DSpace at VNU: An approach to overcoming regeneration recalcitrance in genetic transformation of lupins and other legumes

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

1

Centre for Plant Genetics and Breeding (PGB), School of
Plant Biology M080, The University of Western Australia,
Crawley, WA 6009, Australia

2

School of Plant Biology M090, The University of Western
Australia, 35 Stirling Highway, Crawley, WA 6009, Australia

3

Faculty of Biology, Hanoi University of Science, Hanoi,
Vietnam

4

Department of Agriculture and Environment, Centre for Crop
and Disease Management, Curtin University, Bentley,
WA 6845, Australia

5

Institute of Agriculture M082, The University of Western
Australia, Crawley, WA 6009, Australia

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

RZRib zone
SAMShoot apical meristem
T0Initial generation of transgenic shoot
T1Progeny of T0 generation

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Introduction
Genetic manipulation (GM) of plants has resulted in commercial uptake of the technology that might be compared
to the green revolution. In the 20-year period 1996 to 2015
there were 2.0 billion accumulated hectares of biotech crops
grown globally, of which 1.0 billion hectares were biotech
soybean [Glycine max (L.) Merrill]. The only other significantly cultivated biotech-enhanced legume was alfalfa
(Medicago sativa L.) in the USA (James 2015). Additionally, the importance of Medicago truncatula Gaertn. and
Lotus japonicus L. as genome models has driven development of a functional transformation system for these legume
species. However, despite the importance of pulse legumes
to both human and agroecosystem health, research on any
of these crop species has been hampered by the lack of a
high throughput genetic transformation system (Somers et
al. 2003; Atif et al. 2013; Iantcheva et al. 2013).
In the Mediterranean cropping systems of Australia, the
dominant legume is Lupinus angustifolius L. (narrow leaf
lupin; NLL) (Dracup and Kirby 1996). Widening the NLL
gene pool by GM research has been carried out towards
adding agronomic traits such as herbicide tolerance (Pigeaire et al. 1997; Barker et al. 2016), necrotrophic fungal
pathogen resistance (Wijayanto et al. 2009), value-added
traits such as improved protein quality (Molvig et al. 1997)

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

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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

selection did not materially improve the results from the
current methodology (Barker et al. 2016). However, results
from use of hygromycin as a selectable marker along with
expression of the green fluorescent protein (GFP) led to
the unexpected realisation that transformation of NLL cells
exposed to A. tumefaciens was essentially universal, and
also that the majority of cells that were exposed by the current wounding method did not appear to develop into shoots
(Nguyen et al. 2016). Only development of GFP expressing shoots from deeper tissue could be observed, presumably when stabbing went deeper than originally intended.
We hypothesised that better understanding the structure of
the NLL shoot apical meristem and determination of the
origin of shoots that originated from wounded embryonic
axis whilst following the current methods would provide
information that would enable the design of a more efficient
transformation protocol. The aims of this research were
threefold: first, to significantly improve the frequency of
generation of transgenic NLL shoot materials; second, to
reduce or remove the chimeric structure of transgenic NLL
shoots; third, to determine if the transformation protocol
was transferable to other pulse legume crops.

Materials and methods
Regulatory approval
Approval for this research was obtained from the Office of
the Gene Technology Regulator (Australia) under approval
number NLRD 5/1/406 from the University of Western Australia Institutional Biosafety Committee.
Agrobacterium strain and vector construct
Transformation experiments were carried out using the A.
tumefaciens strain AgL0 (Lazo et al. 1991) harbouring the
binary Ti plasmid clone pH35 (Nguyen et al. 2016). The vector pH35 contained a GFP-GUS fusion for plant expression
under control of CaMV35S eukaryotic promoter with duplicated enhancer region, hygromycin resistance gene (HygR)

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

al. 2016), then transferred to Cc medium and transformed
with AgL0:pH35. Explants were collected from 4 (D4) to
10 (D10) days after transformation for microscopy analysis.
Deep and broad stabbing: The dome of NLL seedlings
was stabbed 1–1.5 mm depth in a wider area but also still
including the SAM. Explants then went into co-cultivation
medium and were transformed with AgL0:pH35. Samples
were collected from D4 to D10 for microscopy analysis.
Other legumes were germinated as described for NLL
and were used for transformation when seed imbibition was
apparent, 2–3 days after initial exposure to moisture. Species treated were white lupin (L. albus L.), pearl lupin (L.
mutabilis L.), L. pilosus L., field pea and faba bean (large
seeded form).

