m of 1 week old etiolated seedling were used as t h e explants. Those explant w e r e inoculated in solid basal medium dispensed in plates. Each p l a t e contained
6 explants. T e n p l a t e s w e r e incubated in darkness while a n o t h e r 1 0 p l a t e s w e r e incubated under 16 hours light period. T h e growth regulators consisted of hours light period. The growth regulators consisted of 10-5M of kinetin, BA, IAA, NAA, 2,4-D and combination of IAAIKIN, 2. 4-DIKIN, NAA/KIN,IAA/BA, 2,4-D/BA and NAAIBA.
2. The e f f e c t of plant growth regulators on t h e growth of petiole explant.
L e a v e petioles measuring 0.5 c m of 2 - 4 months oldgreen house grown plants w e r e inoculated in basal medium incorporated with growth regulators. T e s t t u b e s with a r a t e of 1 e x p l a n t per t u b e r w e r e t o observe f o r e a c h t r e a t m e n t .
3. Selection f o r high yielding indican and anthocyanin callus line.
The r e d d e s t a n d t h e biggest calli grew on t h e medium containing KIN/IAA w e r e used as explants. These petiole derived callus w e r e subcultured t o basal me- dium containing : (a) I O - ~ M K I N / ~ O - ~ M NAA, (b) 106M I K I N / I O - ~ M NAA. A r a t e
of 6 calli p i e c e s w e r e inoculated in o n e plate. The callus c u l t u r e s w e r e incubated at 20°C, a n d 1 6 hours illumination. Two weeks a f t e r incubation half of e a c h callus, a b o u t 0.5 g w e r e analyzed f o r anthocyanin c o n t e n t while t h e o t h e r half w e r e c u t i n t o 4 pieces a n d t r a n s f e r e d t o a fresh medium of t h e s a m e formulation f o r a n - o t h e r 2 weeks. Callus which indicated t h e highest anthocyanin c o n t e n t w e r e t h e n maintained f o r f u r t h e r subculture for t h e callus line selection. The calli of low anthocyanin c o n t e n t w e r e discarded. As a control t r e a t m e n t initial calli grew on medium containing
KINIIAA
w e r e also subcultured a n d analyzed t h e i r anthocyanin c o n t e n t without subjected t o any selection.b. Extraction and estimation of anthocyanin content
Callus t o b e analyzed was t a k e n o u t f r o m p l a t e and cleaned f r o m a g a r with tissue paper and measured its fresh weight. As much as 0.1 g. f.w. callus was sus- pended in 2 ml of 0.1% HCI MeOH overnight, t h e n centrifuged at 2000 rpm for 10 minutes. The s u p e r n a t a n t was measured for i t s adsorbance in a c u v e t t e of 1 c m pathlength with a UV-VIS double b e a m spectrophotometer; Jasco
U
best- 30, at wavelengths ranging f r o m 200-
1100 nm. Absortion peak was reached at 525 nm.c. Extraction and Estimation of Indican
Fresh Calli of 2 g r a m w e r e homogenized and e x t r a c t e d in 1 5 m l cold a c e t o n at 4"C, overnight. The e x t r a c t s w e r e subsequently filtered. The f i l t r a t e s w e r e then, e v a p o r a t e d t o dry at 40°C under vaccuum. These dried e x t r a c t s w e r e sus- pended with 1 m l methanol. Then, t h e solutions w e r e centrifuged at 2000 rpm for 10 minutes. The s u p e r n a t a n t s were finally injected t o HPLC. Indican c o n t e n t was t o be d e t e c t e d at 254 nm with Hitachi L-6000 pump, Hitachi L-4200 UV-VIS de- t e c t o r and Hitachi D-2500 c h r o m a t o integrator. As t h e solvent was 5% CH3CN in 0.1% H3PO4. Indican c o n t e n t was determined by comparing t h e peak of s a m p l e t o t h e peak of indican standard.
