Doubling Acclimatization Survival of Micropropagated American Chestnuts with Darkness and Shortened Rooting Induction Time
One of the most difficult processes of micropropagation is rooting and acclimatizing in vitro shoot cultures, especially for hardwood tree species. As more transgenic lines of potentially blight-resistant American chestnut (Castanea dentata) are developed, we expect to produce thousands of tiny shoots to be rooted, transferred to potting mix, and grown to a large enough size for planting outdoors. Many shoots are lost during rooting and acclimatization, so pinpointing factors that enhance survival is extremely important. Five factors were examined in relation to acclimatization success — light intensity, light color, time in rooting medium, temperature, and presence of activated charcoal. The percentage of plantlets surviving from rooting initiation to 16 weeks in the growth chamber was increased from 33 to 67% by rooting the shoots in darkness instead of on a light bench. The best combination of rooting factors was to place shoots in rooting medium containing activated charcoal in complete darkness for only four days at 25C. This combination of factors increased plantlet survival from approximately 33% using the original rooting protocol to 73%. Finding that American chestnut plantlets have better acclimatization survival after being placed in rooting medium for only four days should enhance many laboratory practices. Shortening the time in rooting medium and including a period in darkness will increase the survival of novel transgenic American chestnut lines, allowing them to be planted in field trials more quickly.
Significance to the Nursery Industry
This study observed the effects of altering the rooting protocol of American chestnut. When micropropagating recalcitrant hardwoods, some of these factors (such as darkness, light color, temperature, activated charcoal) may be reasonable starting points for protocol modification. Transgenic American chestnuts that are resistant to chestnut blight will be of considerable value to nursery growers and foresters.
Introduction
Micropropagation has gained renown in the last 30 to 40 years as a tool for clonally replicating desirable genotypes of plants (8, 19). This technology, combined with genetic transformation, has the potential to bring a famous tree species back to prominence in North American forests.
American chestnut (Castanea dentata) comprised almost 25% of Appalachian timber in the eastern United States pre-1900 (26). These trees once provided strong, rot-resistant lumber used for diverse purposes such as telephone poles, cabins, furniture and fence posts (9). Its nutritious nuts provided food for both domestic and wild animals, and was a staple food for rural Americans (15). Due to the introduction of the chestnut blight (Cryphonectria parasitica) from Asia in the late 1800s, the susceptible American chestnut was nearly wiped out (6, 13). Now the species has been reduced to an understory shrub, sprouting from old stumps and usually killed by blight before reaching maturity (26).
The American Chestnut Research and Restoration Project at SUNY-ESF is currently developing new genotypes of American chestnut by introducing various genes to produce timber-type trees with enhanced resistance to fungal infections such as chestnut blight and Phytophthora (Ink disease) (22). Using an Agrobacterium-mediated transformation protocol, chestnut embryos are transformed with genes encoding putative resistance-enhancing genes (22). The transformed embryos are regenerated to shoots, multiplied, and then individually rooted and acclimatized in a growth chamber and greenhouse before being planted in the field. The sequence of steps from turning a transformed embryo containing the gene of interest into a tree in the field can take as long as two years (unpublished data), and a low success rate in one crucial step can severely slow the process. The most difficult steps in this pathway are rooting the shoots from in vitro shoot culture and acclimatizing them to conditions in a growth chamber or greenhouse.
Rooting shoot cultures of woody species can be a challenging endeavour, especially for chestnut species (20). When rooting chestnut shoots derived from mature trees in vitro, meristemoids fail to develop in the cambial tissues, and high levels of polyamines are also expressed, which may inhibit rooting (2). The SUNY ESF tissue culture laboratory published research on rooting American chestnuts in vitro in 1997, but did not address acclimatization survival rates (31). Our procedure had not been substantially altered in recent years (21); this is the first time we have done a systematic re-evaluation of several of the basic parameters. Serres et al. also examined American chestnut rooting, and determined that without external application of a plant growth regulator all shoots would die; they also did not publish acclimatization survival rates (27).