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Explants were also moved back to Cc3 for 2 weeks to generate more axillary shoots. All surviving shoots were then
subcultured onto micro-propagation media (1X MS salts,
3 % (w/v) sucrose, 0.5 g L−1 MES, pH to 5.7, 0.7 % (w/v)
Phytoblend (Caisson Laboratories I nc.), autoclaved then
1X B5 vitamins, 0.1 mg L−1 BAP, 0.01 mg L−1 NAA,150
mg L−1 Timentin® added on cooling) with 10 mg L−1 hygromycin selection (MPH10) for 2 weeks followed by 2 weeks
on rooting media with 30 mg L−1 hygromycin selection
(RMH30).Rooting medium contains 1X MS salts, 3 % (w/v)
sucrose, 0.5 g L−1 MES, pH to 5.7, 0.6 % (w/v) Phytoblend.
Autoclave, cool, then add 1X B5 vitamins, 0.1 mg L−1 BAP,
0.01 mg L−1 NAA, 150 mg L−1 Timentin®, 3.0 mg L−1 IBA,
0.1 mM aromatic amino acids (phenylalanine, tyrosine, and
tryptophan), 1 mg L−1 ascorbic acid.
Selection prior to MPH10 treatment, by adding a drop of

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

except the BAP and NAA are reduced to 1.0 mg L−1 BAP,
0.1 mg L−1 NAA. After 2 weeks in Rg, emerged shoots
were excised individually from each explant and transferred
back to Cc medium containing 150 mg L−1Timentin (Cc 3).

Putative transformed shoot explants were longitudinal or
cross sectioned to analyse by confocal microscopy. GFP
expression was detected by Nikon Ti-E inverted motorised
microscope with Nikon A1Si spectral detector confocal
system running NIS-C Elements software at the Centre for
Microscopy, Characterisation & Analysis (CMCA), The
University of Western Australia. Images were captured by
confocal system applying objective 4x, 10x and 20x with
laser wavelength 488 nm and 500–550 nm for GFP excitation and emission, respectively.
Surviving shoots from MPH were imaged to detect in
vivo fluorescence using a CRi Maestro 2 in combination
with Maestro software including CPS™ (Compute Pure

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Spectrum) and RCA™ (Real Component Analysis) spectral
library generation tools. For GFP imaging, the samples were
scanned with blue filter, excitation filter 435–480 nm, emission range from 500 to 550 nm.

Plant Cell Tiss Organ Cult

Results

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

formation and emergence of axillary bud from PZ. d–e Axillary bud
formation from vascular tissue in transverse section of NLL shoot (red
circle dashed lines) Bar 200 µm. (Color figure online)

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Plant Cell Tiss Organ Cult

in order to retain as much meristem structure as possible.
However our preliminary results suggested that regeneration competence was restricted to deeper tissues than those
being exposed by the current method. We therefore tested a
deeper stabbing method. Figure 2 illustrates the current and
improved stabbing method target area and the expression of
GFP in the deeper zone. Figure 3 shows the anatomy of GFP
transformed NLL explants following the two wounding
methods from D4 to D10 post-transformation. Observation
of GFP expression revealed that deep and broad stabbing exposed more meristematic cells to Agrobacterium.