Result and Discussion
a. Hypocotyl explant
under n a t u r a l condition, t h e s t e m colour of cultivars 1, 2, 3, 4 a r e g r e e n while of cultivars 5 a n d 6 show r e d pigmentation. Those d i f f e r e n c e In pigmentation was evident e i t h e r under light o r dark. However hypocotyls of cultivars 5 a n d 6 which were exposed t o light evidently showed m o r e pigmentation, as i t was observed vi- sually t h a n t h e ones which w e r e incubated in darkness. Light has been shown t o play a n i m p o r t a n t role in inducing anthocyanin synthesis (Nozue and Yasuda, 1985; Rengel and Kordan, 1987). eventhough in t h e case of vitis culture, light was n o t necessary f o r anthocyanin synthesis. (Yamakawa et al, 1983).
A f t e r o n e week of culture, calli w e r e f o r m e d on t h e hypocotyl explants in- cubated in t h e medium containing (1 - - 5 ~ ) : 2 weeks calli w e r e also formed f r o m t h e hypocotyl explants c u l t u r e d in t h e medium containing BA, IAAIKIN, IAAIBA.
Tabel 1. Pengaruh beberapa zat pengatur tumbuh pada pembentukan antosianin, kalus dan a k a r d a r i eksplan hipokotil d a r i 6 kultivar Yolygonum tinchto- rium Ait yang tumbuh di bawa pencahayaan, sesudah 2 minggu.
Table 1 . The e f f e c t s of various plant growth regulatorson red pigment formation, callus and root format ion of six-cultivars o f Yolygonum tinchtorium Ait hypocotil explants grown under light condition after 2 weeks.
T r e a t m e n t s ( ~ o - ~ M )
No hormon KIN B A I A NAA 294-D IAA-KIN NAA-KIN 294-D-KIN IAA-BA NAA-BA 2,4-D-BA green s t e m
1,2,3,4l (1 00%)2 1,2,3 (1 00%) 1,2,3 (1 00%) 1,2,3,4 (1 00%) 1,2,3,4 (1 00%) 1,2,3,4 (100%) 1,2,3,4 (100%) 1,2,3,4 (1 00%) 1,2,3,4
( 100%) 1,2,3,4 (1 00%) 1,2,3,4 (1 00%) 1,2,3,4 (1 00%)
r e d s t e m
pink callus root
It's obvious t h a t auxin was important f o r callus formation. Table 1 shows t h e res- ponse of t h e hypocotyl explant in culture a f t e r 2 weeks. Calli formed in this ex- periment were generally white. Only t h e one formed in t h e medium containing IAA/KIN showed a l i t t l e pigmentation. Callus formed on t h e medium containing NAA was friable and showed a good appearance while callus formed on medium containing 2,4-D was compact but turned brown (indicated senescense) a f t e r 2 weeks. It is possibly because t h e concentration of t h e 2,4-D in t h e medium was t o o high.
b. Callus formation from petiole explants
Callus for mation from petiole explants required t h e presence of auxin with or without cytokinin in t h e medium. In f a c t calli were not formed in t h e medium containing BA, kinetin o r 4PU, but they were formed in t h e medium containing IAA, NAA or 2,4-D as well as in t h e medium containing both auxin and cytokinin.
The e f f e c t s of plant growth regulators on t h e morphogenetic response of pe- tiole explants could be described as follows.
Tabel 2. Kombinasi tat pengatur tumbuh terbaik untuk menginduksi kalus pada 6 kultivar Yolygonum tinchtorium Ait.
Table 2. The best combination of plant growth regulators t o induce callus in six cultivar o f Yolygonum tinchtorium Ait.
Cultivars Combination of plant growth regulators
1. Shouyouko shirobana 3 ppm BA
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0.5 ppm NAA (++++++) 2. Matsue-Ai 3 ppm BA-
0.5 ppm NAA (++++) 3. Hyakkan 2 ppm BA-
0.5 ppm NAA (++++++) 4. Shouyouko Akabana 1 ppm KIN - 3 ppm 2,4-D (++++++)5. Miyagi Ai 3 ppm BA
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0.5; 1; 2; 3 ppm NAA 0.05 pprn KIN-
1 pprn 2,4-D (+++++)6. Akaguki kosenbon 1 ppm BA
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0.5 ppm NAA (+++++)+
: showed t h e relative s i z e of callus + : menunjukkan ulauvln relatif kalusIn case of combination of KIN-2,4-D; at 2 pprn KIN, in various concentration of 2,4-D, for six cultivars, t h e callus size is t h e lowest. Callus formed at 2 pprn 2,4-D in various concentration of KIN is bigger than t h a t at 1 pprn 2,4-D o r 3 pprn 2,4-D, e x c e p t for cultivar no. 4 (Kojoko Akabana). It seems t h a t 3 pprn 2,4-D was too high t o promote callus growth and 2 pprn kinetin inhibited callus growth.
