in vitro rooting of hybrid hazelnuts (corylus avellana x ......90 temporary immersion system 91 the...
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In vitro Rooting of Hybrid Hazelnuts (Corylus avellana X Corylus americana) in Temporary Immersion System
Journal: Botany
Manuscript ID cjb-2019-0206.R1
Manuscript Type: Article
Date Submitted by the Author: 24-Feb-2020
Complete List of Authors: Nicholson, James; University of Guelph Ontario Agricultural College, Plant AgricultureShukla, Mukund; University of Guelph Ontario Agricultural College, Plant AgricultureSaxena, Praveen; University of Guelph Ontario Agricultural College, plant Agriculture
Keyword: rooting, rocker culture system, acclimatization, plant density, Hazelnut micropropagation
Is the invited manuscript for consideration in a Special
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1 In Vitro Rooting of Hybrid Hazelnuts (Corylus avellana X Corylus americana) in
2 Temporary Immersion System
3 Nicholson James, Shukla Mukund R. and Saxena Praveen K.*
4 Gosling Research Institute for Plant Preservation, Department of Plant Agriculture,
5 University of Guelph, Guelph, ON N1G 2W1, Canada.
6 Nicholson James, Email: [email protected]
7 Shukla Mukund R., Email: [email protected]
8
9 *Corresponding author
10 Saxena Praveen K., Email: [email protected]
11 Gosling Research Institute for Plant Preservation (GRIPP),
12 Department of Plant Agriculture, University of Guelph, ON, Canada N1G 2W1
13 Tel: 519-824-4120 ext 52495; Fax 519-767-0755
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18 Abstract
19 Commercial micropropagation of hybrid hazelnuts (Corylus avellana L. X C. americana
20 Marshall) has been limited due to their poor rooting ability in vitro as well as ex vitro leading to
21 high mortality of plantlets transplanted in the greenhouse. The objective of this study was to
22 develop an efficient and cost-effective protocol for rooting and plantlet acclimation of in vitro
23 grown hazelnut shoots. Efficient in vitro rooting was accomplished in a rocker based temporary
24 immersion bioreactor system. The use of a temporary immersion system (TIS) in combination
25 with the inert substrate Oasis® In Vitro Express (IVE) significantly improved the in vitro rooting
26 efficiency (100%) compared to semi-solid medium (27%) after 4 weeks of culture. A higher
27 density (36 explants/vessel) of shoot explants in the TIS was found to support a significantly
28 greater shoot height, chlorophyll content and longest root length, compared to the lowest density
29 treatment (12 explants/vessel). Efficiency of rooting and the number of roots formed were
30 similar in both high and low density of explants in the culture vessels and the resulting plantlets
31 exhibited >80% survival in the greenhouse. These results demonstrate the usefulness of rocker
32 based TIS for commercial micropropagation of hazelnuts and potentially other tree species.
33 Key words: Hazelnut micropropagation, rooting, rocker culture system, acclimatization, plant
34 density
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38 Introduction
39 Micropropagation has been proven as an effective method to rapidly produce a large number of
40 high-quality plants that are genetically identical and disease-free (Chandra et al. 2010). The
41 success of using micropropagation to mass produce a given plant species on a commercial scale
42 can often be severely limited due to the high plant mortality caused when plantlets are
43 transferred from their in vitro laboratory environment to ex vitro greenhouse or field conditions
44 (Pospíšilová et al. 1999; Chandra et al. 2010; Pérez-Jiménez et al. 2015). This transition from in
45 vitro to ex vitro is particularly stressful for plantlets since their unique in vitro environment (high
46 relative humidity, low light irradiance, reduced gas-exchange and sugar in the culture medium)
47 can cause plantlets to develop an atypical morphology, anatomy and physiology (Pospóšilová et
48 al. 1999; Shin et al. 2014; Pérez-Jiménez et al. 2015; Shekhawat and Manokari 2017; Revathi et
49 al. 2019). To improve survival of plantlets during the acclimation phase in the greenhouse, the
50 plantlets can be hardened in vitro, enhancing their vigour and ability to acclimate ex vitro.
51 Common in vitro hardening techniques include reduction in the concentration of sugar and basal
52 salts, addition of growth regulators, increasing the light irradiance, improving gas-exchange,
53 altering the state of the culture medium (i.e. semi-solid or liquid medium), increasing the culture
54 vessel size and use of an inert substrate during in vitro rooting (McClelland and Smith, 1990;
55 Pospíšilová et al. 1999; Ayenew et al. 2013; Economou 2013; Shin et al. 2013; Rezali et al.
56 2017).
