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Oct 12, 2006 - Abstract Leafless nodal segments (4 ± 1 mm long) of hardy hibiscus were excised from in vitro proliferating microshoots, encapsulated in ...
Plant Cell Tiss Organ Cult (2006) 87:127–138 DOI 10.1007/s11240-006-9146-7

ORIGINAL PAPER

Greenhouse growth and acclimatization of encapsulated Hibiscus moscheutos nodal segments John E. Preece Æ Todd P. West

Received: 1 August 2005 / Accepted: 7 July 2006 / Published online: 12 October 2006  Springer Science+Business Media B.V. 2006

Abstract Leafless nodal segments (4 ± 1 mm long) of hardy hibiscus were excised from in vitro proliferating microshoots, encapsulated in sodium alginate solidified with 50 lM CaCl2, stored under refrigeration for 4 weeks in darkness, and then planted in the greenhouse. Planting in vermiculite and placing under intermittent mist was the best environment tested. If the encapsulated nodal segments were exposed to light for at least 2 weeks while in vitro in the laboratory prior to planting in the greenhouse, all survived, rooted, and produced shoots in the greenhouse. Rooting into the vermiculite was best if the encapsulated nodal segments were planted 1 cm deep and not covered. Anatomically, the new leaves that were produced from shoots that grew under mist in the greenhouse from encapsulated nodal segments were about the same thickness as leaves produced in vitro; had fewer intercellular spaces than the in vitro produced leaves; had palisade cells inter-

mediate in length, and were intermediate for epicuticular wax formation between in vitro produced leaves and leaves on macrocuttings rooted in the greenhouse. The stomates on greenhouse shoots from encapsulated nodal segments closed similar to stomates on leaves on rooted macrocuttings, and were unlike in vitro produced leaves where the stomates remained open even when stressed. Storing and planting encapsulated nodal segments could allow producers to generate sufficient numbers of nodal segments, refrigerate them until needed, and facilitate greenhouse acclimatization and production of plants. Keywords Alginate Æ Hardy hibiscus Æ Hardening-off Æ Microencapsulation Æ Micropropagation Æ Plant anatomy

Introduction

J. E. Preece (&) Department of Plant, Soil and Agricultural Systems, MC 4415, Southern Illinois University, Carbondale, IL 62901, USA e-mail: [email protected] T. P. West Division of Plant and Soil Sciences, West Virginia University, 1090 Agriculture Sciences Building, Morgantown, WV 26506-6108, USA

Plantlets formed in vitro have poor water retention capacity because their leaves have reduced amounts of epicuticular waxes, abnormally functioning stomata (usually they remain open), and abnormal internal anatomy (Preece and Sutter 1991; Pospisilova et al. 1999). The leaves that develop in vitro never return to the normal condition so new leaves with better transpiration control must develop under lower relative

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humidity during acclimatization to the greenhouse or field (Preece and Sutter 1991). Alginate encapsulation, adapted from synthetic seed technology, can improve the success of plantlet acclimatization. Bapat and Rao (1990) sprouted encapsulated Morus indica L. axillary buds in autoclaved soil in petri dishes. Up to 63% of the buds survived when encapsulated in a matrix containing the fungicide carbendenzim. Refouvelet et al. (1998) reported successful greenhouse transfer of encapsulated lilac (Syringa vulgaris L.) axillary buds without a saturated humidity stage. Hardy hibiscus is a popular flowering shrub for landscapes and flower gardens. It is a non-invasive plant that remains in clump growth form and grows well in wet or poorly drained soils. Micropropagation is more efficient than cutting propagation for this species. The ability to encapsulate and store nodal segments and then plant them in the greenhouse could add to the efficiency of micropropagation of Hibiscus moscheutos. This research was conducted to develop a greenhouse planting protocol for alginate encapsulated hardy hibiscus nodal segments by evaluating greenhouse media and planting depths, humidity environments, and light pretreatment. To understand how the new growth from encapsulated nodal segments acclimated to the ex vitro environment, anatomical differences were compared among leaves produced from greenhouseplanted encapsulated nodes, microshoots in vitro, and greenhouse propagated macrocuttings.

