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

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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 .
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
سﻟا نوﻛﺗ لدﻟاقﺗﺷﻣﻟا ً
ﯾﺛارو
ً
ارو وﻟا رﻟا روذﺟﻟا نﻣ وﻛﺗﻣﻟا
ﻠﻋأ يرﻛﺳﻟا رﻟا قاروBeta vulgaris رﺗﻛﺑAgrobacterium rhizogenes
1601 تادزﻼﺑ ﻠﻋ وﻟاRi
ﻟا بﻌﺷ ﺗﻗ حﻟا مﺎﻗ مازﻣ
ﻟا تﻟا ةدﺣوﯾﺣ، ةﺣﻟا موﻠﻋ مﺳﻗ، رﺗﻟا ﻠﻛ، لوﻟا ﺎﺟ
ﺔﺻﻟا
ﻠﻋ رﻌﻟا روذﻟا توﻛﺗ قاروأيرﻟا رﻟا تﺑﻧ )Beta vulgaris(، نﻣ وﻟا ﻣﺎﻧ تﺑﻧ
لﻟا ﻲﻓ، ﻣﻟا ﻲﻓاو رﺑﺑ ﻟاAgrobacterium rhizogenes R1601 . ﺗﻧ تدأو ﻟا لرﺗ
،نورﻻا نﻋ فﺷﻛﻠﻟ رﻟاﻟا ضﻣﺎﺣﻷاﯾﻣ رﯾﻏﻻا ،يدﺗﻋحﺟﻧ ﻩذﻫ ﻲﻓ اروﻟا لوﻟاﺟﺳﻷا .
و ٕاوﻣﻧ وﻬﺳﺑ روذﺟﻟا ذﻫ نﻣ نوﻟا سﻟا زﻣا ﻠﻋطﺳو MS ﻠﻋ يوﺎﺣﻟا بﻟا0.5 رﺗﻟ مﻐﻠﻣ
-1
2,4-D وطﺳو ﻠﻋ B5 بﻟا ﻟاـﺑ زﺟﻣ 0.7 مﻐﻠﻣرﺗﻟ
-1
BA و 0.7 ﻠﻣ مﻐرﺗﻟ
-1
2,4-D وأ فﺻﺗ اوﻘﺑ
ﻟو شﻬﻟا ﻧو طﺳو ﻠﻋ ضﻻاMS وﻟوﻧﺑﻟا طﺳو ﻠﻋB5 .
ً
وﻠﺳ وﻣﻧ ﻲﻓ سﺎﻛﻟا كﺳو
ًﻣﻣ ﻠﻋ
طﺳو B5 لﻟا ﻠﻋ يوﻟا1.0 مﻐﻠﻣرﺗﻟ
-1
Kin. و 1.0 مﻐﻠﻣرﺗﻟ
-1
2,4-D .
ًادﺟ ﻬﻣ ﻟا ﻩذﻫ ﻲﻓ
وﻟا ﻟا تﺟﻣ تﺑﻧﻠنﻻ
ً
وﻠﺧ ً
طﺧ دﻌﺗ وﻟا سﻟا عرازﻣ ﻲﻓ ﻬﻣإجﺗﻧ يرﻛﺳ رﻧﺑ تﺎﺑﻧ
ًارو وﺣﻣ نﻣ ةرﺑﻣ اذﻫسﻟﺎﻟا.
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±C (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
... For example, Transgenic Solanumnigrum L. plants could emerge spontaneously from these induced roots [6]. Moreover, the roots induced by A. rhizogenes 1601 harbouring Riplasmid also stimulated the formation of transgenic callus from sugar beet plants [7]. In addition, the protoplasts were isolated directly from Maesalanceolata L. roots induced by A. rhizogenes strain (LBA 9402/12) showed continued to divide and transform into a callus [7]. ...
... Moreover, the roots induced by A. rhizogenes 1601 harbouring Riplasmid also stimulated the formation of transgenic callus from sugar beet plants [7]. In addition, the protoplasts were isolated directly from Maesalanceolata L. roots induced by A. rhizogenes strain (LBA 9402/12) showed continued to divide and transform into a callus [7]. Furthermore, rather than including the genetic material directly into the protoplast cells, the genetic transformation can be accomplished by fusing the transgenic hairy root protoplast with the receiving protoplast and producing transgenic plants [8]. ...
