PUTATIVE GENETICALLY MODIFIED CALLUS DERIVED FROM TRANSFORMED HAIRY ROOTS INDUCED ON SUGARBEET (BETA VULGARIS) EXPLANTS BY AGROBACTERIUM RHIZOGENES 1601 HARBOURING RI-PLASMID

Abstract

Hairy roots were induced on leaf explants excised from sugarbeet (Beta vulgaris) plants when inoculated by Agrobacterium rhizogenes R1601. Paper electrophoresis proved the presence of agropine, the unusual amino acid in these roots which demonstrated the transformation of these tissues. Transformed hairy roots were easily grown on agar-solidified Murashige and Skoog, 1962 (MS) medium containing 0.5mgl-1 2,4dichlorophenoxy acetic acid (2,4-D) or B5 medium supplemented with a mixture of 0.7 mgl-1 Benzyl Adenine (BA) and 0.7 mgl-1 (2,4-D). Callus produced from these hairy roots grown on MS medium was of friable and white in color, while brown in color on B5 medium. Calli were grown efficiently in liquid B5 medium provided with a mixture of 1.0 mgl-1 kinetine (Kin.) and 1.0 mgl-1 2,4-D. These results may contribute in transformation of sugarbeet plants. 456 ‫اﻟﻛﺎﻟس‬ ‫ﺗﻛوﯾن‬ ‫اﻟﻣﻌدل‬ ‫اﻟﻣﺷﺗق‬ ً ‫اﺛﯾﺎ‬ ‫ور‬ ً ‫اﺛﯾﺎ‬ ‫ور‬ ‫اﻟﻣﺣوﻟﺔ‬ ‫اﻟﺷﻌرﯾﺔ‬ ‫اﻟﺟذور‬ ‫ﻣن‬ ‫اﻟﻣﺗﻛوﻧﺔ‬ ‫ﻋﻠﻰ‬ ‫أ‬ ‫اﻟﺳﻛري‬ ‫اﻟﺑﻧﺟر‬ ‫اق‬ ‫ور‬ Beta vulgaris ‫ﺑ‬ ‫ﺑﻛﺗرﯾﺎ‬ Agrobacterium rhizogenes 1601 ‫ﺑﻼزﻣﯾدات‬ ‫ﻋﻠﻰ‬ ‫اﻟﺣﺎوﯾﺔ‬ Ri ‫اﻟﻧﻌﻣﺔ‬ ‫ﺷﻌﯾب‬ ‫ﻗﺗﯾﺑﺔ‬ ‫اﻟﻣﻼح‬ ‫ﻗﺎﺳم‬ ‫اﺣم‬ ‫ﻣز‬ ‫اﻟ‬ ‫اﻟﺗﻘﻧﯾﺎت‬ ‫وﺣدة‬ ‫ﺣﯾﺎﺗﯾﺔ‬ ، ‫اﻟﺣﯾﺎة‬ ‫ﻋﻠوم‬ ‫ﻗﺳم‬ ، ‫اﻟﺗرﺑﯾﺔ‬ ‫ﻛﻠﯾﺔ‬ ، ‫اﻟﻣوﺻل‬ ‫ﺟﺎﻣﻌﺔ‬ ‫اﻟﺧﻼﺻﺔ‬ ‫ﻋﻠﻰ‬ ‫اﻟﺷﻌرﯾﺔ‬ ‫اﻟﺟذور‬ ‫ﺗﻛوﻧت‬ ‫اق‬ ‫أور‬ ‫اﻟﺳﻛري‬ ‫اﻟﺑﻧﺟر‬ ‫ﻧﺑﺎﺗﺎت‬) Beta vulgaris (، ‫ﻣن‬ ‫اﻟﻣﻔﺻوﻟﺔ‬ ‫ﻧﺎﻣﯾﺔ‬ ‫ﻧﺑﺎﺗﺎت‬ ‫اﻟﺣﻘل‬ ‫ﻓﻲ‬ ، ‫اﻟﻣ‬ ‫ﻓﻲ‬ ‫اﻗﻊ‬ ‫و‬ ‫ﺑﺑﻛﺗرﯾﺎ‬ ‫اﻟﻣﻠﻘﺣﺔ‬ Agrobacterium rhizogenes R1601. ‫ﻧﺗﺎﺋﺞ‬ ‫أﻛدت‬ ‫و‬ ‫اﻟ‬ ‫ﺗرﺣﯾل‬ ‫اﻻﻛروﺑﯾن،‬ ‫ﻋن‬ ‫ﻟﻠﻛﺷف‬ ‫اﻟﻛﻬرﺑﺎﺋﻲ‬ ‫اﻟ‬ ‫ﺣﺎﻣض‬ ‫اﻷ‬ ‫ﻣﯾﻧﻲ‬ ‫ﻏﯾر‬ ‫اﻻ‬ ‫ﻋﺗﯾﺎدي،‬ ‫ﻧﺟﺎح‬ ‫ﻫذﻩ‬ ‫ﻓﻲ‬ ‫اﺛﻲ‬ ‫اﻟور‬ ‫اﻟﺗﺣول‬ ‫اﻷﻧﺳﺟﺔ‬. ‫و‬ ٕ ‫ا‬ ‫ﻧﻣوﻩ‬ ‫ﺑﺳﻬوﻟﺔ‬ ‫اﻟﺟذور‬ ‫ﻫذﻩ‬ ‫ﻣن‬ ‫اﻟﻣﺗﻛون‬ ‫اﻟﻛﺎﻟس‬ ‫اﻣﺗﺎز‬ ‫ﻋﻠﻰ‬ ‫وﺳط‬ MS ‫ﻋﻠﻰ‬ ‫اﻟﺣﺎوي‬ ‫اﻟﺻﻠب‬ 0.5 ‫ﻟﺗر‬ ‫ﻣﻠﻐم‬-1 2,4-D ‫و‬ ‫وﺳط‬ ‫ﻋﻠﻰ‬ B5 ‫اﻟﺻﻠب‬ ‫اﻟ‬ ‫ﺑـ‬ ‫ﻣﺟﻬز‬ 0.7 ‫ﻣﻠﻐم‬ ‫ﻟﺗر‬-1 BA ‫و‬ 0.7 ‫ﻣﻠ‬ ‫ﻐم‬ ‫ﻟﺗر‬-1 2,4-D ‫و‬ ‫أ‬ ‫ﺗﺻف‬ ‫اﻣﻪ‬ ‫ﺑﻘو‬ ‫وﻟ‬ ‫اﻟﻬش‬ ‫وﻧﻪ‬ ‫وﺳط‬ ‫ﻋﻠﻰ‬ ‫اﻻﺑﯾض‬ MS ‫وﻟوﻧﻪ‬ ‫اﻟﺑﻧﻲ‬ ‫وﺳط‬ ‫ﻋﻠﻰ‬ B5. ً ‫ﺳﻠوﻛﺎ‬ ‫ﻧﻣوﻩ‬ ‫ﻓﻲ‬ ‫اﻟﻛﺎﻟس‬ ‫وﺳﻠك‬ ً ‫ﻣﻣﺎﺛﻼ‬ ‫ﻋﻠﻰ‬ ‫وﺳط‬ B5 ‫اﻟﺳﺎﺋل‬ ‫ﻋﻠﻰ‬ ‫اﻟﺣﺎوي‬ 1.0 ‫ﻣﻠﻐم‬ ‫ﻟﺗر‬-1 Kin. ‫و‬ 1.0 ‫ﻣﻠﻐم‬ ‫ﻟﺗر‬-1 2,4-D. ً ‫ﺟدا‬ ‫ﻣﻬﻣﺔ‬ ‫اﻟﻧﺗﺎﺋﺞ‬ ‫ﻫذﻩ‬ ‫ﻓﻲ‬ ‫اﻟﺣﯾوﯾﺔ‬ ‫اﻟﺗﻘﺎﻧﺔ‬ ‫ﻣﺟﺎﻻت‬ ‫ﻟ‬ ‫ﻠﻧﺑﺎﺗﺎت‬ ‫ﻻن‬ ً ‫ﺧﻠوﯾﺎ‬ ً ‫ﺧطﺎ‬ ‫ﺗﻌد‬ ‫اﻟﻣﺗﻛوﻧﺔ‬ ‫اﻟﻛﺎﻟس‬ ‫ارع‬ ‫ﻣز‬ ‫ﻓﻲ‬ ‫ﻣﻬﻣﺎ‬ ‫إ‬ ‫ﻧﺗﺎج‬ ‫ﺳﻛري‬ ‫ﺑﻧﺟر‬ ‫ﻧﺑﺎﺗﺎت‬ ً ‫اﺛﯾﺎ‬ ‫ور‬ ‫ﻣﺣوﻟﺔ‬ ‫ﻣن‬ ‫ﻣﺑﺎﺷرة‬ ‫ﻫذا‬ ‫اﻟﻛﺎﻟس‬ .

