Factors controlling high efficiency adventitious bud formation and plant regeneration from in vitro leaf explants of roses (Rosa hybrida L.)

Scientia Horticulturae 88 (2001) 41±57

Factors controlling high ef®ciency adventitious bud formation and plant regeneration from in vitro leaf explants of roses (Rosa hybrida L.) Rusli Ibrahim*, Pierre C. Debergh1 Division of Agrotechnology and Biosciences, Malaysian Institute for Nuclear Technology Research, MINT 43000 Bangi, Selangor, Malaysia Accepted 30 May 2000

Abstract Studies were conducted to improve adventitious bud regeneration in roses (Rosa hybrida L.), speci®cally to extend the protocol to different genotypes and to initiate production of multiple shoots per explant. The best results were obtained by using a two-stage procedure where excised lea¯ets were incubated on Murashige and Skoog (MS) (1962) induction medium with 6.8 mM TDZ plus 0.49 mM IBA in the dark for 7 days and subsequently transferred to an MS-based regeneration medium with 2.22 mM BA plus 0.049 mM IBA exposed to a PPFD of 15 mmol mÿ2 sÿ1 PAR. Bud formation capacity was also signi®cantly affected by the genotype and the environment, such as the use of bottom cooling creating a lower RH in the vessel. The addition of silver nitrate to the induction medium also signi®cantly improved the percentage of regeneration in three genotypes tested. Regenerated shoots failed to elongate when transferred to MS proliferation medium containing 0.5 mg lÿ1 BA, however maximum bud development and elongation were achieved when kinetin in the range 1±2 mg lÿ1 was used. Elongated shoots were excised and rooted best on zero growth regulator half-strength MS modi®ed medium. Rooted plantlets were acclimatized under greenhouse conditions for evaluation of somaclonal variation. # 2001 Elsevier Science B.V. All rights reserved. Keywords: Bottom cooling; Dark incubation; Rosa hybrida L.; Silver nitrate; TDZ

*

Corresponding author. Fax: ‡6-03-89258262. E-mail address: [email protected] (R. Ibrahim). 1 Present address: Department of Plant Production-Horticulture, University of Gent, Coupure Links 653, B-9000 Gent, Belgium. 0304-4238/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 2 3 8 ( 0 0 ) 0 0 1 8 9 - 8

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1. Introduction The ability to regenerate whole plants from somatic tissues is a prerequisite for Agrobacterium-mediated transformation, one of the most successfully used gene transfer techniques to genetically engineer perennial crops (Dandekar, 1992). The use of Agrobacterium and the selection for antibiotic kanamycin in these protocols severely reduces regeneration (Fiola et al., 1990; Hassan et al., 1993). It is also a prerequisite for ef®cient mutation breeding in vitro. Regeneration of adventitious shoots in Rosa spp. has been reported from immature zygotic embryos (Burger et al., 1990), from in vitro and in vivo grown leaves and shoot explants (Rout et al., 1992; Dubois and De Vries, 1995), and putatively, from roots and leaves of `Mairutral' (Arene et al., 1993), and from stem explants of R. damascena Miller (Ishioka and Tanimoto, 1990). In many regeneration studies, authors focused on determining the optimal composition of the culture media. Thidiazuron, a cytokinin-like compound, was found to be very effective for stimulation of adventitious shoots in Rosaceous crops, including Malus spp. (Fasolo et al., 1989; Swartz et al., 1990; Korban et al., 1992), Pyrus spp. (Chevreau et al., 1989; Leblay et al., 1991), Prunus spp. (Mante et al., 1989) and Rubus spp. (Fiola et al., 1990; Swartz et al., 1990). Among the many other factors that affect organogenesis, dark incubation and photosynthetic radiation are known to be crucial for regeneration and have been studied on leaves of apple rootstock M26 (Predieri and Malavasi, 1989), and on red raspberry (Rubus idaeus L.) leaf tissues (Cousineau and Donelly, 1991). However, few have reported the effect of low relative humidity and light ¯uence rate on regeneration of shoots from leaf tissues. The system for plant regeneration developed here can be exploited for use in mutation breeding by exposing leaf explants to irradiation and capturing the genetic variants through adventitious shoot regeneration. The aim of the present study was to develop an ef®cient system to regenerate adventitious buds from in vitro leaf explants of Rosa hybrida L. at high frequencies by manipulating growth regulator requirements, explant tissue types and culture conditions.