Materials and methods

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Alginate encapsulation, cold storage, and subsequent growth Single nodes, 4 ± 1 mm long with the subtending leaf removed were excised from established stage II axillary shoot cultures. Nodal segments were coated with 2.75% high-viscosity sodium alginate (Product Number A7128, Sigma Chemical Co., Mississauga, ON, Canada) and then placed into sterile 50 lM CaCl2 solution for 30 min, followed by two 5-min sterile deionized water rinses. Encapsulated nodes were then placed in 100 · 15 mm petri dishes with the edges wrapped in Parafilm, and stored under refrigeration (5 ± 1C) for 4 weeks in darkness. Planting depth of encapsulated nodes in vermiculite in the greenhouse After 4 weeks of cold storage, encapsulated nodal segments of ‘Lord Baltimore’ and ‘Southern Belle’ were removed from refrigeration and were planted in medium grade vermiculite either uncovered on the surface, uncovered planted 1 cm deep, or covered with approximately 2–3 mm of medium after being planted 1 cm deep, resulting in a 2 · 3 factorial combination of two cultivars and three planting methods. The planted encapsulated nodal segments were placed under intermittent mist (mist interval of 9 s every 6 min) within a glass-covered greenhouse under natural photoperiod. Data were collected and analyzed on axillary shoot number, shoot length, root number, and root length after 4 weeks of greenhouse growth.

Stage II stock cultures

Effects of in vitro light pretreatment on growth and survival in the laboratory and greenhouse

Nodal explants of two H. moscheutos cultivars, ‘Lord Baltimore’ and ‘Southern Belle’ were placed in vitro using the methods described by West and Preece (2004). Briefly, nodal segments were cultured on Driver and Kuniyuki Walnut (DKW) medium (Driver and Kuniyuki 1984), 3% sucrose, 1 · 10–7 M thidiazuron (TDZ), pH 5.8, and 6.5 g L–1 agar (Phytotechnology Laboratories, KS, USA).

Every week for 5 weeks, nodal segments of ‘Lord Baltimore’ and ‘Southern Belle’ were encapsulated, put in petri dishes and placed 30 cm below cool white fluorescent lamps that provided a photon flux of approximately 40 lmol m–2 s–1 for a 16-h photoperiod at 25C. The first nodal segments were allowed to remain under the lamps for 4 weeks, resulting in light exposure for 0, 1, 2, 3 or 4 weeks. At this time, data were collected

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and analyzed on axillary shoot number, shoot length, root number, and root length. After data collection, the growing nodal segments were planted uncovered and 1 cm deep in medium grade vermiculite. They were placed under intermittent mist in a glass-covered greenhouse under natural photoperiod. After 4 weeks of greenhouse growth, data were collected and analyzed on axillary shoot number, shoot length, root number, and root length. Leaves for anatomical study Mature leaves (the third oldest on each plant) were excised from 6-week-old greenhouse-grown plants produced by rooting 10 cm long macrocuttings, 6-week-old plants that grew from encapsulated nodal segments that sprouted under mist, and 4-week-old shoots that grew in vitro. Source shoots grew from encapsulated nodal segments that had been planted uncovered and 1 cm deep in medium grade vermiculite under intermittent mist (9 s every 6 min) and natural photoperiod following 2 weeks of light pretreatment in the laboratory. After 4 weeks under mist, the plants produced from the nodal segments and those propagated by rooting cuttings were removed from the mist and transferred to 10 cm (height and top diameter) plastic pots containing two sphagnum peat: one vermiculite: one perlite (by volume) medium with 3.75 g 14N–4.2P–11.6K Osmocote. They grew in the greenhouse for another 2 weeks before leaves were excised for analysis. Leaves from in vitro axillary shoots were removed from 4-week-old stage II shoots that were proliferated using the single node subdivision method.

