CYTOLOGICAL ANALYSIS OF ROOT CULTURES OF Artemisia cina

Tri Muji Ermayanti Oktavia Yanti and Erwin AJ Hafiizh

Research Centre for Biotechnology Indonesian Institute of Sciences (LIPI)

lalan Raya Bogor Km 46 Cibinong Indonesia 16911

Phone 62-21-8754587 Fax 62-21-87545 E-mail lmermayanlihormailcom

ABSTRACT

Artemisia cina is a medicinal plant species producing bioactive compounds which are p tential a antitumor antifungal and antibacterial The aim of this ludy was to analyze the stabililY of chromo orne number in root cultures of A cina Transformed root culture was established by infect ion of leaves of A cina with Agrobacterill1l rhizogenes strains 07-20001 ATCC-15834 A4 and A lwnejaciens strain RlOOO Roots isolated from glasshou e plants plantlets grown in solid and liquid MS medium were util ized for investigation of chromo orne examination of unlransfonned roots Chromosome examination was onducled by squashing method and chromosome numbers w re calcululed under microscope The re ults showed that both untransformed and Iransromled root had in lability in the chromosome number but had the modal number of chromosome x=8 with the uiploid number of2n =4x = 32 Roots i olaled rrom glasshou e plants of A ina had 53 7~ of cell with the diploid numbers of 2n = 3_ and 463 of ce lls had chromosome numbers ranged from 2n = 12 LO 2n = 64 Untransformed roots isolated from plantlets cultured in solid medw had only 361 or cells with chromo orne number of 20 = 32 and unlran fomled ro t5 grown in liquid medium had 494 of cells with 20 =32 The chromosomt numbers of A dna transformed roots was affected by trains of Agrobacterillm Ro t lransfonned with the bacterium Slram 07-20001 showed lhe high t in normal chromosome number of 2n = 32 (624) followed by roots lransformed wiLll strains AT C-15834 (6 L9 ) R1000 (436~) and A4 (430) The range of the chromosome number r untransfonned rOOLS was from 2n= 17 10 2n=64 whi l t that of transformed roots was from 2n= 1t to 20=66

Keywords untransformed roots transformed rools chr mo me number genetic stabi lity

Artemisia cilia

ABSTRAK

Arrelllisia cilia merupakan atu jenis tanaman obat yang memproduksi zat bioaklif yang potens iaJ sebagai antitumor anticendawan dan antibakteri Tujuan dari penelitian ini adalah untuk menganali is stabilitas jumlah kromosom pada kullur akar Artemisitl cillll Kultur akar rambut diperoleh dengan menginfeksi daun tltlnaman dengan Agrobacterillnl rhizogeles galur 07-20001 ATCC- 15834 A4 dan Agrobacterf1 tllmejaciens galur R 1000 Akar yang diisolasi dari tanamun yang ditumbuhkan di rumah kaca planlet yang diku llurkan pada media MS padat dan kultur akar pada med ia cair digunakan unluk pengumatan kromo om untuk akar bukan hasil Lransforma i (akur normal) Pengamatan kromosom dilukukan dengan melode stundar dengan pewarna asetoorsein Setelah dihancurkuo akar diamati di bawah mikroskop Hasil penelitian menunjukkan bahwa akar bukan hasil transformasi maupun akar rambut mempunyai ketidakstabilan dalarnjumJuh kromosom tetapi mempunyaijumlah kromosom dengan modal x=8 dan jumlah diploid 2n=4x=32 Akar yang diisoJasi dari tanaman l1lmah kaca mempunyai

50 Annale Bogorien es n Vol 9 no2 2004

537 sel yang berjumlah kromosom normal diploid yai tu 2n = 32 dan 463 sel lainnya mempunyai kisaran jumlah kromosom dari 2n = 22 sampai dengan 2n = 64 Akar yang di isolas i dad pianlel yang tumbuh di media MS padat hanya mempunyai 36 1 sel dengan jum1ah kromosom [lorma l 2n=32 edangkan akar yang ditumbuhkan dalam med ia ca ir mempunya i 494 sel denganjumlah 2n=31 Jumlah

kromosom pada akar rambul bervariasi lergantung dari galur AgrobacleriulII yang dipergunakan untuk infeksi eksplan tanaman Akar rambu t hasil transformasi dengan galur bakteri 07-20001 mempunyai stabiliLas kromo om yang paling linggi dengan jumlah normal diploid 2n = 32 (624) diikuti oleh akar rambul dnri galur ATe -15834 (619) RlOOO (436) dan A4 (430) Kisaran jumlah Icromosom pada akar dan lanaman rumah kaca dan dari media MS padat dan caif adalah 2n=I7-64 sedangkan kisaran jumlah kromosom akar rambut ada lah 2n= 11-66

Kata kunci ku Ilu r akar normal dan akar rambul jumlah kromosom stabilitas genetik Artemisia cillC

INTROD eTION

Variation in chromosome numbers is a common phenomenon in plant cell and tissue cu lture and increases with the age of the culture For example in Vicia faba aneuploidy increa e with the increasing age or cultured callus (Jha amp R y 1982) and in almond increa jng age results in hIgh level of polyploidy as well a aneuploidy (Mchra amp Mehra 1974) SImilar results were ob erved In maize (McCoy amp Phillips 1982) NicOfiana specie (Nuti Ronchi ef al J981) llordeum (Onon 1980) and protoplas tshyderived potato plants (Karp et al 1982) In wheat plants regenerated from cultured immature embryos structural chromosome variation such as Iranslocations was also identified during meiosis (Karp amp Maddock 1984)

Rootculture is usually established in order to investigate the secondary metabolite produced in roots ntransfomled roots or hairy root as a result of transformation with AgrobucteriufII rhizogenes have certa in advantages compared to cell and suspension cultures Root culture of some species have greater level of gen typic and phenotypic stability than dedifferentiated cells so that problems with culture variation and loss of productivity are alleviated (Hamill ef aI 1987)ln ome specie bioactive compounds produced in hairy root are much greater than that in cell suspension cultures (Sasaki et al 1998 Lee el al 1999 Sallo et al 200 1) or

leav s of plantlets (Zarate 1999) and varied depending on the plant genotype (Ermayanti 1999) Aird el al (1988) examined hairy roOl cultures cytologically to assess their chromo orne numbers All hairy root cultures of some species examined had correct 2n diploid number of chromosomes in root tip cells Thi contrasted with their observation that in suspension cells of Nicofiana rust ca and Bera vulgaris the chromosome numbers were variable with both polyploids and aneuploid

Artemisia cina is a medicinal plant that usually used as anthelmintic e pecially for children antibacterial antifungal and antitumor because it produces alkaloids saponin flavonoid dan polyphenols (Syamsuhidayal amp HUlapea 1991 Tan et al 1998) Chromosome number of genus Anenlisa is x = 8 or 9 A cina has unique chromo ome numbers with both modal number of x =8 and x =9 The normal plants of these species have chromosome numbers of diploid 2n =2 x =18 (from modal x = 9) or of polyploid 211 =4x =32 (from modal x =8) (Darlington amp Wylie 1956 dePadua e

al 1999) The aim of th is tudy was to as ess the genetic stability or root cultures of Al1elllisia cilia by assessment of chromosome numbers in both untranslormed and transformed roOl cultures The genetic information between transformed root Jines resu lted afte r transformation with some strains f Agrobacterilll1l i needed for manipulating levels of seconJary products in

nnales Bogorien cs n s Vol 9 no2 2004 5 1

root cullure and of identifying the genes involved

MATERIALS AND METHODS

Roots collected from intact plants of A cina grown in glasshouse (originated from Medicinal Plant Research Divi ion Re earch and Development Centre for Health Tnwangmangu Central Java Indonesia) and tho e i olaled from plantlets resulted from shoot multiplications grown in solid and liquid MS medium (Murashige and Skoog 1962) without hormones were used as cytological analysis of untransformed root cultures A rh izogenes slrains 07-20001 ATCC- lS834 A4 and A lumejaciells strain R 1 000 were used for transformation of A cina after 2 day grown in LB (for A lumejaciens strain RJOOO) or YMB (for A rhiwgenes strains A4 07-20001 andATCCshy15834) medium Sterile leaf blades on MS medium were inoculated by wound ing explants with a scalpel which had been dipped into the bacterial broth then the explants were incubated at 28degC in darkne s After being freed from the bacteria using 100shy200 mgl cefotuxime transf rmed root tips were cut at 1-2 em and cultured in MS liquid medium without additions of exogenous hormones

Cytological analysis was conducted by squash meth d (Karp 1991) Root tip of each treatment were olleeted and immersed in alurated paradichloro-benzene for 3 hours at room temperature followed by overnight fi ation In glacial acetic acid ethanol (13) Subsequently root tips were hydrolyzed in 1 N HCI at room temperature for 5 min then washed in water and stained with 2 aceto orcein in 45 acetic acid Finally root lips were squa hed in 45 acetic acid and examined under microscope Cells at metaphase were analyzed for chromosome number and a maximum of to clear cells from each root tip were cored Cells that were broken or overlapped with the neighbouring

cells were not involved More than 30 root tips of each treatments (untransfonned and each line of transfonned roots) were excised and more than 100 clear cell were exami ned for chromosome counts

RESULTS AND DISCUSSION

The results indicate that the nonn al chromosome number of A cina roots wa polyploid number with 4 x = 2n = 32 It showed that untransformed root (roots of glasshouse plants root middot isolated from plan tl ets cultured in solid and liquid MS medium) had variation in chromosome numbers which ranged from 2n= 17 to 2n=64 From 192 clear metapha e cells of glasshouse plants showed 537 of cells had correct chromosome numbers of 2n=32 152 of cells had chromosome numbers lower than the nonnal 2n number and 313 of cell had higher than normal chromosome numbers (Table 1) The chromo orne numb r of gla shouse plants ranged from 2n=22-64 (Fig 1) Only one cell out of 192 cells examined had polyploid numbers of 4n =64 None of the cells had haploid numbers

