BIOTECHNOLOGY
IN THE 21 ST CENTURY
ENSURING SUSTAINABILITY AND SAFETY IN THE PURSUIT OF
BIOTECHNOLOGY'S ECONOMIC BENEFITS
Proceedings of The 2
nd
Indonesian Biotechnology Conference
Editor in Chief Koesnandar Editorial Board : Soedj atmiko
Inez H. Slamet Loedin
Esti Widjayanti
Yenni Bakhtiar
Widya Asmara
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Roy A. Sparringa
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Bambang Sukmadi
Abu Amar
T echnical Editor: Erwahyuni E. Prabandari
Farida Ro sana Mira
Published by
Indonesian Biotechnology Consortium
,.
;
ADVANCING BIOTECHNOLOGY
IN THE 21 sT CENTURY
ENSURING SUSTAINABILITY AND SAFETY IN THE PURSUIT OF
BIOTECHNOLOGY'S ECONOMIC BENEFITS
Proceedings of The 2nd Indonesian Biotechnology Conference Yogyakarta, 23-26 October 2001
Editor in Chief Koesnandar Editorial Board : Soedjatmiko
Inez H. S lamet Loedin
Esti Widj ayanti
Yenni Bakhtiar
Widya Asmara
Amin Subandrio
Agus Purwantara
Debbie S. Retnoningrum
Nuki Bambang Nugroho
Roy A. Sparringa
Khaswar Syamsu
Setiarti Sukotj o
Bambang Sukmadi
Abu Amar
Technical Editors: Erwahyuni E. Prabandari
Farida Rosana Mira
Cytological analysis of Artemisia cina and Artemisia annua in untransformed and transformed root cultures TRI MUJI ERMAYANTI, LAELA SARI & ERWIN AL HAFIIZH Research Centre for Biotechnology, Indonesian Institute of Sciences-LIPI
Jalan Raya Bogor Km. 46 Cibinong, Indonesia, 1691 1
Phone : 02 1-8754587; Fax . 02 1-8754588; E-mail :
[email protected]. id
Abst ract
Artemisia cina and A. annua are two medicinal species producing bioacti ve compounds whic h are potential as antimalarial , antitumor, antifungal and antibacterial. The ai m of the study was to analyze the stability of chromosome numbers in root cultures of A. cina and A annua. Transformed root culture was established by infection of leaves and stems of A. cina with Agrobacterium rhizogenes strains ATCC-8196, 07 20001, ATCC- 15 834 and A4 and A. tumefaciens strain RIOOO . Roots isolated from plantlet grown in vitro of A. cina and from callus originated from leaves of A. an nua were utilized for investigation of chromosome examination of untransformed roots. Chromosome examination was conducted by isolating root tips, immersed in saturated paradichlorobenzene, followed by evemight fix ation in acetic acid and ethanol. Roots were then hydrolysed and stained with acetoorcein. After squashing, roots were ex ami ned under microscope. The results showed that untransformed roots of A. cina had 60.1 % of cells with the diplo id numbers of 2n = 32, and 25. 7% of cells had chromosome numbers ranged from 2n = 12 to 2n = 36. The chromosome numbers of A. cina transformed ro ots was affected by strains of Agrobacterium. Roots transformed with the bacteria strain ATCC-8196 showed highest normal chromosome numbers of 2n = 32 (78 .6%) follow ed by roots transformed with strains 07-20001 (60. 5%), ATCC 15 834 (58 .2%), , A4 (52.7%), and R IOOO (40.4%). A. annua roots originated from callus showed 56.5% of cells had chromosome nu mbers of 2n = 18, and 43.5% cells had uneuploid number of 14- 17. The chromosome numbers of A. annua untransformed roots will be compared with that of transformed roots. The genetic stability of roots is expected to be related to the production of its bioactive compounds.