2. Materials and methods 2.1. Plant material Resting axillary buds were excised from vigorously ¯owering shoots of three cut roses (Rosa hybrida L.), `Inka'; RUI 317 and RUI 319. Plants were maintained in a heated greenhouse and used as the explant sources.

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2.2. Culture establishment and shoot multiplication Lateral ¯owering shoots were harvested, leaves were removed and the stems were cut into segments of 6±10 cm in length and rinsed under running tap water for about 15±20 min. Surface sterilization was carried out under the laminar ¯ow hood by soaking stem segments in a solution of 0.4% mercury chloride for 3 min followed by three rinses in sterile distilled water. Explants were then immersed in 10% Chlorox (5.25% sodium hypochlorite) supplemented with a few drops of Tween 20 for 10 min followed by three rinses in sterile distilled water. Subsequently, the explants were recut into nodal cuttings of 1.5±2.0 cm in length. One explant was inoculated per 2.5 cm15.0 cm culture tube containing 25 ml of shoot proliferation medium (Table 1), closed with Kap UtsTM. All media components, except for speci®c treatments, were mixed and adjusted to pH 5.8 prior to autoclaving at 1218C, 104 kPa for 20 min. Cultures were incubated at 2328C in a growth room under a 16 h light photoperiod provided by OSRAM (Lumilux Warmton Warm White, L36W/31-830) ¯uorescent tubes with a photosynthetic photon ¯ux density (PPFD) of 40 mmol mÿ2 sÿ1 PAR. After 2±3 weeks, axillary shoots (2±3 cm in length) were excised from the stem tissue and transferred to the same fresh medium for multiplication. Subculturing was carried out every 4 weeks in vessels containing 80 ml medium (80 ml medium in 500 ml glass jars closed with opaque polypropylene caps). Unless otherwise stated, bottom cooling that created 85% relative humidity (Vanderschaeghe and Debergh, 1987) was applied to control humidity within the vessels. A bottom Table 1 Media and culture conditions used for the regeneration of adventitious buds Proliferation (or multiplication) Induction

Regeneration

Composition MS macro ‡ micro MS vitamins 2.22 mM BA 3% Sucrose 100 mg lÿ1 Myo-inositol 100 mg lÿ1 NaFeEDDHA 0.7% Agarmix (ROTH agar ‡ Lab M agar, 0.35% each)

MS macro ‡ micro MS vitamins 6.8 mM TDZ, 0.49 mM IBA 3% Sucrose 100 mg lÿ1 Myo-inositol 100 mg lÿ1 Caseine hydrolysate 0.7% Agarmix (ROTH agar ‡ Lab M agar, 0.35% each) 60 mM AgNO3 (filter-sterilized) pH 5.8

MS macro ‡ micro MS vitamins 2.22 mM BA, 0.01 mM IBA 3% Sucrose 100 mg lÿ1 Myo-inositol 100 mg lÿ1 NaFeEDDHA 0.7% Agarmix (ROTH agar ‡ Lab M agar, 0.35% each)

Darkness 23  28C

16 h Photoperiod 23  28C 15 mmol mÿ2 sÿ1

pH 5.8 Culture conditions 16 h Photoperiod 23  28C 40 mmol mÿ2 sÿ1 Bottom cooling (85% RH)

