Journal
Journal of Applied Horticulture, 14(1): 3-6, 2012
Appl
Increased regeneration ability of transgenic callus of carrot
(Daucus carota L.) on B5-based regeneration medium
Yuan-Yeu Yau1,2* and Kevin Yueju Wang2
1
USDA-ARS Vegetable Research Crops Unit and Department of Horticulture, University of Wisconsin-Madison,
1575 Linden Drive, Madison, WI 53706, USA. 2Present address: Department of Natural Resources,
Northeastern State University, Broken Arrow, OK 74014, USA. *E-mail: yau@nsuok.edu
Abstract
The in vitro development of a whole plant from a single cell is a characteristic feature of plants. Successful embryogenesis and
regeneration during in vitro tissue culture are influenced by different factors including medium components. In this study, we compared
two regeneration media (MSIII, B5) and a mixture of these media (MSIII+B5) for the regeneration of plants from putative transgenic
carrot calli. Seventeen times more plantlets were regenerated on B5 medium than on either MSIII or MSIII+B5 medium. A total of
432 plantlets were regenerated on B5 medium, compared to only 24 and 28 plantlets on MSIII and MSIII+B5, respectively. Plantlets
regenerated on B5 medium were generally healthier and bigger than those regenerated on either MSIII or MSIII+B5 medium. Fiftytwo plantlets, 7-9 cm in length, were observed on the B5 regeneration medium, while no plants having 7-9 cm length were observed
on either MSIII or MSIII+B5 medium after 4 months. This study demonstrated that B5 is a better medium than MSIII or MSIII+B5
medium for carrot callus regeneration and can be used routinely and efficiently for carrot genetic transformation experiments. The
transgenic nature of the regenerated plants was confirmed by both GUS staining assay and Southern hybridization analysis.
Key words: Agrobacterium, carrot, callus, genetic transformation, regeneration
Introduction
Carrot (Daucus carota L.) is one of the major vegetable crops
produced around the world. According to the annual report from
Food and Agriculture Organization (FAO) of the United Nations,
approximately 20 million metric tons of carrots were produced
worldwide in 2005, with China, Russia and the United States
the top three producing countries (http://www.fao.org). Carrots
represent a major source of vitamin A and fiber for human
nutrition (Simon, 1997; Horvitz et al., 2004). The phytochemicals
in carrots such as β-carotene (provitamin A), lutein, lycopene and
anthocyanins play an important nutritional role in human health
(Seddon et al., 1994). In the past decades, traditional breeding
methods have greatly contributed to the improvement of carrot
traits such as root shape, root color, smooth skin, β-carotene
levels and sugar content (Ammirato, 1986; Simon et al., 1989;
Yau and Simon, 2005). However, genetic transformation can be
used as a complementary technology to improve carrot quality
and productivity (Jayaraj et al., 2007). Although carrot is a model
system for tissue culture studies on somatic embryogenesis, carrot
is not considered as a model plant (like Arabidopsis and tobacco)
for genetic transformation due to its prolonged periods of time
for tissue-culturing and development of regenerated plants. The
process of carrot transformation is lengthy and labour intensive.
Current protocols for carrot transformation still have room for
improvement. A more efficient genetic transformation system is
desirable.
Regeneration is an important part of plant tissue culture. A
suitable regeneration medium to regenerate plants from callus
is critical in performing tissue culture or genetic transformation
(Shin et al., 2000; Šuštar-Vozlič et al., 1999; Shao et al., 2000).
For certain in vitro breeding programs, efficient regeneration is
especially important to obtain a large number of healthy plantlets
for growing or evaluation from calli which are cultured for a
prolonged period (Albert et al., 1995; Winicov, 1996). Studies to
improve plant regeneration from long-term cultured callus, such
as in rice, including modifying medium have been reported (Yin
et al., 1993; Yang et al., 1999).
Although researchers have successfully used MS- (Murashige and
Skoog, 1962) and B5- (Gamborg et al., 1976) based or modified
regeneration media for callus induction and regeneration in carrot
transformation to produce a small scale of transgenic carrots for
the purpose of study under laboratory conditions (Wurtele and
Bulka, 1989; Thomas et al., 1989; Gilbert et al., 1996; Hardegger
and Sturm, 1998; Baranski et al., 2006), it will be useful to
know which of these two regeneration media provide a better
regeneration capacity when a large scale plantlet production
is needed for commercial purpose. In addition, carrot is an
outcross species with severe inbreeding depression and one way
of maintaining a specific trait (especially those traits controlled
by multiple genes) in the progeny is through tissue culture. The
information of callus regeneration ability on existing media is
important. A direct comparison of MS- and B5-based media for
carrot callus regeneration has not been reported, even though
B5 medium has been mentioned as the better medium for carrot
callus induction (Hardegger and Sturm, 1998).
