Barley callus: a model system for bioengineering of starch in cereals
© Carciofi et al.; licensee BioMed Central Ltd. 2012
Received: 12 July 2012
Accepted: 4 September 2012
Published: 7 September 2012
Starch is the most important source of calories for human nutrition and the majority of it is produced by cereal farming. Starch is also used as a renewable raw material in a range of industrial sectors. It can be chemically modified to introduce new physicochemical properties. In this way starch is adapted to a variety of specific end-uses. Recombinant DNA technologies offers an alternative to starch industrial processing. The plant biosynthetic pathway can be manipulated to design starches with novel structure and improved technological properties. In the future this may reduce or eliminate the economical and environmental costs of industrial modification. Recently, many advances have been achieved to clarify the genetic mechanism that controls starch biosynthesis. Several genes involved in the synthesis and modification of complex carbohydrates in many organisms have been identified and cloned. This knowledge suggests a number of strategies and a series of candidate genes for genetic transformation of crops to generate new types of starch-based polymers. However transformation of cereals is a slow process and there is no easy model system available to test the efficiency of candidate genes in planta.
We explored the possibility to use transgenic barley callus generated from immature embryo for a fast test of transgenic modification strategies of starch biosynthesis. We found that this callus contains 4% (w/w dw) starch granules, which we could modify by generating fully transgenic calli by Agrobacterium-transformation. A Green Fluorescent Protein reporter protein tag was used to identify and propagate only fully transgenic callus explants. Around 1 – 1.5 g dry weight of fully transgenic callus could be produced in 9 weeks. Callus starch granules were smaller than endosperm starch granules and contained less amylose. Similarly the expression profile of starch biosynthesis genes were slightly different in callus compared with developing endosperm.
In this study we have developed an easy and rapid in planta model system for starch bioengineering in cereals. We suggest that this method can be used as a time-efficient model system for fast screening of candidate genes for the generation of modified starch or new types of carbohydrate polymers.
Starch is a product of plant photosynthetic carbon fixation and is the principal source of energy for human nutrition. Cereal crops produce the majority of starch in agriculture. Starch is also a useful biopolymer used in different industrial sectors . Different crops synthesise structurally and chemically diverse starches with specific physicochemical properties that are suitable for diverse end-uses. However the natural variation found in natural starches does not provide enough different functionalities for the many end-uses. Therefore, after extraction, starch is often processed through a series of chemical or enzymatic modification and physical treatments in order to confer additional properties. Therefore the development of new crops with novel starches, avoiding post-harvest processing, is of economic interest. Genetic modification of the plant starch biosynthetic pathway to design a wide range of tailor made carbohydrates has shown to be a promising strategy [2–6]. The elucidation of the starch biosynthetic mechanism and the characterization of a number of genes, from different origins, involved in polysaccharide modification are offering a rich toolbox for in planta production of improved polymers by transgenic technology [7, 8]. Therefore it is expected that many novel starches or heterogeneous polysaccharides can be engineered in relevant crops, such as cereals. A severe limitation in the process of testing candidate transgenes is the time required to do genetic transformation, regeneration of transformed plants and characterization of the engineered starch stored in the sink organs (e.g. cereal grains). In barley, which is an important crop and a genetically well characterized cereal model, this time is at least 6 months.
A fast model system for genetic transformation of starch would therefore be useful. The model system should permit the high-throughput screening of candidate transgene activities and the preliminary characterization of the generated modified polysaccharide in a time-efficient way. This could allow to test the validity of several potential transformation strategies. The promising transgenic constructs would be selected to be employed in a cycle of plant transformation, full regeneration and propagation. Such in vitro approach may potentially boost the production of new valuable crops synthesizing rationally designed polymers.
Callus tissues of different plants accumulate starch granules especially when they are grown in high sugar media and specific culture conditions, such as controlled temperature, correct hormone treatment and osmotic potential [9–15]. It is not clear why starch is deposited in callus. But it has been suggested that the starch function as an energy reserve for the energy-requiring process of organogenesis (e.g. shoot formation). Alternatively it may play a role in equilibrating the level of free soluble sugars to counterbalance the osmotic potential in the medium [16–20].