Full text

Iraqi J. Biotech.11 (2):455-463(2012) Mzahem K. Al-Mallah and Qutaba Sh. Al-Ne'ma
455
PUTATIVE GENETICALLY MODIFIED CALLUS DERIVED
FROM TRANSFORMED HAIRY ROOTS INDUCED ON
SUGARBEET (BETA VULGARIS) EXPLANTS
BY AGROBACTERIUM RHIZOGENES 1601 HARBOURING
RI-PLASMID
Mzahem K. Al-Mallah Qutaba Sh. Al-Ne'ma
Biotechnology Unit, Department of Biology, College of Education, University of Mosul
ABSTRACT
Hairy roots were induced on leaf explants excised from sugarbeet (Beta vulgaris)
plants when inoculated by Agrobacterium rhizogenes R1601. Paper electrophoresis
proved the presence of agropine, the unusual amino acid in these roots which
demonstrated the transformation of these tissues. Transformed hairy roots were
easily grown on agar-solidified Murashige and Skoog, 1962 (MS) medium
containing 0.5mgl-1 2,4dichlorophenoxy acetic acid (2,4-D) or B5 medium
supplemented with a mixture of 0.7 mgl-1 Benzyl Adenine (BA) and 0.7 mgl-1 (2,4-
D). Callus produced from these hairy roots grown on MS medium was of friable
and white in color, while brown in color on B5 medium. Calli were grown
efficiently in liquid B5 medium provided with a mixture of 1.0 mgl-1 kinetine (Kin.)
and 1.0 mgl-1 2,4-D. These results may contribute in transformation of sugarbeet
plants.
Key words: Agrobacterium rhizogenes, Hairy roots, Callus cultures, Sugarbeet
MSO: MS medium free from growth regulators, B5O: Gamborg medium free from growth
regulators.

