Somatic embryogenesis, secondary somatic embryogenesis, and shoot organogenesis in Rosa

J. Plant Physiol. 159. 313 – 319 (2002)  Urban & Fischer Verlag http://www.urbanfischer.de/journals/jpp

Somatic embryogenesis, secondary somatic embryogenesis, and shoot organogenesis in Rosa Xiangqian Li, Sergei F. Krasnyanski, Schuyler S. Korban* Department of Natural Resources & Environmental Sciences, University of Illinois, 310 ERML, 1201 W. Gregory, Urbana, IL 61801, USA

Received August 15, 2001 · Accepted October 1, 2001

Summary The influence of various 2,4-D concentrations (11.3–181 µmol/L) on induction of callus from leaf tissues of Rosa hybrida cvs. Carefree Beauty and Grand Gala and R. chinensis minima cv. Red Sunblaze was evaluated. Following transfer of callus to a regeneration medium containing different concentrations of thidiazuron (TDZ) (0 – 90.8 µmol/L), 6-benzyladenine (BA) (0 – 44.4 µmol/L), or 2.9 µmol/ L gibberellic acid (GA3), alone or in various combinations, the highest frequency of embryogenic (32 %) and organogenic (55.3 %) callus was induced on ‘Carefree Beauty’. Secondary somatic embryos were also induced on somatic embryos of ‘Carefree Beauty’. The effects of different concentrations of TDZ (2.3 µmol/L), BA (2.2 or 4.4 µmol/L), and abscisic acid (ABA) (3.8 or 7.6 µmol/L), alone or in combinations, on proliferation and germination of secondary somatic embryos were also evaluated. ABA was found to be the most effective in promoting proliferation and germination of somatic embryos. The growth rate of secondary embryogenic callus grown on ABA increased by 36fold, while germination rate of somatic embryos was more than five times higher than those derived from embryogenic callus grown on BA and TDZ. For R. chinensis minima cv. Red Sunblaze, only somatic embryogenesis (6.6 %) was observed; while, for R. hybrida cv. Grand Gala, only shoot organogenesis (3.3 %) was observed. Key words: plant growth regulators – Rosa chinensis minima – Rosa hybrida – secondary somatic embryogenesis – shoot organogenesis – somatic embryogenesis Abbreviations: ABA abscisic acid. – BA 6-benzyladenine. – 2,4-D 2,4-dichlorophenoxyacetic acid. – GA3 gibberellic acid. – MS Murashige and Skoog (1962). – TDZ thidiazuron

Introduction Successful development of regeneration systems for a number of rose species have already been reported. Embryogenic callus has been initiated from in vitro-derived leaf or * E-mail corresponding author: [email protected]

stem segments of Rosa hybrida cv. Carl Red and R. canina (Visessuwan et al. 1997), R. hybrida cv. Carefree Beauty, and R. chinensis minima cv. Baby Katie (Hsia and Korban 1996). Embryogenic callus has been also induced in leaves of R. hybrida cvs. Domingo and Vicky Brown (De Wit et al. 1990), petioles and roots of R. hybrida cvs. Trumpeter and Glad Tidings (Marchant et al. 1996), root explants of both R. hy 0176-1617/02/159/03-313 $ 15.00/0

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brida cv. Moneyway (van der Salm et al. 1996) and R. Heritage × Alista Stella Gray (Sarasan et al. 2001), petals of R. hybrida cv. Arizona (Murali et al. 1996), and immature seeds of R. rugosa (Kunitake et al. 1993). This has also been achieved using immature leaf or stem segments of R. hybrida cv. Landora (Rout et al. 1991), in vivo mature leaves of R. hybrida cv. Soraya (Kintzios et al. 1999), anther filaments of R. hybrida cv. Royalty (Noriega and Söndahl 1991), as well as anthers, petals, receptacles, and leaves of R. hybrida cv. Meirutal (Arene et al. 1993). The wide range of explants and experimental approaches that have been employed with different rose species and cultivars strongly suggest that it is difficult to develop a universal genotype-independent method for the production of embryogenic callus in rose (Marchant et al. 1996). Recent progress on rose regeneration has been reviewed by Rout et al. (1999). Rout et al. (1991) and Hsia and Korban (1996) have observed secondary somatic embryogenesis in rose. Secondary somatic embryogenesis has great potential for largescale micropropagation, which is especially important for those woody plants having long generation cycles, and with low frequencies of somatic embryogenesis. So far, there are no reports on proliferation and germination of secondary somatic embryos in rose. In this study, we investigated effects of various plant growth regulators (PGRs) on regeneration of R. hybrida cvs. Carefree Beauty and Grand Gala, and R. chinensis minima cv. Red Sunblaze, induction of secondary somatic embryogenesis, as well as proliferation, maturation, and germination of secondary somatic embryos in R. hybrida cv. Carefree Beauty.

