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: 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 susan.barker@uwa.edu.au Centre for Plant Genetics and Breeding (PGB), School of Plant Biology M080, The University of Western Australia, Crawley, WA 6009, Australia School of Plant Biology M090, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia Faculty of Biology, Hanoi University of Science, Hanoi, Vietnam Department of Agriculture and Environment, Centre for Crop and Disease Management, Curtin University, Bentley, WA 6845, Australia 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 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 13 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 13 Plant Cell Tiss Organ Cult one percent in the current NLL cultivar (Wijayanto et al 2009; Nguyen et al 2016; Barker et al unpublished results) The difficulty with NLL transformation led to examination of alternative selection methodologies Glyphosate 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 (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) 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), 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 in combination with Maestro software including CPS™ (Compute Pure 13 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  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) 13 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 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 13 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 13 Preliminary observations with other pulse legumes The final aim of this research was to investigate the transferability of the new NLL transformation methodology to other pulse legumes Figure 7 demonstrates that the transformation potential of wounded surface cells of other lupin species, field pea and faba bean is identical to the observations with NLL Furthermore, development of GFP-expressing 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 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 13 13 48 313 N-D18 N total 48 313 144 36 (75) 183** 100 (69.4) 77 (8.6) 42 (33.6) 119* (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 (10.4) 32/288**** 12 12 10 (10.4)x x Shoots (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  0.05) 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