Keywords Narrow leafed lupin · Lupinus angustifolius legume transformation · Regeneration · Agrobacterium tumefaciens · Green fluorescent protein · Shoot axillary bud transformation ·
Trang 1ORIGINAL ARTICLE
Susan J Barker
susan.barker@uwa.edu.au
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
Received: 25 July 2016 / Accepted: 2 September 2016
© Springer Science+Business Media Dordrecht 2016
An approach to overcoming regeneration recalcitrance in genetic
transformation of lupins and other legumes
An Hoai Nguyen 1,2,3 · Leon M Hodgson 1,4 · William Erskine 1,5 · Susan J Barker 2,5
DOI 10.1007/s11240-016-1087-1
Agrobacterium, in combination with delayed selection
proved successful, increasing initial explants transforma-tion 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 con-tent Furthermore, the method was also tested
success-fully 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
Cc Co-cultivation medium
CZ Central zone eGFP Enhanced green fluorescent protein
GM Genetic manipulation;
MPH Micropropagation medium with hygromycin NLL Narrow-leaf lupin
Rg Regeneration medium PPT Phosphinothricin
PZ Peripheral zone
RZ Rib zone SAM Shoot apical meristem T0 Initial generation of transgenic shoot T1 Progeny of T0 generation
Abstract For pulse legume research to fully
capi-talise on developments in plant molecular genetics, a
high throughput genetic transformation methodology
is required In 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
herbi-cide 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 In
this research it was identified that despite ready
trans-formation, apical cells were not competent to
regener-ate However a deep and broad wounding procedure to
expose underlying axillary shoot and vascular cells to
Trang 2one 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 exami-nation 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 cur-rent wounding method did not appear to develop into shoots (Nguyen et al 2016) Only development of GFP express-ing shoots from deeper tissue could be observed, presum-ably 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 Aus-tralia 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 vec-tor pH35 contained a GFP-GUS fusion for plant expression under control of CaMV35S eukaryotic promoter with
dupli-cated 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
tumefa-ciens 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
Introduction
Genetic manipulation (GM) of plants has resulted in
com-mercial 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
sig-nificantly cultivated biotech-enhanced legume was alfalfa
(Medicago sativa L.) in the USA (James 2015)
Addition-ally, the importance of Medicago truncatula Gaertn and
Lotus japonicus L as genome models has driven
develop-ment 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
(Pige-aire 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
mechani-cally 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
agar-based 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
trans-genic 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
culi-naris 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
Trang 3Explants were also moved back to Cc3 for 2 weeks to gen-erate 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 Inc.), 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 hygro-mycin 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 embed-ded 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 sec-tions 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)
GFP imaging and analysing
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 excita-tion 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
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
pres-ent 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
Spe-cies treated were white lupin (L albus L.), pearl lupin (L
mutabilis L.), L pilosus L., field pea and faba bean (large
seeded form)
Sub-culture media and selection protocol
Transformed explants were cultured in Cc media 2 days
in dark conditions, then 2 days under normal light
condi-tions (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)
Trang 4organized 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 cytohistologi-cal 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)
Development of wounded meristem shoots
The hypothesis that wounded apical meristem has capabil-ity to rebuild itself is the basis for the approach taken in previous studies, with the idea that the interference in meri-stem integrity by stabbing will activate new groups of meri-stem cells to produce shoots This method therefore aimed only
to wound the meristem area without significant damage,
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,
emis-sion range from 500 to 550 nm
Results
NLL shoot apical meristem
Analysis of sections from NLL shoots 2–3 days after
ger-mination 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
reproduc-tive organs Histology revealed that cells of the NLL were
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
cen-tral zone, PZ peripheral zone, RZ rib zone, LP leaf primordia b–e are
stained with Toluidine blue, captured by Olympus microscopy b
Lon-gitudinal 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)
Trang 5Moreover, 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 dis-abled by the wounding procedure, releasing axillary meri-stem cells to activate shoot development
Chimerism in transgenic shoots, selection methodology and enhanced explant survival
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 effi-ciency Droplet selection approaches were trialed for trans-formations following co-cultivation of the NLL explants
with Agrobacterium, in combination with the two
stab-bing 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 applica-tion at D4, to 33.6 % after applicaapplica-tion 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 uni-form 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 abun-dance by which transgenic shoots could be generated by the improved wounding methodology in combination with selec-tion on MPH and suggested that further subculturing of such
in order to retain as much meristem structure as possible
However our preliminary results suggested that
regenera-tion 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
stab-bing exposed more meristematic cells to Agrobacterium
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
Trang 6by this method, that calculation is based on the assumption
of a single genetic transformation event having been cap-tured 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
Preliminary observations with other pulse legumes
The final aim of this research was to investigate the transfer-ability of the new NLL transformation methodology to other pulse legumes Figure 7 demonstrates that the transforma-tion potential of wounded surface cells of other lupin spe-cies, 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 opera-tor 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
materials to generate additional axillary buds from each
origi-nal shoot might prove a way to generate more uniformly
trans-genic 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
sub-cultured 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
visu-alised 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
selec-tion step Addiselec-tional 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
transforma-tion at T0 is improved about threefold to approximately 10 %
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
Trang 7which 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 propaga-tion in selecpropaga-tion the chimeric structure of transgenic NLL shoots was reduced, with larger proportion of transgenic tis-sues compared to non transgenic tistis-sues and potential reduc-tion 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
regeneration methodology for future genetic transformation
of a range of pulse legume species
Discussion
The three aims of this research were achieved By
observa-tion of meristem tissues following wounding, a change to
wounding technique and delayed droplet selection enabling
genetic transformation of the deeper meristem cells from
Fig 4 Chimeric structure of original axillary buds following the deep
and broad wounding method Scale bars are all 500 µm a–c
Cryo-stat 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
auxil-lary shoot The SAM of axilauxil-lary 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 paren-chyma 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)
Trang 8Table 1
1 hyg
1 hyg
a O—
b Expl
c All e
2 = 29
2 = 6.
f Shoo
2 = 47
2 = 0.
Trang 9seedling and stem cell populations for the generation of all post-embryonic tissues At the embryo stage, cell prolifera-tion 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 spe-cies 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 Sus-sex 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 characteris-tic 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 pluripo-tent 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 cor-tex 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 orga-nized into three distinct zones of differentiation and func-tion 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 pro-cambium originate from the PZ, while RZ gives rise to pith
reduce chimerism of NLL and other legumes were initiated
following observations by Wijayanto (2007) that
occasion-ally, following early non-transgenic shoot growth, an
appar-ently 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
tis-sues 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
6.8%
18.0%
33.6%
47.9%
69.4%
75.0%
0.0%
10.0%
20.0%
30.0%
40.0%
50.0%
60.0%
70.0%
80.0%
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
Trang 10in 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 origi-nal 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
lay-ers 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
meristem Anticlinal division elongates the bud, while
peri-clinal 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)
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
experimen-tal 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
extend-ing the unexpected observations of Nguyen et al (2016)
However, there was no evidence that the competent cells
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