pH 5.8

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cooling with 85% relative humidity in the culture vessel was created by setting the temperatures of the water from the chiller at 188C and the thermostat at 178C. The temperature in the culture room was maintained at 2328C. 2.3. General methods for adventitious bud regeneration Lea¯et explants were removed from 4-week-old cultures that had been subcultured for at least four micropropagation cycles and maintained on bottom cooling (see Table 1). Only the three uppermost fully expanded and unfurled lea¯ets (10±20 mm long) were excised from donor shoots and the petioluli were discarded. For a reliable regeneration system and a very high ef®ciency in the formation of adventitious buds in roses (Rosa hybrida L.), a modi®ed form of the two-step culture procedure that had been developed by Dubois and De Vries (1995) was adopted. First, whole detached lea¯et explants were inoculated with the abaxial side touching the induction medium (Table 1). The pH of the medium was adjusted to 5.6 with 1 N NaOH solution prior to autoclaving. Nine to twelve lea¯ets, depending on their size, were inoculated in each 90 mm15 mm sterile petri dish containing 25 ml medium and was sealed with para®lm. All cultures were initially incubated in the dark at 238C for 7 days. In the second step, following dark incubation, lea¯et explants with the abaxial face down were transferred to regeneration medium and maintained under culture conditions as in Table 1. The number of lea¯et explants forming shoots and the number of shoots formed per regenerating lea¯et were recorded after 30 days in culture. The following experimental modi®cations in the stock shoot cultures, culture media and environmental conditions were analyzed sequentially. 2.4. Effect of growth regulators Preliminary experiments were carried out in order to ®nd out the effectiveness of three different types of cytokinin, BA, TDZ and zeatin in combination with two types of auxin, IBA and NAA for the induction of adventitious buds. A total of 40 explants were used for each treatment. Later on, the best combination of cytokinin and auxin was selected for the induction medium. Nine to twelve lea¯et explants were cultured per petri dish with ®ve replicates for each treatment. 2.5. Effect of silver nitrate Excised lea¯ets were cultured on induction medium supplemented with various levels (60, 120, 180, or 240 mM) of ®lter-sterilized silver nitrate. Nine to twelve lea¯et explants were cultured per petri dish with ®ve replicates for each treatment.

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2.6. Effect of age of the explants Axillary shoots were initially maintained for 4, 5, 6 and 7 weeks on shoot proliferation medium (Table 1). After each induction time, lea¯et explants were harvested and treated using the two-stage culture procedure described above. Nine lea¯et explants were used per petri dish with ®ve replicates for each treatment. 2.7. Effect of relative humidity in the culture vessel To study the possible variations in leaf size induced by different relative humidities (RHs) in the container, shoot cultures were maintained at 85, 95 and 100% RH for 4 weeks. Lea¯ets were excised from each relative humidity condition and treated using the two-stage culture procedure described above. Nine to twelve lea¯et explants were cultured per petri dish with ®ve replicates for each treatment. 2.8. Effect of duration of initial darkness Lea¯et explants were placed on induction medium and initially cultured for 4, 7, 10 or 15 days in the dark. After each induction period, explants were transferred to regeneration medium and maintained for another 23 days under 15 mmol mÿ2 sÿ1 PAR. For each treatment, nine lea¯et explants were cultured per petri dish with ®ve replicates. 2.9. Effect of PAR After 7 days of induction in the dark, lea¯et explants were subsequently transferred to regeneration medium and exposed to three different light levels (15, 40 or 90 mmol mÿ2 sÿ1 PAR) as described above and maintained for another 23 days. For each treatment, nine lea¯et explants were cultured per petri dish with ®ve replicates. 2.10. Histology After 12±15 days in culture, leaf sections that have been cultured in different regeneration experiments were ®xed in FAA (18:1:1 v/v/v, 50% ethanol/glacial acetic acid/formalin), dehydrated by passage through an ethanol/butyl alcohol series, and embedded in paraf®n wax. Sections 10 mm thick were cut and stained with Safranin-Fast green. 2.11. Elongation of regenerated adventitious shoots After 30 days of culture, explants with regenerated shoot buds were transferred to fresh batches of the regeneration medium supplemented with BA (0.44 or

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0.88 mM), 2iP (4.92 or 9.84 mM) or kinetin (4.65 or 9.30 mM) for elongation. Explants were transferred individually to 2.5 cm15.0 cm culture tube containing 25 ml culture medium and closed with Kap UtsTM. Cultures were incubated at 2328C under a 16 h light photoperiod of 40 mmol mÿ2 sÿ1 PAR. A total of 24 explants with four replicates were cultured for each treatment. 2.12. Root induction Elongated shoots were excised and subcultured every 4 weeks to fresh medium with the same composition as the shoot proliferation medium. Several media based on half-strength MS modi®ed medium (Table 1) supplemented with ®ve levels (0.49, 2.46, 4.92 or 9.84 mM) of IBA were tested for rooting. Explants were transferred individually to 2.5 cm15.0 cm culture tubes containing 25 ml culture medium and closed with Kap UtsTM. Cultures were incubated at 2328C under a 16 h light photoperiod of 40 mmol mÿ2 sÿ1 PAR. A total of 24 explants with four replicates were cultured for each treatment. 2.13. Statistical analysis A general analysis of variance (ANOVA) was conducted for all variables using the Statgraphics program (STSC Inc., 1987), employing a completely random design. The number of leaves forming shoots and the number of shoots formed per regenerating leaf were recorded after 30 days in culture. All experiments were conducted at least twice. 3. Results and discussion 3.1. Shoot proliferation Cultures were initiated and proliferated on the same standard MS medium with 2.22 mM BA. Rinsing explants under running tap water before surface sterilization with 0.3% mercury chloride followed by 10% commercial bleach greatly reduced fungal and bacterial contamination and over 92% of these cultures were successful. Cultures were routinely micropropagated at 4-week intervals for at least four cycles. 3.2. Organogenesis Adventitious buds were observed on lea¯ets of all three genotypes tested. Lea¯et explants responded by internal swelling and developed callus around the cut end at the petiolular base (Fig. 1A) 2±3 days after transfer to the regeneration

