Plant Cell Tiss Organ Cult
DOI 10.1007/s11240-016-1087-1

ORIGINAL ARTICLE

An approach to overcoming regeneration recalcitrance in genetic
transformation of lupins and other legumes
An Hoai Nguyen1,2,3 · Leon M. Hodgson1,4 · William Erskine1,5 · Susan J. Barker2,5

Received: 25 July 2016 / Accepted: 2 September 2016
© Springer Science+Business Media Dordrecht 2016

Abstract  For pulse legume research to fully capitalise on developments in plant molecular genetics, a
high throughput genetic transformation methodology
is required. I n Western Australia the dominant grain
legume is Lupinus angustifolius L. (narrow leafed lupin;
NLL). Standard transformation methodology utilising
Agrobacterium tumefaciens on wounded NLL seedling
shoot apices, in combination with two different herbicide selections (phosphinothricin and glyphosate) is time
consuming, inefficient, and produces chimeric shoots
that often fail to yield transgenic progeny. Investigation
of hygromycin as an alternative selection in combination
with expression of green fluorescent protein indicated
that transformation of NLL apical cells was not the rate
limiting step to achieve transgenic shoot materials. I n
this research it was identified that despite ready transformation, apical cells were not competent to regenerate. However a deep and broad wounding procedure to
expose underlying axillary shoot and vascular cells to


Susan J. Barker







Agrobacterium, in combination with delayed selection
proved successful, increasing initial explants transformation efficiency up to 75 % and generating axillary shoots
with significant transgenic content. Based on knowledge
gained from studies of plant chimeras, further subculture
of these initial axillary shoots will result in development
of low chimeric transgenic materials with heritable content. Furthermore, the method was also tested successfully on other Lupinus species, faba bea and field pea.
These results demonstrate that development of a high
yielding transformation methodology for pulse legume
crops is achievable.
Keywords  Narrow leafed lupin ·
Lupinus angustifolius legume transformation ·
Regeneration · Agrobacterium tumefaciens · Green
fluorescent protein · Shoot axillary bud transformation ·
Mericlinal and periclinal chimera · Delayed selection
methodology
Abbreviations
CcCo-cultivation medium
CZCentral zone
eGFPEnhanced green fluorescent protein
GMGenetic manipulation;
MPHMicropropagation medium with hygromycin
NLLNarrow-leaf lupin
RgRegeneration medium
PPTPhosphinothricin
PZPeripheral zone

and upgraded pod set along with grain yield (Atkins et al.
2011). The basic principle of this method is to mechanically pre-wound the seedling shoot apical meristem (SAM)
to enhance subsequent transformation by Agrobacterium
tumefaciens. The method of Pigeaire et al. (1997) involves
excision of germinated seedling hypocotyls followed by
stabbing the dome several times with a fine needle, adding
a drop of Agrobacterium tumefaciens strain AgL0 to the
damaged surface, then incubation of these explants on agarbased culture media. Transgenic shoots regenerate directly
from transformed totipotent cells existing in the original
explants and are propagated through numerous weeks of
selection and transfer to optimise the proportion of transgenic materials by use of the selectable marker bar gene that
confers tolerance to the herbicide phosphinothricin. This
method has also been successfully applied to yellow lupin
(L. luteus L.; Li et al. 2000) and in our laboratory to other
pulses such as field pea (Pisum sativum L.), faba bean (Vicia
faba L.), chickpea (Cicer arietinum L.) and lentil (Lens culinaris Medik.) (unpublished results). However, as with other
methodologies for different pulses, this NLL transformation
methodology is time-consuming and inefficient. Despite the
lengthy micropropagation regime, the derived shoots are
chimeric, survival of these shoots in the selection process is
of low frequency, and transgene transfer to progeny is less,
resulting in an overall transformation frequency of less than

13

Plant Cell Tiss Organ Cult

one percent in the current NLL cultivar (Wijayanto et al.
2009; Nguyen et al. 2016; Barker et al. unpublished results).
The difficulty with NLL transformation led to examination of alternative selection methodologies. Glyphosate

for plant transformation and spectinomycin/streptomycin
resistance (Sm/SpR) for bacterial transformation (Karami
et al. 2009; Nguyen et al. 2016). To prepare the A tumefaciens for transformation, a fresh plate culture was grown
from − 80 °C glycerol stock storage. An overnight liquid
culture was prepared from a single colony, that was diluted
1/10 on the morning of the transformation and grown with