Iraqi J. Biotech.11 (2):455-463(2012) Mzahem K. Al-Mallah and Qutaba Sh. Al-Ne'ma
456
سﻟﺎﻛﻟا نﯾوﻛﺗ لدﻌﻣﻟاقﺗﺷﻣﻟا ً
ﺎﯾﺛارو
ً
ﺎﯾﺛارو ﺔﻟوﺣﻣﻟا ﺔﯾرﻌﺷﻟا روذﺟﻟا نﻣ ﺔﻧوﻛﺗﻣﻟا
ﻰﻠﻋأ يرﻛﺳﻟا رﺟﻧﺑﻟا قاروBeta vulgaris ﺑ ﺎﯾرﺗﻛﺑAgrobacterium rhizogenes
1601 تادﯾﻣزﻼﺑ ﻰﻠﻋ ﺔﯾوﺎﺣﻟاRi
ﺔﻣﻌﻧﻟا بﯾﻌﺷ ﺔﺑﯾﺗﻗ حﻼﻣﻟا مﺳﺎﻗ مﺣازﻣ
ﻟا تﺎﯾﻧﻘﺗﻟا ةدﺣوﺔﯾﺗﺎﯾﺣ، ةﺎﯾﺣﻟا موﻠﻋ مﺳﻗ، ﺔﯾﺑرﺗﻟا ﺔﯾﻠﻛ، لﺻوﻣﻟا ﺔﻌﻣﺎﺟ
ﺔﺻﻼﺧﻟا
ﻰﻠﻋ ﺔﯾرﻌﺷﻟا روذﺟﻟا تﻧوﻛﺗ قاروأيرﻛﺳﻟا رﺟﻧﺑﻟا تﺎﺗﺎﺑﻧ )Beta vulgaris(، نﻣ ﺔﻟوﺻﻔﻣﻟا ﺔﯾﻣﺎﻧ تﺎﺗﺎﺑﻧ
لﻘﺣﻟا ﻲﻓ، ﻣﻟا ﻲﻓﻊﻗاو ﺎﯾرﺗﻛﺑﺑ ﺔﺣﻘﻠﻣﻟاAgrobacterium rhizogenes R1601 . ﺞﺋﺎﺗﻧ تدﻛأو ﻟا لﯾﺣرﺗ
،نﯾﺑورﻛﻻا نﻋ فﺷﻛﻠﻟ ﻲﺋﺎﺑرﻬﻛﻟاﻟا ضﻣﺎﺣﻷاﻲﻧﯾﻣ رﯾﻏﻻا ،يدﺎﯾﺗﻋحﺎﺟﻧ ﻩذﻫ ﻲﻓ ﻲﺛاروﻟا لوﺣﺗﻟاﺔﺟﺳﻧﻷا .
و ٕاﻩوﻣﻧ ﺔﻟوﻬﺳﺑ روذﺟﻟا ﻩذﻫ نﻣ نوﻛﺗﻣﻟا سﻟﺎﻛﻟا زﺎﺗﻣا ﻰﻠﻋطﺳو MS ﻰﻠﻋ يوﺎﺣﻟا بﻠﺻﻟا0.5 رﺗﻟ مﻐﻠﻣ
-1
2,4-D وطﺳو ﻰﻠﻋ B5 بﻠﺻﻟا ﻟاـﺑ زﻬﺟﻣ 0.7 مﻐﻠﻣرﺗﻟ
-1
BA و 0.7 ﻠﻣ مﻐرﺗﻟ
-1
2,4-D وأ فﺻﺗ ﻪﻣاوﻘﺑ
ﻟو شﻬﻟا ﻪﻧو طﺳو ﻰﻠﻋ ضﯾﺑﻻاMS ﻪﻧوﻟوﻲﻧﺑﻟا طﺳو ﻰﻠﻋB5 .
ً
ﺎﻛوﻠﺳ ﻩوﻣﻧ ﻲﻓ سﻟﺎﻛﻟا كﻠﺳو
ًﻼﺛﺎﻣﻣ ﻰﻠﻋ
طﺳو B5 لﺋﺎﺳﻟا ﻰﻠﻋ يوﺎﺣﻟا1.0 مﻐﻠﻣرﺗﻟ
-1
Kin. و 1.0 مﻐﻠﻣرﺗﻟ
-1
2,4-D .
ًادﺟ ﺔﻣﻬﻣ ﺞﺋﺎﺗﻧﻟا ﻩذﻫ ﻲﻓ
ﺔﯾوﯾﺣﻟا ﺔﻧﺎﻘﺗﻟا تﻻﺎﺟﻣ ﻟ تﺎﺗﺎﺑﻧﻠنﻻ
ً
ﺎﯾوﻠﺧ ً
ﺎطﺧ دﻌﺗ ﺔﻧوﻛﺗﻣﻟا سﻟﺎﻛﻟا عرازﻣ ﻲﻓ ﺎﻣﻬﻣإجﺎﺗﻧ يرﻛﺳ رﺟﻧﺑ تﺎﺗﺎﺑﻧ
ًﺎﯾﺛارو ﺔﻟوﺣﻣ نﻣ ةرﺷﺎﺑﻣ اذﻫسﻟﺎﻛﻟا.

