In Vitro Cellular & Developmental Biology - Plant (2025) 61:672–679
[Link]
BIOTECHNOLOGY
Floral and apical meristems as a target for Agrobacterium‑mediated
transformation of Arabidopsis thaliana
Vladimir Sidorov1,2 · Peizhen Yang1
Received: 14 November 2024 / Accepted: 13 January 2025 / Published online: 31 July 2025 / Editor: Todd Jones
© The Author(s) 2025
Abstract
The majority of transformation protocols for Arabidopsis thaliana are based on Agrobacterium-mediated transformation of
female reproductive cells in vivo, or somatic cells of leaf, stem, hypocotyl, or root explants in vitro. Here, Agrobacterium-
mediated transformation systems using in vitro meristematic floral buds or apical meristems from precultured seedlings
are reported. Arabidopsis floral cultures were established from inflorescences of seed-derived plants grown in vitro. On
cytokinin-containing medium, chopped inflorescence segments quickly produced fast-growing cultures consisting mainly
of real de novo inflorescences. Cytokinin-containing medium was also used for 1-wk preculture of seedlings for induction
of multiple meristems from shoot apical buds. NPTII and GUS were used as selectable and visual markers for transforma-
tion of in vitro floral cultures, and aadA and RUBY were used for transformation of precultured seedlings. Transformation
frequency for floral culture was about 15% (calculated as the number of independent transformants per initial number of
explants). Transformation frequency for precultured seedlings using RUBY was roughly 12% (calculated as the number of
RUBY positive plants per number of seedlings inoculated). Molecular and phenotypic analysis of T 1 progeny confirmed that
the transgenes were stably integrated and inherited in progeny.
Keywords Arabidopsis · Agrobacterium-mediated transformation · Meristem as target
Introduction reproductive cells in vivo followed by pollination and forma-
tion of transgenic seeds. Leaf disks, stem and hypocotyl seg-
Arabidopsis thaliana is a popular model organism from the ments (Lloyd et al. 1986; Sheikholeslam and Weeks 1987),
Brassicaceae family. For many years, it has played an impor- and root explants (Vaivekens et al. 1988; Koncz et al. 1990;
tant role in studying the genetics, epigenetics, physiology, Marton and Browse 1991; Huang and Ma 1992; Gelvin
metabolism, cell biology, and molecular biology of plant 2006; Buck et al. 2009) have been used as target tissues for
traits (Woodward and Bartel 2018). It has a short life cycle transformation. The Agrobacterium-mediated transformation
(5 to 6 wk) and one of the smallest genomes in the plant protocols for these explants typically require from 8 wk to
kingdom (Kramer 2015; Kimball 2022). Arabidopsis was 4 mo and include time for seed germination and explant
the first plant to have its genome sequenced (Arabidopsis preparation, Agrobacterium inoculation, and regeneration of
Genome Initiative 2000; Somerville and Koornneef 2002; transgenic T0 plants. After development of efficient Arabi-
Koornneef and Meinke 2010). Many different transforma- dopsis protoplast techniques (Negrutiu et al. 1975; Sidorov
tion protocols have been developed for Arabidopsis and can et al. 1978; Datum and Willmitzer 1988; Wenck and Marton
be categorized into two types: (a) transformation of somatic 1995; Dovzhenko et al. 2003; Jeong et al. 2021), it became
cells (and protoplasts) and regeneration through organo- feasible to use Arabidopsis protoplasts for transformation
genesis or embryogenesis, (b) transformation of female (Mathur et al. 1995; Mathur and Koncz 1998; Yoo et al.
2007; Jiang et al. 2021).