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Protoplasts represent a unique experimental system for the circulation and formation of genetically modified plants. Here, protoplasts were isolated from genetically modified hairy root tissues of Brassica oleracea L. induced by the Agrobacterium rhizogenes strain (ATCC13332). The concentration of enzyme solutions utilized for protoplast isolation was 1.5 % Cellulase YC and 0.1 % Pectolyase Y23 in 13% mannitol solution, which resulted in high efficiency of isolation within 8 hours, in which the protoplast yield was 2 × 104 cells ml-1 and the percentage of viability was 72%. Each protoplast has one nucleus with a nucleation of 48%. A polymerase chain reaction (PCR) assay verified the presence of rol B and rol C genes in hairy root tissues by detaching a single bundle of DNA replication from these roots using a specific pair of primers. The current study demonstrated that A. rhizogenes strain (ATCC13332) is a vector for the incorporation of T-DNA genes into cauliflower plants, as well as the success of the hairy roots retention of rol B and rol C genes transferred to it.
... Conservation of genetic marker on Ri-plasmid of A. rhizogenes R1601 was carried out through the spread of antibiotic saturated paper disc on the surface of agar-solidified APM medium previously streaked with 0.1 ml of bacterial suspension [11]. Detection of agropine in these tissue was carried out following the procedure mentioned previously [12]. ...
Thesis
Full-text available
انوةدإ دج الد ا رسدة الحالردة نباتدات البروتمدج Brassica oleracea var. italica ةدؽ تطايز تالس أر ا ز باد ا رتيا الةذ ورت السدرقان اولأو ا ر (. قدد نةدح تدالس الاو ا ر والسدرقان الظاةردة عمى وسط ةؾ ا رفدر وسدكؾ) MS - ( الصدم الطددعؼ د 2.5 ةمغدؼ لودر 1 6-Benzyl adenine (BA) - و 2.2 ةمغدؼ لودر 1 Naphthalene Acetic- Acid (NAA) وتدالس الةدذور الظداةج عمدى وسدط MS - الطددعؼ د 2.5 ت 1.5 ت 2.2 ( ةمغدؼ لودر 1 Thidiazuron (TDZ) دج الوكدؾيؽ الومقدايج لأ درا ال ضدرية بطرحمدة واحددة ب ددىا تدؼ تةدذير ىدذه الأ درا بسديؾلة عمدى وسدط MSO الصم ال الج ةؽ ةظغطات الظطدؾ وتأقمطدإ ظةداح. اوسدوحدثإ دج ىدذه الد ا رسدة الأرظدة الةسدطرة Somatic embryos ةؽ تالس السرقان والةذ ور وتذلػ ةؽ قظع السرقان والةذور عظد ز ا رعويا إوداةويددا عمددى وسدط MS - الصدم الطدددعؼ ددد 2.4 و 2.5 ( ةمغددؼ لودر 1 TDZ . وتشدكمإ ةددؽ ز ا رعدة الأرظدة الطسوأ دمة عمدى وسدط MSO الصدم ال دالج ةدؽ ةظغطدات الظطدؾ أ رعداً خضدريةً مددد عددددىا 225 رعداً ردذرت ظةداح وي دلد ىدذا ةسدا ا رً ةظاسدباً لإنودداج أ عددداد تب درة ةددؽ نباتددات البروتمج. تطكظدإ الد ا رسدة الحالردة دج أحدد رؾانبيدا الطيطدة ةدؽ إنوداج البدذور الصدظاةرة synthetic seeds بإسددو داي الأرظددة الةسددطرة والقطددؼ الظاةرددة. أوعيددرت الظودداي ان الورت ددز 2% اللة ظدات الصدؾديؾي Sodium alginate كدان ةلايطداً ردداً دج اعظدا أعمدى نسدبة تحدؾل conversion ) انبا(ث لمبدذور ةدؽ الأرظدة الةسدطرة مغدإ 62 % عظدد خزنيدا ةددة 7 أيداي دج 4° سد م زية دج حد ؽ أعظدى ترت دز 3% اللة ظدات الصدؾديؾي اعمدى نسدبة تحدؾل لمبدذور الحاويدة عمدى القطدؼ الظاةردة و ددمإ إلدددى 96 % عظدددد الغددروف نفسدديا ةددة خدزن 7 أيددداي دددج 4° سددد م زية( وقدددد اعوطدددد الورت دز 2% لة ظدات الصدؾديؾي دج الوةدارب لوغمرد الأرظدة الةسدطرة والقطدؼ الظاةردة تؾندو اعظدى ا ضدي فدكي لمبددذور الصددظاةرة ي ئددة ت ا رك دد ت رويددة Beads ( وداخمددو ةددع 12 / ةمغددؼ لودر - كمؾريد الكالس ؾي الطايج CaCl2. 2H2O الطسو دي عاةي تصم لمبذور. وودد وامدحاً ةدؽ نوداي الد ا رسدة أ ل ن زيدادة ةددة ال دزن ليدذه البدذور الطصدظ ة ان كدس سدمباً ° عمدى نسدبة تحؾليدا الودج مغدإ ادندى ةسدوؾياتيا 28 و 52 % عظدد خزنيدا ةددة 62 يؾةداً دج 4 س م زية لكي ةؽ ذور الأرظدة الةسدطرة والقطدؼ الظاةردة عمدى الورت د . ةدؽ ناحردة أُخدر تدان لزيدادة دررة ح ا ررة ال زن اثرسمبج بالد ج نسبة تحؾل البذور الوج ان دةإ ) %2 ( عظدة خزنيدا ةددة 32 يؾةداً دج حد ا ررة الغر دة 25 ° سد م زية. تطدا ان تزويدد حشدؾة البدذور الصدظاةرة لأرظدة الةسدطرة دد - 1.2 ةمغؼ لور 1 BA - و 2.5 ةمغؼ لودر 1 NAA ر دع تفدا ة انباتيدا لوصدي 68 % عظدد خزنيدا ةددة 7 أياي ج 4° س م زية و 42 % ج دررة ال زن 25 ° س م زية ووذات