In t h e combination of KIN-IAA, t h e increased of KIN concentration f r o m 0.5 pprn t o 3 pprn also caused t h e increased in thecallus size. Callus grown on t h e media containing 5 and 10 pprn KIN has t h e s a m e size as the one grown in t h e presence of 3 pprn KIN. The increased of IAA concentration from 0.5 pprn to 10 pprn at various concentration of KIN did not give any differences in callus size. This indicates t h a t KIN played more important role than IAA t o promote callus
In t h e case of combination of BA-NAA, in t h e a b s e n c e of BA, in a l l concen- t r a t i o n of NAA (from 0.5 ppm t o 3 ppm) only small callus size formed. But when BA was added, t h e callus size increased, while t h e root formation decreased. T h e best combination of BA-NAA to induce callus was d e p e n t on t h e cultivar, as shown in t a b l e 2.
The increased c o n c e n t r a t i o n of 4PU in all c o n c e n t r a t i o n of NAA caused t h e increased of callus size. While t h e increased of NAA concentration f r o m 0.1 ppm t o 5 ppm did n o t c a u s e t h e increased in callus size. This tendency w a s similar to BA-NAA tendency.
Table 2. s u m m a r i z e s t h e best growth regulators f o r t h e biggest petiole de- rived callus size f o r m e d in t h e medium. In s h o r t callus growth is d e p e n t upon t h e genotypes and t h e level of growth regulators c o n c e n t r a t i o n included in t h e me- dium.
c. Selection f o r anthocyanin high yielding callus line
Those t w o previous e x p e r i m e n t s significantly indicated t h a t only c u l t i v a r no. 5 (Miyagi Ai) a n d cultivar no. 6 (Akaguki Kosenbon) are worth t o b e explored f o r anthocyanin production, s i n c e t h e y showed a c l e a r red pigment f o r m a t i o n in a l l combinations of growth regulators. Furthermore, t h e colour f o r m a t i o n of Mi- yagi Ai cultivar is controlable f o r s o m e e x t e n t s i n c e it is d e p e n t upon t h e r a t i o of auxin a n d cytokinin ( t a b l e 3). Therefore f o r t h e callus line selection e x p e r i m e n t only callus derived f r o m petiole explants w e r e s e l e c t e d through subculture. Every subculture indicated o n e passage of selection.
The anthocyanin c o n t e n t of s c r e e n e d callus line was evidently increased with t h e passage of selection, while t h e c o n t e n t of anthocyanin of unscreened callus line remained constant. T h e i n c r e a s e of t h e anthocyanin c o n t e n t s t a r t to b e c o n s t a n t a f t e r 12 passages (fig. 1 and fig. 2.). Miyagi Ai cultivar was of i n t e r e s t t o b e cho- s e n as a model f o r callus line screening s i n c e it has been widely known. This re- s u l t a r e t r u e f o r both callus lines established in bothmedium containing 1 0 - 5 ~ ki- n e t i n 1 1 0 ' ~ ~ NAA a n d ~ o - ~ M k i n e t i n I l 0 - 6 ~ NAA.