The floral dip method revolutionized Arabidopsis trans-
* Vladimir Sidorov formation (Clough and Bent 1998; Chung et al. 2000; Bent
vladimirsdrv@[Link] 2006; Harrison et al. 2006; Tague and Mantis 2006; Zhang
1
Bayer Crop Science AG, St. Louis, MO, USA et al. 2006). The primary transformation target in the floral
2 dip method is the female reproductive organ (Ye et al. 1999;
Chesterfield, USA
Vol:.(1234567890)
AGROBACTERIUM TRANSFORMATION FLORAL APICAL MERISTEM ARABIDOPSIS THALIANA 673
Bechtold et al. 2000; Desfeux et al. 2000). Videos with dif- the need for induction with acetosyringone prior to inocu-
ferent modifications of this method are available on You- lation. AB32 also carries a pVS1-based Ti binary vector
Tube ([Link] (Itoh et al. 1984) that has both nptII and uidA (GUS) cas-
[Link] This settes in its T-DNA region. The nptII gene was driven by the
method allows efficient genetic transformation of Arabi- CaMV 35S promoter, and uidA by an enhanced CaMV 35S
dopsis thaliana without any plant tissue culture, and with promoter. For transformation of apical meristems, a binary
transgenic seed produced within about 3.5 mo of Agrobac- vector containing Ruby and aadA expression cassettes was
terium infection. used. Ruby was driven by the dahlia mosaic virus (DaMV)
Recently, Agrobacterium-mediated transformation of promoter (Sahoo et al. 2014), and aadA was driven by the
vegetative (apical or axillary) and floral meristems has been promoter from the Arabidopsis actin 7 gene located on chro-
demonstrated for quinoa (Sidorov et al. 2024). Transforma- mosome 5. Original expression cassette with the Ruby gene
tion of this starting material, like floral dip transformation, was made by He et al. (2020).
allows the production of transgenic plants without an inter- For the preparation of Agrobacterium, 0.5 mL of glycerol
vening callus stage. stock was inoculated into 200 mL of Luria–Bertani (LB)
Here, we report the development of similar Agrobacte- broth (Lennox) (MilliporeSigma®, L3022) medium with
rium-mediated transformation systems for Arabidopsis floral 50.0 mg L−1 spectinomycin (Spec) and 30 mg L−1 genta-
and shoot apical meristems. These protocols were devel- mycin (Gent) and cultured on a shaker at 200 rpm at 28°C
oped not to replace the widely used floral dip method, but for 20–22 h. The Agrobacterium culture was subsequently
as alternative systems that are simple, efficient, eco-friendly centrifuged at 3000 g at 4°C for 30 min. The pelleted Agro-
(no contamination of environment with Agrobacterium), and bacterium was re-suspended in inoculation medium, and the
greenhouse independent. density was adjusted to 0.8 to 1.0 at OD660. The inoculation
medium contained 2.16 g L −1 Murashige and Skoog (MS;
Murashige and Skoog 1962) basal salt mixture (PhytoTech
Materials and Methods Labs, M524), 1 mL/L MS vitamins 1000 × (PhytoTech Labs,
M553), 68.5 g L−1 sucrose, 36.0 g L−1 glucose, 0.115 g L−1
Initial Material Seeds of Arabidopsis thaliana (L.) Heynh L-proline, and 200.0 μM acetosyringone.
Columbia-0 were used. They were surface sterilized with For floral culture transformation, approximately 200 floral
10.0% sodium hypochlorite for 20 min. After thoroughly culture clumps (approximately 140 g) were transferred to
rinsing with sterile water, seeds were placed into PlantCon™ 250-mL Corning® bottles with 150 mL of Agrobacterium
containers (MP Biomedicals, Solon, OH) and cultivated on suspended in inoculation medium. Bottles were placed in
Driver and Kuniyuki Walnut (DKW) medium (Driver and a vacuum desiccator and vacuum infiltrated for 5 min after
Kuniyuki 1984; PhytoTech Labs®, Lenexa, KS; D2470) bubbling was first observed. Floral culture clumps with
with 20.0 g L −1 sucrose, pH 5.7 (DKW #1), at 25°C 80 μE Agrobacterium suspension were then transferred to 50-mL
−2 −1
m s light intensity, and 16-h day length. tubes and centrifuged for 30 min at 1300 rpm (Eppendorf™