Plant Cell Tiss Organ Cult

agitation until reaching the optimal biomass (optical density
at 550 nm of 0.4–0.8).
Plant material
Growth media were prepared as described by Barker et al.
(2016) except for hygromycin steps which followed Nguyen
et al. (2016). Mature seeds of NLL, cultivar Mandelup, were
surface sterilized, germinated in the dark in a growth room
2–3 days and excised to remove the cotyledons and young
leaves. For early development in normal shoots analysis, the
seedlings were cultivated in co-cultivation (Cc) medium,
consisting of 1X MS salts, 3 % (w/v) sucrose, pH 5.7, 0.3 %
(w/v) Phytagel (Sigma), autoclaved, then added on cooling:
1X B5 vitamins, 10.0 mg L−1 BAP, 1.0 mg L−1 NAA For
transformation shoot developmental analysis, after the seed
coat was removed from the shoot axis, leaf primodia present in the plumule were removed to reveal the apical dome
using a Leica stereo-microscope. The apical dome area was
wounded by the following methods:
SAM wounding only: The NLL SAM was stabbed
with a fine needle 10–12 times following Pigeaire et al.
(1997) and further observations of Wijayanto (Nguyen et

hygromycin 1 mg mL−1 to the apical dome of transformed
explants was trialled based on previous results (Nguyen et
al. 2016), on days 4, 10, 13, 16 and 18 post-transformation.
Numbers of surviving explants were recorded 1 week after
droplet treatment.
Plant tissue fixation, sectioning and imaging
The apical dome was excised from the collected
explants,submerged in 30 % sucrose solution and embedded into optimum cutting temperature (OCT) compound
(TISSUE-TEK®) and frozen at −20 °C in a CM3050 S
Cryostat (Leica) (Tirichine et al. 2009). The frozen block
with the sample was trimmed, cross and longitudinal sections were taken until the region of interest was reached.
Sections (20–40 µm) containing the intact plant material
were placed onto adhesive glass slides (Fischer et al. 2008).
The sections were stained with 10 % toluidine blue for
Olympus BH2 microscopy or 0.1 % Fluorescent Brightener
28 (Calcofluor White) for Nikon A1Si Confocal microscopy
visualization (Yeung et al. 2015).

Sub-culture media and selection protocol

GFP imaging and analysing

Transformed explants were cultured in Cc media 2 days
in dark conditions, then 2 days under normal light conditions (Fluorescent cool white PAR: 100–170 μmol m−2 s−1).
The explant was washed in 100 mg mL−1 Timentin® and
transferred to new Cc media (Cc 2) adding 150 mg L−1
Timentin® to eliminate further growth of Agrobacterium in
the shoots. Two weeks after co-cultivation, the transformed
seedlings were moved to regeneration media (Rg). This
medium contains the same components as Cc2 medium


organized to form a typical tunica and corpus (Fig. 1). The
tunica in NLL is functionally two-layered: protoderm or
primitive epidermal layer (L1) and subepidermal layer (L2).
Figure  1 also shows concordance with the cytohistological zone concept that the shoot apex is organized into three
distinct zones of differentiation and function: central zone
(CZ); peripheral zone (PZ); rib zone (RZ).

NLL shoot apical meristem

Development of wounded meristem shoots

Analysis of sections from NLL shoots 2–3 days after germination showed that the anatomical structure of the shoot
apex comprises 20–25 cell layers in a cone shape (Fig. 1a, b).
This structure initially provides precursors for a primary
shoot that later develops side shoots and the reproductive organs. Histology revealed that cells of the NLL were

The hypothesis that wounded apical meristem has capability to rebuild itself is the basis for the approach taken in
previous studies, with the idea that the interference in meristem integrity by stabbing will activate new groups of stem
cells to produce shoots. This method therefore aimed only
to wound the meristem area without significant damage,