Many factors can have a significant effect on rooting rate and subsequent acclimatization success, including exposure to light during the rooting stage. Some close relatives of American chestnut are best rooted in darkness, such as Japanese chestnut (Castanea crenata) (28), Chinese chestnut (Castanea mollissima) (25), and European chestnut (Castanea sativa) (11). Other colors of light besides white light have also been used to enhance rooting in tree cultures, although the responses between tree species are very different. In pear (3) and cherry (17), the application of red light increased rooting rates in comparison to white light.
Activated charcoal is used in tissue culture medium for a variety of purposes — to induce germination, to remove excess plant growth regulators from the shoots, to provide shade, and to induce rooting (29). Activated charcoal was typically added to American chestnut rooting medium to absorb excess rooting hormones (31).
Lowered temperatures have also been shown to induce rooting in micropropagated trees (4, 18). The time spent exposed to rooting hormones in rooting medium can also affect acclimatization success (14, 23).
Among the many factors which affect the rooting and acclimatization process, we chose to carry out a series of multiple-variable factorial studies to examine five factors — exposure to light, light quality, activated charcoal, temperature, and time in rooting medium. Our overall goal was to test our current procedure (full light, white light, in activated charcoal, 25C (77F), for eight days) to see whether any of these factors could be modified to significantly increase rooting success and acclimatization survival of tissue-cultured American chestnut plantlets.
Materials and Methods
Experiment 1 — Light exposure and presence of charcoal. The first rooting experiment was conducted as a 2 × 2 factorial with two levels of each factor: exposure to light (light and dark) and activated charcoal (with charcoal and without charcoal). Clones Ellis #1 and WB 275-27 blocked the experiment. WB275-27 is a shoot culture of a wild-type somatic embryo line started at SUNY-ESF from a tree at the TACF Meadowview Farm. Ellis #1 is a shoot culture of a wild-type somatic embryo line started at SUNY-ESF from a tree in New York State donated by the New York chapter of The American Chestnut Foundation. Embryo lines were regenerated to axillary shoot cultures according to the procedure in Maynard et al (21).
Plant material. American chestnut shoot cultures of Ellis #1 and WB 275-27 were maintained in Magenta® GA-7 vessels with vented lids containing Chestnut Stage II Multiplication (CHNII) medium [Woody Plant Medium (WPM) salts (Phytotechnology Laboratories), 109 mg·liter−1 Nitsch & Nitsch vitamins (Phytotechnology Laboratories), 6 mM CaCL2, 3 mM MgSO4, 1 μM 6-benzylaminopurine (BA), 0.5 μm indole-3-butyric acid (IBA), 3.5% sucrose, 3.5 g·liter−1 Phytagel (Sigma-Aldrich) and brought to pH 4.5 with 1N KOH]. All media were autoclaved for 20 minutes at 15 psi and 121C (250F). Small axillary shoots were transferred to Magenta® GA-7 vessels with vented lids contained Pre-Rooting Medium [WPM salts, 109 mg·liter−1 Nitsch & Nitsch vitamins, 2.3 mM 2-(N-morpholino)ethanesulfonic acid (MES), 12.5 μM polyvinylpyrrolidone 40000 MW (PVP-40), 0.22 μM BA, 3% sucrose, 3.5 g·liter−1 Phytagel and brought to pH 5.5 with 1N KOH] for elongation. There were 20 shoots per vessel. The vessels were kept on a light bench with a 16-hr photoperiod at a light intensity of 31 μmol·sm−2 for at least six weeks, or two transfers cycles, before the rooting treatments were applied.
In vitro rooting procedure. Shoots chosen for experimentation were required to have a height of 2.5 cm (1 in) and at least one leaf (< 1 cm (0.4 in) in length). Ten shoots were removed from the Pre-Rooting Medium and excised from the basal callus. The basal end was cut at a 30° angle from vertical to increase the exposed surface area of the vascular tissue. The shoots were then dipped into a filter-sterilized 10 mM IBA aqueous solution at a depth of 0.5 cm (0.2 in) for two minutes. Ten shoots of each line were placed into a Magenta® GA-7 vessel with a vented lid containing Rooting With Charcoal Medium (½ strength Murashige and Skoog Basal Salt Mixture (Phytotechnology Laboratories), 3% sucrose, 2 g·liter−1 activated charcoal (separately autoclaved in a 1 liter Pyrex jar) 3.5 g·liter−1 Phytagel and brought to pH 5.5 with 1N KOH). Ten shoots of each line were placed in vessels with Rooting Without Charcoal Medium (½ strength Murashige and Skoog Basal Salt Mixture, 3% sucrose, 3.5 g·liter−1 Phytagel and brought to pH 5.5 with 1N KOH). Activated charcoal must be autoclaved dry, separately from the medium, and added after the medium has cooled to 55C, or it will quickly settle to the bottom and leave the medium clear.