For induction and propagation of in vitro floral cultures, Centrifuge 5810 R). The centrifuged samples were then
inflorescences from in vitro grown plants were chopped poured through a 100-mm stainless steel mesh strainer (0.4
and placed on DKW medium with 20.0 g L −1 sucrose, to 1 mm mesh size; Oneida, New York, NY) and the sepa-
−1 −1
1.0 mg L kinetin (Kin), 0.5 mg L 6-benzylaminopurine rated explants were blotted onto sterile paper towels for 3
(BA), 0.1 mg L −1 gibberellic acid ( GA3), 3.5 g L −1 aga- to 5 min. Lastly, separated and dried tissues were spread on
rose (MilliporeSigma®, Burlington, MA; A6013), pH 5.7 a sterile 8.2-mm Whatman® filter paper (Cat #1001–082),
(DKW #2). All plant growth regulators (PGRs), including prewetted with 0.5 mL of inoculation medium in Petri dishes
α-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic (25 × 85 mm), and co-cultured in a Percival (Percival Sci-
acid (2,4-D), indole-3-butyric acid (IBA), and ancymidol entific Inc., Perry, IA) for 4–5 d under 10 μE m−2 s−1 light
(Ancym), were obtained from PhytoTech Labs. For induc- at 21°C and 65% relative humidity. After co-culture, all flo-
tion of multiple bud cultures from apical meristems, steri- ral culture explants were transferred to semisolid DKW #2
lized seeds were placed into 250-mL Corning® flasks (Corn- selection medium (DKW #1), supplemented with 50.0 mg
ing, NY) with vent caps containing 50 mL liquid DKW #2 L−1 paromomycin (Par), 750.0 mg L −1 carbenicillin (Carb)
medium and cultured on a rotary shaker (20 rpm) at 25°C, (PhytoTech Labs, C346), and 300.0 mg L−1 cefotaxime
30 μE m−2 s−1 light intensity, and 16-h daylength. (Cef) (PhytoTech Labs, C380) and incubated in the growth
chamber set to 25°C, 80 μE m −2 s−1 light intensity and a
Transformation Agrobacterium tumefaciens strain AB32 16 h-day length. The transformed explants were sub-cultured
was used for all transformation experiments. AB32 has a in Petri plates every 12 to 14 d on the same medium but
constitutively active VirG (Ye et al. 2016), which eliminates
674 V. Sidorov and P. Yang
with reduced concentration of Carb (500.0 mg L −1) and Cef at 37°C in X-Gluc, the plant material was de-stained in 70%
−1
(200.0 mg L ). v/v ethanol and examined for GUS expression. The presence
For transformation with the Ruby construct, we used of transgenes was also determined by TAQMAN PCR proce-
seedlings precultured for 7 d on medium inducing formation dure according to the manufacturer’s instructions (Applied
of multiple apical buds (DKW #2). Seedlings were collected Biosystems, Foster City, CA). Chi-square tests were used
from liquid medium and crushed in a blender as described to analyze observed versus expected transgene segregation
previously by Sidorov et al. (2022). Briefly, 7-d-old seed- of T1 seeds.
lings were blended with 30 mL of DKW #2 medium for 5 to
8 s at high speed (approximately 20,000 rpm). The blended
material was then sieved through a commercial strainer Results and Discussion
with 0.4 to 1 mm mesh size (Oneida) washed with DKW #2
medium, pat dried on sterile paper towels, and transferred Initiation and Maintenance of Floral Cultures Sterilized
to a 50-mL tube. Blended seedlings were transformed with seeds of Arabidopsis were sown on DKW #1 medium with-
Agrobacterium similar to floral culture, described above. out PGRs and incubated, as described above, at long-day
After co-culture for 4 to 5 d under the same conditions as (16 h) illumination. Under these conditions, seeds were ger-
for floral cultures, the material was transferred to DKW #2 minated and in 1 mo gave rise to flowering plants. Plants
medium supplemented with 25.0 mg L −1 Spec, 750.0 mg had an indeterminate, raceme-type inflorescence and usu-
−1 −1
L Carb, and 300 mg L Cef and incubated in a growth ally classified as a long-day plant. DKW-based medium was
chamber set to 25°C, 80 μE m −2 s−1 light intensity, and a mainly used, and MS-based medium gave similar results.