The objective of this study was to evaluate the regeneration
ability of transgenic callus tissues derived from carrot line B493
(Simon et al., 1990) on the media MSIII and B5, as well as on
a mixture of MSIII and B5 (referred to as MSIII+B5). Carrot
inbred line B493 was used for this experiment due to its ability in
4
Increased regeneration ability of transgenic carrot callus on B5-based regeneration medium
callus induction (Simon et al. 1990). The number and size of the
regenerated plantlets on these three different regeneration media
were compared. The presence of the transgene in the regenerated
plants from transformed calli was also characterized by GUS
staining assay and Southern blotting.
Materials and methods
Experimental procedures to obtain transgenic callus: To
investigate the recgeneration ability, putative transgenic B493
callus was used (not the non-transgenic material) because we
thought that this would accurately simulate the conditions
for producing transgenic carrot—including the selection of
Agrobacterium-infected callus on a medium with a selective
agent for several months.
Callus induction from explants: Seeds of carrot inbred line B493
were wrapped in two layers of cheese cloth, and the seed surface
was sterilized by treatment with 70% (v/v) ethanol for 2 minutes
at room temperature, then with 5% (w/v) sodium hypochlorite
(NaOCl) containing 0.02% (w/v) Triton X-100 for 15 minutes.
After sterilization, seeds were rinsed several times with sterile
water from a Milli TM-Q UF Plus Water System (Millipore
Corporation, Bedford, MA, USA) and then placed on solid MS
(Murashige and Skoog salt formulation) medium supplemented
with 3% (w/v) sucrose, 1 μg/mL thiamin (B1), 0.1% (w/v) myoinositol (Sigma, St. Louis, MO, USA) and 1% (w/v) agar (Bactoagar, Detroit, MI, USA) and adjusted to pH 5.8 with 0.5N KOH,
termed as MSIII. The medium was autoclaved at 121oC and 1.2
kgs/cm2 for 15 minutes and then cooled for plating into 100 × 15
mm sterile polystyrene petri-dishes (Fisher Scientific, Pittsburgh,
PA, USA). Each plate contained 6 seeds. Seeds were germinated
on plates and grown under fluorescent light. Callus induction
medium, termed MSI [MSIII medium supplemented with 1 mg/L
2, 4-dichlorophenoxyacetic acid (2, 4-D) (Sigma, St. Louis, MO,
USA) and 21.5 μg/L kinetin (Aldrich, Milwaukee, WI, USA)],
was used for callus induction. Under sterile conditions, roots of
the plantlets were cut into 5 mm lengths and placed on the surface
of the MSI medium. Plates were incubated in the dark at room
temperature for callus induction (Fig. 2A).
Agrobacterium-mediated genetic transformation: Induced
calli were then used for genetic transformation. Carrot callus
transformation method described by Wurtele and Bulka (1989)
was followed. Transformation vector pBI121 (Fig. 1) which
comprises T-DNA right border-Nos promoter-nptII gene-Nos
terminator-35S promoter-gus gene-Nos terminator- T-DNA left
border was transferred into Agrobacterium tumefaciens LBA4404
for plant transformation (Thomas et al., 1989). Agrobacteriuminfected calli were selected on MSI medium supplemented with
300 μg/mL kanamycin + 500 μg/mL cefotaxime in dark (Yau et
al., 2008). Putatively transformed callus clumps (Fig. 2B) were
used for evaluating regeneration on the three regeneration media
described below.
Regeneration medium: MS-based regeneration medium, MSIII,
was the same as that for seed germination and was prepared as
described above. B5-based regeneration medium was prepared
according to the protocol reported by Gamborg et al. (1976). To
obtain MSIII+B5 regeneration medium, equal volumes of MSIII
and B5 liquid media were mixed. 1% (w/v) agar was added to
the liquid medium to solidify the medium.
Regeneration of putatively genetic-transformed callus: Four
putatively transformed calli, 3 mm in diameter and derived from
a single callus, were evenly distributed on each plate. Twenty
plates for each medium were labeled and randomly placed on
iron shelves (60 × 120 cm) with constant fluorescent lighting;
subculturing to the same medium was performed every 3 weeks.
