Initial attempts were made to isolate protoplasts from a wide range of American elm tissues including young leaves (1st
position) from actively growing in vitro plants, seedlings, and greenhouse grown plants, as well as cotyledons, hypocotyls, and seedling roots. During these initial trials a number of cell wall degrading enzymes were evaluated at different concentrations and combinations, including the reportedly more active mixture used successfully in several Ulmus
spp. by Dorion et al. [18
] (data not shown). While protoplasts were occasionally obtained, the results were similar to what had been previously reported in that the yields were often very low [15
] and the success rate was inconsistent regardless of composition of the enzyme solution. Sometimes high yields as described by Dorion et al. [18
] were obtained, but this was not consistent even when the protocol was the same between isolation attempts and all reasonable precautions to use uniform plant material were taken. For example, a high yield of protoplasts was obtained from freshly emerged greenhouse leaves on March 30, 2011, but the isolation completely failed 5 days later on April 4, 2011 under same experimental conditions using fresh leaves from the same group of trees. The lack of reproducibility with young freshly emerged leaves and long exposure to a range of enzyme mixtures was deemed insufficiently reliable to proceed with regeneration and fusion experiments and was the impetus for this study.
Washing young in vitro American elm (Ulmus americana
) leaf tissue with water increased protoplast yields from an average of approximately 4000/g to 34 000/g. While this was a statistically significant increase in yield, it was far below the millions per gram reported for other species [22
], it did not work consistently between isolation attempts, and was not a large enough improvement to attempt culture and fusion. The relative ineffectiveness of this procedure for American elm may indicate that the interfering compounds were present in higher amounts, were more difficult to extract, and/or there were other factors inhibiting protoplast isolation in this species. This difference was likely responsible for the difficulties that have been encountered in isolating protoplasts from American elm compared to other species of elm and woody plants [15
]. As such, while this simple washing technique was capable of sufficiently removing the interfering compounds from some woody plants, including Ulmus glabra
], it was insufficient for the more recalcitrant American elm. This study was aimed at elucidating the underlying biochemical mechanisms and developing a systematic approach to circumvent the problem.
The increase in cell wall digestibility of leaves that had been leached with water can be reversed by incubating the leaf in its own extract [22
]. In the current study, this phenomenon was further investigated by incubating tobacco leaf tissue, a species with a readily digestible cell wall, in an aqueous extract of American elm leaves prior to enzymatic degradation. The aqueous extract effectively inhibited cell wall digestion in a dose dependent manner (Figure d), supporting the hypothesis that the resistant nature of the cell wall in woody plants such as American elm was because of a water soluble chemical or group of chemicals [22
]. This inhibiting effect could also be induced by using leachate from young leaves of in vitro American elm plants or leaf derived callus, indicating that this phenomenon occurs in vitro and was not limited to leaf tissues (data not shown). These observations with tobacco indicated that there was no requirement for any specialized enzymes or a fundamental difference in cell wall composition between resistant species such as American elm and a susceptible species such as tobacco. It was likely that the presence of compounds responsible for reduced digestibility would instill this trait in the cell wall of higher plants in general.
Figure 1 Digestibility and protoplast isolation frequencies of tobacco leaf discs treated with elm extract or hydroxycionnamic acids. Leaf discs were vacuum infiltrated (20 min) and incubated for 24 h in various concentrations of aqueous American elm leaf extract, (more ...)
Previous evidence suggests that the resilience of the cell wall in woody plants was because of the presence of the hydroxycinnamic acids, p
-coumaric and ferulic acid [22
]. Many plants are known to release or quickly synthesize hydroxycinnamic acids in response to mechanical damage or microbial attack [25
]. These compounds serve as precursors for the formation of hydroxycinnamoyl-CoAs which contribute to cell wall strengthening and lignification, ultimately reducing cell wall digestibility and inhibiting microbial infection [24
]. Plant tissues are likely to accumulate these compounds throughout their existence as they are continuously exposed to various stresses, which may contribute to the observation that leaves become increasingly resistant to enzymatic degradation with age [18
]. Protoplast isolation typically depends on mechanically wounding the tissue followed by incubation in cell wall degrading enzymes purified from fungi and it was likely that this process elicits the further release or synthesis of hydroxycinnamic acids which then modify the cell wall in some species.
The addition of either p
-coumaric or ferulic acid to washed leaf tissue from woody species has been shown to re-instate resistance to cell wall digestion similar to when incubated in their own leachate [22
]. Similar observations were made in the current study with tobacco where leaf tissue incubated in either compound reduced protoplast isolation in a dose dependant manner similar to American elm leaf extract (Figure a-d). These data support previous studies that suggest hydroxycinnamic acids were involved in re-enforcing the cell walls and increasing resistance to enzymatic degradation. This study also indicates that p
-coumaric and ferulic acid alone were capable of increasing cell wall resilience in a species that was typically easily digested, again suggesting that incorporation into the cell wall was not dependent upon any unique characteristics of woody plants.