Materials and Methods Plant material and culture conditions Proliferating shoot cultures of R. hybrida cvs. Carefree Beauty and Grand Gala, and R. chinensis minima cv. Red Sunblaze were previously established from nodal stem segments of greenhouse-grown plants as described by Hsia and Korban (1996). All basal media containing half- or full-MS salts (Murashige and Skoog 1962), full-MS vitamins, and 87.6 mmol/L sucrose, were adjusted to pH 5.7 with 1 N NaOH prior to autoclaving. All cultures were incubated under a 16 h photoperiod provided by cool-white fluorescent light (60 µmol m – 2 s –1), except for callus induction which was conducted in the dark, and a temperature of 23 ± 1˚C. Each treatment for callus induction, embryogenic callus induction, and shoot organogenesis was replicated three times, and each replication consisted of a single Petri plate containing 10 explants.

Gala’ were quite large, and therefore they were cut into leaflets, and used for culture. All leaf explants were placed with the abaxial surface in contact with the medium. The basal medium containing full-MS salts and MS vitamins was supplemented with different concentrations of 2,4-D, including 11.3, 45.2, 90.5, and 181 µmol/L, and solidified with 7 g TC agar (PhytoTechnology, Shawnee Mission, Kansas). Cultures were incubated in the dark for 4 weeks, and then data on number of explants developing callus were recorded.

Induction of somatic embryogenesis and shoot organogenesis Explants previously incubated on media containing 2,4-D (a concentration of 11.3 µmol/L was used for both ‘Carefree Beauty’ and ‘Grand Gala’; while, a concentration of 45.2 µmol/L 2,4-D was used for ‘Red Sunblaze’) were transferred to a 1⁄2 MS basal medium containing different concentrations of 6-benzyladenine (BA) (0 – 44.4 µmol/L), thidiazuron (TDZ) (0 – 90.8 µmol/L), gibberellic acid (GA3) (2.9 µmol/L), alone or in various combinations. The medium was solidified with 2.5 g gelrite gellan gum (PhytoTechnology), and cultures were grown under light conditions as described above. In one experiment, all leaf explants with and without callus of all three cultivars were incubated for a period of 4 months. In a second experiment, leaf explants of ‘Carefree Beauty’ with callus were transferred to media containing different cytokinins and/or GA3, and incubated for 2 months, and later transferred to a PGR-free medium for an additional 2 months. Explants with callus developing only on the petiole were transferred to a basal medium containing 90.8 µmol/L TDZ, and incubated for 4 months. Data on number of explants developing somatic embryos and/or adventitious shoots were recorded.

Proliferation and maturation of secondary somatic embryos of ‘Carefree Beauty’ Primary embryogenic callus of ‘Carefree Beauty’ induced on a medium containing 2.3 µmol/L TDZ was used for proliferation and maturation of secondary somatic embryos. In one experiment, a total of 32 primary embryogenic calli were transferred into four Petri plates (8 calli per plate per replication) and incubated on a medium containing either 3.8 or 7.6 µmol/L ABA for a period of one month, and then transferred to a PGR-free medium for a period of 5 months. In a second experiment, 70 primary embryogenic calli were transferred into 10 Petri plates (7 calli per plate per replication) and incubated on a medium containing either no PGR, 2.2 or 4.4 µmol/L BA alone, 2.3 µmol/L TDZ alone, or a combination of 2.2 µmol/L BA and 2.3 µmol/L TDZ for a period of 4 months. Explants were then transferred to a PGR-free medium for an additional 2 months. Each of the above two experiments contained two replications per treatment; and organized in a completely randomized design. All basal media contained 1⁄2 MS salts, MS vitamins, and were solidified with 2.5 g gelrite gellan gum. Data were recorded on callus with secondary somatic embryos, number of developing shoots, and/or plantlet regeneration.