Iraqi J. Biotech.11 (2):455-463(2012) Mzahem K. Al-Mallah and Qutaba Sh. Al-Ne'ma
457
INTRODUCTION
Different methods have been developed for introducing foreign DNA into plants, for
example transformation with naked DNA (1) use of virus infection (2), protoplasts
fusion with bacterial spheroplasts (3)and fusion of protoplasts with liposomes (4).
However, the most commonly used method exploits the natural infection mechanism of
the Gram-negative bacterium Agrobacterium mediated transformation(5).
Agrobacterium rhizogenes induces the formation of hairy roots in dicotyledonous plants
by incorporating T-DNA from the Ri-plasmid (Root-inducing plasmids ) into host plant
nuclear DNA (6). These hairy roots are capable of rapid shoot-independent growth with
minimal to no hormone requirements when grown in tissue culture (7). Foreign genes
are inserted in T-DNA through Ri-plasmid, transferred and integrated into the host
genome (8). In this paper, formation of putative genetically engineered callus from
transformed hairy roots produced on sugarbeet induced by A. rhizogenes strain R1601
was reported.
MATERIALS AND METHODS
Direct inoculation and stimulation of hairy roots
Agrobacterium rhizogenes strain R1601 (Agropine-type) was used in this study.
Cultures were grown on APM agar solidified medium (9) supplemented with 100 mgl-1
of both Kanamycin and Carbenciline. For explant inoculation, 48 hrs. cultures grown in
APM liquid medium on shaker incubator at 28±1ºC/150 rpm in dark were used (10).
Leaf and petiole explants were excised from field grown sugarbeet (Beta vulgaris)
plants (Fig. 1, A), after sterilization with 70% ethanol for 2.0 min. then immersed in 3%
NaOCl for5.0 min.(11). After washing 3 times with sterile water, explants were
wounded at 3-5 site/explant. Sterile needle was dipped in bacterial inoculum and
smeared gently over the wound.
Elimination of bacteria from hairy roots cultures
Primary hairy roots, which developed after two weeks, were excised and cultured on
(MSO) agar-solidified MS hormone-free medium (12). Specimen flasks were kept in
diffused light (100 Lux, 25 ºC). Individual roots 1.0-1.5 cm length developed along the
inoculated surface were excised, and each was transferred to agar-solidified MSO
medium containing gradual conc. 150, 300 and 500 mgl-1 of cefotaxime (Cefosam-
Sammara-Iraq) in 9 cm diameter plastic petridishes. Transformed hairy roots free from
bacteria were subcultured every 25 days on agar-solidified MSO as selection medium
without antibiotics (10).
Electrophoresis
Aliquot of 10-20 µl was spotted on Whatman No.3mm paper (15 × 30 cm) and
subjected to electrophoresis 300 V/cm, 60 min. (Esselte Studium, S-11285 Stockhelm,
Sweden). The buffer used: formic acid: acetic acid: water (5:15:80 V:V:V). After
drying, electophortograms in a current of hot air, they were stained with silver nitrate,
and after 15-30 min. submerged with 2% methanolic NaOH, dried and submerged with
5% sodium thiosulphate and washed with running water (13).

Iraqi J. Biotech.11 (2):455-463(2012) Mzahem K. Al-Mallah and Qutaba Sh. Al-Ne'ma
458
Induction of callus from transformed hairy roots in solid cultures
Two cm length of the transformed hairy roots were cultured on the surface of 15 ml
agar-solidified of each MS (12) and B5 medium (14) supplemented with different
concentration of Benzyl Adenine and 2,4-dichlorophenoxy acetic acid as indicated
below:
• MS + 0.0 mgl-1 BA + 0.0 mgl-1 2,4-D (MSO).
• MS + 0.1 mgl-1 BA + 0.5 mgl-1 2,4-D.