Ro t i olated from both plantlets grown in solid MS medium or from untransform d roots grown in liquid medium with no addition of exogenous hormones had greater variation of chromo orne number compared to that ofroots of glasshouse plant (Table 1) Roots in solid medium found to have chromosome numbers ranged from 2n= 17 to 2n=62 (Fig2) whilst roors in liquid culture had 2n= 18-63 chromosomes (Fig3) Roots grown in MS liquid medium was very s low so that the amples taken for chromosome analysi were less than j 00 cells Only few clear root tips which had clear metaphase cells found in thi experiment The lower genetic stabi lity in cultures compared with that of plants grown ex vitro is a common phenomenon many factors ontribute to genetic instability of cultures inc luding cell

Annales Bogoriense n Vo l 9 11022004 52

Table1 Chromo orne number of untran formed and tran formed roots of Artemisia Gina

Source of roots

Number of cells with various chromosome number (2n

Total cells

observed11 (00)

16 (00)

17-31 (00)

32 (00)

33-66 ()

Glasshouse plants 0 0 29 (152)

103 (537)

60 (31 3)

192

Plantlets grown in MS solid medium

0 0 30 (246)

44 (361)

48 (393)

122

Roots grown in MS liquid medium

0 0 25 (31 7)

39 (494)

15 (189)

79

Transformed roots strain 07shy20001

1 (05)

33 (161 )

128 (624)

43 (210)

205

Transformed roots strain ATCCshy15834

0 0 38 (195)

120 (61 9)

36 (186)

194

Transformed rools slrain Rl000 1 (10)

0 39 (386)

44 (436)

17 (168)

101

Transformed roots strain A4 0 0 61 (243)

108 (430)

82 (327)

251

100 90 80 70 60

50

140 30 20 10 o

11 16 21 26 31 36 41 46 51 56 61 66

chromosome numbers (2n)

Figure 1 Cytological analysis of A cina roots isolated from planJets grown in glasshouse

100 90 80 70 60

50 40 30 20 10 o ~

111621263136414851566166

chromosome numbers (2n)

Figure 2 Cytological analy is of A cina roots i olated from planlets cultured in MS solid medium without hormones

100 90 80 70 60

50 40 30 20 10 o -

11 16 21 26 31 36 41 46 51 56 61 66

chromosome numbers (2n)

Figure 3 Cytol gical analy i of A cina untransformed roots cultured in MS liquid medjum without hormone

aging compo ition of mineral elements environmental tress and DNA changes (D Amato 1990) These factors could occur in untran fanned roots ofA cina observed in this experiment

In transformed roots of A cilla genetic tability wa affected by strains of Agrobacterium Root tran fanned with A rhizogenes strain 07-20 0] was the rna t

stable compared to tho e transfonned with other train ofAgroba terium (Table 1) with range of chromosome numbers from 2n= 16 to 2n==43 and one out of 205 cells examined had chromo orne number of 2n=66 One cell

Annales Bogonenses n s Vo l 9 no2 2004 53

had haploid chromosome number of 2n=16 which found in separate root culture (Fig4) Chromo orne numbers of roots tran formed with Agrobacterium strain ATCC-J5834 ranged from 2n= 18 to 2n=60 (FigS) with 619 of them had normal diploid chTOmosome numbers of 2n -32 195 of c lis found to have chromosome numbers lower than the diploid numbers and 186 of cells had higher chromosome number than that of the diploid number Roots transfomled with A rumejaciens strain R1000 had chromosome numbers ranged from 2n=17 to 2n=59 (Fig 6) Roots transformed with A rhizogenes strain A4 was the most unstable line Only 430 of cells had correct number of chromosome 243 of cells had chromosome number lower than diploid and 327 of cells had chromosome number higher than that of diploid numbers The

100~------------------~ 90 80 70

50 60 J ij ~

11 1621 2631 36 41 4651 56 61 66

chromosome numbers (2n)

Figure 4 Cytological analysis of A dna hairy roots lransfonned with A rhizogenes 07-20001

100~------------------~ 90 80 70 60

50 40 30 20 10 ILO~~~~~~~~~=ft~

11 16 21 2631 36 41 46 51 56 61 66

chromosome numbers (2n)

Figure S Cytologi al analysis of A cina hairy roots transfonned with A rhizogenes ATCC-15834

54

distribution of the chromosome number of roots transformed with Agrobacterillm strain A4 is pre ented in Fig 7

The low genetic stability as showed by cytological anaJysis in A cina tran formed roots was in contrast with that of some other species In Swaillson gaegijoia transformed root cultures resultcd after transformation with A rhizogelles strains LBA 9402 and A4 had more than 90 normal diploid chromosome numbers This also the case of untransforrned roots grown in orne dilTerent medium and roots isolated from seedling and germinated seeds The results were in contrary with callus culture and roots regenerated from callus of S galegtjolia (Ennayanti et al 1993) Hairy roots of A anllUa after infection with A rhizogenes strain LBA-9402 had stable chromosome numbers

100 shy90 80 70 60

50 40 30 20 10 bull o

8 14 20 26 32 38 44 50 56 62 68

chromosome numbers (2n)

Figure 6 Cytological analysis of A cina hairy roots transformed with A tumeaciel1s R1000

100r-------------------~ 90 80 70 60

50 40 30 20 10 IIL0 ~~~~~m=~~~~1

8 14 20 26 3238 44 50 56 62 68

chromosome numbers (2n)

Figure 7 Cytological analy is of A cina hairy roots lransfonned with A rhizogeles

A4

Annales Bogoricnses n s Vol 9 no 2 2004

of 2n =18 after 20 months in culture medi urn (Mukherjee et a 1994)

Growth of transformed roots of A cina in MS medium without hormones wa higher than thal of unlransformed roots (Aryanti et a 200 I) However growth of hairy root lines resulted from transformation with AgrobacteriuTII strains RlOOO was lower tban hairy roots transformed with Agrobaclerium strains 07-2000 1 A4 and ATCC-1S834 The root tips of hairy root line R 1000 were very qu ickly turned brown This problem therefore caused the difficulty to find clear metaphase cells for chromo orne xaminaLion Such problem was not found

for hairy roots lines of Agrohacleriltm strains 07-20001 ATCC-lS834 and A4 so that more

lear metapha e cells could be examined for the chromosome analysis

PCR analysis showed that only T L region of T-DN A plasmid was detected (AryanLi el aI 200 L) The integration of the T-DNA which was only for T l-DNA in A cina may be related to the loss of some chromosomes in most root lines This resull is in contrast with Atropa belladollna lran formed with A rhizogenes strain ATCCshy15834 both T

L- and TI-DNAwere integrated

in the transformed roots (Aoki el al J997) In kadiracta illdica roOlS the lransformation event affected the integration of the T-DNA to the plant genome Both T - and TH-DNA

L

regions were detected in two out of three root clones examined whil t one root clone only containedTL-DNA region (Ermayanti el a

2000) The selection of root Jines from each clone of A ina could be investigated to obtain stable root clones which may result in the stable production of bioaclive compounds of the roots

The low tability in transformed roots of A cina may be caused by structural rearrungements as that in Vida faba that found that 50 of transformed roots examined had polyploid (2x-9x) and 6 had aneuploid chromosome numbers or structural reanangements (Ramsay amp Kumar 1990)

Annales Bogorieoses o s Vol 9 no2 2004

The chromosomes become e l im In ated apparently at random In omCltic hybrid cells the chromosomes of one of the parents may be eliminated preferentially This could be the case of A c ina root In hai r y roots cytological changes were also detected in transformed root cultures of Trifolium prarense and in regenerated plants or Lotus cornicHlatus (Webb e t al 1990) The reduction of chromosome numb rs and the loss of regeneration abi lity during subcullUre had occurred in hairy root cultures of Ollobrychisviciaefolia (Xu amp Jia 1996) This may be the case for A cilia that transformed rOOls used in this experiment had been subcultured for 3-6 times the age of the cultures was about 6-12 months To confirm the effect of culture age and subculture on genetic stability or A cina roots cytological examination of germinated seeds and primary roots grown directly at the wounded site of Agrobacterillm infection should be done however seed germination of A cina was very low Instability 0[ phenotype and gene expression in long-term cullure also occurred in carrot h iry root clones (Guivarch et aI 1999) in conlrast with the hairy rOOI clones of tomato wh ich was stable for 50 subcultures over 25 months (Joao amp Brown 1994) Opine and peR analysis should be done to conrirm the gene expres ion in long-term cullure or A cina The facl that untransformed and lran formed root or A ci1la have unstable in chromosome numbers mos t probably i caused by the variation in chromosome numbers of the intact plants themselves (Table 1) Germinated seeds are needed to confirm this genetic variability However the abi Ii t Y ofseed gemi nation was very low None of seeds harvested from three plants grown in th field germinated either in mixture of soil with sand or in a culture medium

The genu Artemisia is placed in the tribe of Anthemideae consists of more than 200 pecles The modal of chromosome numberofgenusArtemisia is x =8 or9 Some species of this genus including A cilia has

55

unique chromosome numbers with both modal number of x = 8 and x = 9 The normal plants of these species may have chromosome numbers of diploid 2n = 2 x= 18 (from modal x - 9) or of polyploid 2n = 4x = 32 (from modal x =8) (Darlington amp Wylie 1956 dePadua et at 1999) The differences in chromosome numbers could rely on the origin and the distribution Most of the species are native of Eurasia and North America These specie are found in central and South-Western Asia which is thought to have been originated here and to have j mmigrmed to North America Some species have been inLr duced in the Male iana area including Indonesia usually as ornamental and medicinal plants (dePadua ef aI 1999) A cina used in this experiment wa originated from Tawangmangu (Central Java Indonesia) There is no report on (he variation of chromosome on A dna grown in indonesia Therefore further investigation on chromosome analysis of this planL grown in Indone ia i needed to confirm whether the variation in chromosome number is a common phenomenon as in [he rool cultures

In conclusion root cultures ofA cilia ha variation in chromosome numbers wiLh basic diploid number of2n=32 Variation in the chromo orne number was also detected in roOls of plant grown in the glasshouse In transformed roots roots transformed with A rhizogefles strain 07-20001 was the most stable whi 1st roots transformed wi th A rhizogenes strain A4 was the most unstable line This information on variation in chromosome number may also contribute LO the further investigation of root culture of A cilla or for the genetic manipulation