Callus induction The top four vigorously-growing leaves were excised from proliferating shoots. Whole leaves of ‘Carefree Beauty’ and ‘Red Sunblaze’ were used as explants for callus induction. However, leaves of ‘Grand

Plantlet development Both regenerated shoots (derived via shoot organogenesis) and plantlets (derived via somatic embryogenesis) were subcultured onto

Somatic embryogenesis in Rosa a shoot elongation medium as described by Hsia and Korban (1996) for a period of one month. Vigorously-growing shoots were transferred to a PGR-free medium for 3 weeks to induce rooting. Rooted plants were transferred to soil mix (1 : 1 : 1 of soil, peat, and perlite) in 4 cm plastic pots for two weeks, and covered with a transparent plastic top. The plastic top was gradually removed, and well-developed plantlets were transferred to the greenhouse and grown at 23 ˚C. Plants were watered daily using a drip-irrigation system, and fertilized once every 2 weeks with 250 ppm of a 20 – 20 – 20 NPK solution (Plant Marvel Laboratories, Chicago, Illinois).

Statistical analysis All data were analyzed using ANOVA and means were compared using the least significant difference (LSD) at the 5 % probability level. All computations were made using the SAS statistical analysis package.

Table 1. Effect of different 2,4-D concentrations on frequency of callus induction from three genotypes of rose. 2,4-D

No. of

% Callus induction

(µmol/L) leaf/leaflet explants

R. chinensis minima

R. hybrida cv.

R. hybrida cv.

cv. Red Sunblazex Carefree Beautyy Grand Galaz

0.0

30

0.0 d

0.0 c

0.0 b

11.3

30

53.3 ab

66.7 a

93.3 a

45.2

30

70.0 a

53.3 b

93.3 a

90.5

30

50.0 b

36.7 b

93.3 a

30

20.0 c

23.3 b

93.3 a

181

Means followed by the same letter within a column were not significantly different as determined by an LSD at 5 % probability level. x

LSD at 0.05 = 18.789.

y

LSD at 0.05 = 18.193.

z

LSD at 0.05 = 9.414.

315

Results Effect of 2,4-D concentration on callus induction Following incubation of whole leaves (with petioles) of ‘Carefree Beauty’ and ‘Red Sunblaze’ and leaflet explants (without petiole) of ‘Grand Gala’ on a medium containing different concentrations of 2,4-D, callus was induced from all three rose genotypes on all 2,4-D concentrations used, but not on 2,4-D-free medium (Table 1). No embryo or embryo-like structures were observed on any of the induced callus. For ‘Red Sunblaze’, the highest frequency of callus induction was observed on a medium containing 45.2 µmol/L 2,4-D, although this was not significantly different from that observed on 11.3 µmol/L 2,4-D (Table 1). For ‘Carefree Beauty’, the highest frequency of callus induction was observed on a medium containing 11.3 µmol/L 2,4-D, and by increasing the level of 2,4-D in the medium, the frequency of callus induction significantly decreased (Table 1). For ‘Grand Gala’, a concentration of 11.3 µmol/L 2,4-D induced the highest frequency of callus induction, which was identical to all other 2,4-D levels used (Table 1). In addition, both the color and texture of the callus derived from these three genotypes of rose were quite different. The callus of ‘Carefree Beauty’ was soft, friable, and opaque-white in color, with very few exceptions of brownish callus observed. However, the callus of both ‘Grand Gala’ and ‘Red Sunblaze’ was hard, compact, and palegreen in color.

Effect of PGRs on somatic embryogenesis and shoot organogenesis All ‘Carefree Beauty’ leaf explants with or without callus induced on a medium containing 2,4-D were transferred to basal medium with different concentrations of TDZ (0 to

Table 2. Effects of different TDZ concentrations on embryogenic and organogenic callus induction of ‘Carefree Beauty’. TDZ (µmol/L)

GA3 (µmol/L)

No. of explants

0 9.1 27.2 54.5 90.8

0 2.9 2.9 2.9 2.9

30 30 30 30 20

% Callus induction Embryogenic callusw

Organogenic callusx

Non-differentiated callus on leaflety

Non-differentiated callus on petiolez

6.7 a 3.3 ab 0b 0b 0b

0a 0a 3.3 a 3.3 a 5.0 a

0a 96.7 b 96.7 b 93.3 b 45.0 c

33.3 a 46.7 ab 53.3 ab 66.6 ab 70.0 b

Means followed by the same letter within a column were not significantly different at P < 0.05. w Embryogenesis observed on callus previously induced on leaf tissue; LSD at 0.05 = 6.664. x Organogenesis observed on callus previously induced on petioles; LSD at 0.05= 8.74. y LSD at 0.05 = 13.7. z LSD at 0.05 = 33.35.