In this study we explore the possibility to use transgenic callus of immature barley embryos as a fast model system to study starch biosynthesis and bioengineering of starch in cereals. We optimized a protocol to purify starch from callus. Transgenic callus induced from immature barley embryo after Agrobacterium-mediated transformation were found to store significant amounts of starch granules, which are comparable to the ones accumulated in barley storage organs. The system was tested by over-expressing the gene encoding granular bound starch synthase Ia (GBSSIa) tagged with green fluorescent protein (GFP). GBSS is known to synthesize amylose. The transgenic protein localized in the starch granules of the callus and increased the content of amylose.
Results and Discussion
Establishing a protocol for generation of transgenic callus to produce starch
Characterization of starch granules in barley callus
Expression of genes involved in starch biosynthesis in callus
The major starch storage tissue in cereal plants is the filial starchy endosperm. However other tissues in the seed and in other organs accumulate starch in a transitory form, such as the aleurone, embryo, pericarp and leaf tissues . The starch is shaped differently in the various tissues due to differential expression of genes, coding for starch biosynthesis enzymes and other factors involved in the shaping of starch .
Overexpression of GFP tagged GBSS in callus: proof-of-concept
Amylose content (%) ± SD
24.5 ± 0.8
Callus Ubi:GFP (control)
2.2 ± 0.2
27.9 ± 0.7
11.7 ± 1.2
Polymorphism in the genes encoding starch synthases have been demonstrated to be associated with variation in starch functionality such as gelatinization temperature and retrogradation [33, 34]. In addition wild relatives of cereals contain a significantly higher diversity of genes encoding enzymes involved in starch biosynthesis than domesticated species. Wild cereals may therefore contribute to the breeding of cereal varieties with new starch functionalities . However, association studies cannot directly demonstrate the effect of amino acid sequence variation in enzymes on starch functionality. We suggest that the callus starch model presented here can be used as a fast screening method to directly evaluate the impact of such known variants of starch synthases in gene pools of breeding populations and wild cereal species.
Binding of GFP tagged GBSS in starch granules
Plant callus tissues have been used as a tool for biochemical studies of the parent tissues [23, 36]. Attempts have been conducted to validate callus and suspension cell cultures as model systems to investigate carbohydrate and starch metabolism in storage organs of different plants, such as maize, rice, potato and sweet potato [10, 21–23, 28, 37].
In our study we aimed to demonstrate that transgenic barley callus may represent a valid model system for starch bioengineering by transgenic transformation of the callus.
We analysed the starch produced by callus generated from immature barley embryos. There is a lower quantity of starch in callus than in endosperm tissue. The starch granules are smaller and contain only little amylose. We examined the expression profile of various genes involved in starch biosynthesis. Callus has a different expression profile than endosperm of the various forms of starch synthases, which is reflected in a different chain length distribution of the starch. We suggest that among the starch synthases the callus system may be particularly well suited for studying the effect of expression of transgenic variants of GBSSIa and SSIa, because these two genes only have very little expression in callus already. We used GFP as a tag for expression of transgenic enzymes in the callus. GFP fluorescence was easily detectable in the callus, and could be used to validate gene transfer to the callus and localize transgenic cells. Fluorescent transgenic cells could be isolated by cutting the callus with a scalpel and sub-culture it to obtain stable transgenic callus (Figure 2). 1 g of dry callus could be obtained after 9 weeks of growth, from which we were able to purify > 5 mg starch. By using GBSSIa as an example we demonstrated that this method could be applied to overexpress genes of enzymes in callus to engineer starch biosynthesis. The transgenic callus had a significantly increased content of amylose, which is expected if the enzyme is active in the callus. In addition we were able to visualize the physical binding of GBSSIa in starch granules. In summary we suggest that the method can be used to do fast in vivo screening of variants of genes encoding enzymes involved in starch biosynthesis or other potential transgenes for bioengineering of starch in cereals. We also suggest that the method can be used to study the binding of proteins such as starch synthases in starch granules.