ACKNOWLEDGEMENT

The authors would like to thank Adelina for the maintenance of root shoot and callus cultures

REFERENCES

Aird ELHJD Hammill amp MJC Rhodes 1988 Cytologica l ana lysis of hairy root cultures from a number of plant specie transformed by Agrobacterium rhizogenes Plant Cell Tiss Org Cult 15 47-57

Aoki T H Matsumoto Y Asako Y Matsunaga amp K Shimomura I 97 Variation of alkaloid productivity among several lones of hairy root and regenerated plants of Atropa beladona transformed with Agrobaclerium rhizogenes 15834 Plant Cell Rep J6 282-286

Aryanti M Bintang TM Ermayanti amp I Mariska 200 l Produclion of antileukemic agent in untransformed and transformed roOl cultures of Artemisia cina Anll Bogorienses 8 (l) 11middot16

DAmato F 1990 Somalic nuclear mutalion in vivo and ill vitro in higher plams Cmyologia 43 (3-4) 191-204

Darlington CD amp AP Wylie J956 Chromosome ALIas of Flowering Plants George Allen amp Unwin Ltd London pp 266-267

DePadua LS N Bunyaprapbatsara amp RHMJ Lemmens 1999 Medical and Poisonous Plants 1 Prosca No 12 (1) Prosea Foundalion Bogor Indonesia Pp 139-147

Ermayanti TM 1999 Genetic transformaLion of Swainsona galegifolia In Bajaj YPS (Ed ) B iOlechnology in Agriculture and Forestry 45 Transgenic Medicinal Plants Springerp 327-335

Ermayaoti TM JA McComb amp PA OBrien 1993 Cytological analysis of seedling rooLS transfonned root cultures and roots regenerated from callus of Swainsona galegifolia (Andr) R Br 1 Exp Bot 44 (259) 375-380

Annales Bogorienses n s Vol 9 no2 20O-t 56

Ermayanti TM L Sari EMR Siregar amp D udrajat 2000 Transformasi mimba (Azadirachla indica A Juss) dengan Agrobacterium rhizogel1es galur ATCCshy15834 Paper presenled al the Kongres dan Seminar Nasional Perhimpunan Bioleknologi Pertanian Indonesia 11 7shy8 Nopember 2000 Yogyakarta Indonesia

Guivarcb A M Boccara M Prouteau amp D Chriqui 1999 Instability of phenotype and gene expression in longshyterm culture of carrot hairy root clones Plant Cell Rep 119 43 -50

Hamill J D AJ Parr MJC Rhodes RJ Robins amp NJ Walton 1987 New routes to plant secondary products Bioi Technol 5 800-804

Jha Tn amp Roy 1982 Chromosomal behaviour in cullure of Vicia faba Cytologia 47 465middot470

Joao KHL amp TA Brown 1994 Long-term stability of root cultures of tomato transformed wilh Agrobacterillm ri1izogellesRI60IJ Exp Bot 274 641 shy647

Karp A 1991 Cytological techniques Plant cell Culture Manual Kluwer Academic Publishers C4 1-13

Karp A amp SE Maddock 1984 Cromosome variation in wheat plants regenerated from cultured immature embryo Theor Appl Genet 67 249-255

Karp A RS Nelson E Thomas amp SWJ Brigbt 1082 Chromosome variation in protoplast-derived potato plants Theor Appl Genet 63 265-272

Lee KT T Suzuki T Yamakawa T Komada Y Igara hi amp K Sbimomura 1999 Production of tropane alkaloids by transformed root cultures of Atropa bellacona in tirred bioreactors with stai nless steel net Plant Cell Rep 18 657-57 1

McCoy TJ amp RL Phillips 1982 Chromosome stability in maize (Zea nwys) tissue cultures and sectoring in some regenerated plants Call 1 Genet Cytol 24 559-565

MehraA amp PM Mehra 1974 Organogenesis and plant let formation in vitro almond Bot Gaz 135 61-73

Mukherjee S Das amp S Jba 1994 Chromosome stability in transformed hairy root cultures of Artemisia anllua L Cell Chrolllos Res 17 7 1-76

Murashige T amp F Skoog 1962 A revised medium for rapid growth and bioassays with tobacco tissue cultures Phisiol Plant 15 473-497

Nuti Ronchi V M Nozzolini amp L Avanti 1981 Chromosomal variation on plants regenerated from two Nicotiall spp Protoplasma 109 433 -444

Orton TJ 1980 Chromosomal variability in tissu cultures and regenerated plants of Hordeum Them Appl Genet 56 101shy112

Ramsay G amp A Kumar 1990 Transformation of Viciafaba cotyledon and stem Ii sue by Agrobacieriu1I rhizogenes infectivity and cytological studies 1 Exp Bot 4 1 (228 ) 841-847

Saito K H Sudo M Yamazaki M Ko eldshyNakamura MKitajima H Takayama amp M Aimi 200 l Feasible production ofcamptothecin by hairy roOl culture of Ophiorrhiza pumila Plant Cell Rep 20 267-27 1

Sasaki K A Udagawa H Ishimaru T Hayashi A W Alfermaoo F Nakanishi amp K Shimomura 1998 High for kolin production in hairy roots of ColellS forskohlii Plant Cell Rep 11 7 457-459

nnales Bogorienses n s Vol 9 no2 2004 57

Syamsuhidayat SS amp JR Hutapea 1991 Invemaris Tanaman Obat Indonesia 1 Badan Penelitian dan Pengembangan Ke ehalan Departemen Kc ehalan Jakarta

Tan RX WE Zbeng amp HO Lang 1998 Biologically active subslrunces rrom the genus Artemisia Plant Med 295-302

Webb KJ S Jones MP Robbin amp FR Minchin 1990 Characterization of transgenic root cultures of Trifolium repeflS Trifolillmprarense and Lotus comicllatus and transgenic plants of LotllS comiculatus Plant Sci 70 243shy254

XU ZQ amp JF Jia 1996 The reduction of chromosome number and loss of regeneration ability during subculture of hairy rOOl cultures on Onobryc7is viciaeolia transformed by Arobacterilll11 rlzizogenel A4 Plant Sci 120 I 07 - 112

Zarate R 1999 Tropane alkaloid production by Arobaclerillm rhizogenes transformed hairy root culture ofAtropa baetica Willk (S lanaceae) Plant Cell Rep 18 418-423

Annales Bugorienses n s Vol 9 no2 2004 58

Table1 Chromo orne number of untran formed and tran formed roots of Artemisia Gina

Source of roots

Number of cells with various chromosome number (2n

Total cells

observed11 (00)

16 (00)

17-31 (00)

32 (00)

33-66 ()

Glasshouse plants 0 0 29 (152)

103 (537)

60 (31 3)

192

Plantlets grown in MS solid medium

0 0 30 (246)

44 (361)

48 (393)

122

Roots grown in MS liquid medium

0 0 25 (31 7)

39 (494)

15 (189)

79

Transformed roots strain 07shy20001

1 (05)

33 (161 )

128 (624)

43 (210)

205

Transformed roots strain ATCCshy15834

0 0 38 (195)

120 (61 9)

36 (186)

194

Transformed rools slrain Rl000 1 (10)

0 39 (386)

44 (436)

17 (168)

101

Transformed roots strain A4 0 0 61 (243)

108 (430)

82 (327)

251

100 90 80 70 60

50

140 30 20 10 o

11 16 21 26 31 36 41 46 51 56 61 66

chromosome numbers (2n)

Figure 1 Cytological analysis of A cina roots isolated from planJets grown in glasshouse

100 90 80 70 60

50 40 30 20 10 o ~

111621263136414851566166

chromosome numbers (2n)

Figure 2 Cytological analy is of A cina roots i olated from planlets cultured in MS solid medium without hormones

100 90 80 70 60

50 40 30 20 10 o -

11 16 21 26 31 36 41 46 51 56 61 66

chromosome numbers (2n)

Figure 3 Cytol gical analy i of A cina untransformed roots cultured in MS liquid medjum without hormone

aging compo ition of mineral elements environmental tress and DNA changes (D Amato 1990) These factors could occur in untran fanned roots ofA cina observed in this experiment

In transformed roots of A cilla genetic tability wa affected by strains of Agrobacterium Root tran fanned with A rhizogenes strain 07-20 0] was the rna t

stable compared to tho e transfonned with other train ofAgroba terium (Table 1) with range of chromosome numbers from 2n= 16 to 2n==43 and one out of 205 cells examined had chromo orne number of 2n=66 One cell

Annales Bogonenses n s Vo l 9 no2 2004 53

had haploid chromosome number of 2n=16 which found in separate root culture (Fig4) Chromo orne numbers of roots tran formed with Agrobacterium strain ATCC-J5834 ranged from 2n= 18 to 2n=60 (FigS) with 619 of them had normal diploid chTOmosome numbers of 2n -32 195 of c lis found to have chromosome numbers lower than the diploid numbers and 186 of cells had higher chromosome number than that of the diploid number Roots transfomled with A rumejaciens strain R1000 had chromosome numbers ranged from 2n=17 to 2n=59 (Fig 6) Roots transformed with A rhizogenes strain A4 was the most unstable line Only 430 of cells had correct number of chromosome 243 of cells had chromosome number lower than diploid and 327 of cells had chromosome number higher than that of diploid numbers The

100~------------------~ 90 80 70

50 60 J ij ~

11 1621 2631 36 41 4651 56 61 66

chromosome numbers (2n)

Figure 4 Cytological analysis of A dna hairy roots lransfonned with A rhizogenes 07-20001

100~------------------~ 90 80 70 60

50 40 30 20 10 ILO~~~~~~~~~=ft~

11 16 21 2631 36 41 46 51 56 61 66

chromosome numbers (2n)

Figure S Cytologi al analysis of A cina hairy roots transfonned with A rhizogenes ATCC-15834

54

distribution of the chromosome number of roots transformed with Agrobacterillm strain A4 is pre ented in Fig 7