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Xiangqian Li, Sergei F. Krasnyanski, Schuyler S. Korban

Figure 1. A – L Somatic embryogenesis, secondary somatic embryogenesis, shoot organogenesis, and plant development in Rosa. A – D Primary somatic embryos of Rosa hybrida cv. Carefree Beauty. A Somatic embryos at the torpedo stage; B somatic embryos at the cotyledonary stage; C a welldeveloped somatic embryo; and D a maturing somatic embryo. E – F Secondary somatic embryogenesis in R. hybrida cv. Carefree Beauty. E Secondary somatic embryos; F maturation of secondary somatic embryos. G Shoot organogenesis in R. hybrida cv. Carefree Beauty. H Primary somatic embryos in R. chinensis minima cv. Red Sunblaze. I Shoot organogenesis in R. hybrida cv. Grand Gala. J – L Regenerated plants. J R. hybrida cv. Carefree Beauty; K R. chinensis minima cv. Red Sunblaze; and L R. hybrida cv. Grand Gala.

90.8 µmol/L) and GA3 (2.9 µmol/L). After 4 months of culture, three morphologically different types of callus were observed: embryogenic, organogenic, and non-differentiated callus. Somatic embryogenesis was observed on callus previously induced from leaf but not from petiole tissues. Somatic embryogenesis was observed on PGR-free medium and on medium containing 9.1 µmol/L TDZ, but not on any of the media containing higher TDZ levels (Table 2). Upon adding TDZ, most callus on leaf explants became hard, compact, and green in color, and did not differentiate. For callus previously induced on petiole explants on medium containing 2,4-D, only shoot organogenesis was observed. This hard green callus produced adventitious shoots on medium containing TDZ at levels of 27.2 µmol/L and higher, although the frequency of shoot organogenesis reached only 5 % (Table 2). Therefore, in a second experiment only callus induced from leaf explants was used to induce somatic embryogenesis, although the incubation period on PGR-containing media was shortened to 2 months. Among various PGRs tested, the highest frequency of somatic embryogenesis (30 – 32 %) was observed for ‘Carefree Beauty’ on medium containing either

2.3 µmol/L TDZ, 2.9 µmol/L GA3, or a combination of 2.9 µmol/ L GA3 with either 2.3 µmol/L TDZ or 2.2 BA (Table 3). Most somatic embryos appeared to have normal morphology with two cotyledons. However, few abnormal somatic embryos were also observed whereby some had either one, three or more cotyledons; while others had cylindrical or plate-like shaped cotyledons. The development of embryogenic callus was asynchronous. Globular, hearted-shaped, and cotyledonary stages were observed throughout the incubation period on PGR-containing media, and later on as these were transferred to PGR-free medium (Fig. 1A – D). The development of bipolar plantlets was also asynchronous and observed throughout the incubation on PGR-free medium, and with continuous subculture, most somatic embryos developed into bipolar plantlets. However, secondary somatic embryogenesis was also observed on ‘Carefree Beauty’ explants (Fig. 1E and F). For ‘Carefree Beauty’, callus induced on petiole of leaf explants on 2,4-D medium were transferred to a basal medium containing only 90.8 µmol/L TDZ. Within 4 months, 21 out of 38 (55.3 %) of callus explants developed shoots (Fig. 1G). On

Somatic embryogenesis in Rosa

317

Table 3. Effect of different TDZ, BA, and GA3 concentrations on embryogenic and organogenic callus induction. TDZ (µmol/L)

0 0 2.3 2.3 4.6 4.6 6.9 27.2 54.5 90.8 0 0 0 0 0 0 0 2.3 4.5 2.3 4.5

BA (µmol/L)

0 0 0 0 0 0 0 0 0 0 2.2 2.2 4.4 4.4 8.8 22.2 44.4 2.2 2.2 4.4 4.4

GA3 (µmol/L)

0 2.9 2.9 0 0 2.9 2.9 2.9 2.9 2.9 0 2.9 0 2.9 0 0 0 0 0 0 0

No. of explants

% Somatic embryogenesis ‘Carefree Beauty’x

‘Red Sunblaze’y

10 bc 30 a 31.6 a 30 a 6.67 bc 20 ab 13.3 bc 10 bc 10 bc 0 c 6.67 bc 32 a 13.3 bc 21.67 ab 10 bc 6.67 bc 0 c 13.3 bc 10 bc 6.67 bc 3.3 c