Three replicates of ten shoots of each of the two clone lines (N = 120) were tested. Three of the Rooting With Charcoal vessels and three of the Rooting Without Charcoal vessels were placed on a light bench with a 16/8 h light/dark photo-period at a light intensity of 31 μmol·m−2·s−1 for eight days. The other three vessels of Rooting With Charcoal vessels and three of the Rooting Without Charcoal vessels were placed in a dark cabinet in the same room with otherwise equal conditions for eight days. On the ninth day, all the shoots were transferred to Post-Rooting Medium (same as Pre-Rooting Medium) and kept on the light bench for 21 days.
Potting. In vitro shoots that had both roots and healthy shoot tips after 21 days in post-rooting medium were carefully removed from the medium and rinsed under distilled water to dislodge excess medium. Stem height, number of roots, length of longest root, number of leaves, and length of longest leaf were recorded at time of potting. The potting mix used was Fafard's Super-Fine Germinating Mix (Conrad Fafard, Inc., Agawam, MA) in 7″ cylindrical black plastic Deepots (Stuewe & Sons, Inc., Tangent, OR). A plastic bag (1.5 Mil, 16.5 × 13.9 × 2.5 cm) was placed over the top of each pot and secured with a large rubber band. All plants were placed into a Conviron CMP5090 growth chamber with a SK 300 steam humidifier (Conviron, Winnipeg, Manitoba, Canada). Chambers were maintained at 80% relative humidity and held at 20C (68F) day temperatures and 16C (61F) night temperatures. The chambers had a 16-hr photoperiod at a light intensity of 100–120 μmol·m−2·s−1. After 14 days, a corner of the plastic bag was cut to increase air flow. At this time the plantlets were watered with a standard N-P-K fertilizer, Miracid (The Scott's Company, LLC, Marysville, OH) (300 mg·liter−1). After 7 days the bags were opened completely at the top to form a collar to maintain high humidity around the plantlet. The pots were watered with 300 mg·liter−1 Miracid as needed to keep the potting mix moist. At 8 weeks the surviving plantlets were measured for stem height, number of leaves, and longest leaf length.
Experiment 2 — Light color. The six light color treatments were white, dim, dark, red, blue, and far-red in a randomized complete block design. The white treatment is the current standard procedure in the lab, as described above. The dark treatment consisted of the shoots being placed in complete darkness in a closed cabinet. The red (530 nm), blue (450 nm), and far-red (630 nm) treatment shoots were placed in a large box separated into three compartments, each completely lined with aluminum foil. Openings in the top of the box allowed for LED lights of each color (Norlux, Carol Stream, IL) to be inserted. All treatments were connected to a timer set for 16 hr photoperiods.
Four blocks were used for this experiment: WB 275-27, a wild type (described above) and parent clone of three transgenic lines; AN-2XG5, a transgenic line containing pTACF3 (VspB promoter and an oxalate oxidase gene); and KS-2PG1 and KS-2PG2, two events containing pTACF7 (an oxalate oxidase gene and an anti-microbial peptide). The preparation of the plant material was the same as described in Experiment 1.
Two vessels of each clone line containing ten shoots were placed in each treatment, for a total of 80 shoots per treatment (N = 480). Shoots chosen for experimentation were standardized by using shoots which reached a height of 2.5 cm with at least one leaf (< 1 cm in length). The in vitro rooting procedure used was the same as described in Experiment 1 except that the shoots were kept in rooting medium for only 6 days instead of 8 days. At 8 weeks the rooted plantlets in the growth chamber were assessed for survival.
