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Keywords Narrow leafed lupin · Lupinus angustifolius legume transformation · Regeneration · Agrobacterium tumefaciens · Green fluorescent protein · Shoot axillary bud transformation ·

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ORIGINAL 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

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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 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

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Explants 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)

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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 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)

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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 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

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by 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

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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 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)

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Table 1

1 hyg

1 hyg

a O—

b Expl

c All e

2 = 29

2 = 6.

f Shoo

2 = 47

2 = 0.

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seedling 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

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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 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

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