0a – – 0a 0a 0a 0a 0a 6.6 b 0a 0a 0a 0a 0a 0a 0a 0a 0a 0a

30 60 60 30 30 30 30 30 30 30 30 50 30 60 30 30 30 30 30 30 30

% Shoot organogenesis ‘Grand Gala’z 0a – – 0a 0a 0a 3.3 b 0a 0a 0a 0a – 0a – 0a 0a 0a 0a 0a 0a 0a

Means followed by the same letter within a column were not significantly different at P < 0.05. x LSD at 0.05 = 16.026 y LSD at 0.05 = 2.324 z LSD at 0.05 = 2.324

average, three shoots per organogenic callus were obtained. Although most shoots were normal, a few shoots were fasciated. For both ‘Red Sunblaze’ and ‘Grand Gala’, callus first induced on 2,4-D media and then transferred to different PGRcontaining media for a period of 4 months turned either hard and green in color on PGR-containing media or brown in color on PGR-free medium. Only 6.6 % of ‘Red Sunblaze’ explants developed somatic embryogenic callus on a medium containing 54.5 µmol/L TDZ and 2.9 µmol/L GA3 (Fig. 1 H; Table 3). One of the two embryogenic calli was transferred to a PGR-free medium, but somatic embryos failed to germinate. Whereas, the other embryogenic callus was transferred to a medium containing 8.8 µmol/L BA, and somatic embryos developed into normal plants. No secondary somatic embryogenesis was observed on either medium. Only 3.3 % of ‘Grand Gala’ explants developed organogenic callus on a medium containing 6.9 µmol/L TDZ (Table 3; Fig. 1I).

lus) and proliferation of secondary somatic embryogenesis (36-fold increase over primary somatic embryos) were observed on a medium containing 3.8 µmol/L ABA (Table 4). These results were not significantly different from those observed on explants incubated on 2.2 µmol/L BA. However, as explants were incubated on a PGR-free medium or other PGR treatments, germination of somatic embryos significantly decreased (Table 4). Whereas, secondary somatic embryogenesis of explants grown on PGR-free medium was not significantly different from that of explants grown on either 3.8 or 7.6 µmol/L ABA. Bipolar plantlets were regenerated on both ABA and PGR-free media. Both shoots and bipolar plantlets were regenerated on 2.2 µmol/L BA treatment. Only shoots were regenerated on explants grown on all other treatments. Proliferation of somatic embryos continued at the same rate for a period of more than one year.

Germination of somatic embryos and proliferation of secondary somatic embryos in ‘Carefree Beauty’

Plantlet development

Both the highest germination rate of somatic embryos (5.25 bipolar plantlets regenerated per primary embryogenic cal-

Following rooting of adventitious shoots, all plantlets derived from both embryogenesis and organogenesis were acclimatized and successfully transferred to a soil mixture, and

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Xiangqian Li, Sergei F. Krasnyanski, Schuyler S. Korban

Table 4. The influence of PGRs on shoot formation and secondary somatic embryogenesis. PGR (µmol/L)

None BA (2.2) BA (4.4 ) TDZ (2.3) BA (2.2) + TDZ (2.3) ABA (3.8) ABA (7.6)

No. of primary No. of regenerated No. of embryogenic callus embryogenic shoots per primary em- with secondary somatic embryos/primary embryocallus bryogenic callusx genic callusy 7 7 7 7 7

2.50 b 4.14 a 0.93 bc 0.72 c 1.07 bc

30.0 a 19.2 ab 12.9 bc 11.4 c 12.0 c

8 8

5.25 a 2.44 b

36.0 a 30.0 ab

Means followed by the same letter within a column were not significantly different at P < 0.05. x LSD at 0.05 = 1.6229; y LSD at 0.05 = 14.62

grown in the greenhouse. All plants flowered normally within a period of 7 months (Fig. 1J, K, and L).