Experiment 3 — Temperature and time of exposure. The experiment was conducted as a 2 × 2 × 2 factorial with two levels for each factor, time (4 days and 8 days), temperature [4C (40F) and 25C (77F)], and presence of activated charcoal (with and without). The experiment was blocked by three transgenic events derived from the Ellis #1 somatic embryo line: LN-4LE4 and LN-4LE9, two events containing the same pyramid vector of laccase, oxalate oxidase, and an anti-microbial peptide; and AD-4XX8, containing oxalate oxidase. The plant material was prepared and rooted according to the procedure in Experiment 1, except that only six shoots were placed in each vessel. All of the treatments were applied in darkness. Two vessels (one with and one without activated charcoal) of each clone line were placed in 25C (77F) for 4 days, 25C (77F) for 8 days, 4C (40F) for 4 days, or 4C (40F) for 8 days. There were 18 shoots in each treatment, for a total of 144 shoots. After the indicated number of days in the Rooting Medium, the shoots were moved into Post-Rooting Medium (described in Experiment 1) for three weeks. They were then potted according to the procedure in Experiment 1, and after two weeks in the growth chamber they were assessed for survival.
Statistical analysis. In each experiment, the different clone lines were treated as blocks in a randomized complete block design. The data were analyzed using PROC GLM in SAS 9.0, and means were tested with Tukey analysis. Additionally, during Experiment 1, two sample t-tests were performed on variables (number of roots, number of leaves, length of roots, and height) which were measured at potting; then the initial values for the plantlets that survived to 16 weeks were tested against those of the plantlets that died.
Results and Discussion
Exposure to light and presence of charcoal. Keeping shoots in the dark while on Rooting Medium improved rooting success; 89% of shoots kept in the dark formed roots, compared to 67% of those kept in light (F = 19.16, p < 0.001). The presence or absence of activated charcoal in the Rooting Medium did not have a significant effect on rooting (F = 0.66, p = 0.427) (Fig. 1). There was no significant interaction between these two variables (F = 1.29, p = 0.271).
Citation: Journal of Environmental Horticulture 31, 2; 10.24266/0738-2898.31.2.77
The number of roots per shoot was significantly increased by the dark treatment compared to the light treatment (F = 15.47, p < 0.001). Activated charcoal had no effect on either treatment (Fig. 2). Again, there was no significant interaction between these two variables (F = 2.18, p = 0.141). The main effect of the light/dark treatment was significant (F = 15.47, p < 0.001) while the main effect of the activated charcoal treatment was not (F = 2.70, p = 0.102).
Citation: Journal of Environmental Horticulture 31, 2; 10.24266/0738-2898.31.2.77
Shoot-tip necrosis at time of potting was doubled in the light without charcoal treatment compared to the other three treatments (F = 6.76, p = 0.011). The other three treatments were not significantly different in levels of shoot tip necrosis (Fig. 3).
Citation: Journal of Environmental Horticulture 31, 2; 10.24266/0738-2898.31.2.77
From potting to 8 weeks in the growth chamber, the dark with charcoal plantlets had 95% acclimatization survival, followed by light with charcoal with 90% survival, then dark without charcoal with 70% survival, and light without charcoal with 49% survival.
A higher number of roots at time of potting (p = 0.07) was less important in predicting 8-week survival than a higher number of leaves at time of potting (p < 0.01) (Fig. 4). More height (p < 0.01) was also an excellent indicator of future survival, while longer root length was less useful (p = 0.03).
Citation: Journal of Environmental Horticulture 31, 2; 10.24266/0738-2898.31.2.77
Light quality. The color of light used during rooting did not significantly affect the percentage of shoots which formed roots: 100% in dark, 94% in dim and far-red, 91% in blue and red, and 87% in white (F = 0.86, p = 0.512).
The number of roots per shoot was highest in the dark treatment and lowest in the white light treatment (Fig. 5); the treatments were significantly different overall (F = 3.66, p = 0.004). Shoots cultured in the dark had significantly higher numbers of roots than in the red, blue and white treatments, but not in the dim and far-red treatments.