Keywords: untransformed roots, transformed roots, chromosome number, genetic stability, Artemisia cina , Artem isia annua
INTRODUCTION Varia tion in chromosome numbers is a common phenomenon in plant cell and tissue culture and increases w ith the age of the culture. For example, in Vicia Jaba, aneuploidy increases w ith increasing age of cu ltured callus (fua & Roy, 1982), and in almond, increasin g age results in high level of polyploidy as well as aneuplo idy (Mehra & M ehra, 1974) . Similar results w ere observed in maize (McCoy and Phillips, 1982), Nicotiana species (Nuti R onchi et al . 1981) , Hordeum (Orton, 1980) and protoplast-derived potato plants (Karp et al.,
1982). In wheat plants regenerated from cultured immature embryos, structural chromosome variations sucr. as trans locations were also identified during meiosis (K arp and Maddock, 1984). Root culture is usually established in order to investigate the secondary metabolite produced in ro ots. Untransformed roots or hairy roots as a results of the transformation with Agrobacterium rh izogenes have certain advantages compared to cell and suspension cultures. Root cultures of some species have greater leve l of genotypic and phenotypic stability than dedifferentiated cells so that problems with culture variation and loss
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productivity are alleviated (Hamill et al., 1987). Aird et al (1988) examined hairy root cultures cytologically to assess their chromosome numbers. All hairy root cultures of some species examined had correct 2n diploid number of chromosomes in root tip cells. This contrasted with their observation that in suspension cells of Nicotiana rustica and Beta vulgaris the chromosome numbers were variable with both polyploids and aneuploids. The aim of the study was to assess the genetic stability of root cultures of Artemisia cina and A. annua . These species are medicinal plants, A. cina is usually used as anthelmintics especially for children, antibacterial, antifunga l and antitumor because it produces alkaloids, saponin, flavonoid dan polyphenols (Syamsuhidayat and Hutapea 1991 ; Tan et al. 1998.), whilst A. annua which produces artemisinin, is commonly used "s antimalaria and more recently the artemisinin is also potensial as an tic~-tlce r drug. The genetic differences between transformed root lines resulted after transformation with some strains of Agrobacterium may provide a way of manipulating levels of secondary products in root cultures and of identifying the genes involved.
METHODOLOGY Roots collected from plantlets grown in vitro of A. cina and from callus originated from leaves of A. annua were used as cyto logical analysis of untransformed root cultures. A. rhizogenes strains ATCC-8196, 07-2000 1, ATCC- 15834 and A4 and A. tumefaciens strain RIOOO were used for transformation of A. cina after 2 days growth in LB (for A. rhizogenes strain ATCC-8 196 and A. tu mef aciens strain RlOOO) or YMB (for A. rhizogen es strains A4, 07- 20001, and ATCC- 1583 4) media. Steril e young stems or leaf blades on MS (Murashige and Skoog, 1962) medium were inoculated by wounding explants with a scalpe l which had been dipped into the bacteria l broth, then explants were incubated
at 28°C in darkness. After being freed from the bacteria using 100-200 mg/l cefotaxime, transformed root tips were cut at 1-2 em and cultured in MS liquid medium without additions of exogenous hormones. of transforma tion Confirmation between plants with Agrobacterium was carried out using PCR analysis (Hamill et al., 1991), and the DNA isolation of hairy roots was conducted according to Sambrook et al. (1989) . More than thirty root tips of each treatments (untransformed and each line of transformed roots) were excised and 1- 10 clear cells from each root tip was examined for chromosome count. Cytological analysis was conducted by squash method (Karp, 1991). Root tips of each tretament were collected and immersed in saturated paradichloro-benzene for 3 hours at room temperature follo wed by overnight fixation in glacial acetic acid : ethanol (I :3). Subsequently, root tips were hydrolyzed in 1 N HCl at room temperatu re for 5 min for A. cina and 10 min for A. annua and then washed in water and stained with 2% aceto orcein in 45% acetic acid. Finally root tips were squashed ill 45% acetic acid and examined under microscope. Cells at metaphase were ana lyz~d for chromosome number and a maximum of 10 clear cells from each root tip was scored. Cells that were broken or overlapping with neighbouring cells were not involved.