To provide space for continued plantlet growth, regenerated
plantlets reaching 5 mm in length were removed from the callus to
freshly prepared plates for continued growth during subculturing.
The numbers and lengths of plantlets were determined after 6
subcultures, when few new regenerants were observed from
the calli. The length from the stem above the tap root to the tip
of the leaves of each healthy plant was measured as an index
of regeneration ability after 4.5 months of growth. “Healthy”
plants are defined as regenerated plants with green leaves,
normal stems and normal roots. Plants were grouped into five
length categories: 0.5-1, 1-2, 3-4, 5-6 and 7-9 cm. Besides, the
numbers of regenerants after 2-cm dramatically dropped from
MSIII plates or MSIII+B5 plates-some with only 1 or 0 plants.
For each medium, the number of plantlets in each length category
was recorded.
GUS histochemical assay: Histochemical staining of carrot
leaf tissues was performed according to Jefferson et al. (1987).
Leaf tissues were harvested and vacuum-filtered for 10 min in
5-bromo-4-chloro-3-indoxyl-β-D-glucuronide (X-gluc) (Gold
BioTechnology, Inc., St. Louis, MO, USA) staining solution and
then stained overnight at 37°C. To check GUS staining, leaves
were first de-chlorophylled by repeated washing in 70% ethanol
until all the tissue was bleached.
Fig. 1. T-DNA construct of binary vector pBI121 used for Agrobacterium-mediated genetic transformation of carrot
callus. Probe used for Southern hybridization was indicated with a bar (diagram is not drawn to scale).
Increased regeneration ability of transgenic carrot callus on B5-based regeneration medium
Southern hybridization: Southern hybridization was carried out
according to the standard protocol described by Sambrook et al.
(1989). Mature carrot plant leaves were collected, lyophilized for
two days and used for total genomic DNA isolation with 2x CTAB
extraction solution (Murray and Thompson, 1980). Genomic DNA
of 5 μg was digested with BbuI (isoschizomer of SphI) restriction
enzyme overnight at 37°C. Digested DNA was run on a 0.8%
TAE gel and transferred to a membrane for Southern hybridization
according to Yau et. al. (2008). Hybridization was performed
with a nptII gene-derived P32-labelled probe which was produced
according to the protocol described by Yau et al. (2008).
Statistical analysis: To determine whether the regeneration of
the plantlets was influenced by the medium, statistical tests were
performed to compare the number of plantlets on one medium
to the number of plantlets on another. Since the data consisted
of discrete counts, a Poisson distribution (Mendes et al., 1999)
was used to model the number of plantlets produced on a given
medium in a given length category. The following null hypotheses
were tested:
Ho: λB5 = λMSIII;
Ho: λB5 = λMSIII+B5; Ho: λMSIII = λMSIII+B5
where, λB5, λMSIII, and λMSIII+B5 denote the population mean of the
Poisson distribution for each of the three mediums. Each null
hypothesis was tested against the two-sided alternative hypothesis
that the two means were not equal. The three tests mentioned
above were performed for all plantlets (irrespective of length
category). Likewise, the three tests were performed separately to
compare the media for each length category. It bears mentioning
that this test is exact, in the sense that it does not depend upon
large sample size asymptotics to provide accurate P-values. The
probabilities for the P-values were calculated using S-PLUS
(Venables and Ripley, 1997).
5
better medium than MS-based medium, MSIII, or MSIII+B5 for
carrot callus regeneration. B5-based medium was also reported to
be a better callus-inducing medium earlier (Hardegger and Sturm,
1998). Taken together, B5-based medium should be used routinely
in carrot genetic transformation to improve its efficiency. Further
analysis may be required if other genotypes of carrot are utilized
for genetic transformation.
To check the transgenic nature of the regenerated plants, two
plants were randomly chosen for a GUS histochemical assay
and Southern hybridization analysis. For histochmical assay for
GUS activity, the transgenic leaf tissue showed GUS staining with
blue color, while the non-transgenic leaf tissue showed no GUS
staining (Fig. 2D and 2E). For Southern analysis, genomic DNA
of transgenic plants was digested with BbuI and hybridized with
a nptII gene-derived P32-labelled probe. Identical hybridization
banding pattern was observed for the two transgenic plants,
and confirmed that these two lines are siblings which derived
from a single callus (Fig. 2F lanes 1 and 2). The wild type (nontransformed) plant exhibited no hybridization signal (Fig. 2F lane
3). Both results from GUS assay and Southern analysis suggest
that the T-DNA is present in the transgenic lines. Since the plant
lines were derived from the same callus, the signal patterns from
Southern analysis showed the same.