Citation: Journal of Environmental Horticulture 31, 2; 10.24266/0738-2898.31.2.77
The dark treatment had the least shoot-tip necrosis at 22% (Fig 6.). The white treatment had the most shoot tip necrosis, 84%, but was not significantly different from either blue or red (both 75%) (F = 8.10, p < 0.001).
Citation: Journal of Environmental Horticulture 31, 2; 10.24266/0738-2898.31.2.77
For acclimatization survival, far-red had the highest 8 week survival rate at 90% (Fig. 7). This was not significantly different from blue (73%) or dark (70%), but was significantly higher than red (51%) and white (43%). The dim treatment plantlets became severely contaminated with fungal gnats and were not included in the survival assessment.
Citation: Journal of Environmental Horticulture 31, 2; 10.24266/0738-2898.31.2.77
Temperature and time of exposure.The presence of activated charcoal was not significant in any of the treatments, so the data were combined over the other two factors.
After two weeks, survival was highest in the 4-day, 25C (77F) plantlets with 73% survival, and lowest in the 8-day, 25C (77F) plantlets with 7% survival (Fig. 8). The difference between the 4-day and 8-day factors was significant (F = 29.49, p < 0.001), while the difference between the 25C (77F) and 4C (40F) treatments was not (F = 0.02, p = 0.890). Interaction was present (F = 8.01, p = 0.011) due to the difference of survival between the 4-day and 8-day treatments at 25C (77F).
Citation: Journal of Environmental Horticulture 31, 2; 10.24266/0738-2898.31.2.77
Activated charcoal. Activated charcoal has been shown to enhance rooting in many species (29). In this experiment, the addition of activated charcoal to the culture medium had a large positive effect on plantlet survival in the growth chamber, without significantly changing rooting rate or number of roots in vitro. While the dark without charcoal treatment had a slightly higher rooting percentage and higher number of roots per shoot, the dark with charcoal plantlets had a higher rate of survival. The higher rate of survival was due to the activated charcoal in the medium, which can absorb excess plant growth regulators and phenolic compounds (29). Due to this slight improvement in acclimatization, we decided to conduct the rest of the light experiments using activated charcoal in the Rooting Medium. We examined the presence of activated charcoal again during the exposure time and temperature experiment, but its presence had no statistical effect.
Exposure to light. A period of darkness at the onset of rooting combined with exposure to an auxin has been shown to be beneficial for rooting many woody plant species. Chinese chestnuts (16), Japanese chestnuts (28), and hybrid walnuts (7) were all placed in darkness while in rooting medium. Our findings agreed with these methods, since the dark treatments consistently outperformed the light treatments in percent rooting, number of roots, and survival.
We were surprised to find that there was no significant difference in rooting between the light color treatments. In other studies, rooting varied widely under different light color treatments, such as in pear (3), and cherry (17).
For our American chestnuts, the dark and far-red treatments appear to be the most effective for rooting success, with the highest number of roots and least shoot-tip necrosis. Both treatments also had high acclimatization survival rates, along with, surprisingly, the blue treatment (Fig. 7). In black cherry, blue light strongly inhibited root growth (10). In peach, rooting with far-red light had the worst acclimatization rate (1), while in plum irradiating with far-red light had the most successful acclimatization rates (12). White light exposure during rooting strongly inhibited root formation in black cherry (10). This range of results demonstrates that findings from other species cannot be relied on to predict behaviour in chestnuts, but that examining a factor that produced so much variability can be advantageous. Due to the expense of setting up a LED-lit far-red chamber, it is more cost-efficient to use the dark treatment for subsequent rooting protocols as the two treatments were not significantly different for survival at eight weeks even at α = 0.01.