Fig. 1  Shoot apical meristem (SAM) of narrow leafed lupin (NLL).
a Longitudinal section of NLL SAM stained with Calcofluor White,
captured by Nikon A1Si confocal microscopy. Bar 100 µm. CZ central zone, PZ peripheral zone, RZ rib zone, LP leaf primordia. b–e are
stained with Toluidine blue, captured by Olympus microscopy. b Longitudinal section of NLL SAM. Bar 20 µm. L1 layer one, L2 layer

two, white arrows cells of L1, yellow arrows cells of L2, red arrows
direction of development of meristem cell derivatives. c Longitudinal
section of NLL SAM. Bar 100 µm. Red circle dashed lines show the

by the transformed explant were apparently generated from
unwounded area or cells at the base or side of a deeper
wound. It appeared that the dominance of the SAM was disabled by the wounding procedure, releasing axillary meristem cells to activate shoot development.
Chimerism in transgenic shoots, selection methodology
and enhanced explant survival

Fig. 2  Shoot wounding method. a, c, e Original (shallow) stabbing.
b, d, f Broad and deeper wounding method. a, b Germinated seedling
with plumule excised to expose the SAM at D0. Black arrows show the
zone that was targeted in the original method. bBlack and white arrows
show the zone to target. c, d Transgenic explant after 7 days (D7)
showing where stabbing has occurred in the two methods. Arrows as
for a and b. e, f Longitudinal section of NLL germinated seedling with
plumule excised at D0, after wounding has occurred; e has undergone
the original stabbing and has some shallow damage to the SAM; f has
undergone the broad and deep wounding method. Scale bar 500 µm

The second aim of this research was to determine the
genetic structure of shoots that developed following NLL
transformation in order to develop an approach to reduce
the chimerism that has been apparent in the outcomes of
the current method. Observation of longitudinal and cross
sections of putative transformed axillary shoots after droplet
selection, by use of confocal microscopy confirmed that a
range of different chimeric structures were being generated,
but also showed that transgenic cells were abundant, being
present in many parts of the stem. Some shoots appeared to
have uniform expression of GFP (Fig. 4).
Our previous study showed that delayed droplet selection
post-transformation might enhance the transformation efficiency. Droplet selection approaches were trialed for transformations following co-cultivation of the NLL explants

materials to generate additional axillary buds from each original shoot might prove a way to generate more uniformly transgenic materials. Clumps of axillary shoots that were obtained
from one round of subsequent subculturing on Cc media are
shown in Fig. 6b. Visualisation of eGFP expression in subcultured clumps showed variation of expression (Fig. 6c). All
shoot clumps on the plate originated from a single original
shoot and segregation of GFP expression levels was clearly
visible. Figure 6d is a cross section through the base of a piece
from a subcultured shoot clump with eGFP expression visualised by confocal microscopy. Only some vascular tissue in
the primary (central) axillary shoot showed eGFP expression,
but both secondary axillary shoots showed abundant GFP in
vascular tissue. Together these results support the hypothesis
that with appropriate subculturing steps genetically uniform
transgenic shoots can be generated. As seen in Fig. 6b, shoot
clumps are not healthy in appearance although these have
not yet been exposed to selection beyond the droplet selection step. Additional improvement to the methodology is also
therefore likely to be achieved by reducing the exposure to
growth regulators during subculturing. These results also
show that, although the calculated frequency of transformation at T0 is improved about threefold to approximately 10 %

by this method, that calculation is based on the assumption
of a single genetic transformation event having been captured within each explant. The variation in eGFP expression
observed within shoot clumps (Fig. 6c, d) is indicative of
multiple events. However this interpretation will require DNA
analysis of T1 generation materials to be confirmed.