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57 In vitro propagation methods are highly influenced by the physical state of the medium, with
58 significant implications for commercial production. Semi-solid medium is commonly used for
59 micropropagation especially for in vitro rooting to provide support for root development
60 (Wawrosch et al. 2005; Jones et al. 2007; Pati et. al. 2011; Latawa et al. 2016). However, gelling
61 agent must be removed from the in vitro grown roots before transplanting in the
62 greenhouse/growth facility to prevent root decay. This process can damage the roots and increase
63 the chances of infection. Thus, a low efficiency of plantlet formation combined with high cost of
64 propagation limit the commercialization of economic important crops such as hazelnut using
65 micropropagation (Simonton et al. 1991).
66 Micropropagation with liquid medium has been suggested since the beginning of plant tissue
67 culture studies as it supports an easy handling of shoots and roots, frequent addition or exchange
68 of fresh medium, and less oxidative stress on the explant (Caplin and Steward 1949). The
69 usefulness of the liquid medium compared to the semi-solid medium has been reported for a
70 number of species including hazelnut (Wawrosch et al. 2005; Jones et al. 2007; Pati et. al. 2011;
71 Latawa et al. 2016). A major issue in liquid culture systems is keeping individual micro-shoots in
72 an upright position during in vitro rooting. There are several reports for in vitro rooting in liquid
73 medium using artificial support like glass beads or coir (Gangopadhyay et al. 2002; Santos Diaz
74 and Carranza Alvarez 2009; Shukla et al. 2019). However, these approaches have not been tested
75 for hazelnut micropropagation. In the present study, the effectiveness of OASIS® IVE foam has
76 been evaluated for in vitro rooting of hazelnut. The autoclavable cellular foam with dibble hole
77 allows easy insertion of microshoots for root development in the liquid medium and can
78 effectively replace the gelling agent. Another factor limiting the success of commercial
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79 micropropagation is the labor-intensive nature of the technique. Optimized plant population
80 within the culture vessel can reduce labour and maintenance costs associated with media
81 preparation, multiplication, and culture storage in several species (Adelberg 2005; Chen 2016; El
82 Boullani et al. 2017; Guranna and Sathyanarayana 2017).
83 The main objective of this study was to develop an efficient technique to induce a high rate of
84 rooting in hybrid hazelnut shoots to improve the acclimatization of in vitro raised plantlets in the
85 greenhouse. It was hypothesized that the use of liquid medium within a rocker based temporary
86 immersion bioreactor system in combination with Oasis® IVE foam substrate and a high plant
87 density will produce plantlets with a greater vigour compared to the traditional method of using a
88 semi-solid medium.
89 Material and Methods
90 Temporary immersion system
91 The rocker based TIS (Culture shift; VRE System, ON, Canada) was used for shoot
92 multiplication and rooting experiments. This rocker is programmable for controlling the
93 immersion interval as well as rocking speed with a separate timer for the light duration. The
94 rocker accommodates five layers and each layer (122 cm X 65.5 cm) is adjustable to
95 accommodate 18 to 24 culture vessels depending upon their size (Fig. 1). This system also has
96 light reflectors that allow the light to be more uniformly distributed across both ends of the
97 vessels with an average light intensity of 50 µmol/m2/s (Fig. 1).
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98 Plant Material
99 Hybrid hazelnut (Corylus avellana L. X Corylus americana Marshall) cultures of the variety
100 'Norfolk', obtained from the hazelnut collection maintained at the Gosling Research Institute for
101 Plant Preservation (GRIPP), University of Guelph, were used for all experiments. Plant material
102 was multiplied as described earlier (Latawa et al. 2016) to get enough number of shoots using 10
103 to 15 explants per bioreactor vessel containing 50 mL of liquid multiplication medium. The
104 bioreactor vessel (Shukla et al., 2017) was 85 mm wide, 235 mm long, and 80 mm high with a
105 lid that was 85 mm wide, 235 mm long, and 12 mm high. The multiplication medium consisted
106 of modified DKW with vitamins (Driver and Kuniyuki 1984) and 460 µM Fe-EDDHA, 17.6 µM
107 BA, 0.29 µM GA3, 0.14 µM IBA, 3% glucose and 2 mL/L Plant Preservative Mixture (PPMTM)
108 (PhytoTechnology Laboratories®, KS, USA) with a pH of 5.7. Liquid cultures were placed on
109 the TIS with an immersion interval of 25 seconds and a 7-second transfer time in between the
110 immersion and removal periods as determined earlier (Latawa et al. 2016). Plantlets were grown
111 under cool white fluorescent lamps (EiKO®, KS, USA) with an intensity of 40-60 µmol/m2/s at
112 22oC with a 16 h photoperiod. After 3 weeks, hazelnut explants were sub-cultured into fresh
113 bioreactor vessels containing 50 mL of multiplication medium. Explant cultures were sub-
114 cultured at 3-week-intervals until enough hazelnut shoots were produced for the experiments. All
115 shoots with 5-6 nodes were used for in vitro rooting experiments.