RESULT AND DISCUSSION The genus Artemisia is placed in the tribe of Anthemideae, consists of more than 200 species. The modal of chromosome number of genus Artemisia is x = 8 or 9. Some species of this genus including A. cina and A. annua hav e uniq ue chromosome numbers with both modal number of x = 8 and x = 9. The normal plants of these species ma y have chromosome number of diplOid 2n = 2 x = 18 (from modal x = 9) or of polyploid 2n = 4x = 32 (from moda l x =8) . (Darlington and Wylie, 1956; dePaclua et al., 1999). The di fferenc es in chromosome numbers could be rely on the origin and the
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distribution. Most of the species are native of Eurasia and North America. These species are fou nd in central and south western Asia which is thought to have originated here, and to have imigrated to North America. Some species have been introduced in the Malesiana area, including Indonesia, usua lly as ornamental and medicinal plants (dePaclua et at., 1999). We found in this study that the normal chromosome number of A. cina roots was polyploid number with 4x = 2n = 32. It
showed that most of the cells in roots of A. cina untransformed roots had chromosome number of2n = 32 (60.1%), 23 .5% of cells had aneuploid chromosome number below 32, and 16.4% of cells had chromosome number higher than the normal cells (Table I). Only one out of 153 cells observed found to have chromosome number half of the normal cells, 2n = 16. The distribution of the chromosome number examined from 153 cells is presented in Figure. I.
Table!. Chromosome number of untransformed and transformed roots of Artemisia cina. Strain of
Source of roots
Agrobac/erium
Un transformed
Number of cells with various chromosome number (2n) 16-23 24-31 8-15 32 33 -64
(%)
(%)
(%)
2
7 (4.6) 9 (21 .4) 9 (5 .4) 18 (10.9) II (14 .9) 16 (30.7)
27 (17.6) 0
(1.3) Transformed
ATCC-8 196 07-20001
0 6 (3.6)
ATCC-1583 4 A4
0 3 (4.1 ) 7 . (13.5)
RI000
27 (1 6.2) 36 (21.8) 7 (9.5) 4 (7.7)
(%) 92 (60.1) 33 (78.6) 101 (60.5) 96 (5 8.2) 39 (52.7) 21 (40.4)
Total cells observed
(%) 25 ( 16.4) 0
153
24 ( 14.3 ) 15 (9 .1 ) 14 (18 .8) 4 (7 .7)
167
42
165 74 52
80 ~----~~~~~~77~~~-'
70
60 50
40 30
20 10 O~~~~~~~~~~~~~ 8
Figure I .
Cytological analysis of A. cina roots isolated from plantlets grown in MS solid medium with no hormones
In transformed roots of A. cina , genetic stability was affected by strains of Agrobacterium. Roots transformed with A. rh izogenes A TCC-8196 was the most stable
compared to roots transformt"d with other strains of Agro bacterium (Table 1; Figure. 2-6). No roots were found to have higher number of the normal chromoso:nes, and
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21.4% of cells had chromosome number of 2n = 22 which found in a separate root culture (Figure 2). Chromosome numbers of roots transformed with Agrobacterium strain 07-25001 ranged from 8 to 42 (Figure.3), with strain ATCC-15 834 ranged from 16-64 (Figure.4), with strain A4 ranged from 8-58 (Figure . 5) and with strain RIOOO ranged from 8-50. Roots transformed with A. tumefaciens shain RIOOO was the most unstable line. Only 40.4% of cells had correct number of chromosomes (Figure. 6). The ·low genetic stability as showed by cytological analysis in A. Gina transformed roots was not a common phenomenon. In Swainsona galegifolia for example, transformed root cultures resulted after transformation with A. rhizogenes strains LBA 9402 and A4 had more than 90% normal diploid chromosome numbers. This also the case of untransformed roots grown in some different media and roots isolated from seedlings and germinated seeds. The results was in contrast with callus culture and roots regenerated from callus (Ermayanti et al., 1993). Hairy roots of A. annua after infection with A. rhizogenes strain LBA-9402 had stable chromosome number of 2n = 18 after 20 months in culture (Mukherjee et al. , 1994). Growth of transformed roots of A . cina in MS medium with no hormones was higher than that of untrans formed roots (Aryanti et al., 2001). However, growth of hairy root lines resulted from transformation with Agrobacterium strains A TCC-8 196, A4 and R 1000 was lower than hairy roots resulted from Agrobacterium line 07-20001 and ATCC-15834. The root tips of hairy roots lines A TCC-8196, A4 and R I 000 were very quick to tum brown. These problems, therefore, caused the dificulty to find clear metaphase cells for chromosome examination. Such problems were not found fo r hairy roots line of Agrobacterium strains 07-20001, ATCC-15834 and untransformed roots, so that more than a hundred clear metaphase cells could be examined for the chromosome analysis.