The low genetic stability as showed by cytological anaJysis in A cina tran formed roots was in contrast with that of some other species In Swaillson gaegijoia transformed root cultures resultcd after transformation with A rhizogelles strains LBA 9402 and A4 had more than 90 normal diploid chromosome numbers This also the case of untransforrned roots grown in orne dilTerent medium and roots isolated from seedling and germinated seeds The results were in contrary with callus culture and roots regenerated from callus of S galegtjolia (Ennayanti et al 1993) Hairy roots of A anllUa after infection with A rhizogenes strain LBA-9402 had stable chromosome numbers

100 shy90 80 70 60

50 40 30 20 10 bull o

8 14 20 26 32 38 44 50 56 62 68

chromosome numbers (2n)

Figure 6 Cytological analysis of A cina hairy roots transformed with A tumeaciel1s R1000

100r-------------------~ 90 80 70 60

50 40 30 20 10 IIL0 ~~~~~m=~~~~1

8 14 20 26 3238 44 50 56 62 68

chromosome numbers (2n)

Figure 7 Cytological analy is of A cina hairy roots lransfonned with A rhizogeles

A4

Annales Bogoricnses n s Vol 9 no 2 2004

of 2n =18 after 20 months in culture medi urn (Mukherjee et a 1994)

Growth of transformed roots of A cina in MS medium without hormones wa higher than thal of unlransformed roots (Aryanti et a 200 I) However growth of hairy root lines resulted from transformation with AgrobacteriuTII strains RlOOO was lower tban hairy roots transformed with Agrobaclerium strains 07-2000 1 A4 and ATCC-1S834 The root tips of hairy root line R 1000 were very qu ickly turned brown This problem therefore caused the difficulty to find clear metaphase cells for chromo orne xaminaLion Such problem was not found

for hairy roots lines of Agrohacleriltm strains 07-20001 ATCC-lS834 and A4 so that more

lear metapha e cells could be examined for the chromosome analysis

PCR analysis showed that only T L region of T-DN A plasmid was detected (AryanLi el aI 200 L) The integration of the T-DNA which was only for T l-DNA in A cina may be related to the loss of some chromosomes in most root lines This resull is in contrast with Atropa belladollna lran formed with A rhizogenes strain ATCCshy15834 both T

L- and TI-DNAwere integrated

in the transformed roots (Aoki el al J997) In kadiracta illdica roOlS the lransformation event affected the integration of the T-DNA to the plant genome Both T - and TH-DNA

L

regions were detected in two out of three root clones examined whil t one root clone only containedTL-DNA region (Ermayanti el a

2000) The selection of root Jines from each clone of A ina could be investigated to obtain stable root clones which may result in the stable production of bioaclive compounds of the roots

The low tability in transformed roots of A cina may be caused by structural rearrungements as that in Vida faba that found that 50 of transformed roots examined had polyploid (2x-9x) and 6 had aneuploid chromosome numbers or structural reanangements (Ramsay amp Kumar 1990)

Annales Bogorieoses o s Vol 9 no2 2004

The chromosomes become e l im In ated apparently at random In omCltic hybrid cells the chromosomes of one of the parents may be eliminated preferentially This could be the case of A c ina root In hai r y roots cytological changes were also detected in transformed root cultures of Trifolium prarense and in regenerated plants or Lotus cornicHlatus (Webb e t al 1990) The reduction of chromosome numb rs and the loss of regeneration abi lity during subcullUre had occurred in hairy root cultures of Ollobrychisviciaefolia (Xu amp Jia 1996) This may be the case for A cilia that transformed rOOls used in this experiment had been subcultured for 3-6 times the age of the cultures was about 6-12 months To confirm the effect of culture age and subculture on genetic stability or A cina roots cytological examination of germinated seeds and primary roots grown directly at the wounded site of Agrobacterillm infection should be done however seed germination of A cina was very low Instability 0[ phenotype and gene expression in long-term cullure also occurred in carrot h iry root clones (Guivarch et aI 1999) in conlrast with the hairy rOOI clones of tomato wh ich was stable for 50 subcultures over 25 months (Joao amp Brown 1994) Opine and peR analysis should be done to conrirm the gene expres ion in long-term cullure or A cina The facl that untransformed and lran formed root or A ci1la have unstable in chromosome numbers mos t probably i caused by the variation in chromosome numbers of the intact plants themselves (Table 1) Germinated seeds are needed to confirm this genetic variability However the abi Ii t Y ofseed gemi nation was very low None of seeds harvested from three plants grown in th field germinated either in mixture of soil with sand or in a culture medium

The genu Artemisia is placed in the tribe of Anthemideae consists of more than 200 pecles The modal of chromosome numberofgenusArtemisia is x =8 or9 Some species of this genus including A cilia has

55

unique chromosome numbers with both modal number of x = 8 and x = 9 The normal plants of these species may have chromosome numbers of diploid 2n = 2 x= 18 (from modal x - 9) or of polyploid 2n = 4x = 32 (from modal x =8) (Darlington amp Wylie 1956 dePadua et at 1999) The differences in chromosome numbers could rely on the origin and the distribution Most of the species are native of Eurasia and North America These specie are found in central and South-Western Asia which is thought to have been originated here and to have j mmigrmed to North America Some species have been inLr duced in the Male iana area including Indonesia usually as ornamental and medicinal plants (dePadua ef aI 1999) A cina used in this experiment wa originated from Tawangmangu (Central Java Indonesia) There is no report on (he variation of chromosome on A dna grown in indonesia Therefore further investigation on chromosome analysis of this planL grown in Indone ia i needed to confirm whether the variation in chromosome number is a common phenomenon as in [he rool cultures

In conclusion root cultures ofA cilia ha variation in chromosome numbers wiLh basic diploid number of2n=32 Variation in the chromo orne number was also detected in roOls of plant grown in the glasshouse In transformed roots roots transformed with A rhizogefles strain 07-20001 was the most stable whi 1st roots transformed wi th A rhizogenes strain A4 was the most unstable line This information on variation in chromosome number may also contribute LO the further investigation of root culture of A cilla or for the genetic manipulation

ACKNOWLEDGEMENT

The authors would like to thank Adelina for the maintenance of root shoot and callus cultures

REFERENCES

Aird ELHJD Hammill amp MJC Rhodes 1988 Cytologica l ana lysis of hairy root cultures from a number of plant specie transformed by Agrobacterium rhizogenes Plant Cell Tiss Org Cult 15 47-57

Aoki T H Matsumoto Y Asako Y Matsunaga amp K Shimomura I 97 Variation of alkaloid productivity among several lones of hairy root and regenerated plants of Atropa beladona transformed with Agrobaclerium rhizogenes 15834 Plant Cell Rep J6 282-286

Aryanti M Bintang TM Ermayanti amp I Mariska 200 l Produclion of antileukemic agent in untransformed and transformed roOl cultures of Artemisia cina Anll Bogorienses 8 (l) 11middot16

DAmato F 1990 Somalic nuclear mutalion in vivo and ill vitro in higher plams Cmyologia 43 (3-4) 191-204

Darlington CD amp AP Wylie J956 Chromosome ALIas of Flowering Plants George Allen amp Unwin Ltd London pp 266-267

DePadua LS N Bunyaprapbatsara amp RHMJ Lemmens 1999 Medical and Poisonous Plants 1 Prosca No 12 (1) Prosea Foundalion Bogor Indonesia Pp 139-147

Ermayanti TM 1999 Genetic transformaLion of Swainsona galegifolia In Bajaj YPS (Ed ) B iOlechnology in Agriculture and Forestry 45 Transgenic Medicinal Plants Springerp 327-335

Ermayaoti TM JA McComb amp PA OBrien 1993 Cytological analysis of seedling rooLS transfonned root cultures and roots regenerated from callus of Swainsona galegifolia (Andr) R Br 1 Exp Bot 44 (259) 375-380

Annales Bogorienses n s Vol 9 no2 20O-t 56

Ermayanti TM L Sari EMR Siregar amp D udrajat 2000 Transformasi mimba (Azadirachla indica A Juss) dengan Agrobacterium rhizogel1es galur ATCCshy15834 Paper presenled al the Kongres dan Seminar Nasional Perhimpunan Bioleknologi Pertanian Indonesia 11 7shy8 Nopember 2000 Yogyakarta Indonesia

Guivarcb A M Boccara M Prouteau amp D Chriqui 1999 Instability of phenotype and gene expression in longshyterm culture of carrot hairy root clones Plant Cell Rep 119 43 -50

Hamill J D AJ Parr MJC Rhodes RJ Robins amp NJ Walton 1987 New routes to plant secondary products Bioi Technol 5 800-804

Jha Tn amp Roy 1982 Chromosomal behaviour in cullure of Vicia faba Cytologia 47 465middot470

Joao KHL amp TA Brown 1994 Long-term stability of root cultures of tomato transformed wilh Agrobacterillm ri1izogellesRI60IJ Exp Bot 274 641 shy647

Karp A 1991 Cytological techniques Plant cell Culture Manual Kluwer Academic Publishers C4 1-13

Karp A amp SE Maddock 1984 Cromosome variation in wheat plants regenerated from cultured immature embryo Theor Appl Genet 67 249-255

Karp A RS Nelson E Thomas amp SWJ Brigbt 1082 Chromosome variation in protoplast-derived potato plants Theor Appl Genet 63 265-272

Lee KT T Suzuki T Yamakawa T Komada Y Igara hi amp K Sbimomura 1999 Production of tropane alkaloids by transformed root cultures of Atropa bellacona in tirred bioreactors with stai nless steel net Plant Cell Rep 18 657-57 1

McCoy TJ amp RL Phillips 1982 Chromosome stability in maize (Zea nwys) tissue cultures and sectoring in some regenerated plants Call 1 Genet Cytol 24 559-565

MehraA amp PM Mehra 1974 Organogenesis and plant let formation in vitro almond Bot Gaz 135 61-73

Mukherjee S Das amp S Jba 1994 Chromosome stability in transformed hairy root cultures of Artemisia anllua L Cell Chrolllos Res 17 7 1-76

Murashige T amp F Skoog 1962 A revised medium for rapid growth and bioassays with tobacco tissue cultures Phisiol Plant 15 473-497