116 In vitro rooting in stationary and temporary immersion systems
117 The in vitro rooting efficiency for the hazelnut variety 'Norfolk' was compared among three
118 different vessel treatments that consisted of bioreactor vessels containing either a: i) liquid
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119 rooting medium with Oasis® IVE foam (Oasis® Grower Solutions, OH, USA) placed stationary,
120 ii) liquid rooting medium with Oasis® IVE foam placed onto the TIS, and iii) semi-solid
121 medium (control). The rooting medium (pH 5.7) consisted of 2% sucrose, 1/2 strength DKW
122 with vitamins, and 10 µM IBA. The semi-solid medium was prepared with PhytagelTM (2.2 g/L).
123 Both Oasis® IVE treatments contained two pieces of foam (each with 25 cells and approximate
124 density of 0.012 gcm-3) placed into each bioreactor vessel and 250 mL of liquid medium was
125 applied to the foam pieces (i.e. 125 mL/foam) before autoclaving at 121° C and 118 kPa for 20
126 min. The bioreactor vessels with Oasis® IVE foam fully absorbed all the liquid medium within
127 10 minutes and an additional 25 mL of the same medium was added to each vessel to ensure that
128 there was enough liquid to flow in the vessel. For the semi-solid treatment, each bioreactor
129 vessel contained 250 mL of rooting medium. For each treatment, 12 shoot explants were placed
130 into the bioreactor vessels (6 explants/foam for the Oasis® IVE foam treatments) and were
131 evenly spaced apart from each other. All treatments were grown on the rocker by placing the TIS
132 treatment on the rocker and the stationary and semi-solid treatment on a flat, non-moving surface
133 at the bottom of the rocker. Each bioreactor vessel was considered one replicate and each
134 treatment contained four vessels that were randomly placed under the cool white fluorescent
135 lamps with a 16 h photoperiod and 50 µmol/m2/s light intensity.
136 The number of shoot explants with roots was recorded after 3, 4, 5 and 6 weeks of culture for
137 each treatment to determine the percent rooting. Plantlets from each treatment with well-formed
138 roots and shoots longer than 2 cm with healthy green leaves and minimal browning were
139 considered vigorous enough to be transplanted to the greenhouse. These plantlets were removed
140 from their vessel at either week 3, 4 or 5, and at week 6, all remaining plantlets from a given
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141 treatment were transplanted into the greenhouse. The percentage of plantlets deemed greenhouse
142 ready was recorded at each harvest week and their survival was recorded after 8 weeks as the
143 percentage of the plantlets surviving transplanting ex vitro. The shoot height (mm), number of
144 primary roots and longest root (mm) were recorded for all plantlets from each treatment before
145 transplanting in the greenhouse. Plantlets were transplanted into 5x10 cell (each cell 4.83 cm in
146 length, 4.83 cm in width and 6.06 cm in height) plug trays (T.O Plastics®, MN, USA) containing
147 Sunshine® Mix #4 growth medium (Sungro® Horticulture, MA, USA). The Sunshine® Mix #4
148 growth medium (Canadian Sphagnum peat moss, coarse perlite, dolomitic limestone, wetting
149 agent and Resilience®) has coarse particle size with high drainage capacity. Plantlets were
150 transplanted into growth media and placed within a mist bed for 2 weeks (80% relative humidity,
151 sprayed with water for 15 seconds every 35 minutes during the day and every 4 hours at night).
152 After 2 weeks, the plantlets were grown under greenhouse conditions consisting of an average
153 day temperature of 23oC and night temperature of 18oC under high pressure sodium lamp
154 (Philips, ON, Canada) with a light intensity of 200 µmol/m2/s and 14 h photoperiods.
155 Effect of plant density on in vitro rooting
156 The effect of plant density on the in vitro growth and rooting ability of shoot explants was
157 evaluated using a TIS with Oasis® IVE foam within the culture vessels placed on the rocker.
158 Three plant density treatments of 12 shoot explants/vessel (6 explants/foam), 24 shoot
159 explants/vessel (12 explants/foam) and 36 shoot explants/vessel (18 explants/foam) were
160 evaluated for root development. There were five bioreactor vessels per treatment with each
161 vessel representing a replicate. After 3 weeks of in vitro rooting, plantlets from all treatments
162 were harvested. The observations were recorded for the percentage of explants with roots, the
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163 shoot height (mm), longest root length (mm), and the number of primary roots. The chlorophyll
164 content (mg/m2) of the 3 uppermost leaves from 12 selected plantlets from 3 bioreactors was
165 measured using a CCM-300 Chlorophyll Content Meter (Opti-Sciences® Inc, NH, USA) for
166 each treatment. All the parameters measured in this experiment were selected to provide an
167 overall evaluation of the plantlet vigour in each density treatment.