PCR analysis showed that only Tl region of T-DNA plasmid was detected (Aryanti et al., 200 I) . This results is in contrast with A. bel/adona transformed with A. rhizogenes strain ATCC- 15834, both Tl and Tr-DNA were integrated in the transformed roots (Aoki et al., 1997). In Azadiracta indica roots, the transformation event affected the integration of the T-DNA to the plant genome . Both Tl- and T r-D NA regions were detec ted in two out of three root clones examined , whilts one root clone only contained Tl-DNA region (Ermayanti et at., 2000). The integration of the T-DN wich was only for Tl-DNA in A. cina may be related to the loss of some chromosomes in most root lines. The selection of the line of roots from each clone could be invenstigated to obtain stable root clones which may result in the stable production of bioactive compound of the roots. The low stability in transfonned root~ of A. cina is also the case of Vicia faba that found that 50% of transforrned roots examined had polyploid (2x-9x) and 6% had aneuploid chromosome numbers or structural rearrangements (Ramsay and Kumar, 1990). The chromosomes become eliminated apparently at random. In somatic hybrid cells, the chromosomes of one of the parents may be eliminated preferentially. In hairy roots , cytological changes were also detected in transfonned root cultures of Trifolium pratense and in regenerated plants of Lotus corniculatus (Webb et al., 1990) . The reduction of chromosome numbers and the loss of regeneration ability during subculture had occurred in hairy root cultures of Onobrychis viciaefolia (Xu and Jia, 1996). This may be the case for A. cina, that transformed roots used in this experiment had been subcultured for 3-6 times, the age of the cultures were ab0ut 6 12 months. To confirm the effect of culture age and subculture on genetic stability of A. Gina and A. annua roots , cytological examination of germinated seeds and primary roots grown directly at the wounded site of Agrobacteriu1I1 infec tion shuuld be done.
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80 ~------~~~
__~__~____~~
70 60 50 40 30 20 10 O ~~~~~~~~~~~~~~ 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64
chromosome numbers (2n) Figure 2.
Cytological analysis of A. cina hairy roots transfonned with A. rhizogenes ATCC-8196 80 ~~--~~~~--~~~--~~~
70 60 50 40 30 20 10 O ~~~~~~~~~~~~""~~ 8 12 16 20 24 28 32 36 40 '14 48 52 56 60 64
chromosome numbers (2n)
Figure 3.
Cytological analysis of A. cina hairy roots transfOlmed with A. rhizogenes 07 2000 1
Figure 4.
Cytological analysis of A. dna hairy roots transformed with A. rhizogenes ATCC-15834
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~-~.-.-- --. ~~
. --------.