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Fig. 1. Induction of adventitious buds and plantlet regeneration from lea¯et explants of Rosa hybrida L. (A) Callus developed at the cut end of the petiolular base after 2±3 days exposure to low light PAR (15 mmol mÿ2 sÿ1) on regeneration medium containing 2.22 mM benzyladenine (BA)‡0.049 mM indole-3-butyric aid (IBA). Initially, the explants had been placed in the dark for 7 days on induction medium containing 6.8 mM thidiazuron (TDZ)‡0.49 mM IBA. (B) Clusters of green pointed adventitious buds formed in the calli on the adaxial surface of a detached lea¯et at the petiolular end on regeneration medium after 12±15 days in culture. (C) High regeneration rate of multiple shoots developed on lea¯ets of Rosa hybrida L. genotype RUI 317 cultured for 30 days using a two-stage procedure.

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medium and exposure to the light conditions. No callus formation was observed while explants were placed on induction medium in the dark. Calli were compact, whitish-yellow, and with a nodular appearance. Less frequently, globular structures formed directly on the lea¯et surface without previous callus proliferation. In most cases, green pointed adventitious buds (Fig. 1B) were ®rst observed on the adaxial side of the lea¯et explants after 13±15 days of culture, and sometimes, leafy shoots were already formed. After 21±30 days of culture, treatments with a higher regeneration rate tended to have more buds per regenerating lea¯et explant (Fig. 1C). 3.3. Effect of growth regulators on regeneration Preliminary experiments clearly indicated that TDZ was signi®cantly more effective in inducing adventitious bud formation than BA or zeatin. In addition, adventitious bud-forming capacity was also signi®cantly more effective when TDZ was combined with IBA. The best concentration of TDZ and IBA for all the three genotypes tested is as presented in Table 2. We therefore used 6.8 mM TDZ plus 0.49 mM IBA as the induction medium for all other experiments. 3.4. Effect of silver nitrate on regeneration The bene®cial effects of AgNO3 on shoot proliferation in several Rosaceae species due to inhibition of ethylene action have been reported (Escalettes and Dosba, 1993; Dubois and De Vries, 1995). Incorporation of silver nitrate in the range 60±240 mg lÿ1 in the induction medium containing 6.8 mM TDZ plus 0.49 mM IBA clearly improved the percentage of regenerating lea¯ets in all the genotypes tested. The best concentration of AgNO3 was 60 mM when rate of regeneration was signi®cantly increased up to 20% for RUI 317, 25% for RUI 319 and 27% for `Inka', in comparison to their respective controls (Table 3). However, a signi®cant variation in the number of regenerating buds could only be observed with RUI 317. Table 2 Effects of a selected induction medium containing 6.8 mM TDZ with 0.49 mM IBA on three genotypes (RUI 317, RUI 319 and `Inka') on the rate of regeneration and average number of buds formed per regenerating explanta Cultivar

Regenerating leaflets (%)

Number of buds per explant

RUI 317 RUI 319 `Inka'

93.5c 76.5a 85.5b

3.8b 2.4a 2.6a

a Values in the same column followed by the same letter are not signi®cantly different from each other (least signi®cant differences test, p<0.05).

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Table 3 Effects of the best concentration of silver nitrate (60 mM) in comparison to the control on percentage of regeneration and number of adventitious buds per regenerating explant after 30 days of culturea AgNO3 (mM)

Control 60

Regenerating leaflets (%)

Number of buds per regenerating explant

RUI 317

RUI 319

`Inka'

RUI 317

RUI 319

`Inka'

78.0a 97.8b

48.8a 73.6c

60.4a 87.2c

2.9a 4.0b

2.9a 3.1a

2.9a 2.7a

a

Values in the same column followed by the same letter are not signi®cantly different from each other (least signi®cant differences test, p<0.05).