16-h day length. Then after 2 wk of selection, explants were The conversion of shoot apical meristem to inflorescence
transferred into Petri plates with the same medium but with meristem has been described in detail previously (Bertero
reduced concentrations of Carb (500.0 mg L −1) and Cef et al. 1996; Hake and Wilt 2003; Gaarslev et al. 2021). A
−1
(200.0 mg L ). flower can be considered as a modified shoot wherein the
shoot apical meristem changes into a floral meristem (Taiz
Protoplast Isolation and Culture The protocol for protoplast et al. 2015). The similar nature of apical meristem and floral
isolation was the same as described previously (Sidorov et buds, which are also highly meristematic, highlights one
al. 2022) except the enzyme composition, which was modi- of the difficulties of working with floral cultures—specifi-
fied to contain 2.0% Cellulase RS, 2.0% Cellulysin, 0.5% cally, transformation of highly organized meristematic tis-
Driselase, and 0.5% Macerozyme R-10 (all from Millipore- sue requires a prolonged selection period to obtain com-
Sigma®). Plant material was incubated for 3 h at 28°C. After plete sorting out of non-transformed tissue from transformed
protoplast isolation and washing, protoplasts were embedded tissue.
into a thin alginate layer and cultured in Kao and Michyluk For induction and propagation of in vitro floral cultures,
(KM) modified medium (see Sidorov et al. 2022) containing inflorescences from in vitro grown plants were chopped
0.2 mg L−1 NAA, 0.2 mg L−1 BA, and 1.0 mg L−1 2,4-D. and placed on DKW #2. Floral cultures propagated in Petri
dishes grew rapidly and were sub-cultured every 12 to 14
Analysis of Plant Material and Statistics Plant explants and d (Fig. 1A). To obtain more compact inflorescences, GA3
seedlings were stained with X-Gluc according to the pub- was removed from the medium and DKW #2 medium with
lished protocol (Jefferson 1989). After overnight incubation 2.0 mg L−1 ancymidol (Ancym), which is an inhibitor of
Figure 1. In vitro floral culture of Arabidopsis and its propagation on rose; pH 5.7. (B) DKW medium with 20.0 g L−1 sucrose, 1.0 mg L−1
different media. (A) 8.5-cm Petri dish with floral culture on Driver Kin, 0.5 mg L−1 BA, and 3.5 g L−1 agarose; pH 5.7. (C, D) DKW
and Kuniyuki Walnut (DKW) medium with 20.0 g L −1 sucrose, medium with 20.0 g L −1 sucrose, 1 mg L −1 Kin, 0.5 mg L −1 BA,
1.0 mg L−1 Kin, 0.5 mg L −1 BA, 0.1 mg L −1 GA3, and 3.5 g L
−1 aga- 2.0 mg L−1 ancymidol, and 3.5 g L−1agarose; pH 5.7. Bar = 4 mm.
AGROBACTERIUM TRANSFORMATION FLORAL APICAL MERISTEM ARABIDOPSIS THALIANA 675
GA3 biosynthesis, was used (Fig. 1B, C, D). Arabidopsis of floral organs has been demonstrated in vivo in different
plants on such medium look stunted, compact, and pheno- mutants (Okamuro et al. 1993; Ung et al. 2011). Multiple
typically similar to Landsberg erecta ecotype (Rédei 1992). pistil cultures on DKW #2 medium or on the same medium
This medium was periodically used for maintenance of floral without GA3 formed normal inflorescences. Pistil cultures
culture. Arabidopsis floral culture to some extent was differ- were used for Agrobacterium-mediated transformation. GUS
ent from floral culture of quinoa, described earlier (Sidorov expression in stably transformed pistil cultures is shown in
et al. 2024). Floral cultures of Arabidopsis consisted entirely Fig. 3D. Since this culture is differentiated and maintained
of inflorescences, while quinoa floral cultures mainly con- on medium without 2,4-D, relatively little if any somaclonal
tained flowers at different stages of development. variation is anticipated.
On the medium for propagation, it was possible to iden-
tify abnormal development of pistils. Distinct stages of Transformation of Floral Cultures and Precultured Seed‑
abnormal pistil development are presented in Fig. 2A, B, C. lings In each treatment, approximately 100 floral cultured
Media used for propagation could also induce shoot regen- clumps were inoculated with Agrobacterium (AB32 carry-
eration from different parts of the inflorescence (Fig. 2D, E). ing pVS1 binary plasmid with nptII and uidA markers). The
The most interesting observation is that media DKW #2 and essential steps of transformation were the vacuum infiltration
DKW #2 without GA3 also induced multiple pistil formation of explants in Agrobacterium-solution and centrifugation.