168 Acclimatization of in vitro grown plantlets and field transfer
169 Plantlets developed in vitro within 2 pieces of Oasis® IVE foam per vessel with densities of
170 either 12, 24 or 36 shoots/bioreactor vessel were removed from the foam and transplanted
171 directly into 5x10 cell T.O Plastics® plug trays containing Sunshine® Mix #4. Plantlets (30 per
172 in vitro density treatment) were transplanted into growth media and placed within a mist bed for
173 2 weeks (80% relative humidity, sprayed with water for 15 seconds every 35 minutes during the
174 day and every 4 hours at night). After 2 weeks, the plantlets were placed into two Conviron® E8
175 (Conviron®, MB, Canada) growth chambers (each chamber representing a replication). The
176 chambers were programmed to have a constant temperature of 23oC during the day and 18oC at
177 night with a photoperiod of 16 h, 70% relative humidity, and a light intensity of 250 µmol/m2/s.
178 Plants were grown in the growth chambers for 9 weeks, and growth measurements were recorded
179 for the survival rate and shoot height. Plants were systematically rotated within the growth
180 chambers every 2 weeks to reduce any potential positional effects. Plants were transferred to the
181 greenhouse after 9 weeks and three month old acclimatized plants were transferred to the field in
182 the month of May 2019. Plants were transplanted in the field nursery area at a distance of about
183 30-35 cm away from each other.
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184 Statistical analysis
185 All experiments were subjected to an analysis of variance (one-way ANOVA test) using a
186 generalized linear mixed model (PROC GLIMMIX) procedure in SAS software version 9.4.
187 Each experiment was designed with a randomized complete block design. Prior to the analyses,
188 data were transformed to a beta distribution and checked for normality. All ANOVA results that
189 were significant were subjected to the Tukey-Kramer Honest Significant Test to determine which
190 means were significantly different from each other. Different letters or asterisks in the figures
191 indicate a significant difference at P
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205 week 3, 4 and 5, however there was no significant difference after 6 weeks of rooting. There was
206 no significant difference in the percentages of rooting of shoot explant between the Oasis® IVE
207 foam TIS and stationary treatments on weeks 3, 4, 5 and 6. The number of plantlets deemed
208 greenhouse ready was also the most time efficient in the Oasis® IVE foam treatment within the
209 TIS allowing 66.7% and 100% of plants to be transplanted into the greenhouse after 3 and 4
210 weeks, respectively (Fig. 3b). The number of plantlets transplanted to the greenhouse was similar
211 or slightly reduced for the stationary treatment and greatly reduced for the semi-solid treatment
212 (Fig. 3b). The semi-solid rooting treatment had a significantly lower number of plantlets ready
213 for the greenhouse than the Oasis® IVE foam TIS and stationary treatment on weeks 3, 4 and 5.
214 There was no significant difference for the number of plantlets ready for greenhouse transplant
215 between the Oasis® IVE foam TIS and stationary treatments on weeks 3, 4 and 5. No plantlets
216 from the semi-solid medium treatment were transplanted to the greenhouse after 3 weeks of in
217 vitro rooting since the majority of plantlets had not developed roots and the shoots which
218 developed roots (< 28%) had pale green leaves with extensive browning. Therefore, at week 3
219 these plantlets were not considered vigorous enough to survive transplanting to ex vitro
220 conditions. Rooted plantlets from the TIS treatment appeared to have healthy, green leaves at
221 week 3, while leaves from the stationary treatment were of a light, pale green colour with some
222 signs of browning at week 3, and the leaves from the semi-solid treatment after 4 weeks of
223 rooting were light green with signs of browning (Figs. 2a-b).
224 The mean shoot height, number of adventitious roots and longest root for greenhouse ready
225 plantlets after 3 weeks of in vitro rooting was 29.8 mm, 7.3, and 25.7 mm for the TIS treatment
226 and 27.9 mm, 8.4, and 21.4 mm in the stationary treatment, respectively. Greenhouse ready
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227 plantlets harvested at 4 weeks of in vitro rooting had a mean shoot height, number of
228 adventitious roots and longest root of 26.4 mm, 6.8, and 27.2 mm for the TIS treatment; 30.5
229 mm, 7.5, and 22.2 mm for the Oasis® IVE foam stationary treatment; and 24.3 mm, 6.7, and
230 29.1 mm for the semi-solid medium treatment, respectively. Greenhouse ready plantlets
231 harvested at 5 weeks of in vitro rooting had a mean shoot height, number of adventitious roots,
232 and longest root of 27.8 mm, 4.1, and 17.5 mm for the stationary treatment and 28.6 mm, 4.8,
233 and 21.4 mm for the semi-solid media treatment, respectively. In comparison, the greenhouse
234 ready plantlets from the semi-solid media treatment harvested at 6 weeks of in vitro rooting had a
235 mean shoot height, number of adventitious roots, and longest root of 26.5 mm, 0.3, and 0.8 mm,
236 respectively. The percentage of survival 8 weeks after transplanting greenhouse ready plantlets
237 from the TIS and the stationary treatment was higher after 3, 4 and 5 weeks, in the range of 87.5
238 to 100%. However, the semi-solid rooting treatment, the percentages of surviving plantlets
239 transferred to the greenhouse on weeks 4, 5 and 6 were low, in the range of 44.4 to 70.6.