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with 2% osmium tetroxide (OsO4) solution for 1 h at 25C, followed by three deionized water rinses. After fixation the leaf sections were dehydrated by placing into an ethanol series (25, 50, and 75%) for 10 min each followed by three separate 10-min soaks in 100% ethanol. After dehydration, the leaf sections were infiltrated for 24 h each in 25, 50, and 75% Spurr’s low viscosity resin (Spurr 1969) followed by two 8 h infiltrations in fresh 100% Spurr’s resin. Leaf sections were then embedded in 100% Spurr’s resin in a flat mold and polymerized for 72 h at 62C. Following polymerization, the leaf sections were cut at a thickness of 0.5–1 lm using an ultramicrotome and stained with paragon-1301. Data were collected and analyzed on length of palisade, upper epidermal, and lower epidermal cells (ten random cells of each cell type) from each plant leaf. Leaf epicuticular wax quantification The third oldest leaf was collected from each of five different plants per treatment and from each leaf a 20 · 25 mm interveinal section was excised for leaf area standardization. The epicuticular waxes were extracted according to the method of Flore and Bukovac (1974) by dipping leaf sections in 100% chloroform three times for 10 s each. The chloroform solutions were then transferred into a shallow tared dish and weighed. The dish was then placed into a chemical fume hood to allow the chloroform to evaporate overnight. The dish was then weighed again after all the chloroform evaporated. The difference in weight between the chloroform solution and final weight is the amount of epicuticular waxes from the leaf section.

Internal leaf anatomy evaluation Stomates One leaf was excised from three different plants (or in vitro shoots) from each treatment and prepared for fixation, dehydration, embedding, sectioning, and staining for examination with the light microscope following the methods of Spurlock et al. (1966). Leaves were first cut into 2 mm2 interveinal sections, then fixed with 2% glutaraldehyde in aqueous 0.05 M phosphate buffer solution (pH 7.2) for 1 h at 25C, then post-fixed

To evaluate the stomatal condition, epidermal impressions were obtained from the abaxial surface of the third oldest leaf on intact plants or on intact microshoots from stage II cultures (nonstressed leaves) and from the same leaves that were then excised from the plants or shoot cultures and allowed to dry at 25C for 5 min (stressed leaves). The selected leaves represented

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five replications of a 2 · 3 factorial combination of two levels of stress and three types of treatment plants (macrocutting propagated, derived from encapsulated nodal segments, and stage II microshoots). For non-stressed tissue culture leaves, the silicone sealant was applied and the microshoots placed back into their 25 · 150 mm borosilicate glass culture tubes capped with autoclavable plastic caps and wrapped with Parafilm to ensure proper humidity. Epidermal impressions were made to a different location on the same leaf as when it was attached to the plant and again 5 min after removal and stress. This allowed for comparisons between stomatal responses on the same leaf during both stressful and non-stressful conditions. Epidermal impressions were made by applying 100% silicone clear sealant (Silicone II Sealant, GE Silicones, Waterford, NY, USA) to selected leaves and allowing it to solidify for 12 h (Sampson 1961). At this time the silicone was removed TM and clear nail polish (Cutex Brand, CP Inc., New York, NY, USA) was applied to a 75 · 25 mm glass microscope slides and the dried silicone impressions placed so that the negative epidermal impressions were placed in direct contact with the nail polish. The nail polish was allowed to dry and harden for 24 h and the section of silicone sealant was removed to produce a positive impression of the leaf abaxial surface on the microscope slides. To evaluate stomatal density (no. of stomata per mm2), leaf epidermal impressions were observed with a light microscope at 400· . Stomates were counted in the field of view and data converted to number of stomates/mm2 (no. of stomata in field view divided by 0.152 mm2 (the field of view area)). Data were collected and analyzed on stomatal density, the number of open stomates related to treatment. Data collection and statistical analysis All experiments were arranged as completely random designs and conducted twice. All data were analyzed using the General Linear Model (GLM) of SAS (SAS Institute Inc. 1999). Data set columns having 50% or more zeros, were

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normalized using a square root transformation (y + 0.5) (Steel and Torrie 1980).