Nuti Ronchi V M Nozzolini amp L Avanti 1981 Chromosomal variation on plants regenerated from two Nicotiall spp Protoplasma 109 433 -444

Orton TJ 1980 Chromosomal variability in tissu cultures and regenerated plants of Hordeum Them Appl Genet 56 101shy112

Ramsay G amp A Kumar 1990 Transformation of Viciafaba cotyledon and stem Ii sue by Agrobacieriu1I rhizogenes infectivity and cytological studies 1 Exp Bot 4 1 (228 ) 841-847

Saito K H Sudo M Yamazaki M Ko eldshyNakamura MKitajima H Takayama amp M Aimi 200 l Feasible production ofcamptothecin by hairy roOl culture of Ophiorrhiza pumila Plant Cell Rep 20 267-27 1

Sasaki K A Udagawa H Ishimaru T Hayashi A W Alfermaoo F Nakanishi amp K Shimomura 1998 High for kolin production in hairy roots of ColellS forskohlii Plant Cell Rep 11 7 457-459

nnales Bogorienses n s Vol 9 no2 2004 57

Syamsuhidayat SS amp JR Hutapea 1991 Invemaris Tanaman Obat Indonesia 1 Badan Penelitian dan Pengembangan Ke ehalan Departemen Kc ehalan Jakarta

Tan RX WE Zbeng amp HO Lang 1998 Biologically active subslrunces rrom the genus Artemisia Plant Med 295-302

Webb KJ S Jones MP Robbin amp FR Minchin 1990 Characterization of transgenic root cultures of Trifolium repeflS Trifolillmprarense and Lotus comicllatus and transgenic plants of LotllS comiculatus Plant Sci 70 243shy254

XU ZQ amp JF Jia 1996 The reduction of chromosome number and loss of regeneration ability during subculture of hairy rOOl cultures on Onobryc7is viciaeolia transformed by Arobacterilll11 rlzizogenel A4 Plant Sci 120 I 07 - 112

Zarate R 1999 Tropane alkaloid production by Arobaclerillm rhizogenes transformed hairy root culture ofAtropa baetica Willk (S lanaceae) Plant Cell Rep 18 418-423

Annales Bugorienses n s Vol 9 no2 2004 58

had haploid chromosome number of 2n=16 which found in separate root culture (Fig4) Chromo orne numbers of roots tran formed with Agrobacterium strain ATCC-J5834 ranged from 2n= 18 to 2n=60 (FigS) with 619 of them had normal diploid chTOmosome numbers of 2n -32 195 of c lis found to have chromosome numbers lower than the diploid numbers and 186 of cells had higher chromosome number than that of the diploid number Roots transfomled with A rumejaciens strain R1000 had chromosome numbers ranged from 2n=17 to 2n=59 (Fig 6) Roots transformed with A rhizogenes strain A4 was the most unstable line Only 430 of cells had correct number of chromosome 243 of cells had chromosome number lower than diploid and 327 of cells had chromosome number higher than that of diploid numbers The

100~------------------~ 90 80 70

50 60 J ij ~

11 1621 2631 36 41 4651 56 61 66

chromosome numbers (2n)

Figure 4 Cytological analysis of A dna hairy roots lransfonned with A rhizogenes 07-20001

100~------------------~ 90 80 70 60

50 40 30 20 10 ILO~~~~~~~~~=ft~

11 16 21 2631 36 41 46 51 56 61 66

chromosome numbers (2n)

Figure S Cytologi al analysis of A cina hairy roots transfonned with A rhizogenes ATCC-15834

54

distribution of the chromosome number of roots transformed with Agrobacterillm strain A4 is pre ented in Fig 7

The low genetic stability as showed by cytological anaJysis in A cina tran formed roots was in contrast with that of some other species In Swaillson gaegijoia transformed root cultures resultcd after transformation with A rhizogelles strains LBA 9402 and A4 had more than 90 normal diploid chromosome numbers This also the case of untransforrned roots grown in orne dilTerent medium and roots isolated from seedling and germinated seeds The results were in contrary with callus culture and roots regenerated from callus of S galegtjolia (Ennayanti et al 1993) Hairy roots of A anllUa after infection with A rhizogenes strain LBA-9402 had stable chromosome numbers

100 shy90 80 70 60

50 40 30 20 10 bull o

8 14 20 26 32 38 44 50 56 62 68

chromosome numbers (2n)

Figure 6 Cytological analysis of A cina hairy roots transformed with A tumeaciel1s R1000

100r-------------------~ 90 80 70 60

50 40 30 20 10 IIL0 ~~~~~m=~~~~1

8 14 20 26 3238 44 50 56 62 68

chromosome numbers (2n)

Figure 7 Cytological analy is of A cina hairy roots lransfonned with A rhizogeles

A4

Annales Bogoricnses n s Vol 9 no 2 2004

of 2n =18 after 20 months in culture medi urn (Mukherjee et a 1994)

Growth of transformed roots of A cina in MS medium without hormones wa higher than thal of unlransformed roots (Aryanti et a 200 I) However growth of hairy root lines resulted from transformation with AgrobacteriuTII strains RlOOO was lower tban hairy roots transformed with Agrobaclerium strains 07-2000 1 A4 and ATCC-1S834 The root tips of hairy root line R 1000 were very qu ickly turned brown This problem therefore caused the difficulty to find clear metaphase cells for chromo orne xaminaLion Such problem was not found

for hairy roots lines of Agrohacleriltm strains 07-20001 ATCC-lS834 and A4 so that more

lear metapha e cells could be examined for the chromosome analysis

PCR analysis showed that only T L region of T-DN A plasmid was detected (AryanLi el aI 200 L) The integration of the T-DNA which was only for T l-DNA in A cina may be related to the loss of some chromosomes in most root lines This resull is in contrast with Atropa belladollna lran formed with A rhizogenes strain ATCCshy15834 both T

L- and TI-DNAwere integrated

in the transformed roots (Aoki el al J997) In kadiracta illdica roOlS the lransformation event affected the integration of the T-DNA to the plant genome Both T - and TH-DNA

L

regions were detected in two out of three root clones examined whil t one root clone only containedTL-DNA region (Ermayanti el a

2000) The selection of root Jines from each clone of A ina could be investigated to obtain stable root clones which may result in the stable production of bioaclive compounds of the roots

The low tability in transformed roots of A cina may be caused by structural rearrungements as that in Vida faba that found that 50 of transformed roots examined had polyploid (2x-9x) and 6 had aneuploid chromosome numbers or structural reanangements (Ramsay amp Kumar 1990)

Annales Bogorieoses o s Vol 9 no2 2004

The chromosomes become e l im In ated apparently at random In omCltic hybrid cells the chromosomes of one of the parents may be eliminated preferentially This could be the case of A c ina root In hai r y roots cytological changes were also detected in transformed root cultures of Trifolium prarense and in regenerated plants or Lotus cornicHlatus (Webb e t al 1990) The reduction of chromosome numb rs and the loss of regeneration abi lity during subcullUre had occurred in hairy root cultures of Ollobrychisviciaefolia (Xu amp Jia 1996) This may be the case for A cilia that transformed rOOls used in this experiment had been subcultured for 3-6 times the age of the cultures was about 6-12 months To confirm the effect of culture age and subculture on genetic stability or A cina roots cytological examination of germinated seeds and primary roots grown directly at the wounded site of Agrobacterillm infection should be done however seed germination of A cina was very low Instability 0[ phenotype and gene expression in long-term cullure also occurred in carrot h iry root clones (Guivarch et aI 1999) in conlrast with the hairy rOOI clones of tomato wh ich was stable for 50 subcultures over 25 months (Joao amp Brown 1994) Opine and peR analysis should be done to conrirm the gene expres ion in long-term cullure or A cina The facl that untransformed and lran formed root or A ci1la have unstable in chromosome numbers mos t probably i caused by the variation in chromosome numbers of the intact plants themselves (Table 1) Germinated seeds are needed to confirm this genetic variability However the abi Ii t Y ofseed gemi nation was very low None of seeds harvested from three plants grown in th field germinated either in mixture of soil with sand or in a culture medium

The genu Artemisia is placed in the tribe of Anthemideae consists of more than 200 pecles The modal of chromosome numberofgenusArtemisia is x =8 or9 Some species of this genus including A cilia has

55

unique chromosome numbers with both modal number of x = 8 and x = 9 The normal plants of these species may have chromosome numbers of diploid 2n = 2 x= 18 (from modal x - 9) or of polyploid 2n = 4x = 32 (from modal x =8) (Darlington amp Wylie 1956 dePadua et at 1999) The differences in chromosome numbers could rely on the origin and the distribution Most of the species are native of Eurasia and North America These specie are found in central and South-Western Asia which is thought to have been originated here and to have j mmigrmed to North America Some species have been inLr duced in the Male iana area including Indonesia usually as ornamental and medicinal plants (dePadua ef aI 1999) A cina used in this experiment wa originated from Tawangmangu (Central Java Indonesia) There is no report on (he variation of chromosome on A dna grown in indonesia Therefore further investigation on chromosome analysis of this planL grown in Indone ia i needed to confirm whether the variation in chromosome number is a common phenomenon as in [he rool cultures

In conclusion root cultures ofA cilia ha variation in chromosome numbers wiLh basic diploid number of2n=32 Variation in the chromo orne number was also detected in roOls of plant grown in the glasshouse In transformed roots roots transformed with A rhizogefles strain 07-20001 was the most stable whi 1st roots transformed wi th A rhizogenes strain A4 was the most unstable line This information on variation in chromosome number may also contribute LO the further investigation of root culture of A cilla or for the genetic manipulation

ACKNOWLEDGEMENT

The authors would like to thank Adelina for the maintenance of root shoot and callus cultures

REFERENCES

Aird ELHJD Hammill amp MJC Rhodes 1988 Cytologica l ana lysis of hairy root cultures from a number of plant specie transformed by Agrobacterium rhizogenes Plant Cell Tiss Org Cult 15 47-57

Aoki T H Matsumoto Y Asako Y Matsunaga amp K Shimomura I 97 Variation of alkaloid productivity among several lones of hairy root and regenerated plants of Atropa beladona transformed with Agrobaclerium rhizogenes 15834 Plant Cell Rep J6 282-286