3.5. Effect of age of lea¯et explants on regeneration Age of the explant tissue had a signi®cant effect on bud formation, and in general, the highest number of buds per regenerating explant and a higher percentage of regenerating lea¯ets were obtained on young tissues and the least on old tissues. In general, explants from 4-week-old shoots gave the highest percentage of regenerating lea¯ets, ranging from 84.6 to 97.8% with 2.8±4.1 buds/explant (Table 4). Variation in the number of regenerated buds was also observed between shoots of different ages, especially in RUI 317. Lea¯ets from younger shoots (4±5-week-old) often produced multiple shoot buds, while older explants (more than 6-week old) formed mainly single shoot buds. However, bud regeneration ability of the lea¯ets decreased dramatically when the age of the donor shoots exceeded 6 weeks. Thus, 4-week-old shoots proved to be the best source of explants. Similar ®ndings have also been reported in Prunus (Mante et al., 1989), Lachenalia (Niederwieser and Van Staden, 1990), Cydonia oblonga (Baker and Bhatia, 1993), Aegle marmelos (Islam et al., 1993), Malus (Famiani et al., 1994). In general, the younger the material, the easier the organ formation occurs in vitro (Thorpe and Patel, 1984), which might be because the youngest Table 4 Effects of age of donor shoots on percentage of regenerating lea¯ets and number of adventitious buds per regenerating explant after 30 days of culturea Age (weeks)

4 5 6 7

Regenerating leaflets (%)

Number of buds per regenerating explant

RUI 317

RUI 319

`Inka'

RUI 317

RUI 319

`Inka'

97.8c 91.2bc 86.8b 62.6a

84.6c 73.6b 73.6b 48.8a

89.0b 84.6b 80.2b 55.8a

4.1b 3.8b 2.8a 2.5a

2.8b 2.4b 2.3ab 1.8a

2.8b 2.4ab 2.4ab 2.1a

a Values in the same column followed by the same letter are not signi®cantly different from each other (least signi®cant differences test, p<0.05).

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lea¯ets have more metabolically active cells with a more suitable hormonal and nutritional situation that could improve organogenesis (Famiani et al., 1994). 3.6. Effect of relative humidity on regeneration Relative humidity in the culture vessel is considered one of the critical factors that determine high percentage of regenerating explants in roses. According to Maene and Debergh (1987) and Vanderschaeghe and Debergh (1987), reduction of RH in the culture vessel can be achieved by placing the cultures on a cooled plate, which causes the water vapor to condense on the agar medium thus reducing hyperhydricity. Lea¯ets from donor shoots maintained on bottom cooling at 85 and 95% RH are 3±6-fold larger compared to lea¯ets from shoots maintained at 100% RH. In general, lea¯et explants taken from donor shoots maintained at 100% RH had a signi®cantly reduced bud-forming capacity both in terms of percentage of regenerating explants and average number of buds per regenerating explant (Table 5). However, there was no signi®cant difference in terms of adventitious bud-forming capacity between 85 and 95% RH. Relative humidity, resulting from temperature gradients in the container and the medium's water status, plays a major role in controlling hyperhydricity (Debergh, 1987). At the same time, it was also observed that maintaining cultures on bottom cooling led to the production of stronger shoots and more waxy leaves. Similar observations have been reported in carnation (Ziv, 1986) and cabbage plants (Sutter and Langhans, 1982) by reducing the RH in the culture atmosphere through use of desiccants. In apple, exposure to low RH improved plant survival ex vitro by improving normal leaf development (Brainerd and Fuchigami, 1981). 3.7. Effect of duration of initial darkness on regeneration In the present study, the caulogenic competence of rose lea¯et explants was signi®cantly affected both by the duration of culture in the dark and the exposure Table 5 Effects of relative humidity (RH%) in the culture vessel containing donor shoots on regeneration and the number of adventitious buds per regenerating explant after 30 days of culturea RH (%)

85 95 100

Regenerating leaflets (%)

Number of buds per regenerating explant

RUI 317

RUI 319

`Inka'

RUI 317

RUI 319

`Inka'

100.0b 95.6b 48.8a

78.0b 71.4b 26.4a

91.2b 82.4b 37.4a

4.1b 3.2b 2.3a

3.0b 2.6b 1.8a

3.8b 3.0ab 2.1a

a Values in the same column followed by the same letter are not signi®cantly different from each other (least signi®cant differences test, p < 0.05).