Results and discussion In a preliminary experiment, encapsulated nodal segments did not survive in the greenhouse under fog or with hand watering regardless of the growing medium. Additionally all encapsulated nodal segments died when under intermittent mist in the greenhouse when planted in perlite or a two sphagnum peat: one vermiculite: one perlite (by volume) medium. However, 20% survived when planted in medium grade vermiculite under intermittent mist. Therefore, all other experiments utilized vermiculite and intermittent mist. Planting depth of encapsulated nodes in vermiculite in the greenhouse None of the encapsulated hibiscus nodal segments that were covered with medium survived (Table 1). There were no significant differences between shoot and root growth of encapsulated nodes that were planted and not covered with vermiculite; both had 20% survival. Sarkar and Naik (1998) reported similar findings with encapsulated nodal segments of potato (Solanum tuberosum L.), only 5% of their encapsulated nodal segments survived if covered with 2–3 mm of growing medium. Whereas potato nodal segments that were planted 3–4 mm deep in the soil and not covered had a survival rate of 52%. This indicates that some factor associated with covering encapsulated nodal segments leads to low survival. One possibility is that covering blocks light from reaching the encapsulated nodal segments. Although there were no significant differences between shoot and root growth of encapsulated nodal segments that were planted uncovered on the medium surface or planted 1 cm deep (Table 1), the developing roots from nodes that were planted on the medium surface had a tendency to grow along the surface of the vermiculite without penetration (Fig. 1). However when the nodal segments were planted 1 cm deep but not

Plant Cell Tiss Organ Cult (2006) 87:127–138 Table 1 Effect of planting depth on node survival with subsequent shoot and root growth and development of direct greenhouse planting (in vermiculite medium under

131 intermittent mist) of ‘Lord Baltimore’ and ‘Southern Belle’ hardy hibiscus encapsulated nodal segments after 4 weeks of growth in greenhouse

Planting depth

Mean shoot number

Mean shoot length (mm)

Root number

Mean root length (mm)

% Survival

Above surface At surface Below surface Significance 5% t-testa

1.0 1.0 0.0 * 0.1

31.2 32.6 0.0 * 15.9

2.4 2.2 0.0 * 0.2

51.4 53.6 0.0 * 25.5

20.0 20.0 0.0 – –

Data were transformed for analysis using (y + 1/2), non-transformed means are presented. Each datum is the mean of 40 replications. The cultivar main effect and the cultivar · planting depth interaction were not significant a

t-test for paired comparisons

* Significant main effect of planting depth at the 5% level according to the F-test with 2 and 161 df

covered, their roots penetrated the vermiculite and grew within the medium, which we considered desirable. Effects of in vitro light pretreatment on growth and survival in the laboratory and greenhouse Pretreating the encapsulated nodal segments with light in the laboratory had a significant impact on shoot and root growth. All of the encapsulated ‘Lord Baltimore’ nodal segments that were placed under lights for at least 2 weeks while in vitro developed one elongating axillary shoot and at least one adventitious root, whereas encapsulated nodal segments of ‘Southern Belle’ required 3 weeks of in vitro light treatment for axillary shoot elongation and 4 weeks of light pretreatment for adventitious root formation (Table 2, Fig. 2). The growth responses to light were quadratic, with a lag time of 2–3 weeks while under

light before growth began on encapsulated nodal segments of both cultivars. Sarkar and Naik (1998) reported using a light pretreatment varying from 3 to 9 days prior to greenhouse planting based on the time it took for the potato shoots to elongate out of the encapsulation medium. They also reported that potato nodal segments that were pretreated with light for longer than 3 days had a decrease in survival rate because of desiccation of the encapsulation medium and nutritional leakage. Encapsulated nodal segments of hardy hibiscus do not decline with increasing exposure to light like the potatoes; in fact, all of the lighted, encapsulated hibiscus nodal segments survived with good green color and no obvious detrimental effects. Following the in vitro light pretreatments, the nodal segments from the above experiment were planted into vermiculite and placed under intermittent mist in the greenhouse (Fig. 3). After 4 weeks under mist in the greenhouse there were

Fig. 1 Encapsulated nodal segments direct planted on the vermiculite surface with roots growing along the surface of the medium without penetration into the medium