An alternative approach to evaluate the role of hydroxycinnamic acids in cell wall digestibility and develop an efficient approach to isolate protoplasts from American elm was to inhibit their biosynthesis. Hydroxycinnamic acids are lignin precursors produced through the phenylpropanoid pathway. While cultural factors such as light exposure, temperature, plant nutrition, and ontological development are known to influence this pathway, numerous factors were evaluated in the current study and were insufficient to facilitate a reproducible protoplast isolation protocol from American elm (data not shown). A number of competitive PAL inhibitors, namely 2-aminoindane-2-phosphonic acid (AIP [30
)-2-aminooxy-3-phenylpropionic acid (AOPP, notation (S
) and L are equal [32
]) and O
-benzylhydroxylamine (OBHA [33
]) have been shown to significantly reduce the production of phenylpropanoids in a variety of species. In a previous study with Lycopersicon esculentum
suspension cultures, the addition of AIP to the medium effectively reduced the cells ability to accumulate wall bound phenolics when challenged with a fungal elicitor [34
]. As such, these three inhibitors were included in the growth medium to assess their influence on cell wall digestibility and protoplast isolation frequency in American elm suspension cultures.
Initial studies indicated that all three PAL inhibitors were deleterious to the growth of in vitro elm plants, and insufficient leaf tissue was produced for further experiments. Similar observations have been made for birch (Betula pubescens
) seedlings where growth was almost completely inhibited in the presence of 30 μM AIP [35
]. Consequently, further experiments were conducted using a two phase suspension culture system in which leaf tissue was embedded in alginate beads suspended in liquid culture medium (Figure a-f) to produce the source callus tissue. Of the three inhibitors, AIP was the most effective and facilitated cell wall digestion and protoplast isolation from American elm callus in a dose dependant manner, increasing the digestion rate from 11.8% (±3.27) in the controls to 65.3% (±4.60) in callus grown in 150 μM AIP (Figure c). Addition of AOPP resulted in a modest increase in protoplast isolation while OBHA had no beneficial effect (data not shown). These data support previous studies that found AIP to be more effective at inhibiting PAL than the other two compounds [31
Figure 2 Two-phase suspension culture of American elm (Ulmus americana) with and without AIP. Cultures were started with leaf tissue embedded in alginate beads and cultured in liquid MSO media supplemented with 5 μM BA, 1 μM NAA: a; Freshly prepared (more ...)
Figure 3 Protoplast isolation rates of American elm callus grown in various concentrations of AIP. American elm callus cells were grown on MSO basal medium supplemented with 5 μM BA, 1 μM NAA, and various levels of AIP and the percentage that developed (more ...)
The increase in cell wall digestibility with increased levels of AIP was accompanied by a reduction in the flavonoid content in the tissue when stained with NPR (Figure ). While the flavonoid content in and of its self was unlikely to influence cell wall digestibility, flavonoids are also synthesized through the phenylpropanoid pathway and have been used as an indicator for the activity of the pathway [36
]. While total phenol content had been used in this capacity in previous reports, AIP was found to interfere with this assay at the concentrations used in this study (data not shown). The callus produced in the presence of AIP also remained creamy white in colour (Figure e-f). This was in stark contrast to the brown coloration observed in the control callus (Figure c-d), indicating that AIP was inhibiting the accumulation of polyphenols. Together, the inverse relationship between both flavonoid and polyphenol contents with protoplast isolation rates strongly suggests that the factor inhibiting enzymatic digestion of the cell wall in American elm was a product of the phenylpropoanoid pathway. Cell wall digestibility was significantly increased by selectively inhibiting this pathway which facilitated the development of an efficient, reproducible protocol for the isolation of American elm protoplasts.
Figure 4 Visualization of flavonoids in American elm callus. American elm callus was grown in liquid MSO basal medium supplemented with 5 μM BA, 1 μM NAA, and various levels of AIP. The callus was then stained with natural product reagent (NPR) (more ...)