5

Moreover, following the deep and broad wounding method,
vascular cells were more frequently transformed than in the
conventional method.
Observation of the development of GFP-expressing
shoots following both wounding methods determined that
meristem cells along the damaged areas were disabled in
their meristematic activities. There was no evidence that
new meristem cells were generated or differentiated from
wounded shoot apical meristem. Axillary shoots produced

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

with Agrobacterium, in combination with the two stabbing methods. The summary of results is shown in Table 1
and Fig. 5. A comparison of the two wounding methods
showed that with delayed droplet application, the survival
of explants increased dramatically, from 6.8 % after application at D4, to 33.6 % after application at D16 for the original
wounding method, and up to 75 % when the new wounding
method was employed and droplet application was delayed
to D18. Analysis of these data indicated that the trend for
differences in explant survival between the old and new
wounding methods were statistically significant, (Table 1).
Visualisation in vivo of whole axillary shoots that had
emerged from different explants and had survived further
propagation on MPH suggested that these were quite uniform in eGFP expression within one shoot, but showed some
variation between shoots from different explants (Fig. 6a).
These results were the initial confirmation of the abundance by which transgenic shoots could be generated by the
improved wounding methodology in combination with selection on MPH and suggested that further subculturing of such

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Plant Cell Tiss Organ Cult

Fig. 3  Development of explant SAM after original (a–c) and new (d–
f) wounding methods. Images are cryostat sections at the designated
days after treatment with A. tumefaciens AgL0:pH35. GFP was imaged

by confocal microscopy. Scale bars are all 500 µm. a, d D4 samples. b,
e D7 samples. c, f D10 samples

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.

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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

axillary bud was observed in white lupin, L. pilosus, and
field pea. The results shown for faba bean and field pea are
from a single experiment performed by a second operator who had not previously performed the deep and broad
wounding method; furthermore all results in Fig. 7 are the
outcome of treatment of fewer than ten germinated seedling
explants for each species. This result confirmed that the data
obtained with NLL were reproducible and provided a robust


Plant Cell Tiss Organ Cult

7

Fig. 4  Chimeric structure of original axillary buds following the deep
and broad wounding method. Scale bars are all 500 µm. a–c Cryostat sections. d–i Hand sections of living tissue. GFP fluorescence in
these sections is green and red fluorescence is chlorophyll. a eGFP
expressed in leaf axil but not in axillary shoots. b Transformed cells
located in vascular tissue of the explants meristem, and a lateral auxillary shoot. The SAM of axillary bud was a mericlinical chimera. c GFP
in axillary shoot showed that the outer layer (L1) of the shoot received

the gene. dArrows indicate GFP expression in L1 (epidermal cells) and
in vascular tissue (L3). e Initial formation of axillary shoot with GFP
in L2 (arrow) and scattered in vascular tissue. f GFP in L2 (group of
parenchyma cells is green) and L3 (xylem is green) as indicated with
arrows. g A vascular bundle with GFP expression (arrow) and parenchyma cells (L2) (arrow). h GFP expression in L3 pith cells (arrow),
i GFP expressed in the whole cross section of the shoot (this shoot
apparently only contains transgenic tissues). (Color figure online)

regeneration methodology for future genetic transformation
of a range of pulse legume species.

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

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13

48
313

N-D18
N total

48
313

144
36 (75)

183**

100 (69.4)

77 (8.6)
42 (33.6)
119*
1 (4.0)
46 (47.9)

Explants

Rgc

35 (72.9)

69

99

61

28 (29.2)
82 (56.9)

79

Shoots

34 (27.2)

Explants

Cc 3d

35

82

28

34

Explants

MPH 10e

79

136

65

85

Shoots

31

17 (11.8)x

5 (10.4)
32/288****

12

12

10 (10.4)x
x

4

Shoots

4 (3.2)
4/125***

Explants

RMH 30f

Explants at the start of treatment following application of AgL0:pH 35 (Cc) and transfer to Cc2 4 days after application (100 % survival)

Shoots/shoot clumps (as shown in Fig. 6b) were moved to RMH30 after 2 weeks on MPH10. Shoot tally is the total still surviving 2 weeks after transfer. Tally of explants is the number of
explants from which the surviving shoots originated. ***, ****O-D16 explant survival data are statistically significantly different from the combined N-[D13–D18] (χ2 = 47.49; p < 0.001).
x
N-D13, N-D16 and N-D18 data are not significantly different from each other (χ2 = 0.14; p > 0.05)

f

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 < 0.001). The O-D16 data are also significantly different from the N-D13 to N-D18 data combined (χ2 = 6.86; p < 0.01)

c

b

a
O—original wounding method, N—broad and deep wounding method. D4, D13, D16 and D18 are days after transformation at which the droplet selection was implemented D4 data are
extracted from Nguyen et al. (2016)