240 Effect of plant density on in vitro rooting
241 Shoot explants from bioreactor vessels containing Oasis® IVE foam in the TIS with densities of
242 12, 24, and 36 shoot explants/vessel (Fig. 2c) all exhibited a high rooting ability that did not
243 significantly differ after 3 weeks of culture. Cultures grown with 12 shoot explants/vessel
244 exhibited a rooting of 80.0%, followed by 93.4% for 24 shoot explants/vessel and 88.9% for 36
245 shoot explants/vessel (Table 1). However, the rooting vigour of plantlets varied among the
246 density treatments (Table 1). Plantlets from a density of 12 produced roots with a longest mean
247 length of 14.5 mm and a mean of 5.28 adventitious roots. Plantlets from densities of 24 and 36
248 both produced roots that were significantly greater in longest root length than plantlets from a
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249 density of 12 (21.7 mm and 20.5 mm, respectively). The 24 explant density cultures produced a
250 significantly greater number of adventitious roots (9.39) than the 12 explant density cultures, but
251 the number of adventitious roots from a 36 explant density (6.78) did not significantly differ
252 from either the 12 or 24 explant density treatments. The shoot height of plantlets from the 36-
253 explant density (28.2 mm) cultures was found to be significantly greater than both the 12 (24.7
254 mm) and 24 explant density cultures (24.7 mm) which did not significantly differ from each
255 other (Table 1). Leaves of plantlets in the 36 explants density treatment visually appeared to be
256 dark green in colour, while those from 24 explant density treatment appeared light green
257 compared to the 12 explants density treatment in which leaves appeared to be pale green (Fig.
258 2c). Leaves from the 12 explants density treatment exhibited symptoms of browning compared to
259 other treatments. Regular visual inspection of cultures revealed that as the density increased, the
260 degree of browning declined. The chlorophyll content in the leaves of plantlets was significantly
261 different across all treatments. The chlorophyll content in the leaves of plantlets from 12 explants
262 density had the lowest content at 275.2 mg/m2, followed by 322.5 mg/m2 from the leaves of 24
263 explant density cultures. The greatest chlorophyll content was observed in 36 explant density
264 cultures at 354.9 mg/m2 (Table 1). The 36 explants density treatment produced the most vigorous
265 plantlets among the treatments after 3 weeks of in vitro rooting.
266 Acclimatization efficiency of in vitro grown plantlets
267 After 9 weeks within the controlled environment growth chamber, the survival rate of plantlets
268 grown in the peat-based growth medium was 81%, 87% and 84% for the 12, 24 and 36 explant
269 density treatments, respectively. After 9 weeks of growth, the mean shoot height and leaf area of
270 transplanted plantlets were not significantly different in all density treatments (Fig. 2f). Of the
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271 200 acclimatized plants transplanted in the field, 97% survived and continued to grow after 8
272 months (Figs. 2g-h). About 2-3% of the plants were severely damaged or eaten by herbivores.
273 Discussion
274 Micropropagation is an effective technique to rapidly produce genetically identical plant
275 material. However, its use on a commercial scale can be severely limited for difficult-to-root
276 species such as hybrid hazelnuts. Micropropagated plantlets that do not form roots or develop a
277 weak root system in vitro or ex vitro conditions are often less vigorous during the acclimation
278 phase resulting in poor survival and development ex vitro. The use of micropropagation on a
279 commercial scale can be further limited due to the high cost of labor-intensive production. The
280 objective of this study was to develop an in vitro rooting protocol that meets both the commercial
281 requirements of (i) inducing a high frequency of rooting and providing plantlets with a well-
282 developed root system for better acclimation, and (ii) being cost-effective and time efficient. In
283 vitro and ex vitro rooting protocols for hazelnuts have been reported earlier (Yu and Reed 1995;
284 Nas and Read 2004; Damiano et al. 2005, Caboni et al. 2009; Ellena et al. 2018), however the
285 use of a liquid medium in a TIS in combination with an inert foam substrate to enhance its
286 rooting has not been evaluated. Yu and Read (1995) reported significant cultivar response for in
287 vitro and ex vitro rooting in hazelnut and achieved a survival rate in the range of 78 – 100%.