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Table 6 Effects of duration of initial darkness (4, 7, 10 or 15 days) on percentage of regenerating lea¯ets and number of adventitious buds per regenerating explant after 30 days of culturea Duration (days)

4 7 10 15

Regenerating leaflets (%)

Number of buds per regenerating explant

RUI 317

RUI 319

`Inka'

RUI 317

RUI 319

`Inka'

51.2b 97.8c 95.6c 39.6a

35.2a 73.6b 75.8b 37.6a

44.2b 84.6c 84.6c 28.6a

2.3a 4.0b 3.7b 2.3a

1.5a 2.5c 2.4bc 1.9b

2.2a 2.6a 2.5a 2.1a

a

Values in the same column followed by the same letter are not signi®cantly different from each other (least signi®cant differences test, p<0.05).

to TDZ in the induction medium as well as by the genotype. Effect of the length of the dark pretreatment was variable depending on the genotype. In our experiments, the percentage of regenerating explants was highest after 7 or 10 days of darkness (signi®cantly better than 4 and 15 days), and the number of buds per regenerating explant was greatest after 7 days (Table 6). While the frequency of callus formation was not affected, cultures in prolonged darkness not only resulted in low regenerability, but also promoted the formation of small and stunted buds that were very dif®cult and took much longer time to elongate. Percentage of regenerating explants as high as 73.6±97.8% (2.5±4.0 buds/explant) were obtained after 7 days of dark pre-treatment. In preliminary experiments, direct exposure of lea¯et explants to light (40 mmol mÿ2 sÿ1 PAR) without prior incubation in the dark failed to stimulate bud formation (data not presented). Incubation in the dark has been reported to increase shoot regeneration in various plant species, such as blueberry (Billings et al., 1988), pear (Chevreau et al., 1989), apple (Fasolo et al., 1989) and quince (Baker and Bhatia, 1993). Our results indicated that an incubation period of 7 days in the dark is optimal for shoot regeneration of rose. 3.8. Effect of PAR on regeneration A low PPFD (15 mmol mÿ2 sÿ1 PAR) during exposure to regeneration medium signi®cantly improved the percentage of regenerating explants. However, over the range of genotypes tested in this experiment, lower PAR (15 mmol mÿ2 sÿ1) had no signi®cant effect on the number of buds per regenerating explant (Table 7). Genotype RUI 317 seemed to be the most sensitive, followed by `Inka' and RUI 319, the least. It was also observed that high PAR (90 mmol mÿ2 sÿ1) not only signi®cantly reduced the percentage of regenerating explants but also produced stunted and hyperhydric adventitious shoot buds for all the genotypes.

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Table 7 Effects of different levels of PAR (15, 40 and 90 mmol mÿ2 sÿ1) on percentage of regenerating leaves and number of adventitious buds per regenerating explant after 30 days of culturea PAR (mmol mÿ2 sÿ1)

Regenerating leaflets (%)

Number of buds per regenerating explant

RUI 317

RUI 319

`Inka'

RUI 317

RUI 319

`Inka'

15 40 90

97c 67b 54a

73c 50b 32a

85c 62b 41a

4.0a 3.8a 3.6a

2.5a 2.6a 2.4a

2.8b 2.7ab 2.3a

a

Values in the same column followed by the same letter are not signi®cantly different from each other (least signi®cant differences test, p<0.05).