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Table 2 Effect of number of weeks of in vitro light pretreatment in the laboratory on shoot and root growth of encapsulated hardy hibiscus nodal segments as preparation

for planting in vermiculite and placing under intermittent mist in greenhouse

Cultivar

Weeks

Shoot number

Shoot length (mm)

Root number

Root length

Lord Baltimore

0 1 2 3 4 La Q 0 1 2 3 4 L Q

0.0 0.0 1.0 1.0 1.0 ** ** 0.0 0.0 0.0 1.0 1.0 ** ** ** 0.12 0.16

0.0 0.0 3.3 3.9 3.7 ** ** 0.0 0.0 0.0 3.0 3.7 ** ** ** 1.93 2.56

0.0 0.0 1.0 1.4 1.7 ** ** 0.0 0.0 0.0 0.0 2.1 ** ** ** 0.36 0.48

0.0 0.0 3.0 13.7 18.6 ** ** 0.0 0.0 0.0 0.0 9.2 ** ** ** 5.71 7.54

Southern Belle

b Significance t-testc

5% 1%

Data were transformed for analysis using (y + ½), non-transformed means are presented. Each datum is the mean of 20 replications a

L - linear, Q - quadratic effects

b

**Significant at the 1% level according to F-test

c

t-test for paired comparisons

** Significant interaction between cultivar and light treatment at the 1% level, respectively, according to the F-test with 4 and 189 df

highly significant interactions between light and cultivar on shoot and root growth. One hundred percent of encapsulated nodal segments of both cultivars survived in the greenhouse if they had received at least 2 weeks of light pretreatment in the laboratory before being planted in the greenhouse (Table 3). The growth responses to Fig. 2 ‘Lord Baltimore’ encapsulated node exposed to in vitro light pretreatment for 0, 1, 2, 3 or 4 weeks (40 lmol m–2 s–1 of cool white fluorescent light and a 16-h photoperiod at 25C)

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light were mostly quadratic, with a light pretreatment of 0 (no light pretreatment) or 1 week being insufficient, and the most growth if the segments were pretreated with light for at least 2 weeks. Growth of the cultivars was somewhat different, depending on the length of light pretreatment, with ‘Lord Baltimore’ producing

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Fig. 3 Encapsulated ‘Lord Baltimore’ hardy hibiscus nodal segments planted in medium grade vermiculite and placed under intermittent mist for 4 weeks. After 4 weeks under mist, the sprouted nodal segments were transplanted

into a two sphagnum peat: one vermiculite: one perlite (by volume) medium. The 0 week control was newly encapsulated for comparison purposes with segments in the greenhouse for 4 or 8 weeks

longer roots than ‘Southern Belle.’ However, unlike ‘Southern Belle,’ ‘Lord Baltimore’ encapsulated nodal segments declined in length as the light pretreatment reached 3–4 weeks. The tips of roots that grew from ‘Lord Baltimore’ segments

while in vitro had black tips, likely because of dessication when the roots emerged from the encapsulation medium. Because growth was good, 100% of the nodal segments survived, and root tips did not dry out, we used a 2 weeks in

Table 3 Effect of number of weeks of in vitro light pretreatment in the laboratory on shoot and root growth of encapsulated hardy hibiscus nodal segments 4 weeks after

planting in vermiculite and placing under intermittent mist in a greenhouse

Cultivar

Weeks

Shoot number

Shoot length (mm)

Root number

Root length (mm)

Percent survival

Lord Baltimore

0 1 2 3 4 Linear Quadratic 0 1 2 3 4 Linear Quadratic

0.0 0.2 1.0 1.0 1.0 ** ** 0.0 0.2 1.0 1.1 1.0 ** ** ** 0.2 0.2

0.0 0.8 6.9 7.0 5.2 ** ** 0.0 1.0 6.4 12.0 5.9 ** ** ** 1.9 2.5

0.0 0.1 1.4 2.1 1.4 ** ** 0.0 0.2 1.2 2.0 2.2 ** ns ** 0.4 0.5

0.0 2.0 37.2 24.6 18.3 ** ** 0.0 1.7 11.2 19.9 12.6 ** ** ** 4.0 5.3

0 2 100 100 100 – – 0 10 100 100 100 – – – – –

Southern Belle

Significanceb t-testa

5% 1%

a

t-test for paired comparisons. Each datum is the mean of 20 replications

b

ns Non-significant

** Non-significant or significant interaction between cultivar and in vitro light pretreatment at the 1% level, respectively, according to the F-test with 4 and 189 df

