Plant Regeneration from Callus of Australian Fan Flower, Scaevola

• JOUR.AL OF •

J Plant Physiol Vol. 154. pp. 374-378 (1999)

Plani: Physjoloar © 1999 URBAN & FISCHER

Plant Regeneration from Callus of Australian Fan Flower, Scaevola PREM

L.

BHALLA

and HUlLING Xu

Plant Molecular Biology and Biotechnology Research Laboratory, Institute of Land & Food Resources, The University of Melbourne, Parkville, Victoria, 3052 Australia Received March 18, 1998 ·Accepted June 25, 1998

Summary

Plant regeneration from callus of fan flower, Scaevola phlebopetala F. Muell., an Australian native species of horticultural significance has been achieved. The effects of coconut milk, kinetin, 2,4-D and BAP on callus and shoot induction were investigated. Coconut milk promoted callus induction. Kinetin was not required for callus induction and 2,4-D induced a poor quality callus. BAP had a significant effect on callus induction, growth and appearance of callus. BAP also induced shoot initiation from callus produced from root and leaf explants. Shoot regeneration was achieved on a medium containing 5.0 o/o coconut milk, 60 g/L sucrose and 3 mg/L BAP. Shoot growth required a hormone free medium containing 20 g/L sucrose. Regenerated shoots were rooted on the medium supplemented with 4 mg/L of IBA. Ninety six percent of the regenerated plants survived when transferred to soil under glasshouse conditions.

Key words: Scaevola phlebopetala, Australian fan flower, Goodeniaceae, regeneration, tissue culture. Abbreviations: BAP = 6-benzyl aminopurine; IBA = indole-3-butyric acid; 2,4-D = 2,4-dichlorophenoxyacetic acid; SDS =sodium dodecyl sulphate. Introduction

Plants of genus Scaevola ( Goodeniaceae) commonly known as "fan flowers" are mostly endemic to Australia. Fan flowers are relatively new to ornamental horticulture. They are used as ground cover in Australia and as hanging basket, window box and garden bed plants in Europe and America (Steffen, 1989; von Hentig and Ehlers, 1991; von Hentig, 1993). Breeding of superior fan flowers of compact form with long flowering periods is desirable. The existing variability in a breeding population may not be sufficient for modern plant breeding purposes, thus an effort has to be made to broaden the existing gene pools of crops. In addition, Scaevola seeds are known to be difficult to germinate (Wrigley and Fagg, 1988). Thus, application of biotechnology may lead to alternative methods for propagation and genetic modification of Scaevola. Introducing genetically defined traits into plants is possible using molecular biology and gene transfer techniques, which must be coupled with reproducible and efficient plant regen-

eration methods. To date, no work has been reported on plant regeneration of Australian fan flowers (Johnson and Burchett, 1996). In this study, we report successful and efficient plant regeneration from callus of Scaevola phlebopetala F. Muell. Materials and Methods

Plant material Scaevola phlebopetala F. Mud!. plants were grown and maintained under normal glasshouse conditions. Explants Shoot cuttings 5-6 em in length were used as a source of explants. These shoot cuttings were surface-sterilised using 1.25 o/o sodium hypochlorite with detergent (0.5 mL of 20 o/o SDS, sodium dodecyl sulphate, in 50 mL) in 50 mL sterile plastic tubes (Greiner) by continuous shaking for 20 min. A maximum of five shoot cut0176-1617/99/154/374 $ 12.00/0

Plant Regeneration of Scaevola tings per tube were used for sterilisation. After rinsing three times in sterile distilled water, the shoot cuttings were cultured on MS medium (Murashige and Skoog, 1962) containing 6.0gL-1 agar for 6-8 weeks to ensure sterility of explant source.

Callus induction The basal medium for callus induction consisted of major elements, minor elements, vitamines as described in Murashige and Skoog (1962). The medium was solidified with 0.6 o/o agar (Cat #A9915, Sigma Chemical Company, St. Louis). This medium was supplemented with various amounts of sucrose (20-120 g/L) and coconut milk. (2.5-15 %) for callus induction. Various concentrations of kinetin (2-10 mg/L, dissolved in 1 N HCl) and 2,4-D (0.5-3.0 mg/L, dissolved in ethanol) were also tested for callus induction. Coconut milk was prepared as described in Bhojwani and Razdan (1983) and added before autodaving. pH of the medium was adjusted to 6.0 before autodaving at 121 ·c for 20 min. Aqueous solutions of growth hormones were filter-sterilised (0.22 mm millipore filter) and then added to the autodaved medium. The cultured Petri dishes were sealed using micropore surgical tape (3M) and incubated at 25 ·c with 16 h light (General Electric cool white tubes) of 50 J.tEm -Z s-1 at Petri dish level. The number of explants producing callus was recorded 4 weeks after culture initiation. Each experiment was repeated three times.

