In vitro Rooting of Hybrid Hazelnuts (Corylus avellana X ......90 Temporary immersion system 91 The rocker based TIS (Culture shift; VRE System, ON, Canada) was used for shoot 92 multiplication

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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

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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

14

15

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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

419

420

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421

422

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550

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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

Documents

In vitro Rooting of Hybrid Hazelnuts (Corylus avellana X ......90 Temporary immersion system 91 The rocker based TIS (Culture shift; VRE System, ON, Canada) was used for shoot 92 multiplication