13

Preliminary observations with other pulse legumes
The final aim of this research was to investigate the transferability of the new NLL transformation methodology to other
pulse legumes. Figure 7 demonstrates that the transformation potential of wounded surface cells of other lupin species, field pea and faba bean is identical to the observations
with NLL. Furthermore, development of GFP-expressing


which axillary buds develop, we significantly improved the
frequency of generation of transgenic NLL shoot materials
(Table 1; Figs. 1, 2, 3, 5). Second, by subsequent propagation in selection the chimeric structure of transgenic NLL
shoots was reduced, with larger proportion of transgenic tissues compared to non transgenic tissues and potential reduction of multiple chimeric events (Table 1; Figs. 4, 6). Third,
the enhanced frequency of generating transgenic shoots
was demonstrably transferable to other pulse legume crops
(Fig.  7). Efforts to improve transformation frequency and

Discussion
The three aims of this research were achieved. By observation of meristem tissues following wounding, a change to
wounding technique and delayed droplet selection enabling
genetic transformation of the deeper meristem cells from

13


13

48
313

N-D18
N total

48
313

144
36 (75)


Explants

Cc 3d

35

82

28

34

Explants

MPH 10e

79

136

65

85

Shoots

31

17 (11.8)x

Number of shoots is the total number of individual or clumped shoots produced after incubation of explants and original excised shoots on Cc3 for two more weeks. Number of explants
remained the same as the previous step

e

d
After 2 weeks on Rg, explants and excised shoots were moved back to Cc (Cc3). Number of explants is the number that were producing shoots. Number of shoots is the total number of separate
shoots excised from explants at time of transfer to Cc3. These shoots were also propagated on Cc3 for a further 2 weeks

All explants were moved to Rg at D18. Number of explants is those surviving 7 days after droplet treatment, which was applied from D13 to D18 as indicated. These data are a subset of those
shown in Fig. 5. *, **Significant difference between the combined data for old versus new wounding method following droplet selection (χ2 = 299.37; p 
Plant Cell Tiss Organ Cult


Plant Cell Tiss Organ Cult

9

80.0%

75.0%
69.4%

70.0%

60.0%

47.9%

50.0%

40.0%
33.6%
30.0%

18.0%

20.0%

10.0%


gene were components of an hypothesis-driven approach to
tackle regeneration recalcitrance of NLL. During that study
it was found that shoots were developing from deeper tissues than previously understood (Nguyen et al. 2016) which
led to the present accompanying study.
NLL shoot apical meristem structure
Transformation recalcitrance was investigated initially by
determination of the structure of the NLL SAM (Fig. 1) to
discover from which zone new shoot development occurred
following wounding. The development of plants is mainly
divided into two stages: embryonic and post-embryonic.
Embryogenesis in plants provides a basic body plan for the

seedling and stem cell populations for the generation of all
post-embryonic tissues. At the embryo stage, cell proliferation occurs throughout the body, while in the latter phase
many regions discontinue cell division and become more
specialized. Described as the centre of post-embryonic
organ formation in the shoot, the shoot apical meristem
(SAM) first produces the plumule, which develops into the
vegetative and reproductive components of the plant body
(Chien et al. 2011). The literature on plant anatomy has
largely focused on tobacco and tomato species, and some
fruit trees and ornamental horticulture species. Researchers
have taken advantage of the ease with which the former species undergo growth in tissue culture and the existence of
genetic mutations across this range of plants that allow the
layers of the SAM to be distinguished. (Steeves and Sussex 1989; Tilney-Bassett 1986; Szymkowiak and Sussex
1996). No similar information about pulse legumes could be
found. However the similarity of the SAM of NLL (Fig. 1)
to reports from species in other dicot plant clades, and
the subsequent observations about GFP-expressing organ
development indicate that interpretation of our results was

10

Plant Cell Tiss Organ Cult

Fig. 6  Subculture propagation to reduce chimerism of shoots. a–c
Plate cultures. a In vivo imaging of GFP fluorescence in transgenic
shoots visualized using Maestro. Shoots were derived from several
explants following one round of media selection. b Typical shoot
clumps that develop following 2 weeks of subculture on Cc3, c plate

of distinct shoots separated after micropropagation of a single original
axillary shoot such as those shown in a, with different eGFP abundance
apparent in different subcultured shoots and in sectors of shoot clumps.
d Transverse cryostat section of the base of a subcultured shoot, with
eGFP expression detected by confocal microscopy. Scale bar 500 µm

meristem. Anticlinal division elongates the bud, while periclinal division expands the diameter of the shoot. Leaves and
axillary buds arise from the PZ although lateral buds usually
originate from deeper layers and thus slightly deeper initials
in the corpus, than the leaves (Tilney-Bassett 1986; Steeves
and Sussex 1989; Bowman and Eshed 2000; Evert 2006).