288 Similarly, the response to IBA treatments varied widely among cultivars (Bassil et al. 1991) as
289 Damiano et al. (2005) reported highest in vitro rooting percentage (70%) with IBA treatments in
290 the liquid medium. Ellena et al. (2018) reported 100% in vitro rooting in semi-solid medium;
291 however, poor survival rates (53 and 63 %) were observed for two cultivars used in their study.
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292 Our earlier study demonstrated the beneficial effects of a rocker based temporary immersion
293 bioreactor system (TIS) on shoot multiplication of hazelnut cultivars (Latawa et al. 2016). In this
294 study, a newly designed TIS with added features was used for both shoot multiplication and root
295 formation from these differentiated shoots to develop a liquid based system for complete plantlet
296 recovery. The results of this study using the improved rocker and TIS demonstrated that the
297 transition from semi-solid medium to TIS can significantly improve the effectiveness of in vitro
298 rooting and subsequent growth of the plantlets. The plantlets resulting from TIS showed higher
299 survival rate during acclimatization and field transplant. Both the TIS containing the liquid
300 rooting medium and Oasis® IVE foam, as well as, the stationary rooting treatments were found
301 to be effective methods to propagate a high number of plantlets in a much shorter period
302 compared to the traditional technique of rooting in the semi-solid medium (Fig. 3). The
303 improvements in the in vitro rooting ability seen in both treatments can be attributed in part to
304 the use of the inert substrate Oasis® IVE foam. The Oasis® IVE foam is a specially designed
305 block of inert, phenolic foam that has been etched and grooved into multiple cube blocks that
306 shoot explants can be inserted into (Naylor-Adelberg et al. 2016). The foam is placed in the
307 liquid medium and it can hold shoot explants in an upright position during the rooting stage. The
308 main advantage of Oasis® IVE foam is that it allows the shoot explants to easily interact with
309 the liquid rooting medium while also acting as a well aerated substrate to enhance oxygen
310 availability and cellular respiration of the roots (Adelberg 2017). Therefore, both the use of
311 liquid medium in the TIS and stationary treatments along with Oasis® IVE foam may have
312 improved the interaction of rooting medium and oxygen with the hazelnut shoot explants
313 resulting in a rapid growth of roots and robust plantlet formation.
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314 The use of inert substrates (e.g. foam, rockwool, vermiculite and perlite) to enhance aeration has
315 been reported to be a very effective technique for improving in vitro rooting in many woody
316 species compared to semi-solid based rooting (Economou 2013). The use of wide pore phenol
317 resin foam for in vitro rooting in red raspberry (Rubus idaeus cv. ‘Gigant’) shoots resulted in
318 plants with a more vigorous root system and greater shoot growth (Gebhardt 1985). The use of
319 vermiculite containing 1/4 strength DKW, 24.6 µM IBA and gelrite improved the rooting and
320 number of primary roots in hybrid walnuts (Jay-Allemand et al. 1992). Similarly, vermiculite in
321 combination with 1/2 strength DKW, 50 µM IBA and Phytagel™ also helped improve rooting in
322 Black Walnut (Juglans nigra) shoot explants (Stevens and Pijut 2018). The rooting response of
323 the hazelnut cultivars 'Montebello' and 'Tonda Gentile Romana' also improved when basal shoot
324 tips were placed in 80 ppm IBA for 1 day in darkness and then transferred to an in vitro rooting
325 medium consisting of vermiculite and agar (Caboni et al. 2009).
326 Even though hybrid hazelnut shoot explants rooted in Oasis® IVE foam placed either in
327 stationary or within TIS exhibited a similar enhanced rooting efficiency, the TIS treatment was
328 more effective than the stationary treatment. The TIS treatment allowed for the highest mean
329 rooting rate (100% of explants with roots), rapid root development (within 3 weeks), and the
330 greatest mean number of vigorous plantlets ready for the greenhouse (100% of plantlets within 4
331 weeks). Furthermore, the leaves of the plantlets from TIS were healthier, darker green in colour
332 with high chlorophyll content than those from the stationary and semi-solid treatments. The
333 prevention of hyperhydricity was characterized by high chlorophyll in oregano shoots and helped
334 the establishment of clonal plants in the greenhouse (Hazarika 2006). Higher chlorophyll content
335 contributes to the development of photosynthetic system in the transplanted plants and reduces
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336 foliage deterioration (Grout and Donkin 1987; Smith et al. 1990). On a similar note, in our
337 experiments higher survival rate was observed in the TIS plants during acclimatization compared
338 to stationary and semi-solid system due to lack of hyperhydricity and higher chlorophyll content.