Aryanti M Bintang TM Ermayanti amp I Mariska 200 l Produclion of antileukemic agent in untransformed and transformed roOl cultures of Artemisia cina Anll Bogorienses 8 (l) 11middot16

DAmato F 1990 Somalic nuclear mutalion in vivo and ill vitro in higher plams Cmyologia 43 (3-4) 191-204

Darlington CD amp AP Wylie J956 Chromosome ALIas of Flowering Plants George Allen amp Unwin Ltd London pp 266-267

DePadua LS N Bunyaprapbatsara amp RHMJ Lemmens 1999 Medical and Poisonous Plants 1 Prosca No 12 (1) Prosea Foundalion Bogor Indonesia Pp 139-147

Ermayanti TM 1999 Genetic transformaLion of Swainsona galegifolia In Bajaj YPS (Ed ) B iOlechnology in Agriculture and Forestry 45 Transgenic Medicinal Plants Springerp 327-335

Ermayaoti TM JA McComb amp PA OBrien 1993 Cytological analysis of seedling rooLS transfonned root cultures and roots regenerated from callus of Swainsona galegifolia (Andr) R Br 1 Exp Bot 44 (259) 375-380

Annales Bogorienses n s Vol 9 no2 20O-t 56

Ermayanti TM L Sari EMR Siregar amp D udrajat 2000 Transformasi mimba (Azadirachla indica A Juss) dengan Agrobacterium rhizogel1es galur ATCCshy15834 Paper presenled al the Kongres dan Seminar Nasional Perhimpunan Bioleknologi Pertanian Indonesia 11 7shy8 Nopember 2000 Yogyakarta Indonesia

Guivarcb A M Boccara M Prouteau amp D Chriqui 1999 Instability of phenotype and gene expression in longshyterm culture of carrot hairy root clones Plant Cell Rep 119 43 -50

Hamill J D AJ Parr MJC Rhodes RJ Robins amp NJ Walton 1987 New routes to plant secondary products Bioi Technol 5 800-804

Jha Tn amp Roy 1982 Chromosomal behaviour in cullure of Vicia faba Cytologia 47 465middot470

Joao KHL amp TA Brown 1994 Long-term stability of root cultures of tomato transformed wilh Agrobacterillm ri1izogellesRI60IJ Exp Bot 274 641 shy647

Karp A 1991 Cytological techniques Plant cell Culture Manual Kluwer Academic Publishers C4 1-13

Karp A amp SE Maddock 1984 Cromosome variation in wheat plants regenerated from cultured immature embryo Theor Appl Genet 67 249-255

Karp A RS Nelson E Thomas amp SWJ Brigbt 1082 Chromosome variation in protoplast-derived potato plants Theor Appl Genet 63 265-272

Lee KT T Suzuki T Yamakawa T Komada Y Igara hi amp K Sbimomura 1999 Production of tropane alkaloids by transformed root cultures of Atropa bellacona in tirred bioreactors with stai nless steel net Plant Cell Rep 18 657-57 1

McCoy TJ amp RL Phillips 1982 Chromosome stability in maize (Zea nwys) tissue cultures and sectoring in some regenerated plants Call 1 Genet Cytol 24 559-565

MehraA amp PM Mehra 1974 Organogenesis and plant let formation in vitro almond Bot Gaz 135 61-73

Mukherjee S Das amp S Jba 1994 Chromosome stability in transformed hairy root cultures of Artemisia anllua L Cell Chrolllos Res 17 7 1-76

Murashige T amp F Skoog 1962 A revised medium for rapid growth and bioassays with tobacco tissue cultures Phisiol Plant 15 473-497

Nuti Ronchi V M Nozzolini amp L Avanti 1981 Chromosomal variation on plants regenerated from two Nicotiall spp Protoplasma 109 433 -444

Orton TJ 1980 Chromosomal variability in tissu cultures and regenerated plants of Hordeum Them Appl Genet 56 101shy112

Ramsay G amp A Kumar 1990 Transformation of Viciafaba cotyledon and stem Ii sue by Agrobacieriu1I rhizogenes infectivity and cytological studies 1 Exp Bot 4 1 (228 ) 841-847

Saito K H Sudo M Yamazaki M Ko eldshyNakamura MKitajima H Takayama amp M Aimi 200 l Feasible production ofcamptothecin by hairy roOl culture of Ophiorrhiza pumila Plant Cell Rep 20 267-27 1

Sasaki K A Udagawa H Ishimaru T Hayashi A W Alfermaoo F Nakanishi amp K Shimomura 1998 High for kolin production in hairy roots of ColellS forskohlii Plant Cell Rep 11 7 457-459

nnales Bogorienses n s Vol 9 no2 2004 57

Syamsuhidayat SS amp JR Hutapea 1991 Invemaris Tanaman Obat Indonesia 1 Badan Penelitian dan Pengembangan Ke ehalan Departemen Kc ehalan Jakarta

Tan RX WE Zbeng amp HO Lang 1998 Biologically active subslrunces rrom the genus Artemisia Plant Med 295-302

Webb KJ S Jones MP Robbin amp FR Minchin 1990 Characterization of transgenic root cultures of Trifolium repeflS Trifolillmprarense and Lotus comicllatus and transgenic plants of LotllS comiculatus Plant Sci 70 243shy254

XU ZQ amp JF Jia 1996 The reduction of chromosome number and loss of regeneration ability during subculture of hairy rOOl cultures on Onobryc7is viciaeolia transformed by Arobacterilll11 rlzizogenel A4 Plant Sci 120 I 07 - 112

Zarate R 1999 Tropane alkaloid production by Arobaclerillm rhizogenes transformed hairy root culture ofAtropa baetica Willk (S lanaceae) Plant Cell Rep 18 418-423

Annales Bugorienses n s Vol 9 no2 2004 58

of 2n =18 after 20 months in culture medi urn (Mukherjee et a 1994)

Growth of transformed roots of A cina in MS medium without hormones wa higher than thal of unlransformed roots (Aryanti et a 200 I) However growth of hairy root lines resulted from transformation with AgrobacteriuTII strains RlOOO was lower tban hairy roots transformed with Agrobaclerium strains 07-2000 1 A4 and ATCC-1S834 The root tips of hairy root line R 1000 were very qu ickly turned brown This problem therefore caused the difficulty to find clear metaphase cells for chromo orne xaminaLion Such problem was not found

for hairy roots lines of Agrohacleriltm strains 07-20001 ATCC-lS834 and A4 so that more

lear metapha e cells could be examined for the chromosome analysis

PCR analysis showed that only T L region of T-DN A plasmid was detected (AryanLi el aI 200 L) The integration of the T-DNA which was only for T l-DNA in A cina may be related to the loss of some chromosomes in most root lines This resull is in contrast with Atropa belladollna lran formed with A rhizogenes strain ATCCshy15834 both T

L- and TI-DNAwere integrated

in the transformed roots (Aoki el al J997) In kadiracta illdica roOlS the lransformation event affected the integration of the T-DNA to the plant genome Both T - and TH-DNA

L

regions were detected in two out of three root clones examined whil t one root clone only containedTL-DNA region (Ermayanti el a

2000) The selection of root Jines from each clone of A ina could be investigated to obtain stable root clones which may result in the stable production of bioaclive compounds of the roots

The low tability in transformed roots of A cina may be caused by structural rearrungements as that in Vida faba that found that 50 of transformed roots examined had polyploid (2x-9x) and 6 had aneuploid chromosome numbers or structural reanangements (Ramsay amp Kumar 1990)

Annales Bogorieoses o s Vol 9 no2 2004

The chromosomes become e l im In ated apparently at random In omCltic hybrid cells the chromosomes of one of the parents may be eliminated preferentially This could be the case of A c ina root In hai r y roots cytological changes were also detected in transformed root cultures of Trifolium prarense and in regenerated plants or Lotus cornicHlatus (Webb e t al 1990) The reduction of chromosome numb rs and the loss of regeneration abi lity during subcullUre had occurred in hairy root cultures of Ollobrychisviciaefolia (Xu amp Jia 1996) This may be the case for A cilia that transformed rOOls used in this experiment had been subcultured for 3-6 times the age of the cultures was about 6-12 months To confirm the effect of culture age and subculture on genetic stability or A cina roots cytological examination of germinated seeds and primary roots grown directly at the wounded site of Agrobacterillm infection should be done however seed germination of A cina was very low Instability 0[ phenotype and gene expression in long-term cullure also occurred in carrot h iry root clones (Guivarch et aI 1999) in conlrast with the hairy rOOI clones of tomato wh ich was stable for 50 subcultures over 25 months (Joao amp Brown 1994) Opine and peR analysis should be done to conrirm the gene expres ion in long-term cullure or A cina The facl that untransformed and lran formed root or A ci1la have unstable in chromosome numbers mos t probably i caused by the variation in chromosome numbers of the intact plants themselves (Table 1) Germinated seeds are needed to confirm this genetic variability However the abi Ii t Y ofseed gemi nation was very low None of seeds harvested from three plants grown in th field germinated either in mixture of soil with sand or in a culture medium

The genu Artemisia is placed in the tribe of Anthemideae consists of more than 200 pecles The modal of chromosome numberofgenusArtemisia is x =8 or9 Some species of this genus including A cilia has

55

unique chromosome numbers with both modal number of x = 8 and x = 9 The normal plants of these species may have chromosome numbers of diploid 2n = 2 x= 18 (from modal x - 9) or of polyploid 2n = 4x = 32 (from modal x =8) (Darlington amp Wylie 1956 dePadua et at 1999) The differences in chromosome numbers could rely on the origin and the distribution Most of the species are native of Eurasia and North America These specie are found in central and South-Western Asia which is thought to have been originated here and to have j mmigrmed to North America Some species have been inLr duced in the Male iana area including Indonesia usually as ornamental and medicinal plants (dePadua ef aI 1999) A cina used in this experiment wa originated from Tawangmangu (Central Java Indonesia) There is no report on (he variation of chromosome on A dna grown in indonesia Therefore further investigation on chromosome analysis of this planL grown in Indone ia i needed to confirm whether the variation in chromosome number is a common phenomenon as in [he rool cultures