in NLL meristem tissue generated any new shoots. Instead,
these results were consistent with the reassessment of plant
regeneration proposed by Sugimoto et al. (2011), the original observations of Pigeaire et al. (1997) that transformants
were generated from axillary buds, the report by Babaoglu
et al. (2000) that genetic manipulation without apical layers of L. mutabilis was more likely to generate transgenic
shoots and the study of Sena et al. (2009) showing that
regeneration of new organs does not require a functional
apical meristem. All the observations about axillary shoot

sectioned. GFP fluorescence is green, whilst chlorophyll fluorescence
is red. g–i Samples were cryostat sectioned. All scale bars are 500 µm.
a–c White lupin. Boxed region in a is an axillary bud enlarged in b
and c. b and c are z sections through the axillary bud showing eGFP
fluorescence in different layers. b White arrow is GFP expression in
epidermis (L1). cWhite arrow points to vascular tissue (L3) expressing

eGFP, double ended white arrow points to PZ tissue and expression
in RZ is circled. d Pearl lupin showing eGFP expression along the
wounded areas e–f L. pilosus e is section through the centre of the
SAM. f is a section through an axillary bud showing expression of
eGFP in the epidermis and deeper tissues. g Faba bean section showing e GFP expression on wounded areas. h–i Field pea. Axillary shoot
development (boxed region from h) is enlarged in i, showing extensive
GFP expression as was observed for axillary shoots of NLL

on chimeric plants to the development of axillary buds and,
subsequently to the gametes (Tilney-Bassett 1986). Successful selection of T0 transgenic shoots requires a combination of resistance across layers. Shoots that developed
from the original wounding method and early selection,
were commonly observed to have multiple small sectors
of transgenic cells and a very low survival rate (Wijayanto
2007; Nguyen et al. 2016; Nguyen unpublished results). We
propose that such shoots originated from non-transgenic

axillary shoots regenerating from below the damaged section of the SAM, with transgenic cells being recruited during early shoot development in the explants, resulting in
the observed pattern of transgenic cells in predominantly
non-transgenic shoot tissues. In contrast, the broad and deep
wounding method results in transformation of cells that are
competent to develop into shoots such that the majority of
shoots that are generated contain significant proportions of
transgenic tissue (Figs. 4, 6, 7).

chimerism
Although GFP-expressing shoots were abundant, different
extents of chimerism amongst the transgenic shoots investigated were still observed (Figs. 4, 6). Indeed, in terms of
generating transgenic shoot material, the outcome of this
research has been a good example of moving “from rags
to an embarrassment of riches”. A further step in the propagation of transgenic shoots was trialed to reduce multiple
transgenic cell chimerism. Figure 6 shows visual evidence
that this can be achieved. It is clear that there will be abundant transgenic L2 cells in the NLL shoots that have been
generated following culture selection. Future work with T1
materials to examine heritability and DNA structure will
however be necessary to finalise this aspect of the transformation methodology. Once generated, periclinal chimeras (where one or more layers are uniformly genetically

13

Plant Cell Tiss Organ Cult

distinct) are stably maintained during propagation, and a
mericlinal chimera (sectoral through layers) can be stabilised as a periclinal chimera by propagation from axillary
buds (Szymkowiak and Sussex 1996). Two to three rounds
of regenerations were recommended to reduce chimeras
in tobacco (Maliga and Nixon 1998) and in strawberry
(Mathews et al. 1995).
Transformation of other pulse legume species
The results reported here demonstrate that a high frequency
of transgenic shoots can be produced with less effort across
pulse legume species by following the methodology as
described, compared to the earlier protocol. Our results and
observations are closely aligned with those described for
cereal seedling shoot apical meristem transformation, which
has been applied with success across a range of cereal crop