• MS + 0.5 mgl-1 BA + 0.1 mgl-1 2,4-D.
• MS + 0.0 mgl-1 BA + 0.5 mgl-1 2,4-D.
• MS + 0.1 mgl-1 BA + 0.0 mgl-1 2,4-D.
• MS + 0.7 mgl-1 BA + 0.7 mgl-1 2,4-D.
• MS + 0.2 mgl-1 BA + 0.0 mgl-1 2,4-D.
• MS + 0.0 mgl-1 BA + 0.3 mgl-1 2,4-D.
• MS + 0.4 mgl-1 BA + 0.0 mgl-1 2,4-D.
Also the same combinations of 2,4-D and BA using B5 medium were tested. All
samples were kept in culture room at 25±2 ºC in light and dark (16/8 hrs, respectively)
of intensity 800 Lux.
Induction of callus from transformed hairy roots in liquid cultures
Single cluster of hairy roots was cultured in 9 cm diameter petridishes containing
10 ml of half-strength liquid B5 medium as bellow:
B5 + 5 mgl-1 BA + 5 mgl-1 2,4-D.
B5 + 1 mgl-1 Kin. + 1 mgl-1 2,4-D.
B5 + 1 mgl-1 Kin. + 0.05 mgl-1 2,4-D.
In other experiments single pieces of hairy roots were cultured in 250 ml conical
flasks contained 50 ml of half-strength B5 liquid medium previously mentioned and
kept on incubating shaker (GFL., Germany) at 90 rpm/min., 25±2ºC (15) with
fluorescent tubes at 16 hr./8 hr. light/dark photoperiod and a light intensity of 800 Lux.
Other pieces of hairy roots were transferred on half strength B5O liquid medium and
incubated at the same conditions.
RESULTS AND DISCUSSON
Production of bacterial-free hairy root cultures
Hairy roots formed on explants at inoculation sites by A. rhizogenes. The development
of transformed hairy roots was 9 roots on each petiole after 4 wks of inoculation
Fig.(1, B), They were negatively geotropism in their growth on solid MSO medium
with dense of root hairs Fig. (1,C).
Presence of Agropine in hairy roots
Electrophoretic analysis of extracts from primary hairy roots induced by A. rhizogenes
strain R1601 gave a positive silver nitrate reaction; therefore, plant materials containing

Iraqi J. Biotech.11 (2):455-463(2012) Mzahem K. Al-Mallah and Qutaba Sh. Al-Ne'ma
459
'agropine' were classified as ' agropine type' Fig.(2). This result proved the incidence of
transformation of these tissues.
Figure(1):Callus derived from transformed hairy roots induced on sugarbeet (Beta
vulgaris) explants by Agrobacterium rhizogenes 1601
Culture of hairy rootshaving
dense root hairs on solidified
MSO medium
Hairy roots developed on
petiole
(4weeks old) on solidified MSO
medium
Field sugarbeet plants
(10 weeks old)
Callus produced from hairy roots
on solidified B5+ 0.7 mgL-1 BA +
0.7mgL-1 2,4-D medium(6weeks)
weeks)
Callus produced from hairy roots
on solidified MS+0.5mgL-12,4-D
medium (12 weeks)
Culture of transformed hairy
roots on half-strength B5O liquid
medium (2 weeks old)
A B C
D E F

Iraqi J. Biotech.11 (2):455-463(2012) Mzahem K. Al-Mallah and Qutaba Sh. Al-Ne'ma
460
Hairy root normal root normal root standard Agropine
Figure(2): Electrophoretogram showing the presence of agropine in hairy roots induced on
sugarbeet explants by Agrobacterium rhizogenes 1601
Production of callus on solid cultures
Results showed that supplementation of agar-solidified MS medium with BA and
2,4-D gave weak response to initiate callus from transformed hairy roots compared with
medium supplied with kinetine and 2,4-D table (1). The best period involved for callus
initiation directly from hairy root, as callus formation was completed at 8 wks was on
MS medium containing 0.5 mgl-1 2,4-D. This callus was slow growing, friable and
white in color Fig. (1, D). Moreover, B5 solid medium encouraged callus formation
from hairy roots at 0.7 mgl-1 BA + 0.7 mgl-1 2,4-D, and required 3 wks. This callus
was friable and brown in color Fig. (1, E), converted to black when it stayed in the same
medium for another week. All the remaining media did not stimulate the initiation of
callus.