In conclusion root cultures ofA cilia ha variation in chromosome numbers wiLh basic diploid number of2n=32 Variation in the chromo orne number was also detected in roOls of plant grown in the glasshouse In transformed roots roots transformed with A rhizogefles strain 07-20001 was the most stable whi 1st roots transformed wi th A rhizogenes strain A4 was the most unstable line This information on variation in chromosome number may also contribute LO the further investigation of root culture of A cilla or for the genetic manipulation

ACKNOWLEDGEMENT

The authors would like to thank Adelina for the maintenance of root shoot and callus cultures

REFERENCES

Aird ELHJD Hammill amp MJC Rhodes 1988 Cytologica l ana lysis of hairy root cultures from a number of plant specie transformed by Agrobacterium rhizogenes Plant Cell Tiss Org Cult 15 47-57

Aoki T H Matsumoto Y Asako Y Matsunaga amp K Shimomura I 97 Variation of alkaloid productivity among several lones of hairy root and regenerated plants of Atropa beladona transformed with Agrobaclerium rhizogenes 15834 Plant Cell Rep J6 282-286

Aryanti M Bintang TM Ermayanti amp I Mariska 200 l Produclion of antileukemic agent in untransformed and transformed roOl cultures of Artemisia cina Anll Bogorienses 8 (l) 11middot16

DAmato F 1990 Somalic nuclear mutalion in vivo and ill vitro in higher plams Cmyologia 43 (3-4) 191-204

Darlington CD amp AP Wylie J956 Chromosome ALIas of Flowering Plants George Allen amp Unwin Ltd London pp 266-267

DePadua LS N Bunyaprapbatsara amp RHMJ Lemmens 1999 Medical and Poisonous Plants 1 Prosca No 12 (1) Prosea Foundalion Bogor Indonesia Pp 139-147

Ermayanti TM 1999 Genetic transformaLion of Swainsona galegifolia In Bajaj YPS (Ed ) B iOlechnology in Agriculture and Forestry 45 Transgenic Medicinal Plants Springerp 327-335

Ermayaoti TM JA McComb amp PA OBrien 1993 Cytological analysis of seedling rooLS transfonned root cultures and roots regenerated from callus of Swainsona galegifolia (Andr) R Br 1 Exp Bot 44 (259) 375-380

Annales Bogorienses n s Vol 9 no2 20O-t 56

Ermayanti TM L Sari EMR Siregar amp D udrajat 2000 Transformasi mimba (Azadirachla indica A Juss) dengan Agrobacterium rhizogel1es galur ATCCshy15834 Paper presenled al the Kongres dan Seminar Nasional Perhimpunan Bioleknologi Pertanian Indonesia 11 7shy8 Nopember 2000 Yogyakarta Indonesia

Guivarcb A M Boccara M Prouteau amp D Chriqui 1999 Instability of phenotype and gene expression in longshyterm culture of carrot hairy root clones Plant Cell Rep 119 43 -50

Hamill J D AJ Parr MJC Rhodes RJ Robins amp NJ Walton 1987 New routes to plant secondary products Bioi Technol 5 800-804

Jha Tn amp Roy 1982 Chromosomal behaviour in cullure of Vicia faba Cytologia 47 465middot470

Joao KHL amp TA Brown 1994 Long-term stability of root cultures of tomato transformed wilh Agrobacterillm ri1izogellesRI60IJ Exp Bot 274 641 shy647

Karp A 1991 Cytological techniques Plant cell Culture Manual Kluwer Academic Publishers C4 1-13

Karp A amp SE Maddock 1984 Cromosome variation in wheat plants regenerated from cultured immature embryo Theor Appl Genet 67 249-255

Karp A RS Nelson E Thomas amp SWJ Brigbt 1082 Chromosome variation in protoplast-derived potato plants Theor Appl Genet 63 265-272

Lee KT T Suzuki T Yamakawa T Komada Y Igara hi amp K Sbimomura 1999 Production of tropane alkaloids by transformed root cultures of Atropa bellacona in tirred bioreactors with stai nless steel net Plant Cell Rep 18 657-57 1

McCoy TJ amp RL Phillips 1982 Chromosome stability in maize (Zea nwys) tissue cultures and sectoring in some regenerated plants Call 1 Genet Cytol 24 559-565

MehraA amp PM Mehra 1974 Organogenesis and plant let formation in vitro almond Bot Gaz 135 61-73

Mukherjee S Das amp S Jba 1994 Chromosome stability in transformed hairy root cultures of Artemisia anllua L Cell Chrolllos Res 17 7 1-76

Murashige T amp F Skoog 1962 A revised medium for rapid growth and bioassays with tobacco tissue cultures Phisiol Plant 15 473-497

Nuti Ronchi V M Nozzolini amp L Avanti 1981 Chromosomal variation on plants regenerated from two Nicotiall spp Protoplasma 109 433 -444

Orton TJ 1980 Chromosomal variability in tissu cultures and regenerated plants of Hordeum Them Appl Genet 56 101shy112

Ramsay G amp A Kumar 1990 Transformation of Viciafaba cotyledon and stem Ii sue by Agrobacieriu1I rhizogenes infectivity and cytological studies 1 Exp Bot 4 1 (228 ) 841-847

Saito K H Sudo M Yamazaki M Ko eldshyNakamura MKitajima H Takayama amp M Aimi 200 l Feasible production ofcamptothecin by hairy roOl culture of Ophiorrhiza pumila Plant Cell Rep 20 267-27 1

Sasaki K A Udagawa H Ishimaru T Hayashi A W Alfermaoo F Nakanishi amp K Shimomura 1998 High for kolin production in hairy roots of ColellS forskohlii Plant Cell Rep 11 7 457-459

nnales Bogorienses n s Vol 9 no2 2004 57

Syamsuhidayat SS amp JR Hutapea 1991 Invemaris Tanaman Obat Indonesia 1 Badan Penelitian dan Pengembangan Ke ehalan Departemen Kc ehalan Jakarta

Tan RX WE Zbeng amp HO Lang 1998 Biologically active subslrunces rrom the genus Artemisia Plant Med 295-302

Webb KJ S Jones MP Robbin amp FR Minchin 1990 Characterization of transgenic root cultures of Trifolium repeflS Trifolillmprarense and Lotus comicllatus and transgenic plants of LotllS comiculatus Plant Sci 70 243shy254

XU ZQ amp JF Jia 1996 The reduction of chromosome number and loss of regeneration ability during subculture of hairy rOOl cultures on Onobryc7is viciaeolia transformed by Arobacterilll11 rlzizogenel A4 Plant Sci 120 I 07 - 112

Zarate R 1999 Tropane alkaloid production by Arobaclerillm rhizogenes transformed hairy root culture ofAtropa baetica Willk (S lanaceae) Plant Cell Rep 18 418-423

Annales Bugorienses n s Vol 9 no2 2004 58

unique chromosome numbers with both modal number of x = 8 and x = 9 The normal plants of these species may have chromosome numbers of diploid 2n = 2 x= 18 (from modal x - 9) or of polyploid 2n = 4x = 32 (from modal x =8) (Darlington amp Wylie 1956 dePadua et at 1999) The differences in chromosome numbers could rely on the origin and the distribution Most of the species are native of Eurasia and North America These specie are found in central and South-Western Asia which is thought to have been originated here and to have j mmigrmed to North America Some species have been inLr duced in the Male iana area including Indonesia usually as ornamental and medicinal plants (dePadua ef aI 1999) A cina used in this experiment wa originated from Tawangmangu (Central Java Indonesia) There is no report on (he variation of chromosome on A dna grown in indonesia Therefore further investigation on chromosome analysis of this planL grown in Indone ia i needed to confirm whether the variation in chromosome number is a common phenomenon as in [he rool cultures

In conclusion root cultures ofA cilia ha variation in chromosome numbers wiLh basic diploid number of2n=32 Variation in the chromo orne number was also detected in roOls of plant grown in the glasshouse In transformed roots roots transformed with A rhizogefles strain 07-20001 was the most stable whi 1st roots transformed wi th A rhizogenes strain A4 was the most unstable line This information on variation in chromosome number may also contribute LO the further investigation of root culture of A cilla or for the genetic manipulation

ACKNOWLEDGEMENT

The authors would like to thank Adelina for the maintenance of root shoot and callus cultures

REFERENCES

Aird ELHJD Hammill amp MJC Rhodes 1988 Cytologica l ana lysis of hairy root cultures from a number of plant specie transformed by Agrobacterium rhizogenes Plant Cell Tiss Org Cult 15 47-57

Aoki T H Matsumoto Y Asako Y Matsunaga amp K Shimomura I 97 Variation of alkaloid productivity among several lones of hairy root and regenerated plants of Atropa beladona transformed with Agrobaclerium rhizogenes 15834 Plant Cell Rep J6 282-286

Aryanti M Bintang TM Ermayanti amp I Mariska 200 l Produclion of antileukemic agent in untransformed and transformed roOl cultures of Artemisia cina Anll Bogorienses 8 (l) 11middot16

DAmato F 1990 Somalic nuclear mutalion in vivo and ill vitro in higher plams Cmyologia 43 (3-4) 191-204

Darlington CD amp AP Wylie J956 Chromosome ALIas of Flowering Plants George Allen amp Unwin Ltd London pp 266-267

DePadua LS N Bunyaprapbatsara amp RHMJ Lemmens 1999 Medical and Poisonous Plants 1 Prosca No 12 (1) Prosea Foundalion Bogor Indonesia Pp 139-147

Ermayanti TM 1999 Genetic transformaLion of Swainsona galegifolia In Bajaj YPS (Ed ) B iOlechnology in Agriculture and Forestry 45 Transgenic Medicinal Plants Springerp 327-335

Ermayaoti TM JA McComb amp PA OBrien 1993 Cytological analysis of seedling rooLS transfonned root cultures and roots regenerated from callus of Swainsona galegifolia (Andr) R Br 1 Exp Bot 44 (259) 375-380