These t w o media w e r e s e l e c t e d s i n c e callus grown o n t h o s e media showed b e t t e r pigmentation. T h e anthocyanin c o n t e n t of t h e s c r e e n e d callus increased u p t o 4 t i m e s fold a f t e r screening of 14 passages. Another interesting thing t o n o t e is t h a t anthocyanin c o n t e n t of callus line grown in t h o s e t w o d i f f e r e n t media we- r e also d i f f e r e n t eventhough t h e i r sizes w e r e a l m o s t t h e same. However it is di- .
f f i c u l t to conclude whether t h e d i f f e r e n c e is simply hormon dependence o r callus line variant dependence o r both, inherents with t h e selection itself. Nonetheless t h e selection of callus line resulted in yielding anthocyanin callus line.
d. Indican c o n t e n t of s e l e c t e d callus line
During t h e c o u r s e of selection, a n intriguing phenomenon was observed. The s c r e e n e d callus, in addition t o forming callus of red pigmentation,also f o r m e d ca- llus of g r e e n colour in t h e s a m e culture.
t h e r e d callus. This result suggest t h a t indican and anthocyanin h a v e d i f f e r e n t biosynthetic pathway. Consequently, higher indican production m a y require a di- f f e r e n t biochemical trlgger f o r c e l l s t o d i f f e r e n t i a t e toward indican systhesis.
CONCLUDING REMARKS
T h e a b o v e e x p e r i m e n t shows t h a t a g r e a t variability o c c u r s on callus culture. This variation, in o n e hand, c a n b e d i r e c t e d f o r yield improvement of c e r t a i n se- condary m e t a b o l i t e s as well as f o r biosynthesis of novel compund, but in t h e o t h e r hand c a n c a u s e a serious problem if i t is applied for l a r g e s c a l e production of se- condary metabolite. However, callus line selection c a n b e a n a l t e r n a t i v e method of improving c e r t a i n secondary m e t a b o l i t e yield by amplyfying t h e t a r g e t cells t o g e t s t a b l e lines.
REFERENCES
Constabel, F., J.P. Shyluk a n d O.L. Gamborg. 1971. T h e e f f e c t s of hormones on anthocyanin accumulation in c e l l c u l t u r e s of Haplopappus gracilis. P l a n t a 9 6 : 317
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325.Nozue, M a n d Yasuda, H. 1985. O c c u r e n c e of anthocyanoplast on cell suspension c u l t u r e s of s w e e t potato. P l a n t C e l l R e p o r t s 4: 252 - 255.
Ozeki, Y a n d A. Komamine. 1981. Inducton of anthocyanin synthesis in relation t o embryogenesis in a c a r r o t suspension culture: corelation of metabolic di- f f e r e n t i a t i o n with morphologica differentiation. Plantarum. 53 : 570
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577.Rengel. Z. a n d H e b e r t A. Kordan. 1978. E f f e c t s of growth regulators o n light- dependent anthocyanin production in Z e a Mays Seedlirigs. Physiol. Plantarum 6 9 : 511
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516.Tabata, M. a n d Y. Fijita. 1985. Biotechnology in plant science. Ed. by Zaitlin. M., Day. P. a n d Hollander, A. Academic Press. New York.
Yamakawa. T., S. Kato. K. Ishida, T. Kodama and Y. Minoda. 1983. Production of anthocyanins by Vitis c e l l s in suspension culture. Agric. Biol. Chem. 47 (10): 2185
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2191.Number of passsages
Gambar 1. Perubahan kandungan antosianin (mg/g berat kering) selama seleksi kalus yang tumbuh pada media yang mengandung KIN 1 0 - 5 ~ NAA 1 0 - 6 ~ . Data adalah rata-rata dari 3 ulangan contoh yang masing- masing dianalisa secara triplo.
Figure 1 . Change in anthocyanin content (mg/g dry weight) durin selection o f
B
Number of passages
Gambar 2. Perubahan kandungan antosianin (mg/g berat kering) selama seleksi kalus yang tumbuh pada media yang mengandung
KIN
1 0 - 6 ~ NAA M. Data adalah rata-rata dari 3 ulangan contoh yang masing- masing dianalisa secara triplo.Figure 2. Change in anthocyanin content (mg/g dry weight) durin selection o f
B
callus grown on media containing KIN 1 O ' ~ M NAA 10'M.
The dataRetention time ( m i d
G a m b a r 3. Kromatografi c a i r kinerja tinggi fraksi yang l a r u t MeOH dari e k s t r a k a c e t o n d a r i kalus hijau Yolygonum tinchtorium Ait.