Results and discussion
More regenerated plants (432) were obtained from the calli
regenerating on B5 regeneration medium (Fig. 2C) as compared
to MSIII (24) and MSIII+B5 (28) (Table 1). Each length category
for B5 regeneration medium had 41 or more healthy, regenerated
plantlets. Regenerated plantlets 5.0-6.0 cm long were the most
numerous (175), followed by plantlets with lengths between
3.0-4.0 cm (94 plantlets) (Table 1). In contrast, few carrot
plantlets were regenerated on MSIII or MSIII+B5 regeneration
medium and no plantlets over 7.0 cm were observed. The overall
tests which compared media irrespective of plantlet length
demonstrated that B5 produced significantly more plantlets than
MSIII (P < 0.000001). Likewise, B5 produced significantly more
plantlets than MSIII+B5 (P < 0.000001). There was no significant
difference between MSIII and MSIII+B5 (P-value: 0.678). These
results demonstrated that B5-based regeneration medium is a
Table 1. Total number and length of regenerated plants from B493 calli
on B5, MSIII and MSIII+B5 media
Medium
Length (cm)
Total
0.5-1.0 1.0-2.0 3.0-4.0 5.0-6.0 7.0-9.0 number
B5
41a
70a
94a
175a
52a
432
b
b
b
b
MSIII
16
6
1
1
0b
24
MSIII + B5 12b
8b
8b
0b
0b
28
Within same column, numbers with the same superscript are not
significantly different at P=0.05.
Fig. 2. Regeneration and characherization of carrot plantlets regenerated
from putative Agrobacterium-transformed callus. (A) Soft and yellowish
callus induced from carrot seedling hypocotyls (see the representative
one by an arrow), (B) Putative transformed callus clumps (light color)
emerge from old callus (dark color) under antibiotic kanamycin selection
(see the representative one by an arrow), (C) A regenerated plantlet on
B5 medium, (D-E) GUS staining of non-transgenic control (D) and
transgenic (E) plants, (F) Southern analysis of two transgenic plants
(lanes 1 and 2) and a non-transgenic plant (lane 3).
Acknowledgements
We thank Dr. Phil Simon for providing carrot materials for this
experiment, and Dr. Roger Thilmony and Dr. Ludmila Tyler for
carefully reading the manuscript and giving suggestions. Authors
also wish to thank Landon Sego for statistical analysis. Research
was funded by the Fresh Carrot Board (N328).
6
Increased regeneration ability of transgenic carrot callus on B5-based regeneration medium
References
Albert, H., E.C. Dale, E. Lee and D.W. Ow, 1995. Site-specific integration
of DNA into wild-type and mutant lox sites placed in the plant
genome. Plant J., 7: 649-659.
Ammirato, P.V. 1986. Carrot. In: Handbook of Plant Cell Culture, Volume
4, D.A. Evans, W.R. Sharp and P.V. Ammirato (eds.). Macmillan
Press, New York. p.457-499.
Baranski, R., E. Klocke and G. Schumann, 2006. Green fluorescent
protein as an efficient selection marker for Agrobacterium rhizogenes
mediated carrot transformation. Plant Cell Rep., 25: 190-197.
Gamborg, O.L., T. Murashige, T.A. Thorpe and I.K. Vasil, 1976. Plant
tissue culture media. In vitro, 12: 473-478.
Gilbert, M.O., Y.Y. Zhang and Z.K. Punja, 1996. Introduction and
expression of chitinase encoding genes in carrot following
Agrobacterium-mediated transformation. In Vitro Cell. Dev. Biol.Plant, 32: 171-178.
Hardegger, M. and A. Sturm, 1998. Transformation and regeneration of
carrot (Daucus carota L.). Mol. Breed., 4: 119-127.
Horvitz, M.A., P.W. Simon and S.A. Tanumihardjo, 2004. Lycopene and
β-carotene are bioavailable from lycopene ‘red’ carrots in humans.
Eur. J. Clin. Nutr., 58: 803-811.
Jayaraj, J. and Z.K. Punja, 2007. Combined expression of chitinases
and lipid transfer protein genes in transgenic carrot plants enhances
resistance to foliar fungal pathogens. Plant Cell Rep., 26: 15391546.
Jefferson, R.A., R.A. Kavanagh and M.W. Bevan, 1987. GUS-fusions:
β-glucuronidase as a sensitive and versatile fusion marker in higher
plants. EMBO J., 6: 3901-3907.