Cc cocultivation media, Rg regeneration media, MPH 10 micropropagation media with 10 mg L−1 hygromycin, RMH 30 rooting media with 30 mg L−1 hygromycin

In columns Rg, Cc3 and RMH30, value in parentheses is the percent of explants that produced transgenic shoots, also described as frequency of transformation events as it is assumed for that
calculation that only one genetic transformation occurs per explants

144

N-D16

895

125
1020
25
96

Explants

Explants

895
125
1020
25
96

Cc 2b

Ccb

O-D4
O-D16
O total
N-D4
N-D13

Experimenta

Table 1  Transformation efficiency at T0 in media selection following delayed droplet selection

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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%

0.0%

6.8%

D4

D10

D13

D16

D18

Fig. 5  Explant survival 1 week after hygromycin droplet selection
with the two wounding methods. Dark bars are data for the original
(SAM only) wounding method. Light bars are data for the new (broad
and deep) wounding method

reduce chimerism of NLL and other legumes were initiated
following observations by Wijayanto (2007) that occasionally, following early non-transgenic shoot growth, an apparently non-chimeric axillary bud could be achieved from the
current methodology. Initially attention was focused on the
wounding method. The observation that excessive damage
destroyed the SAM led us to reduce the extent and depth
of SAM stabbing. However this initiative did not improve
transformation frequency either with the original bar gene
selection (unpublished results) or with glyphosate as a novel
selection methodology (Barker et al. 2016). Investigation of
other factors affecting transformation using hygromycin as
a selectable marker and eGFP as a highly sensitive reporter

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

consistent with the published studies.
Our observation of NLL SAM structure are consistent
with the published SAM structure of eudicot plants that
follows the tunica-corpus configuration as the characteristic of angiosperm shoot apices (Satina et al. 1940; Steeves
and Sussex 1989; Barton and Poethig 1993; Bowman and
Eshed 2000; Lenhard et al. 2002; Evert 2006; Murray et al.
2012). The tunica consists of small populations of pluripotent undifferentiated meristematic cells. Anticlinal division
and differentiation of tunica cells give rise to lateral organs
and provide distal meristematic growth, whereas corpus cell
division is responsible for formation of the stem. The outer
tunica layer (L1) produces shoot epidermal cells, whereas
the inner layer (L2) forms the other tissues, including cortex and undifferentiated germline cells. The vascular tissues
and pith comprise L3 of the developed stem; these tissues
form subsequent to initiation of floral bud development in
tobacco explants (Wilms and Sassen 1987). The majority of
cells remain associated with their originating layer, however
some mixing can occur so that occasionally there can be
contribution of the L1 to the germ cells. Periclinal division
of the corpus or layer three (L3) results in mixing with L2,
creating structural integrity among lateral appendages and
the stem (Satina et al. 1940; Tilney-Bassett 1986).
The NLL shoot apex also shows concordance with the
cytohistological zone concept that the shoot apex is organized into three distinct zones of differentiation and function. CZ cells divide anticlinally, producing the initial cells
for the PZ and RZ, whilst cells in the PZ and RZ combine
periclinal, anticlinal and oblique divisions (Fig. 1c). PZ and
RZ divisions help to form the main stem. Cortex and procambium originate from the PZ, while RZ gives rise to pith

13

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

development following SAM wounding are consistent with
the concept that damage to the apical meristem causes loss
of apical dominance. The new deep and broad wounding
method in addition to that outcome creates the opportunity
for cells around the vascular tissue to be transformed, which
as summarised by Sugimoto et al. (2011) is the origin of
cells that are competent to regenerate.
Also of significance to the aims of this research is the
contribution of the distinct layers identified from research