60 50
40 30 20 10 O~~~~~~~~~~~~~~~
8 12 16 20 24 28 32 36 40 44 48 52 56 60 64
chromosome numbers (2n) ----------------------------~
Figure 5. Cytological analysis of A. cina hairy roots transformed with A. rhizogenes A4 80 ~------~------------~~--~
70 60
50 40 30
20 10 O~~~~~~~~~""~~~~~
812 16 2024283236 40 444852 5660 64
chromosome numbers (2n)
Figure 6. Cytological analysis of A. cina hairy roots transformed with A. tum efaciens R I000 In A. annua untransfomed roots had 56.5 % of the cells with expected chromosome number of 2x = 2n = 18 but 43. 5% cells had uneuploid numbers of 14 17. This is similar to the report of Mukherjee et al. (1994) that after transformation with A. rhizogenes strain LBA 9402 hairy root culture of A. annuli has stable in chromosome number (2n = 18), whilst cell suspension culture derived from hairy root line has variation in chromosome number between 2n = 18 to 48 . The original plants of A. annua also has normal diploid number of 2x = 2n = 18 (Darlington and Wy lie, 195 6; dePlauca et al., 1999). The A. annua is now being transformed with some strains of Agrobacteriwn to establish hai ry root cu ltures. The chromosome analysis of transfo rmed roots will be compared with
that of untransformed roots to assess the genetic stability as well as the production of artemis in in in vitro. Although loss of particular chromosomes in A. dna and A. ann ua might result in biochemical differences leading to higher secondary metabolite production, it is more likely that roots with higher ploidy would have higher level of secondary metabolites . In attempt to obtain polyploid lines of these species, colchicine treatment of plants could be tested.
ACKNO\VLEDGEME NTS The authors would like to Oktavia Yanti for technical assistance in cytological ana lysis , to Adelina for the maintenance of root, shoot and callus cultures, to S.
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Rahmawati and Aryanti for the assis tance work on PCR analysis and to Dr. MA . Subroto and D . Sudrajat for allowing us in using primers for PCR ana lysis .
REFERENCES Aird, E.L.H ., 1.0. Hammill and M.J.C . Rhodes. 1988 . Cytological analysis of hairy root cultures from a number of plant species transfo rmed by Agrobacterium rhizogenes. Plant Cell Tissue Organ Cult. 15 : 47-57. Aoki, T ., H . Matsumoto, Y. Asako, Y. Matsunaga and K. Shimomura. 1997. Variation of alkaloid productivity among several clones of hairy roots and regenerated plants of Atropa transformed with belladona Agrobacterium rhizogenes 15834. Plan t Cell Rep . 16 : 282-286. Aryanti, M. Bintang, T. M. Ermayanti and I. Mariska. 2001 . Production of antileukemic agent in untransformed and transformed root cultures of Artemisia cina. Paper presented in the Indones ian Biotechnology Conference. 24 -26 October 2001. Yogyakarta . Indonesia. Darlington, C.D . and A. P. Wylie. 1956. Chromosome Atlas of Flowering Plants. George A llen & Unwin Ltd . London. Pp. 266-267. DePlauca. L.S. , N . Bunyapraphatsara and R.H.M.J. Lemmens. 1999. Medical and Poisonous Plants 1. Prosea No. 12 ( 1). Prosea Foundation. Bogor. Indonesia. Pp. 139- 147. Ermayanti T.M., L. Sari, E.M .R. Siregar and D. Sudrajat. 2000. Transformasi mimba (A zadirachta indica A . Juss.) dengan Agrobacterium rhizogenes galur A TCC-15834. Paper presented in Kongres dan Seminar Nasional Perhimpuna n Bioteknologi Pertanian Indonesia II , 7-8 No pember 2000. Yogyakarta. Indonesia. (in Indonesian) Ermayanti , T.M., J.A . McComb and P.A. O ' Brien. 1993. Cytological analysis of seedling roots, transformed root