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The clones (C1, C2, C3 and C4) of sugarbeet leaves express their capability to induce the adventitious roots as a result of their injection with suspension of Agrobacterium rhizogenes R1601. Clear differences were found in the response and transgenesis of these explants. The result indicated that C3 was the best in this case, but hairy roots produced from all clones have similar phenotype. Generally, numerous cultures of agropine-positive and rapid propagation hairy roots were obtained.
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Genetic engineering requires a procedure for introducing DNA into host cells, followed by integration into the host genome and gene expression. Although several procedures for DNA-medi-ated gene transfer in mammalian cells, yeast and bacteria have been reported, no such methods are yet available for plant cells. The major obstacle to DNA uptake in plant cells is the cell wall, but this can be circumvented by using plant protoplasts, cells freed of their cell walls by enzymatic digestion. However, none of the reports on the uptake of DNA into plant protoplasts1,2 has produced conclusive evidence for the integration of DNA into the host genome, that is, that stable transformation occurs. The tumour-inducing bacterium Agrobacterium tumefaciens, which causes crown gall disease, is a natural system for the introduction of foreign DNA into plants. This bacterium introduces part of its tumour-inducing (Ti) plasmid, called T-DNA, into plant cells, where it becomes integrated into the nuclear DNA of the host3,4 and is transcribed into mRNA5,6. T-DNA encodes tumour-specific enzymes responsible for the formation of amino acid derivatives such as octopine or nopaline7, which the bacterium can use as a sole source of carbon and nitrogen. The transformed cells have also acquired the ability to grow in the absence of phytohormones (autotrophy). An in vitro system for infection of Nicotiana tabacum protoplasts by A. tumefaciens has already been reported8. Transformants are selected by their ability to divide and grow in tissue culture without the addition of plant phytohormones to the synthetic culture medium. Here, we report a reproducible method for the stable transformation of tobacco protoplasts with Ti-plasmid DNA, using a similar selection procedure.
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Vinca rosea protoplasts and Agrobacterium tumefaciens spheroplasts harboring octopine-type Ti plasmid were mixed and treated with polyethylene glycol or polyvinyl alcohol, which facilitated the introduction of spheroplasts into plant protoplasts. After the protoplasts had been kept at 40 C for 4 days, bacteria were found to be completely eliminated from the medium. Among treated protoplasts 1–2 per 1,000 formed colonies on the Murashige and Skoog medium (1962) lacking hormones. When the colonies were isolated and subcultured, they could be maintained as clones. Octopine, an amino acid specific to crown gall, was detected in half of these clones. The phenotypic features of these putative transformants were compared but did not show any coincidental tendencies in relation to color, hardness, form, growth rate, or octopine production. The significance of this system in transformation of higher plant cells is discussed.