Broad and deeper wounding and chimerism
in transgenic shoots expressing eGFP
The use of eGFP as a marker gene proved a significant source
of relevant information to assist testing of the experimental hypotheses, as would be expected from the wide range
of successful applications that have been reported (Voss et
al. 2013). Transformation with Agrobacterium was clearly
observed essentially for all exposed cells in every species
that we examined (Figs. 3, 7), confirming and extending the unexpected observations of Nguyen et al. (2016).
However, there was no evidence that the competent cells

13


Plant Cell Tiss Organ Cult

11

Fig. 7  GFP expression imaged by confocal microscopy in explants
of various legumes 11 days (a–f) or 13 days (g–i) following the deep
and broad wounding transformation method. a–f Samples were hand

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).

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12

Transfer of a transgene through NLL and other legume
gametes usually requires L2 to be transgenic (Tilney-Bassett 1986). Nguyen et al. (2016) identified that vascular
tissue damage as well as SAM destruction resulted from
hygromycin treatment of explants; with this selection
therefore L3 tolerance is also essential. Theoretically, lateral organs originate from procambium in PZ of SAM and
vascular cambium in stem of plants (Evert 2006). Visualising the cross section of NLL showed that the axillary buds
originated from a group of cells in vascular tissue (Fig. 1d,
e). The wide and deep wounding method (Fig. 2) achieved
these objectives.
Delayed selection improved transformation efficiency
Early selection in combination with the original wounding method was the least successful approach; deep and
broad stabbing combined with delayed hygromycin droplet selection was the most effective method of achieving
significantly transgenic shoot materials (Fig.  5; Table 1).
This result supports the observed effect of delayed selection in the accompanying paper (Nguyen et al. 2016), where
improved survival of explants was observed with PPT selection as well as hygromycin, when selection was delayed.
A moderately enhanced frequency of heritable transgenic
shoots was achieved using the original wounding and selection method by Wijayanto et al. (2009). I n that research,
transgenic NLL that had less susceptibility to fungal pathogen due to expression of the anti-apoptotic baculovirus gene
P35 was generated. It was not possible to separate operator
from transgene effect in that project. However these new
results are consistent with the suggestion that reduced plant
cell stress due to inhibition of apoptosis may have enhanced
the frequency of transgenic shoot survival in that example.
Selection methodology and propagation to reduce

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

species. In the case of cereals, transformability of apical cells
also has proven not to be the rate limiting issue as initially
reported, but as with pulse crops, selectable marker use has
proven difficult and chimerism of primary transformants has
been reduced significantly by producing and multiplying
shoots without selection for several weeks prior to transfer to
selection media (Sticklen and Oraby 2005). We propose from
our experiences, that the same successful outcome by application of this general methodology is also likely for other dicot
species where transformation has proven difficult to achieve.
The frequency of events observed in our research means
that approaches to GM crop development that avoid the
retention of foreign DNA, such as the CRISPR-Cas9 methodology (Quétier 2016) could be applied to achieve tailored
genetic events that meet the regulatory requirements of
the international community, opening the way for a range
of legume crop improvements that currently are not being
attempted. In conclusion, if combined with further propagation and selection, it is feasible that this approach to transgenic shoot regeneration can provide an affordable means
of high throughput genetic transformation that is within the
capabilities of personnel in any pulse research or improvement program.
Acknowledgments  The first author acknowledges with deep gratitude the Australian Award Scholarship funded by the Australian Government. The authors acknowledge the facilities of CELLCentral,
School of Anatomy Physiology & Human Biology, The University of
Western Australia and the facilities, scientific and technical assistance
of the Australian Microscopy & Microanalysis Research Facility at
the Centre for Microscopy, Characterisation & Analysis (CMCA), The
University of Western Australia, a facility funded by the University,
State and Commonwealth Governments.
Author contrib utions  Project design and data analysis AHN, SJB
and WE, transformations AHN and LH, plant tissue culture AHN,
microscopy AHN, figure preparation AHN and SJB, manuscript preparation AHN, SJB, WE and LH.

Plant Cell Tiss Organ Cult

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