While the approach of using AIP has dramatically increased our ability to consistently obtain large numbers of protoplasts from American elm, inhibiting the phenylpropanoid pathway with AIP is known to reduce the accumulation of biomass in a number of species [35
]. In order for this technology to have practical application in developing protoplast regeneration systems in difficult woody species, it was critical to examine the viability and growth potential of the resulting protoplasts. In the current study, protoplasts obtained from tissue cultured in the presence of 100 μM AIP had a relatively high viability, typically ranging from about 80% to over 90% based on fluorescein diacetate (FDA) staining (Figure a). The viability observed in the protoplasts derived from this system was comparable to the upper levels observed in other species of Ulmus
where protoplast regeneration has been successful [18
Figure 5 Protoplasts and developing cells obtained from American elm callus. Images depict American elm tissue cultured in MSO basal medium supplemented with 5 μM BA, 1 μM NAA, and 100 μM AIP after a 4 h digestion in cell wall degrading (more ...)
This relatively high viability may be a consequence of obtaining protoplasts from young actively growing callus tissue, the reduced duration of exposure to the potentially deleterious cell wall degrading enzyme solution, or a combination of these and other factors. Whereas most protoplast isolation protocols in American elm require 4 to 48 h of incubation in enzyme solution [12
], in our system this can be reduced to 1-4 h. While successful protoplast isolation from American elm was generally inconsistent and often worked less than half of the time in previous attempts [15
], the suspension cultures grown in the presence of AIP have reliably yielded viable protoplasts on a bi-weekly basis for more than 5 months and continue to be productive.
Protoplasts isolated using this system and suspended in low melting point agarose beads cultured in liquid Kao and Michayluk [37
] medium supplemented with 5 μM NAA and 5 μM BA started to re-develop cell walls within 2 days and showed early signs of cell division (Figure b,c). Efficient re-synthesis of the cell wall is a pre-requisite for cytokinesis in protoplasts and is influenced by the conditions used for cell wall digestion and isolation [38
]. Previous studies have reported that cell wall formation in American elm protoplasts occurs sporadically and starts later, between 4 and 21 days post-culture [15
]. After 6 days of culture in the current system, 28.5% (±3.59) of the protoplasts had initiated cell division and well developed cell walls were present (Figure d-g). This compares favourably to previous studies where American elm protoplasts had much lower division rates, as low as 1%, was only observed in some preparations, and started 9-21 days after initial culture [15
]. In previous studies where cell division was observed, the cells failed to continue to divide and there has been no previous report of protoplast-derived callus regeneration in this species [15
]. In the current system, the cells continued to divide and protoplast-derived calli were produced by day 14 (Figure h-k). As such, this protocol represents the first report of callus regeneration from American elm protoplasts more than 30 years after the first attempt.
The consistent supply of protoplasts has also facilitated initial studies into protoplast fusion technologies to realize the long term-goal of producing interspecific elm hybrids which may exhibit resistance to DED [12
]. Thus far, we have optimized various electrofusion parameters to conduct fusion experiments with American elm protoplasts. The process of somatic fusion using electroporation can be detrimental to protoplasts, particularly those that are less viable and unable to withstand the repeated centrifugation and culture manipulations required. Stable heterokaryons have been observed (Figure c-g), putative hybrid cells remained viable after being transferred into agarose beads (Figure g), and initial signs of cell division have been observed in protoplasts that have been exposed to the electrofusion procedure. While electrofusion parameters need further optimization to maximize fusion while minimizing cell lysis, these preliminary results indicate that protoplasts produced through this system were sufficiently robust to survive electrofusion and will lay the foundation to initiate somatic fusion with DED-resistant species of elm.
Figure 6 Electrofusion of American elm protoplasts. American elm protoplasts, a; aligned in AC current, b; shortly after DC pulses, c; fused together after DC pulses, d-f; fused heterokaryon with nuclei stained with DAPI with UV excitation and decreasing light (more ...)
It was difficult to make direct comparisons between this study and previous efforts to regenerate U. americana
protoplasts because of different source material, media, plating densities, and culture systems. Nevertheless, sustained proliferation of protoplasts (derived through this protocol) into calli compared to previous unsuccessful attempts may be a result of the reduction of polyphenols in the source tissue, which are known to inhibit subsequent protoplast division [39
]. As well, a shorter period of incubation with much less stress on the protoplasts may contribute to their higher growth response. Regardless of the mechanisms, the reproducibility of the current system in providing regenerable protoplasts represents a significant step forward and a solid foundation to develop a protoplast manipulation system for American elm. This development could ultimately facilitate the development of DED resistant somatic hybrids, cybrids, and provide an alternate avenue to insert large segments of DNA. Protoplast based systems have been used to generate novel germplasm with disease or insect resistance in a range of species such as potato [40
], tobacco [41
], and citrus [7
]. Given the multigenic response involved in DED resistance, together with the long life span of U. americana
relative to the rapidly evolving pathogen, protoplast technologies are particularly appealing to integrate stable disease resistance traits found in some Asiatic Ulmus
species into U. americana