Production of callus in stationary liquid cultures
Callus formation from hairy roots grew slowly on solid culture. Stationary liquid
culture was more suitable for callus formation on half-strength B5 medium. The
required period for callus formation on B5 liquid medium with 1.0 mgl-1 Kin.+1.0 mgl-
1 2,4-D and B5 medium with 1.0 mgl-1 Kin. + 0.05 mgl-1 2,4-D was best response.
Weak response occur in B5 medium containing 5.0 mgl-1 BA + 5.0 mgl-1 2,4-D. Callus
was friable in texture and brown in color table(1).
Spot of
transformed
hairy root
spot of
standard
Agropine

Iraqi J. Biotech.11 (2):455-463(2012) Mzahem K. Al-Mallah and Qutaba Sh. Al-Ne'ma
461
Table (1):Callus production from transformed hairy roots induced on
sugarbeet Beta vulgaris explants inoculated by Agrobacterium rhizogenes
*10 replicates / treatment
Production of callus on shaking liquid cultures
Hairy root samples grew on the same liquid cultures mentioned above with shaking
gave similar results when it grew on stationary liquid medium. It failed to form callus in
B5 medium provided with 5.0 mgl-1 BA and 5.0 mgl-1 2,4-D only. The control samples
of hairy roots grown on a half-strength B5 liquid medium was not forming which
exhibited rapid growth under these conditions Fig. (1, F). The appearance of hairy
roots at inoculation sites with A. rhizogenes R1601, on varios explants attributed to the
transition of a segment of Ri-T-DNA to the genetic material of the plant cell (16), and
for the expression of genes to show the first signs of genetic transformation of plant cell
by formation of transformed hairy roots (17). The development of hairy roots on
explants, inoculated with A. rhizogenes, grown in MS medium free from growth
regulator may due to the disturbance of phytohormons content in these tissues. This
situation led to the vast increase of auxins which encouraged the formation of these
adventitious roots in the absence of such auxin in the culture medium used. The
presence of agropin in hairy roots confirms the genetic transformation of these tissue
(18). The synthesis of this unusual amino acid occur as a result of the transition of T-
DNA segment of Ri-plasmid containing the genes that controlled the formation of
agropin (19). The rapid growth of transformed hairy roots and its dense content of
root hairs, may due to the length of apical meristem for these roots compared with
untransformed roots(20), as well as, to the increased rates of cell division (21). These
transformed hairy root represent an efficient alternative approach to regenerate into
plants as occur in Solanum nigrum (22) or through callus-derived from hairy roots (23).
Our conclusion , this transformed tissue may represent a short cut to obtain genetically
modified plants. Moreover, they could be a good source for protoplasts isolation, cell
suspension formation and for secondary metabolic substances.
Media Callus
induction % Callus texture
and colour
Liquid B5O 0*
culture B5+1.0 mgl-1 Kin. +1.0 mgl-1 2,4-D 97 Friable/brown
B5+1.0 mgl-1 Kin.+0.05 mgl-1 2,4-D 90 Friable/brown
B5+5.0 mgl-1 BA + 5.0 mgl-1 2,4-D 66 Friable/brown
Solid MSO 0
culture MS + 0.5 mgl-1 2,4-D 10 Friable/white
B5+0.7 mgl
-
1 BA + 0.7 mgl
-
1 2,4
-
D
43
Friable/brown

Iraqi J. Biotech.11 (2):455-463(2012) Mzahem K. Al-Mallah and Qutaba Sh. Al-Ne'ma
462
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Spot of standard
Spot of transformed
Hairy root