Shoot initiation Regenerated callus from above was used for shoot initiation. Shoot initiation medium consists of basal medium supplemented with 50 mLIL coconut milk, 60 g/L sucrose and various concentrations ofBAP (0.5-5.0mg/L, dissolved in ethanol). The number of shoots produced from callus was counted after 6-8 weeks of culture.

Shoot growth and root induction Once shoots reached 2-3 leaf stage (I em in length), 4-5 shoots were transferred to a plastic transparent cup containing hormonefree medium (basal medium) with reduced sucrose (20 g/L). Welldeveloped shoots (-4 weeks) were then transferred to rooting medium. Rooting medium was basal medium containing 20 g/L sucrose and IBA (4mg/L, dissolved in ethanol).

Transfer ofplantlets to soil Shoots producing at least 5 roots of 2-3 em in length and 8-10 leaves were transferred to a mist chamber in a cutting mix (10 L pearlite, 4 L sieved peat, 36 L of 6 mm pine bark and 37.5 g dolomite) for two weeks before transferring to commercial potting mix under glasshouse conditions.

Statistical analysis The data from experiments were analysed statistically using a one way analysis of variance.

Results and Discussion

Callus induction Sucrose concentrations ranging from 20 g/L to 120 g/L were first evaluated to determine the level of carbohydrate requirement for callus induction (Fig. 1). In general, leaf ex-

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plants produced very small callus regions at cut surfaces in low sucrose concentrations (20 g/L and 40 g/L). The explants looked dark brown and dead at 100 g/L and 120 g/L of sucrose after two weeks and no callus formation was observed in these treatments. No response from root explants was observed in these treatments. Various concentrations (0, 2.5, 5, 10, 15 %) of coconut milk were added to the medium. Significant improvement in callus induction from root explants occurred when 2.5% coconut milk was added (Fig. 2). When 5% coconut milk was used, callus produced from both leaf and root explants increased significantly, but higher concentrations (10 and 15 %) of coconut milk had no significant effect on callus induction (Fig. 2). The requirement of coconut milk for growth and maintenance of callus was evident when coconut milk was ommitted from callus induction medium; without coconut milk callus turned brown and died within 10-15 days. Based on these observations, 5.0% of coconut milk was added in callus induction medium routinely. Various concentrations of kinetin (2, 4, 6, 8, 10 mg/L) were tested without effect on callus induction and callus growth of leaf and root explants (data not shown). The effect of 2,4-D (0, 0.5, 1, 2, 3 mg/L) on callus formation from root and leaf explants was also compared. The results showed that callus induction from root and leaf explants was of a lower quality when 2,4-D was used to replace cocunut milk. Colour and appearance of callus were also influenced by this hormone. 2,4-D induced callus from both leaf and root explants was harder, smoother and paler than the callus produced on medium containing coconut milk. With leaf explants, callus production was observed all over the explants. The callus appeared very hard and white in 1-3 mg/L treatments: whereas,

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Fig. 2: Effect of coconut milk on callus induction of leaf and root explants. Coconut milk was added in the basal medium containing 20 g/L sucrose. callus at the 0.5 mg/L treatment was green. No significant difference was evident in the number of callus produced. The response from root explants was slower, and callus induction was less than from leaf explants. The callus induced from both leaf and root explants in response to 2,4-D did not regenerate into shoots. From this study, it was apparent that 2,4-D alone is not sufficient for induction of good quality callus in Scaevola. The effect of BAP on callus induction was also studied using leaf and root explants (Table 1 A and 1 B). Addition of BAP to the medium containing coconut milk had a significant effect on callus induction, growth and appearance of the callus from root explants (Fig. 3 A). Leaf explants also responded on this medium and produced very small callus. These small calli started to produce shoots after a very brief period {2 weeks) of callus production. This effect was ob-

Table lA: Effect ofBAP on callus induction and shoot regeneration

from leaf explants. The medium contains 60 g/L sucrose and 5 % coconut milk.

Explants No. callus No. BAP con- No. producing callus producing shoots shoots per centration explants No. % No. % explant mg/L 0 0.5 1 3 4 5

63 62 62 44 60 60

26 55 62a 38 58 a 58 a

41 89 100 86 97 97

0 8 19 27a 43a 48

0 5 31 71 74 83

0 6-11 11-42 12-52 15->30 15->30

Means within each column followed by the same letter are not significantly different at P= 0.05.

Table lB: Effect ofBAP on callus induction and shoot regeneration from root explants. The medium contains 60 g/L sucrose and 5 % coconut milk. BAP con- No. Explants No. callus No. centration exproducing callus producing shoots shoots per mg/L plants No. No. explant % % 0 1 3 4 5

67 64 67 60 60

12 34a 38a 35a 58

19 53 57 58 97

0 4 23 35 21

0 11 60 100 36

0 1-5 6->20 6->20 3-10

Means within each column followed by the same letter are not significantly different at P= 0.05.