Discussion The first successful rose regeneration via in vitro shoot organogenesis was reported by Hill (1967). Since then, several regeneration protocols have been reported both on shoot organogenesis and/or somatic embryogenesis of roses. Among these protocols, the presence of 2,4-D alone or in combination with other PGRs in the culture medium has been reported to be essential for inducing somatic embryogenesis in rose (Rout et al. 1991, Kunitake et al. 1993, Hsia and Korban 1996, Marchant et al. 1996, van der Salm et al. 1996, Visessuwan et al. 1997). However, the effect of 2,4-D on callus induction of different rose cultivars has been quite variable. In this study, increasing 2,4-D concentration from 11.3 to 181 µmol/L resulted in a decrease in frequency of callus induction in one cultivar of R. hybrida, ‘Carefree Beauty’, while it did not affect callus induction in another cultivar, ‘Grand Gala’. As for R. chinensis minima cv. Red Sunblaze, there was an increase in frequency of callus induction as the 2,4-D increased from 11.3 to 45.2 µmol/L, but then followed by a significant reduction. Interestingly, we observed two types of callus induced on our explants: one type of callus was observed on the leaf tissue, while the other was observed on the petiole. Callus induced on leaf tissue was larger in size than that on the petiole, and when transferred to a basal medium in the absence of or at low levels of PGRs, the callus developed somatic embryos. Whereas, callus induced on petioles and transferred to a basal medium containing high PGRs (in particular TDZ) promoted only shoot organogenesis. Hsia and Korban (1996) have obtained a 6.6 % frequency of somatic embryogenesis in leaf sections of ‘Carefree Beauty’ incubated on a medium containing 23 µmol/L TDZ and 3 µmol/L GA3. In this study, among various PGRs investigated, the highest frequency of somatic embryogenesis

(31 %) has been observed on explants grown on a medium containing a lower concentration of TDZ (2.3 µmol/L) and supplemented with 2.9 µmol/L GA3. The incubation period of callus induced on leaf explants, 2 months, on TDZ-containing medium appears to be critical for induction of somatic embryogenesis as both longer incubation periods and higher TDZ levels promoted the development of undifferentiated hard-green callus instead. Interestingly, somatic embryogenesis has been induced on PGR-free medium, and on medium containing 2.9 µmol/L GA3 alone at frequencies of 10 % and 30 %, respectively (Table 3). GA3 has been previously reported to induce somatic embryogenesis in several rose cultivars (Rout et al. 1991, Marchant et al. 1996, Kintzios et al. 1999). GA stimulates the production of numerous enzymes, notably α-amylase, in germinating cereal grains (Davies 1995). In addition, GAs promote seed germination in some species that otherwise require cold stratification and/or light for inducing seed germination (Davies 1995). Overall in this study, TDZ has been more effective than BA in inducing somatic embryogenesis. However, adding 2.9 µmol/L GA3 to either TDZ or BA containing media at any level has improved the frequency of somatic embryogenesis. Combining BA and TDZ in the same medium has been less effective in inducing somatic embryogenesis than either PGR used alone. In addition, differences in genotypic response for somatic embryogenesis have been observed. Among the three cultivars used, somatic embryogenesis was observed in ‘Carefree Beauty’ and ‘Red Sunblaze’, while only shoot organogenesis was observed in ‘Grand Gala’. These genotypic differences in responses to exogenous PGRs and their undergoing of different cellular differentiation pathways have been previously noted by others (Rout et al. 1999). Secondary somatic embryogenesis has been observed in rose by Rout et al. (1991) and Hsia and Korban (1996). However, in this study, both proliferation and germination of secondary somatic embryos are successfully reported for the first time. It is found that ABA is the most effective PGR treatment for both secondary somatic embryo induction and germination. Although TDZ is more effective than BA in inducing embryogenic callus, it is less effective than BA in inducing secondary embryogenesis and promoting germination of somatic embryos. Visessuwan et al. (1997) have also found TDZ more effective than BA in inducing embryogenic callus, although the number of regenerated shoots per somatic embryo was lower than that reported for BA. The high proliferation and regeneration rate of secondary somatic embryos in ‘Carefree Beauty’ are very useful for genetic improvement of this cultivar. Somatic embryos are reportedly derived either from single cells or from single cells within a proembryonic mass (Litz and Gray 1995). Somatic embryogenic cells can act independently from neighboring cells and undergo somatic embryogenesis or they can continue to differentiate into secondary embryogenesis (Raemakers et al. 1995). This continuous proliferation of somatic em-

Somatic embryogenesis in Rosa bryos via secondary embryogenesis is both cost and time effective, and is independent of the explant source. Therefore, this is a useful system for rose micropropagation and for future genetic transformation. Acknowledgements. This research was funded by a grant received from the Fred C. Gloeckner Foundation and a grant received from the Horticultural Research Institute.

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