(Fig. 3A, B, C). In vitro pistil culture is a unique phenom- For selection of transformants, we used the same medium
enon not described before, although abnormal development as for propagation, DKW #2 medium supplemented with
Figure 2. In vitro pistil development and regeneration from various L−1 BA, and 0.1 mg L−1 GA3. Bar = 2.5 mm. (D, E) Regeneration of
parts of Arabidopsis inflorescence. (A, B, C) Abnormal growth of shoots. Bar = 6 mm. (F) Diagram of Arabidopsis flower showing pis-
pistils on DKW medium supplemented with 1.0 mg L −1 Kin, 0.5 mg til.
Figure 3. Establishment, propagation, and transformation of Arabi- plemented with 1.0 mg L −1 Kin, 0.5 mg L −1 BA, and 0.1 mg L−1
dopsis in vitro pistil culture with NPTII/GUS construct. (A, B, A3; 8.5-cm plate. (D) Stable GUS expression in transformed pistil
G
C) Establishment of in vitro pistil culture. (A) Bar = 2.5 mm. (B) culture after 25 d of selection. Bar = 3 mm.
Bar = 4 mm. (C) Propagation of pistil culture on DKW medium sup-
676 V. Sidorov and P. Yang
50.0 mg L−1 Par. Selection was applied without delay and the medium used for selection (DKW #2, 25.0 mg L −1
−1 −1
was stringent enough to cause bleaching and prevent growth Spec, 500.0 mg L Carb, 200.0 mg L Cef), very fast
of non-transformed tissue. Transient and stable GUS expres- shoot germination from meristems was observed. In pre-
sion shows that the target for transformation was flowers and liminary experiments, it is also possible to use PGR-free
different parts of the inflorescence (Fig. 4A, B); however, medium; however, the number of germinated shoots was
we were mainly interested in isolating transformants from smaller. In an experiment with approximately 150 seeds
meristematic tissue of inflorescences. During each sub-cul- on DKW #2 selection medium, 16 pink plants, 2 green
ture, only the green viable tissue was transferred to a new plants with pink flowers, and 6 green plants (Fig. 5D, E)
selection medium. Approximately 45 d after transformation, were obtained. Eighteen plants expressing Ruby were ana-
clumps of what appeared as non-chimeric tissue were clearly lyzed by TAQMAN PCR (Table 1). All of them except one,
visible on the selection medium (Fig. 4C); however, after analysis of which was failed, were aadA positive, and 80%
staining with X-Gluc, several of these clumps were still chi- of events had single aadA copy.
meric. Regeneration of chimeric plants is always a concern Time frame for production of transgenics with the
in meristem explant-based transformation systems (Ye et al. described method can be considerably short in comparison
2022, 2023). To address these concerns, extended selection to the floral dip method. Especially Ruby expression allowed
time (up to 3 mo) was used. After the extended selection to identify the transformant very early. The described meth-
period, 24 lines resistant to paromomycin were isolated. ods based on using floral culture and shoot apical meristem
TAQMAN PCR analysis showed that out of 24 selected also have some important benefits over the widely used floral
lines, 20 lines were nptII positive and 4 were negative, 12 dip method. In floral dip, transgenic T1 progeny are hemizy-
lines had 1 copy of nptII, and 7 lines had 2 copies of nptII gous, and must be grown to the T 2 generation to produce
(Table 1). All nptII positive lines were also stained with homozygous progeny. With currently recommended meth-
X-Gluc and were identified as GUS positive (Fig. 4D, E). ods, some homozygous progeny are produced immediately
In the current work with germinated seed transfor- in the T1 generation.
mation, seeds after sterilization were precultured in liq-
uid medium (Fig. 5A); seedlings were blended and then Production of Seeds and Analysis of T1 Progeny Seeds from
transformed. The target for transformation was the shoot transgenic inflorescences were produced in vitro on DKW
apical meristem, however, small pieces of seedlings were #1 medium supplemented with 2.0 mg L −1 IBA. On this
also transformed and initiated pink callus (Fig. 5B). On medium, the inflorescences produced normal flowers, which
Figure 4. Transformation and selection of Par-resistant inflores- (C) 1-mo selection on medium with 50.0 mg L−1 Par. (D, E) 2-mo
cence shoots of Arabidopsis. Transient and stable GUS expression in selection; transformed and non-transformed, bleached shoots. (D)
plant material during selection. (A) 1-d culture after transformation. Bar = 10 mm. (E) Bar = 3.4 mm.