Plant Cell Tiss Organ Cult

References
Atif RM, Patat-Ochatt EM, Svabova L, Ondrej V, Klenoticova H,
Jacas L, Griga M, Ochatt SJ (2013) Gene transfer in legumes. In:
Lüttge U, Beyschlag W, Francis D, Cushman J (eds) Progress in
Botany. Springer, Berlin, pp 37–100
Atkins CA, Emery RJN, Smith PMC (2011) Consequences of transforming narrow leafed lupin (Lupinus angustifolius [L.]) with an
ipt gene under control of a flower-specific promoter. Transgenic
Res 20:1321–1332
Babaoglu M, McCabe MS, Power JB, Davey MR (2000) Agrobacterium-mediated transformation of Lupinus mutabilis L. using
shoot apical explants. Acta Physiol Plant 22:111–119
Barker SJ, Si P, Hodgson L, Ferguson-Hunt M, Khentry Y, Krishnamurthy P, Averis S, Mebus K, O’Lone C, Dalugoda D, Koshkuson N, Faithfull T, Jackson J, Erskine W (2016) Regeneration
selection improves transformation efficiency in narrow-leaf lupin.
Plant Cell Tissue Org Cult. doi:10.1007/s11240-016-0992-7
Barton MK, Poethig RS (1993) Formation of the shoot apical meristem in Arabidopsis thaliana: an analysis of development in the
wild type and in the shoot meristemless mutant. Development
119:823–831
Bowman JL, Eshed Y (2000) Formation and maintenance of the shoot
apical meristem. Trends Plant Sci 5:110–115
Chien CT, Chen SY, Tsai CC, Baskin JM, Baskin CC, Kuo-Huang LL
(2011) Deep simple epicotyl morphophysiological dormancy in
seeds of two Viburnum species, with special reference to shoot
growth and development inside the seed. Ann Bot 108:13–22
Evert RF (2006) Esau’s plant anatomy. Wiley, Hoboken
Fischer AH, Jacobson KA, Rose J, Zeller R (2008) Preparation of
slides and coverslips for microscopy. CSH Protoc. doi:10.1101/
pdb.prot4988
Iantcheva A, Mysore KS, Ratet P (2013) Transformation of leguminous
plants to study symbiotic interactions. Int J Dev Biol 57:577–586

Satina S, Blakeslee AF, Avery AG (1940) Demonstration of the three
germ layers in the shoot apex of Datura by means of induced
polyploidy in periclinal chimeras. Am J Bot 27:895–905
Sena G, Wang X, Liu H-Y, Hofhuis H, Birnbaum KD (2009) Organ
regeneration does not require a functional stem cell niche in
plants. Nature 457:1150–1154
Somers DA, Samac DA, Olhoft PM (2003) Recent Advances in
legume transformation. Plant Physiol 131:892–899
Steeves TA, Sussex I M (1989) Patterns in plant development. Cambridge University Press, Cambridge
Sticklen MB, Oraby HF (2005) Shoot apical meristem: a sustainable
explant for genetic transformation of cereal crops. In Vitro Cell
Dev Biol 41:187–200
Sugimoto K, Gordon SP, Meyerowitz EM (2011) Regeneration in
plants and animals: dedifferentiation, transdifferentiation or just
differention? Trends Cell Biol 21:212–218
Szymkowiak EJ, Sussex IM (1996) What chimeras can tell us about
plant development. Ann Rev Plant Physiol Plant Mol Biol
47:351–376
Tilney-Bassett RAC (1986) Plant chimeras. Edward Arnold Ltd,
London
Tirichine L, Andrey P, Biot E, Maurin Y, Gaudin V (2009) 3D fluorescent in situ hybridization using Arabidopsis leaf cryosections and
isolated nuclei. Plant Methods 5:11
Voss U, Larrieu A, Wells DM (2013) From jellyfish to biosensors: the
use of fluorescent proteins in plants. Int J Dev Biol 57:525–533
Wijayanto T (2007) Genetic manipulation of programmed cell death
(PCD) for reduced susceptibility to necrotrophic fungi in narrowleafed lupin (Lupinus angustifolius). PhD Thesis, The University
of Western Australia
Wijayanto T, Barker SJ, Wylie SJ, Gilchrist DG, Cowling WA (2009)
Significant reduction of fungal disease symptoms in transgenic
lupin (Lupinus angustifolius) expressing the anti-apoptotic baculovirus gene p35. Plant Biotechnol J 7:778–790