Vector engineering and transgenic callus generation
Transgenic callus cultures were induced from immature embryos of Golden Promise, by Agrobacterium-mediated transformation as described previously . Three plasmid vectors were used for transformation: a control vector (pUCEUbi:GFP-NOS) and two vectors designed to manipulate starch synthesis altering amylose content (pUCEUbi:TP-GFP-HvGBSSIaΔTP:NOS and pUCEUbi:HvGBSSIa-GFP:NOS). They were engineered by using the pUCE tool box for construction of cereal transformation vectors as described previously . They all contain the Hygromycin phosphotransferase selection marker gene (HPT), and the common maize ubiquitin promoter for constitutive expression of the transgene. In addition green fluorescent protein (GFP) was used as a marker. The control vector (pUCEUbi:GFP:NOS) was engineered for constitutive expression of GFP without an enzyme . The other binary vectors were designed to overexpress barley granule bound starch synthase Ia. (HvGBSSIa), In the case of pUCEUbi:TP-GFP-HvGBSSIaΔTP:NOS the transit peptide (TP) from GBSSIa was attached in front of GFP. The GBSSI-GFP and GFP-GBSSI sequences were synthesized artificially by DNA2.0 (http://www.dna20.com) fusing together the coding sequence of GFP with the coding sequence of HvGBSS with or without the native transit peptide, respectively. The two constructs were provided by the company in their cloning vectors and they were both digested with PacI (Fermentas, FastDigest), and re-cloned into the pUCE vectors Ubi:USER:NOS and Ubi:TP-USER:NOS (PacI digested and treated with calf intestine alkaline phosphatase) respectively. The two binary vectors are described in details in .
Transgenic callus maintenance and culture procedures
After two days on callus induction media the embryos were transferred to callus production media as described in the protocol in . After three weeks of culturing on production media the calli were examined with a ‘Wild MZ8 Leica’ stereo microscope, equipped with a GFP fluorescence detection system and calli pieces of full transgenic tissues were excised and sub-cultured on fresh production media to obtain homogeneous fully transgenic calli. Callus cultures were sub-cultured one additional time after 3 weeks, and after 9 weeks total the transgenic calli tissues were harvested and snap-freezed in liquid nitrogen and stored in a freezer (−80) to be directly used for analysis or for starch granules purification.
Light microscopy observations
For light microscopy analysis, 30 μm sections were cut from fresh frozen callus tissues using a HM 550 OM Cryostat microtome. Callus tissue sections, mashed tissue and purified starch granules were stained with Lugol´s iodine stain solution (250 mg I2, 2.5 g KI, 125 mL ddH2O) and mounted on a Zeiss Axioplan 2 Imaging microscope. Pictures were taken using the software provided by the manufacturer.
Measurement of starch content
Callus tissue was freeze-dried using a Heto LyoPro 6000 freeze dryer and ground using mortar and pestle. Pulverized material was used for starch content analysis. Starch content was determined using the ‘Total starch AOAC Method 996.11/AACC Method 76.13’ kit from Megazyme International Ltd. (Wicklow, Ireland) using the protocol recommended by the manufacturer for samples containing also d-glucose and/or maltodextrins.
Extraction of total RNA and quantitative real-time PCR
Total RNA was purified from transgenic callus tissues, barley endosperm 15–20 DAP and barley leaves; and cDNA was synthesised and quantitative real-time PCR (RT qPCR was conducted as described in . Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was co-amplified as housekeeping gene to calculate relative quantification of expression . All the analyses were conducted in three biological replicates each with three technical replicates using specific primers targeting the following starch biosynthetic genes: starch branching enzyme I (SbeI), starch branching enzyme IIa (SbeIIa), starch branching enzyme IIb (SbeIIb), starch synthase I (SSI), starch synthase IIa (SSIIa), starch synthase IIIa (SSIIIa), starch synthase IV (SSIV), granule bound starch synthase Ia (GBSSIa), granule bound starch synthase Ib (GBSSIb), glucan water dikinase I (GWDI). The primer sequences are described in Additional file 2: Table S1. Student’s t-test was used to compare the levels of expression of the same genes among the different tissues (with 95% confidence level).
Starch extraction and purification
Starch purification form barley grain was conducted according to the protocol described in Additional file 3: Protocol.
Scanning electron microscopy
Scanning electron microscopy was carried out as described in .
The amylose content was determined by iodine colorimetrics following the method described in .
Chain-length distribution of starch
This work was funded by The Danish Council for Independent Research Technology and Production Sciences and by Graduate School of Agriculture, Food and Environment (SAFE), Aarhus University. We are thankful to Monica Palcic and colleagues at The Carlsberg Laboratory, Denmark, for ideas and suggestions to this work.
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