Bar = 4.2 mm. (B) 12-d culture after transformation. Bar = 3 mm.
Table 1 TAQMAN PCR Selectable/ Selective agent Total # events # nptII # events with # aadA # events with
analysis of Arabidopsis events visual mark- positive 1 nptII copy positive 1 aadA copy
obtained after transformation ers events events
with constructs containing
nptII/GUS and aadA/Ruby as nptII/GUS Paromomycin 24 20 12
selectable and visual markers
aadA/Ruby Spectinomycin 18 18 14
AGROBACTERIUM TRANSFORMATION FLORAL APICAL MERISTEM ARABIDOPSIS THALIANA 677
Figure 5. Application of the RUBY visual marker in Arabidopsis C) 19-d culture after transformation of blended seedlings and selec-
transformation. (A) Germination (1 wk) Arabidopsis seeds in vented tion on DKW medium, 1.0 mg L −1 Kin, 0.5 mg L
−1 BA, and 0.1 mg
flask with liquid DKW medium, supplemented with 1.0 mg L−1 Kin, −1
L GA3 and supplemented with 25.0 mg L −1
Spec. (C) Germination
0.5 mg L−1 BA, and 0.1 mg L −1 GA3. Single seedling with shoot api- of Ruby shoots. Bar = 2.5 mm. (D, E) Selected flowering plants on 35
cal meristem after 7-d culture presented in insertion. Bar = 4 mm. (B, d after transformation. Bar = 10 mm.
Figure 6. In vitro seed production and analysis of different trans- Seed germination of lines #4 and #12 on DKW PGR-free medium
genic lines of Arabidopsis. (A) Induction and maturation seeds in with 50.0 mg L −1 Par; arrows indicate Par-sensitive seedlings.
vitro on DKW medium supplemented with 2.0 mg L −1 IBA. (B, C) Bar = 17 mm. (D) GUS expression in seedlings of line #12.
were spontaneously self-pollinated and produced siliques two failed to germinate. Similar results were obtained with
full of seeds (Fig. 6A). Culture conditions for seed pro- event #12. Segregation of transgenes in the T 1 progeny for
duction were the same as for growing plants (25°C, 80 μE events 4 and 12 was statistically analyzed using a chi-square
m−2 s−1 light intensity, 16-h day length). test, which confirmed a statistical fit to a 3:1 segregation
Interestingly, seeds could not be obtained on DKW #1 ratio as expected for self-pollinated progeny of a single copy
(PGR-free) medium. Seeds of Arabidopsis produced in vitro T-DNA insertion. Production of seeds from RUBY transgen-
were germinated on screening medium. No refrigeration ics was more difficult. On DKW #1 medium with 2 mg L −1
was required for seed germination (there appeared to be no IBA, pink inflorescences were weak and produced siliques
dormancy period). Seeds produced in vitro were placed on without normal seeds. Medium used for seed production also
DKW #1 medium with 50.0 mg L−1 Par for germination and usually induced multiple roots. So, plants could be trans-
analyzed for segregation in progeny (Fig. 6B, C). planted to soil for production of seeds in vivo. Unfortunately,
Analysis of T 1 progeny from two initial proof-of-concept it was not checked. Earlier, He et al. (2020) also could not
(POC) events (#4 and #12), which had single copy for nptII show seed set in pink Arabidopsis plants, expressing Ruby
confirmed normal Mendelian inheritance of transgenes in in leaves and stem, growing in soil but they demonstrated
progeny. Green seedlings germinated on PGR-free medium production of clearly pink seeds in green normal-looking
with 50.0 mg L−1 Par were classified as resistant ( NPTII+), Ruby positive plants transformed with seed-specific pro-
while yellow, smaller seedlings were designated as Par- moter At2S3.
sensitive (NPTII−). As shown in Fig. 6D, most seedlings
were GUS positive. In event #4, out of 140 seeds tested, 105 Protoplast Isolation and Culture Different Arabidopsis
seedlings were green ( NPT+), 33 were yellow ( NPTII−), and explants have been used for protoplast isolation, culture,
678 V. Sidorov and P. Yang
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