cultures and roots regenerated from callus of Swainsona galegifolia (Andr.) R. Br. 1. Exp. Bot. 44 (259) : 375-3 80. Hamill, J.D., A.J. Parr, M .J.C. Rhodes, R.J. Robins and N.J. W alton. 1987 . New routes to plant secondary products . Biotechnology. 5 : 800-804. Hamill, J.D ., S. Rounsley, A. Spenser, G . Todd and M .J.C. Rhodes. 1991 . The use of the polymerase chain reaction in plant transformation studies. Plant Cell. Rep. 10 : 221-224. Jha, T.B. and Roy, 1982. Chromosomal behaviour in culture of Vicia fa ba . Cytologia. 47 : 465-470. Karp, A. 199 1. Cytological techniques . Plant cell Culture manual. C4 : 1-13. Karp, A . and S.E. Maddock. 1984. Cromosome vanatIOn in wheat plants regenerated from' cultured immature embryo. Theor. Appl. Genet. 67 : 249-255. Karp, A. , R.S. Nelson, E. Thomas and S. W.J. Bright. 1082. Chromosome variation in protoplast-derived potato plants. Theor. Appl. Genet. 63 : 265 272. McCoy, T .J. and R.L., Phillips. 1982. Chromosome stability in maize (Zea mays) tissue cultures and sectoring in some rege nerated plants. Can. 1. Genet. Cwol. 24 : 559-565 . Mehra, A. and P.M. Mehra. 1974. Organogenesis and plan let formation in vitro almond. Bot. Gazette. 135 : 61 -73. Mnkherj ee, S., S. Das and S. Jha. 1994. Chromosome stability in transformed hairy root cultures of Artemisia annua L. Cell. Chromo Res. 17 : 71 76. Murashige, T . and F. Skoog. 1962. A revised medium for rapid growth and tobacco tissue bioassays with cultures . Phisio. Plant. 15: 473-497. Nuti Ronc hi, V., M . Nozzolini and L. A vanti. 1981. Chromosoma 1 variation on plants regenerated from two Nicotiana spp. Protoplasma. 109 : 433-444 .
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Orton, TJ. 1980. Chromosomal variability in tissue cultures and regenerated plants of Hordeum. Theor. Appl. Genet. 56 : 101-11 2. Sambruok, 1, E.F. Fritsch and T. Maniatis. 1989. Molecular Cloning, a Laboratory Manual. 2 nd Ed., Cild Spring Harbor Laborato ry Press. Syamsuhidayat, S.S. and J.R. Hutapea. 1991 . Inventaris Tanaman Obat Indonesia 1. Badan Perielitian dan Pengembangan Kesehatan. Departemen Kesehatan. Jakarta (in Indonesian) Tan. R.X., W.E. Zheng and H. O. Lang. 1998 Biologically active subs trances from the genus Artemisia. Plant Medica. 295 -302. Webb, K.1 ., S. Jones, M .P. Robbins and F.R. Minchin. 1990. Characterization of transgenic root cultures of Trifolium rep ens, Trifoliumpratense and Lotus corniculatus and trangenic plants of Lotus corniculatus. Plant Sci. 70 : 24 3-254. XU, Z.Q. and J.F. Jia. 1996. The reduction of chromosome number and loss of regeneration ability during subculture of hairy root cultures on Onobrychis viciaefolia transformed by Agrobacterium rhizogenes A4. Plant Sci . 120: 107- 11 2. QUESTIONS AND ANSWERS Nuri ta (Res. Unit for Estate Crop) Q I. I think your experiment is quite interesting bec ause you have very nice chromosome . But, I want to remind you that chromosomal variation is sometime not because of the change in the number of the chromosome, but it is rather caused by several changes of the nucleotides. Which one do you think is quite better for this case, using chromosome analysis or DNA ana lysis?
the line of the culture, or the species of your Artemisia? Q3. Which one do you also thi nk that stabilizes the genetic causing variation in the number of chromosome? For example is X-8 more stable than X-9? Al&A2 : The research just began. In the fu ture , I would like to develop strain collection with same chromosome number, and then the concentration of bioactive compound will be detected. So I did not choose any special strain for that, but I prefer to choose one that gives the highest bioactive compound concentration. Sometime species which do have haploid chromosome appeared to be more stable. In this case, the important thing we have to see is the involvement of Agrobacterium itself. Agrobacterium, sometimes, is stable on several subcultures and lines , but I wonder that Artemisia would be more vary in number. I have observed out of about 160 ce11l and I found various number of chromosome. The problem on determining control that confirming if they are stable or not, is that Artemicia's growth is variation is generated from seed on not later. A3 : From literatures, sometimes they are stable at 36 and sometime at 18. But, there is no report if this gives such positive implications to the Artimycin content or not. This is important to be answered. I am afraid I cannot answer whether X-8 is better than X-9.
Q2 . Whic h one do you think has the strong influence, the strain of Agrobacterium, THE
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