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Fig. 4 Hibiscus moscheutos cv. Lord Baltimore leaf cross sections from the third oldest node of A shoot grown in vitro, B from the new growth from an encapsulated nodal

segment under intermittent mist in the greenhouse, and C a rooted macrocutting in the greenhouse

vitro light pretreatment for all encapsulated nodal segments that were planted in the greenhouse for our anatomical studies.

significant difference was observed in the palisade cell layer(s). Leaves of young plants that grew in the greenhouse from encapsulated nodal segments and that formed in vitro had a single layer of palisade cells, whereas leaves from macrocuttings had a triple layer of palisade cells (Fig. 4). Additionally, the length of the palisade cells was longest in leaves of rooted macrocuttings and shortest in the in vitro-produced leaves, with the new leaves forming from encapsulated nodal segments intermediate. This indicates a gradual change in leaf anatomy from in vitro shoots to the new growth under mist from encapsulated nodal segments as they adapted from the in vitro environment to the greenhouse environment. Brainerd et al. (1981) reported similar results in plum (Prunus insititia L. cv. Pixy) stating that the length of palisade cells was significantly less in tissue culture plants as compared to greenhouse and field-grown plants. The palisade layer of the in vitro leaves had large

Internal leaf anatomy evaluation We are aware of no other published studies examining the anatomy or epicuticular wax amounts of leaves emerging from encapsulated nodal segments during acclimatization under mist in the greenhouse and comparing them to in vitro shoots and rooted macrocuttings. The origin and growing conditions had a significant impact on the internal leaf anatomy of hardy hibiscus depending on whether the leaves formed on rooted macrocuttings, from greenhouse-planted encapsulated nodal segments that developed under intermittent mist, or from microshoots that formed in vitro (Table 4). Of the three cell types measured (palisade, upper epidermal, and lower epidermal cells), the only

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intercellular spaces and the cells were wider at the top than at the bottom. This same irregular palisade layer cell shape and density was observed in raspberry (Rubus idaeus L.) leaves from plants grown in vitro (Donnelly and Vidaver 1984). The similar epidermal cell sizes among the three types of hardy hibiscus leaves were also

reported for plum leaves that were from tissue culture, greenhouse-grown or field-grown (Brainerd et al. 1981). Greenhouse-grown hardy hibiscus leaves were thicker than either leaves produced from tissue-cultured plants or greenhouse-planted encapsulated nodal plants (Fig. 4). Lee et al. (1988) reported that in vitro developed sweetgum (Liquidambar styraciflua L.) leaves

Fig. 5 Stomatal impressions of the different leaf types and conditions of ‘Lord Baltimore’ hardy hibiscus A nonstressed from rooted macrocuttings in the greenhouse, B dehydrated from rooted macrocuttings in the green-

house, C non-stressed from encapsulated nodal segments, D dehydrated from encapsulated nodal segments, E nonstressed from in vitro shoots, and F dehydrated from in vitro shoots

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were also thinner than leaves that developed in the greenhouse. Leaf epicuticular wax quantification

ns Non-significant

** Non-significant or significant at the 1% level, respectively, according to the F-test with 2 and 81 df

c

t-test for paired comparisons

The amount of epicuticular waxes differed among the three hardy hibiscus leaf types with greenhouse leaves producing the most and tissue cultured leaves producing the least and the new greenhouse growth from encapsulated nodal segments intermediate (Table 4). Several studies have shown that leaves formed in vitro have low amounts of epicuticular waxes and abnormally functioning stomata (Sutter and Langhans 1982; Zaid and Hughes 1995; Correll and Weathers 2001).