339 Additionally, the enhanced root growth and plantlet survival in the greenhouse in our study is
340 also likely a result of the improved absorption of nutrients by shoot explants as well as better
341 aeration due to consistent movement of the liquid medium within the bioreactor vessel placed in
342 the TIS. The TIS is utilized in micropropagation to improve the growth and development of
343 explants by periodically submerging them partially into liquid medium to maximize their
344 interaction with the nutritional milieu. This improved media interaction enhances the explant's
345 ability to uptake water, nutrients and plant growth regulators (Ascough et al. 2004). The TIS also
346 separates the explants from the liquid medium for a given period, which increases the gaseous
347 exchange to further improve the growth and development of cultured tissues and organs.
348 Improving the gas exchange potential in vitro is important as it helps improve the explant's
349 ability to perform cellular respiration and photosynthesis (McAlister et al. 2005; Jackson 2005).
350 The combination of the rocker system with the Oasis® IVE foam immersed in liquid medium
351 may have provided a more intense aeration and liquid medium interaction with the shoot
352 explants than that achieved with the Oasis® IVE foam placed stationary, thus resulting in a more
353 efficient rooting and greater vigour of hybrid hazelnut plantlets.
354 Once it was determined that using Oasis® IVE foam in combination with a TIS was an effective
355 method for in vitro rooting of hazelnut, the system was further optimized to increase the
356 efficiency of micropropagation for commercial applications. For this, the influence of the density
357 of explants in the culture vessel was investigated. The highest density of 36 shoot explants/vessel
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358 resulted in an efficient rooting frequency of approximately 89% of explants in 3 weeks, as well
359 as a significantly greater shoot height and chlorophyll content than the 12 and 24 explant density
360 treatments along with a significantly greater longest root length (Table 1). These results revealed
361 that the medium in bioreactor vessels with Oasis® IVE foam can effectively support a high
362 density of 36 explants/vessel for 3 weeks and this increase in the density did not compromise the
363 quality of the plantlets and also improved their overall growth. Optimizing the medium use
364 efficiency is important since the medium components (sugar, basal salts, distilled water, and
365 plant growth regulators) can make up 5-35% of the total production costs associated with
366 commercial micropropagation (Prakash et al. 2004; Chen 2016; Guranna and Sathyanarayana
367 2017). Guranna and Sathyanarayana (2017) found that the cost per plantlet during the in vitro
368 rooting phase could be reduced by 50% by doubling the explant density in Musa spp. Increasing
369 the in vitro density also increases the number of explants and the pace with which they could be
370 placed into culture vessels for initiating the rooting phase, thus improving labour efficiency.
371 Reduced labour cost is an important factor in the success of commercial micropropagation as
372 labour can account for 35% of the total production cost in developing countries and 60-70% in
373 the developed countries (Savangikar 2002; Tomar et al. 2008). Adelberg (2005) reviewed the
374 labour efficiency of 22 technicians in micropropagation of 40 varieties of Hosta over 6 months
375 and found that the number of plants harvested from the vessel per hour by the technicians greatly
376 increased with increased density of explants per vessel, thus improving the work efficiency. The
377 labour efficiency can be further improved using a high explant density within Oasis® IVE foam
378 as observed in the present study in which the incorporation of foam effectively improved the
379 transplant, survival, and growth of the plantlets in the greenhouse. Adelberg et al. (2017) found
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380 that Oasis® IVE foam greatly reduced the time it takes to unwrap the vessels, rinse off the
381 medium and plant Echinacea x Sombrero® 'Salsa Red' rooted plantlets in the greenhouse by
382 approximately 66% when compared to plantlets grown on agar medium. An additional advantage
383 of using Oasis® IVE foam in micropropagation is a superior growth of plantlets in the
384 greenhouse due to a strong root system protecting them from transplant related damage
385 (Adelberg et al. 2015).
386 The exact mechanism why a higher shoot explant density per vessel resulted in rooted hazelnut
387 plantlets with a greater vigour than those from lower density cultures remains unknown.
388 However, it is likely that the explants cultured at a higher density may potentially release growth
389 promoting compounds at a higher concentration due to the larger number of explants within the
390 culture vessel. Further, it may also be speculated that a higher explant density could reduce
391 toxicity caused by potentially suboptimal concentrations of salts, sugar, and other medium
392 components including nitrogen and plant growth regulators (Desjardins et al. 2009; Adelberg et
393 al. 2013). It is therefore logical to assume that the effect of explant density is likely to be
394 genotype-specific due to their often-unique nutritional requirements which can influence growth
395 of the cultures positively or negatively. For example, El Boullani et al. (2017) found that
396 increasing the density of globe artichoke (Cynara cardunculus L.) from 3 to 7 shoot explants per
397 vessel reduced both their in vitro multiplication and ex vitro survival in the greenhouse. It was
398 concluded that the explants in a lower density treatment benefitted from a greater availability of
399 nutrients in the medium (El Boullani et al. 2017). Thus, the explant density needs to be
400 optimized for each species and cultivar and more research is needed to elucidate the mechanisms
401 behind this phenomenon (Sarkar et al. 1997; Guranna and Sathyanarayana 2017).