Predicting acclimatization success. Surprisingly, we found that the number of roots per shoot was much less significant in predicting acclimatization success than the number of leaves per shoot (Fig. 4.). Generally, the number of roots is considered a key indicator of future plantlet survival; higher number of roots significantly predicted acclimatization success in micropropagated teak (5). However, our finding agrees with studies observing transgenic plum, where root number did not correlate with acclimatization success, but leaf number and shoot height were critical (12). In an earlier study performed on American chestnut seedlings, we found that seedlings with more initial aboveground biomass outperformed seedlings with larger initial root systems after 3 years in the field (unpublished data). Potting every rooted shoot without regard for other characteristics such as height and leaf number results in a waste of time, labor and materials. Based on this finding, we now recommend that in vitro shoots should be chosen for rooting only if they have more than 6 leaves prior to rooting and are greater than 3 cm in height so they will be large enough to survive after potting.
Time spent in rooting medium. Decreasing the time spent in rooting medium from 8 to 4 days greatly increased plantlet survival in the growth chamber. In contrast to these shorter rooting durations, 14 days was the original recommendation for rooting American chestnuts (31), and a revised version recommended 10–12 days (21). Our finding that 4 days in rooting media increases survival rates is supported by protocols used to root other Castanea species; Chinese chestnuts (Castanea mollissima) are placed in rooting medium for only 5 days before being transferred to a post-rooting medium (16), as are Japanese chestnuts (28), European chestnuts (11), and more distantly related hybrid walnuts (7). An alternate protocol recommends leaving Castanea sativa shoots in a high-auxin concentration rooting medium for 24 hours (30), as does another American chestnut micropropagation protocol (27).
Shortening the duration in rooting medium from 10 to 4 days and placing shoots in darkness while in rooting medium more than doubled rooting percentage and eight-week acclimatization survival. These improvements in the micro-propagation protocol of the American chestnut will reduce loss of plantlets during the post-rooting and acclimatization, speeding production and increasing the number of field-ready plants for the 2013 planting season. These advancements will assist the American Chestnut Foundation's goal of eventually reintroducing blight-resistant American chestnut back into its native range. Potential benefits of this re-introduction include carbon sequestration, production of rot-resistant hardwood timber and increased food for wildlife (9).
Exposure to light and presence of charcoal: Percentage of rooted shoots per treatment after 3 weeks in Post-Rooting at time of potting medium. Treatments with same letter are not significantly different by Tukey at α = 0.10. Error bars indicate +/− one standard error of the mean.
Exposure to light and presence of charcoal: Number of roots per shoot after 3 weeks in Post-Rooting medium at time of potting. Treatments with same letter are not significantly different by Tukey at α = 0.10. Error bars indicate +/− one standard error of the mean.
Exposure to light and presence of charcoal: Percent shoot-tip necrosis after 3 weeks in Post-Rooting medium at time of potting. Treatments with same letter are not significantly different by Tukey at α = 0.10. Error bars indicate +/− one standard error of the mean.
Exposure to light and presence of charcoal: Factors measured at potting which influenced survival after 16 weeks in the growth chamber. Error bars indicate +/− one standard error of the mean.
Light quality: Number of roots per shoot by light treatment after 3 weeks in Post-Rooting medium at time of potting. Treatments with same letter are not significantly different by Tukey at α = 0.05. Error bars indicate +/− one standard error of the mean.
Light quality: Percent shoot-tip necrosis after 3 weeks in Post-Rooting medium at time of potting. Treatments with same letter are not significantly different by Tukey at α = 0.10. Error bars indicate +/− one standard error of the mean.
Light quality: Plantlet survival at 8 weeks in growth chamber, by light color rooting treatment. Treatments with same letter are not significantly different by Tukey at α = 0.10. Error bars indicate +/− one standard error of the mean.
Temperature and time of exposure: Overall plantlet survival at 2 weeks in growth chamber. Treatments with same letter are not significantly different by Tukey at α = 0.10. Error bars indicate +/− one standard error of the mean.
Contributor Notes
This research was supported by The Consortium for Plant Biotechnology Research, Inc. (CPBR), Forest Health Initiative (FHI), The New York Chapter of The American Chestnut Foundation (TACF), and ArborGen LLC. Many thanks and much appreciation are due to Linda M. McGuigan, who was instrumental in developing the original American chestnut rooting protocol and also keeps a spotless, incredibly well organized lab.
2Department of Environmental Forest Biology. Corresponding author: allison.dt.oakes@gmail.com.
3Department of Forest and Natural Resources Management.