Mendes, B.M.J., S.B. Filippi, C.G.B. Demetrio and A.P.M. Rodriguez,
1999. A statistical approach to study the dynamics of micropropagation
rate, using banana (Musa spp.) as an example. Plant Cell Rep., 18:
967-971.
Murashige, T. and F. Skoog, 1962. A revised medium for rapid growth
and bioassays with tobacco tissue culture. Physiologia Plantarum,
15: 473-497.
Murray, M. and W. Thompson, 1980. Rapid isolation of high-molecular
weight plant DNA. Nucleic Acids Res., 8: 4321-4325.
Sambrook, J., E.F. Fristh and T. Maniatis, 1989. Molecular cloning: A
Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory Press,
New York.
Seddon, J.M., U.A. Ajani, R.D. Sperduto, R. Hiller, N. Blair, T.C. Burton,
M.D. Farber, E.S. Gragoudas, J. Haller, D.T. Miller, L.A. Yannuzzi
and W. Willet, 1994. Dietary carotenoids, vitamins A, C and E, and
advanced age-related mascular degeneration. J. Am. Med. Assoc.,
272: 1413-1420.
Shao, C.Y., E. Russinova, A. Iantcheva, A. Atanassov, A. McCormac,
D.F. Chen, M.C. Elliott and A. Slater, 2000. Rapid transformation
and regeneration of alfalfa (Medicago falcata L.) via direct somatic
embryogenesis. Plant Growth Regul., 31: 155-166.
Shin, D.H., J.S. Kim, I.J. Kim, J. Yang, S.K. Oh, G.C. Chung and K.H.
Han, 2000. A shoot regeneration protocol effective on diverse
genotpypes of sunflower (Helianthus annuus L.). In Vitro Cell. Dev.
Biol.-Plant, 36: 273-278.
Simon, P.W. 1997. Plant pigments for color and nutrition. HortScience,
32: 12-13.
Simon, P.W., C.E. Peterson and W.H. Gabelman, 1990. B493 and B9304,
carrot inbreds for use in breeding, genetics, and tissue culture.
HortScience, 25: 815.
Simon, P.W., X.Y. Wolff, C.E. Peterson, D.S. Kammerlohr, V.E.
Rubatzky, J.O. Strandberg, M.J. Bassett and J.M. White, 1989. High
carotene mass carrot population. HortScience, 24: 174-175.
Šuštar-Vozlič, J., B. Javornik and B. Bohanec, 1999. Studies of
somaclonal variation in hop (Humulus lupulus L.). Phyton (Austria),
39: 283-287.
Thomas, J.C., M.J. Guiltinan, S. Bustos, T. Thomas and C. Nessler,
1989. Carrot (Daucus carota L.) hypocotyl transformation using
Agrobacterium tumefaciens. Plant Cell Rep., 8: 354-357.
Venables, W.N. and B.D. Ripley, 1997. Modern Applied Statistics with
S-PLUS. Second Edition, Springer.
Winicov, I. 1996. Characterization of rice (Oryza sativa L.) plants
regenerated from salt-tolerant cell lines. Plant Sci., 113: 105-111.
Wurtele, E. and K. Bulka, 1989. A simple, efficient method for the
Agrobacterium-mediated transformation of carrot callus cells. Plant
Sci., 61: 253-262.
Yang, Y.S., Y.D. Zheng, Y.L. Chen and Y.Y. Jian, 1999. Improvement of
plant regeneration from long-term cultured calluses of Taipei 309,
a model rice variety in in vitro studies. Plant Cell Tiss. Org. Cult.,
57: 199-206.
Yau, Y.Y., S.J. Davis, A. Ipek and P.W. Simon, 2008. Early identification
of stable transformation events by combined use of antibiotic
selection and vital detection of green fluorescent protein (GFP) in
carrot (Daucus carota L.) callus. Agricultural Sciences in China,
7: 664-671.
Yau, Y.Y. and P.W. Simon, 2005. Molecular tagging and selection for
sugar type in carrot roots using co-dominant, PCR-based markers.
Mol Breed., 16: 1-10.
Yin, Y., S. Li, Y. Chen, H. Guo, W. Tian, Y. Chen and L. Li, 1993. Fertile
plants regenerated from suspension culture-derived protoplasts of
an indica type rice (Oryza sativa L.). Plant Cell Tiss. Org. Cult.,
32: 61-68.
Received: November, 2011; Revised: December, 2011; Accepted: January, 2012