3.9. Histology Histological studies showed that callus tissue was initiated at the cut edge of the lea¯ets, and that subsequently buds formed in these calli after 10±12 days of culture (Fig. 2A). Usually several buds can be observed in close proximity (Fig. 2B), representing possible proliferation of axillary buds from the ®rst initiated bud or the formation of multiple adventitious buds. The formation of multiple adventitious shoots (Fig. 2C) may indicate the presence of a number of competent cells close to each other and possibly of common origin. Alternatively, the ®rst bud may stimulate the formation of additional buds. 3.10. Elongation of adventitious buds Attempts to micropropagate and multiply regenerated shoot buds using the proliferation medium were not very successful since most shoot buds became necrotic and later died. Therefore, the best chance of survival is to ®rst transfer the lea¯et explants with multiple shoot buds to an elongation medium. Cytokinin commonly stimulates shoot proliferation and inhibits their elongation. Huetteman and Preece (1993) reported that the inhibition of shoot elongation by TDZ might be consistent with its high cytokinin-like activity. In contrast, the present study indicated that both BA (0.44 and 0.88 mM) and kinetin (4.65 and 9.30 mM) were able to elongate shoots signi®cantly better than 2iP (4.92 or 9.84 mM) (Table 8). However, on medium containing kinetin at 4.65 mM, it was also observed that elongation was the best among all the cultivars tested and most of the shoots elongated after 2 weeks reaching up to 1.5±3.0 cm in height. 3.11. Rooting of regenerated shoots Elongated shoots measuring between 1.5 and 3.0 cm in height were used for the rooting experiment. Half-strength modi®ed MS medium with zero growth

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Fig. 2. Histology of morphogenetic responses on leaf segments of in vitro produced shoot buds of Rosa hybrida L. (A) Callus and bud primordia (BP) emerging at the petiolular end of a lea¯et explant after 12 days of culture. (B) Petiolular-stub callus showing bud clusters (BCs) and leaf primordia (LP) formed after 15 days of culture. (C) Formation of multiple shoots after 21 days of culture.

regulators induced root formation more effectively than IBA containing medium at all concentrations tested (Table 9). The average number of roots per rooted shoot was variable (2.4±5.5) and the cumulative length of roots varied from 0.5 to 3.5 cm. In some treatments, roots were visible after 10±15 days, but the ®nal observation was made after 30 days. Nevertheless, a low concentration of IBA (0.49 mM) was signi®cantly more effective than higher concentrations (2.46± 9.84 mM). However, higher concentrations of IBA (4.92±9.84 mM), although

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Table 8 Effect of cytokinins BA (0.44 and 0.88 mM), 2iP (4.92 and 9.84 mM) and kinetin (4.65 and 9.30 mM) on percentage of elongation of regenerated adventitious shoots after 30 days of culturea Cytokinins

Concentration (mM)

RUI 317

RUI 319

`Inka'

BA

0.44 0.88 4.92 9.84 4.65 9.30

87.2b 91.5b 66.8a 58.5a 100b 95.7b

75.0b 83.2b 54.2a 50.0a 91.5b 79.2b

72.7bc 83.2cd 62.7ab 54.2a 95.7d 83.2cd

2iP Kinetin a

Values in each column followed by the same letter are not signi®cantly different from each other (least signi®cant difference test, p<0.05). Table 9 Effects of different concentrations of indole-3-butyric acid (IBA) on percentage of rooting and average number of roots formed after 30 days of culturea IBA (mM)

Control 0.49 2.46 4.92 9.84

Rooting (%)

Number of roots per explant

RUI 317

RUI 319

`Inka'

RUI 317

RUI 319

`Inka'

92.0c 84.0c 68.0b 44.0a 48.0a

88.0c 76.0c 64.0b 32.0a 36.0a

100.0c 88.0c 72.0b 48.0a 52.0a

2.4a 3.5c 3.2b 4.0d 4.8e

2.7a 3.1b 3.1ab 4.1c 4.9d

3.9a 4.0a 3.6a 4.4b 5.5c

a

Values in the same column followed by the same letter are not signi®cantly different from each other (least signi®cant differences test, p<0.05).

producing many roots, inhibited shoot growth and produced darker roots and a substantial amount of callus at the base of the shoots. On zero growth regulator medium, fewer but healthier and more vigorously growing roots were formed directly at the base of the shoots with no callus formation. 4. Conclusion An ef®cient adventitious bud regeneration system from lea¯ets of in vitro micropropagated shoots was developed for three rose cultivars: `Inka', RUI 317 and RUI 319, using a two-step culture procedure. In the ®rst step, lea¯et explants were initially cultured on MS induction medium containing 4.6±9.2 mM TDZ with 0.49 mM IBA and incubated in the dark for 7 days. At this stage, beside the incorporation of growth regulators TDZ plus IBA and silver nitrate (in the range 60±180 mM) in the induction medium and pre-incubated under darkness, other