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402 In conclusion, our results demonstrate that a significant improvement in the in vitro rooting of
403 the hybrid hazelnut can be obtained with a rocker based temporary immersion bioreactor system
404 in combination with Oasis® IVE foam placed within the culture vessels. The optimization of
405 explant density can further improve the entire process of hazelnut micropropagation from the
406 laboratory to the greenhouse at reduced labour and supply costs than traditional
407 micropropagation methods. It will be useful to test customized larger vessels to increase the
408 density of shoots for root induction and for further improvement in growth and acclimatization of
409 plantlets. Introduction of photoautotrophic conditions in the greenhouse or controlled
410 environment chambers may further facilitate efficient field transplantation of micropropagated
411 hazelnut plants. Overall, the results from this study offer an efficient and effective liquid culture
412 based micropropagation protocol to rapidly produce high quality hazelnut trees for field
413 cultivation.
414 Acknowledgements
415 This research was supported by the grants from Ferrero Canada and the Gosling Foundation,
416 Guelph, Canada, to the Gosling Research Institute for Plant Preservation (GRIPP).
417
418
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420
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421
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549
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552 Table 1 Comparison of the rooting response, shoot height, number of adventitious roots, longest
553 root, and leaf chlorophyll content after 3 weeks of in vitro rooting in bioreactor vessels
554 containing Oasis® IVE foam placed in a TIS with densities of 12, 24 or 36 shoot explants per
555 bioreactor vessel. Columns with the same letter indicate no significant difference (P
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562 Fig 1. The temporary immersion system (Culture shift; VRE system, ON, Canada) used for
563 micropropagation of hazelnut.
564
565 Fig 2. Hybrid Hazelnut 'Norfolk' shoot explants within bioreactor vessels containing (a) Oasis®
566 IVE foam after 3 weeks in a TIS (b) and semi-solid medium after 4 weeks placed stationary.
567 Plantlets rooted after 3 weeks in vitro at densities of 12, 24 and 36 (left to right) shoot explants
568 per bioreactor vessels using Oasis® IVE foam placed in a TIS (c and d) and plants rooted in vitro
569 with a density of 12, 24 or 36 shoot explants/vessel after 9 weeks (e and f) of growth ex vitro in a
570 peat-based growth medium within a controlled environment growth chamber. Micropropagated
571 plants were growing in the field after 3 months (g) and 8 months (h) of transplanting.
572
573 Fig 3. In vitro rooting percentage over 6 weeks (A) and percentage of rooted plantlets of hybrid
574 hazelnut 'Norfolk' transplanted to the greenhouse over 5 weeks (B) using the in vitro rooting
575 methods of bioreactor vessels containing semi-solid medium (control), Oasis® IVE foam
576 containing liquid medium placed stationary, and Oasis® IVE foam containing liquid medium
577 placed in a TIS. Asterisk indicates a significant difference (P
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The temporary immersion system (Culture shift; VRE system, ON, Canada) used for micropropagation of hazelnut.
254x190mm (300 x 300 DPI)
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Hybrid Hazelnut 'Norfolk' shoot explants within bioreactor vessels containing (a) Oasis® IVE foam after 3 weeks in a TIS (b) and semi-solid medium after 4 weeks placed stationary. Plantlets rooted after 3 weeks in
vitro at densities of 12, 24 and 36 (left to right) shoot explants per bioreactor vessels using Oasis® IVE foam placed in a TIS (c and d) and plants rooted in vitro with a density of 12, 24 or 36 shoot
explants/vessel after 9 weeks (e and f) of growth ex vitro in a peat-based growth medium within a controlled environment growth chamber. Micropropagated plants were growing in the field after 3 months
(g) and 8 months (h) of transplanting.
254x190mm (300 x 300 DPI)
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In vitro rooting percentage over 6 weeks (A) and percentage of rooted plantlets of hybrid hazelnut 'Norfolk' transplanted to the greenhouse over 5 weeks (B) using the in vitro rooting methods of bioreactor vessels containing semi-solid medium (control), Oasis® IVE foam containing liquid medium placed stationary, and Oasis® IVE foam containing liquid medium placed in a TIS. An asterisk indicates a significant difference
(P