b

The third leaf from the bottom (fully expanded) was collected and analyzed from macrocutting-propagated plants, new growth in the greenhouse under intermittent mist from encapsulated nodal segments, and from shoots that proliferated in vitro

a

426.4 ** 34.0 44.7 1.5 ** 0.4 0.5 64.2 ns 22.4 ** 3.2 4.2

45.3 ns

161.8 2.6 64.5 45.6

45.6

149.4 3.7 89.7

Macrocuttings in greenhouse Encapsulated in greenhouse In vitro Significanceb 5% t-testc 1% t-test

46.1 65.1

Palisade cell length (lm)

Upper epidermal cell length (lm)

Lower epidermal cell length (lm)

Epicuticular wax (mg)

Number of stomata per mm2

Plant Cell Tiss Organ Cult (2006) 87:127–138

Leaf origina

Table 4 Effect of propagation method and acclimatization on leaf cell size, epicuticular wax formation and number of stomata of Hibiscus moscheutos ‘Lord Baltimore’

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Stomatal density and condition The number of stomata per mm2 was similar between the leaves that developed in the greenhouse from rooted macrocuttings and from encapsulated nodal segments, with the in vitro produced leaves having more than 2.6 times more stomata per mm2 (Table 4). In contrast, in vitro plum shoots developed leaves that had a reduced stomatal density compared to greenhouse-grown plants (Brainard et al. 1981). However, an increase in stomatal density was also reported for in vitro developed leaves of L. styraciflua (Lee et al. 1988). Leaves that develop on microshoots in vitro have been reported to function abnormally with stomata remaining open during stress situations when normally they would close, making acclimatization difficult (Brainerd and Fuchigami 1982). Stomatal function of hardy hibiscus was significantly affected by leaf type but not leaf condition (fresh or dehydrated) with the stomata open on in vitro produced leaves, regardless of whether they were stressed or not (tabular data not presented, Fig. 5). Leaves on rooted cuttings and new shoots that grew in the greenhouse from encapsulated nodal segments had stomata that were closed when fresh imprints were made (unstressed) and after 5 min of dehydration (stressed). Leaves developed in tissue culture were found to have 85% of the stomata in the open condition in the fresh imprints and after

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5 min of dehydration they were found to be 76% open. Therefore an advantage of planting encapsulated hardy hibiscus nodal segments in the greenhouse is that they produce their leaves in this environment and the stomatal apparatus functions normally, unlike microshoots produced in vitro.

Conclusions Leaves on microshoots that develop in vitro are poorly developed for survival in greenhouse or field conditions, therefore many plants die during the acclimatization stage of micropropagation. Because the leaves that form in vitro do not change during acclimatization, it is essential that microplants grow new leaves in the ex vitro environment that are adapted to the lower relative humidity levels of the greenhouse or field. Alginate encapsulated nodal segments that lack leaves may be ideal for ex vitro establishment since most or all of their leaves will develop in the greenhouse or field. This system will work if the encapsulated nodal segments will produce adventitious roots, their axillary buds will elongate and new leaves will be produced ex vitro. This can be done with hardy hibiscus because the encapsulated nodal segments easily root and produce shoots. For success, it is essential that the encapsulated nodal segments receive 2–3 weeks of light pretreatment while in vitro before being planted in the greenhouse. When placed in the greenhouse, the encapsulated nodal segments must be planted in vermiculite one cm deep, but not covered with medium and placed under intermittent mist. The new leaves that develop show signs of acclimatization. While having only a single palisade layer, and having a thickness similar to in vitro produced leaves, the leaves from the elongating shoot from the encapsulated segments could close their stomata and had fewer intercellular spaces compared to in vitro produced leaves. These new leaves showed signs of gradually becoming more like leaves produced on macrocuttings produced in the greenhouse, in that they could close their stomata, their palisade cells were longer than in vitro leaves, and they

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produced more epicuticular waxes than in vitro leaves. Therefore, if handled properly, encapsulated hardy hibiscus nodal segments can be stored under refrigerated conditions until needed, pretreated with light, and then planted in vermiculite and placed under intermittent mist in the greenhouse. This will allow propagators to scale up the number of nodal segments needed to meet sales demand, store them, and then root and acclimatize them in sufficient time for customer needs.

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