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factors were also considered as equally critical for the successful initiation of adventitious buds, such as: age of the donor shoots (4-week-old), position of lea¯ets on the stem (fully expanded from top three nodes), and source of donor shoots (maintained on bottom cooling with 85% RH in the culture vessel). Factors such as darkness, silver nitrate and age of the explants have been reported by many investigators to affect the regeneration of adventitious buds/shoot in roses. However, this is the ®rst report that incubating donor shoots on bottom cooling at 85% RH in the culture vessel were used in order to reduce hyperhydricity and thereby produce stronger shoots with more waxy and bigger lea¯ets and a high percentage of regenerating lea¯et explants in Rosa hybrida L. was achieved. In the second step, after dark incubation, explants were subsequently subcultured on MS regeneration medium containing 2.22 mM BA with 0.049 mM IBA and placed in the presence of light for the remaining 23 days. In this experiment, we have demonstrated that low PAR (15 mmol mÿ2 sÿ1) signi®cantly improved the percentage of regenerating lea¯et explants as compared with medium (40 mmol mÿ2 sÿ1) or high (90 mmol mÿ2 sÿ1) PAR. Regenerating adventitious shoot buds failed to survive when excised and transferred directly on the multiplication medium. Therefore, in order to achieve a high rate of survival, shoot buds which were still attached to the original lea¯et explant were ®rst transferred to an elongation medium containing 4.65 mM kinetin and thereby maximum elongation as high as 100% was obtained. Elongated shoots rooted best on modi®ed half-strength MS medium without any growth regulator. This system resulted in a recovery percentage of responsive explants that was suf®ciently high (75±96%) and with suf®cient shoots per explant (2.4±3.7) to be used for future Agrobacterium-mediated transformation and in vitro mutagenesis work. Since mutation and transformation are regarded as single-cell events (Srikandarajah et al., 1990), the more the regeneration sites per explant, the better are the chances of transformation. References Arene, L., Pellegrino, C., Gudin, S.A., 1993. Comparison of the somaclonal variation level of Rosa hybrida L. cv. Meirutral plants regenerated from callus or direct induction from different vegetative embryogenic tissues. Euphytica 71, 83±90. Baker, B.S., Bhatia, S.K., 1993. Factors affecting adventitious shoot regeneration from leaf explants of quince (Cydonia oblonga). Plant Cell Tiss. Org. Cult. 35, 273±277. Billings, S.G., Chin, C.K., Jelenkovic, G., 1988. Regeneration of blueberry plantlets from leaf segments. HortScience 23, 763±766. Brainerd, K.E., Fuchigami, L.H., 1981. Acclimatization of aseptically cultured apple plants to low relative humidity. J. Am. Soc. Hort. Sci. 106, 515±518. Burger, D.W., Liu, L., Zary, K.W., Lee, K.W., Lee, C.I., 1990. Organogenesis and plant regeneration from immature embryos of Rosa hybrida L. Plant Cell Tiss. Org. Cult. 21, 147± 152.

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Srikandarajah, S., Skirvin, R.M., Abu-Qaoud, H., Korban, S.S., 1990. Factors involved in shoot elongation and growth of adventitious and axillary shoots of three apple scion cultivars in vitro. J. Hort. Sci. 65, 113±121. STSC Inc., 1987. STATGRAFICS Users' Guide. Sutter, E.G., Langhans, R.W., 1982. Formation of epicuticular wax and its effect on water loss in cabbage plants regenerated from shoot-tip cultures. Can. J. Bot. 60, 2896±2902. Swartz, H.J., Bors, R., Mohamed, F., Naess, S.K., 1990. The effect of in vitro pretreatments on subsequent shoot organogenesis from excised Rubus and Malus leaves. Plant Cell Tiss. Org. Cult. 21, 179±184. Thorpe, T.A., Patel, K.R., 1984. Clonal propagation: adventitious buds. In: Vasil, I.K. (Ed.), Cell Culture and Somatic Cell Genetics of Plants, Vol. 1. Academic Press, New York, pp. 49±60. Vanderschaeghe, A.M., Debergh, P.C., 1987. Technical aspects of the control of the relative humidity in tissue cultured containers. In: Ducate, G., Jacob, M., Simeon, A. (Eds.), Plant Micropropagation in Horticultural Industries. Presses Universitaires, Liege, Belgium, pp. 68±76. Ziv, M., 1986. In vitro hardening and acclimatization of tissue culture plants. In: Withers, L.A., Alderson, P.G. (Eds.), Plant Tissue Culture and its Agricultural Applications. Butterworths, London, pp. 187±196.