Annales Bogorienses n s Vol 9 no2 20O-t 56

Ermayanti TM L Sari EMR Siregar amp D udrajat 2000 Transformasi mimba (Azadirachla indica A Juss) dengan Agrobacterium rhizogel1es galur ATCCshy15834 Paper presenled al the Kongres dan Seminar Nasional Perhimpunan Bioleknologi Pertanian Indonesia 11 7shy8 Nopember 2000 Yogyakarta Indonesia

Guivarcb A M Boccara M Prouteau amp D Chriqui 1999 Instability of phenotype and gene expression in longshyterm culture of carrot hairy root clones Plant Cell Rep 119 43 -50

Hamill J D AJ Parr MJC Rhodes RJ Robins amp NJ Walton 1987 New routes to plant secondary products Bioi Technol 5 800-804

Jha Tn amp Roy 1982 Chromosomal behaviour in cullure of Vicia faba Cytologia 47 465middot470

Joao KHL amp TA Brown 1994 Long-term stability of root cultures of tomato transformed wilh Agrobacterillm ri1izogellesRI60IJ Exp Bot 274 641 shy647

Karp A 1991 Cytological techniques Plant cell Culture Manual Kluwer Academic Publishers C4 1-13

Karp A amp SE Maddock 1984 Cromosome variation in wheat plants regenerated from cultured immature embryo Theor Appl Genet 67 249-255

Karp A RS Nelson E Thomas amp SWJ Brigbt 1082 Chromosome variation in protoplast-derived potato plants Theor Appl Genet 63 265-272

Lee KT T Suzuki T Yamakawa T Komada Y Igara hi amp K Sbimomura 1999 Production of tropane alkaloids by transformed root cultures of Atropa bellacona in tirred bioreactors with stai nless steel net Plant Cell Rep 18 657-57 1

McCoy TJ amp RL Phillips 1982 Chromosome stability in maize (Zea nwys) tissue cultures and sectoring in some regenerated plants Call 1 Genet Cytol 24 559-565

MehraA amp PM Mehra 1974 Organogenesis and plant let formation in vitro almond Bot Gaz 135 61-73

Mukherjee S Das amp S Jba 1994 Chromosome stability in transformed hairy root cultures of Artemisia anllua L Cell Chrolllos Res 17 7 1-76

Murashige T amp F Skoog 1962 A revised medium for rapid growth and bioassays with tobacco tissue cultures Phisiol Plant 15 473-497

Nuti Ronchi V M Nozzolini amp L Avanti 1981 Chromosomal variation on plants regenerated from two Nicotiall spp Protoplasma 109 433 -444

Orton TJ 1980 Chromosomal variability in tissu cultures and regenerated plants of Hordeum Them Appl Genet 56 101shy112

Ramsay G amp A Kumar 1990 Transformation of Viciafaba cotyledon and stem Ii sue by Agrobacieriu1I rhizogenes infectivity and cytological studies 1 Exp Bot 4 1 (228 ) 841-847

Saito K H Sudo M Yamazaki M Ko eldshyNakamura MKitajima H Takayama amp M Aimi 200 l Feasible production ofcamptothecin by hairy roOl culture of Ophiorrhiza pumila Plant Cell Rep 20 267-27 1

Sasaki K A Udagawa H Ishimaru T Hayashi A W Alfermaoo F Nakanishi amp K Shimomura 1998 High for kolin production in hairy roots of ColellS forskohlii Plant Cell Rep 11 7 457-459

nnales Bogorienses n s Vol 9 no2 2004 57

Syamsuhidayat SS amp JR Hutapea 1991 Invemaris Tanaman Obat Indonesia 1 Badan Penelitian dan Pengembangan Ke ehalan Departemen Kc ehalan Jakarta

Tan RX WE Zbeng amp HO Lang 1998 Biologically active subslrunces rrom the genus Artemisia Plant Med 295-302

Webb KJ S Jones MP Robbin amp FR Minchin 1990 Characterization of transgenic root cultures of Trifolium repeflS Trifolillmprarense and Lotus comicllatus and transgenic plants of LotllS comiculatus Plant Sci 70 243shy254

XU ZQ amp JF Jia 1996 The reduction of chromosome number and loss of regeneration ability during subculture of hairy rOOl cultures on Onobryc7is viciaeolia transformed by Arobacterilll11 rlzizogenel A4 Plant Sci 120 I 07 - 112

Zarate R 1999 Tropane alkaloid production by Arobaclerillm rhizogenes transformed hairy root culture ofAtropa baetica Willk (S lanaceae) Plant Cell Rep 18 418-423

Annales Bugorienses n s Vol 9 no2 2004 58

Ermayanti TM L Sari EMR Siregar amp D udrajat 2000 Transformasi mimba (Azadirachla indica A Juss) dengan Agrobacterium rhizogel1es galur ATCCshy15834 Paper presenled al the Kongres dan Seminar Nasional Perhimpunan Bioleknologi Pertanian Indonesia 11 7shy8 Nopember 2000 Yogyakarta Indonesia

Guivarcb A M Boccara M Prouteau amp D Chriqui 1999 Instability of phenotype and gene expression in longshyterm culture of carrot hairy root clones Plant Cell Rep 119 43 -50

Hamill J D AJ Parr MJC Rhodes RJ Robins amp NJ Walton 1987 New routes to plant secondary products Bioi Technol 5 800-804

Jha Tn amp Roy 1982 Chromosomal behaviour in cullure of Vicia faba Cytologia 47 465middot470

Joao KHL amp TA Brown 1994 Long-term stability of root cultures of tomato transformed wilh Agrobacterillm ri1izogellesRI60IJ Exp Bot 274 641 shy647

Karp A 1991 Cytological techniques Plant cell Culture Manual Kluwer Academic Publishers C4 1-13

Karp A amp SE Maddock 1984 Cromosome variation in wheat plants regenerated from cultured immature embryo Theor Appl Genet 67 249-255

Karp A RS Nelson E Thomas amp SWJ Brigbt 1082 Chromosome variation in protoplast-derived potato plants Theor Appl Genet 63 265-272

Lee KT T Suzuki T Yamakawa T Komada Y Igara hi amp K Sbimomura 1999 Production of tropane alkaloids by transformed root cultures of Atropa bellacona in tirred bioreactors with stai nless steel net Plant Cell Rep 18 657-57 1

McCoy TJ amp RL Phillips 1982 Chromosome stability in maize (Zea nwys) tissue cultures and sectoring in some regenerated plants Call 1 Genet Cytol 24 559-565

MehraA amp PM Mehra 1974 Organogenesis and plant let formation in vitro almond Bot Gaz 135 61-73

Mukherjee S Das amp S Jba 1994 Chromosome stability in transformed hairy root cultures of Artemisia anllua L Cell Chrolllos Res 17 7 1-76

Murashige T amp F Skoog 1962 A revised medium for rapid growth and bioassays with tobacco tissue cultures Phisiol Plant 15 473-497

Nuti Ronchi V M Nozzolini amp L Avanti 1981 Chromosomal variation on plants regenerated from two Nicotiall spp Protoplasma 109 433 -444

Orton TJ 1980 Chromosomal variability in tissu cultures and regenerated plants of Hordeum Them Appl Genet 56 101shy112

Ramsay G amp A Kumar 1990 Transformation of Viciafaba cotyledon and stem Ii sue by Agrobacieriu1I rhizogenes infectivity and cytological studies 1 Exp Bot 4 1 (228 ) 841-847

Saito K H Sudo M Yamazaki M Ko eldshyNakamura MKitajima H Takayama amp M Aimi 200 l Feasible production ofcamptothecin by hairy roOl culture of Ophiorrhiza pumila Plant Cell Rep 20 267-27 1

Sasaki K A Udagawa H Ishimaru T Hayashi A W Alfermaoo F Nakanishi amp K Shimomura 1998 High for kolin production in hairy roots of ColellS forskohlii Plant Cell Rep 11 7 457-459

nnales Bogorienses n s Vol 9 no2 2004 57

Syamsuhidayat SS amp JR Hutapea 1991 Invemaris Tanaman Obat Indonesia 1 Badan Penelitian dan Pengembangan Ke ehalan Departemen Kc ehalan Jakarta

Tan RX WE Zbeng amp HO Lang 1998 Biologically active subslrunces rrom the genus Artemisia Plant Med 295-302

Webb KJ S Jones MP Robbin amp FR Minchin 1990 Characterization of transgenic root cultures of Trifolium repeflS Trifolillmprarense and Lotus comicllatus and transgenic plants of LotllS comiculatus Plant Sci 70 243shy254

XU ZQ amp JF Jia 1996 The reduction of chromosome number and loss of regeneration ability during subculture of hairy rOOl cultures on Onobryc7is viciaeolia transformed by Arobacterilll11 rlzizogenel A4 Plant Sci 120 I 07 - 112

Zarate R 1999 Tropane alkaloid production by Arobaclerillm rhizogenes transformed hairy root culture ofAtropa baetica Willk (S lanaceae) Plant Cell Rep 18 418-423

Annales Bugorienses n s Vol 9 no2 2004 58

Syamsuhidayat SS amp JR Hutapea 1991 Invemaris Tanaman Obat Indonesia 1 Badan Penelitian dan Pengembangan Ke ehalan Departemen Kc ehalan Jakarta

Tan RX WE Zbeng amp HO Lang 1998 Biologically active subslrunces rrom the genus Artemisia Plant Med 295-302

Webb KJ S Jones MP Robbin amp FR Minchin 1990 Characterization of transgenic root cultures of Trifolium repeflS Trifolillmprarense and Lotus comicllatus and transgenic plants of LotllS comiculatus Plant Sci 70 243shy254

XU ZQ amp JF Jia 1996 The reduction of chromosome number and loss of regeneration ability during subculture of hairy rOOl cultures on Onobryc7is viciaeolia transformed by Arobacterilll11 rlzizogenel A4 Plant Sci 120 I 07 - 112

Zarate R 1999 Tropane alkaloid production by Arobaclerillm rhizogenes transformed hairy root culture ofAtropa baetica Willk (S lanaceae) Plant Cell Rep 18 418-423

Annales Bugorienses n s Vol 9 no2 2004 58