Table 2: Effect of BAP + 2,4-D on callus production and shoot initiation. Callus induction medium contained 5 % coconut milk,

60 giL sucrose, 1 mg!L BAP and 0.5 mg/L 2,4-D. Shoot initiation medium contained 5 % coconut milk, 60 g/L sucrose and 1 mg/L BAP. No. Explants No. exNo. shoots exproducing callus plants/callus per callus plants No. % inBAP producing shoots

Leaf explants 64 Root explants 61

64 52

100 85

64 52

1-3 24->50

served in all the concentrations of BAP tested {0.5, 1, 3, 4, 5 mg/L). Addition of BAP to the medium also resulted in explants remaining healthy during callus induction and growth phase. We also studied the combined effect of 2,4-D {0.5 mgl L), BAP (1.0 mg/L) and coconut milk (5 %) on callus induction of leaf and root explants (Table 2). A lower concentration of 2,4-D (0.25 mg/L) in the medium containing BAP {1.0 mg/L) and coconut milk (5 %) had similar effect, but higher concentrations {1.0 mg!L and above) resulted in callus turning brown and dead. These explants produced callus within 10 days of culture. Callus was induced all over the root explants and looked healthy and friable. These calli produced a large number of shoots when transferred to shoot regeneration medium after 3-4 weeks. Extended incubation on this medium resulted in calli turning brown and losing its ability to regenerate shoots. Synthetic auxin, 2,4-D is known to induce good quality callus and somatic embryogenesis or organogenesis in a number of ornamental horticulture plants (Dillen et al., 1996; Salm et al., 1996). Our results showed that 2,4-D alone had no effect on callus induction and shoot initiation of Scaevola. In this context, this Australian native plant differs from other ornamental plants, such as rose, cyclamen and Inca Lily in its response to 2,4-D (Carla et al., 1996). Whether 2,4-D has a similar effect on other related species of Australian ornamental plants remains to be tested. Kinetin in the medium appeared to have no effect either on callus induction or maintenance of Scaevola. In contrast, BAP had a significant effect on callus induction, maintenance and shoot initiation for this species. Superiority of BAP over other cytokinins has been re-

Plant Regeneration of Scaevola ported and discussed in relation to shoot proliferation in cultures of trees (Bonga and von Aderkas, 1992).

Plant Regeneration For plant regeneration, 5-10 calli of 1-1.5 em in length obtained from root explants or 0.5 cm2 from leaf explants were incubated in a 8 em Petri dish for a period of 4 weeks on a fresh medium containing 5.0% coconut milk, 60 giL sucrose and varying concentrations of BAP (0.5, 1, 3, 4, 5 mg/ L). Shoot primordia were observed 2-3 weeks after culture (Fig. 3 B) and developed into growing shoots in 2 to 5 weeks. Presence of BAP in the medium stimulated callus growth and induction of organised structures (Fig. 3 B). Shoots were initiated earlier from callus of root explants than leaf explants, and number of shoots regenerated per callus was also significantly higher (Table 2). On average, callus from root explants produced 24-50 shoots/callus while callus from leaf explants produced 1-3 shoots/callus (Table 2). Once shoots started to grow on shoot initiation medium, they were transferred to a hormone free medium (shoot growth medium). After four weeks on shoot growth medium (Fig. 3 C), the shoots were transferred to root induction medium containing 4 mg/L of IBA. Because of the large number of shoots regenerated, two hundred twenty nine regenerated shoots (54, 82, 93 shoots from three different experiments) picked at random were transferred to root induction medium. Two hundred nineteen shoots (52, 78, 89 shoots, respectively) produced well-devel-

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Fig. 3: Callus induction and plant regeneration of Scaevola phlebopetala F. Muell. A BAP (3 mg!L) induced callus from root explants showing friable and healthy callus. Bar scale = 6 mm. B Shoot development. Bar scale = 6 mm. C Developing shoots on shoot growth medium. Bar scale = 12.5 mm. D Developed shoots in rooting medium. Bar scale = 12.5 mm.

377

Table 3: Callus and shoot regeneration media for Scaevola phlebopetala F. Muell.

Media

Components

Basal medium

Murashige and Skoog (1962): major elements, minor elements and vitamines (2.0 mg/L Glycine 100 mg/L myo-lnosito! 0.5 mg/L Nicotinic acid 0.5 mg/L Pyridoxin HCl 0.1 mg/L Thiamin HCI) 6 giL Agar

Callus induction and Leafexplants Shoot initiation medium Basal medium 5 o/o coconut milk 60 g/L sucrose 3 mg!L BAP Shoot growth medium

Basal medium 20 g/L sucrose

Rooting medium

Basal medium 20 g/L sucrose 4 mg!L IBA

Root explants

Basal medium 5 o/o coconut milk 60 g/L sucrose 1 mg!L BAP 0.5 mg/L 2,4-D (callus induction only)

oped roots (Fig. 3D) within 6 weeks. A sample of 128 rooted shoots (45, 38, 45 from three independent experiments) were further transferred to soil under glasshouse conditions and 123 plantlets (43, 36, 44 respectively from three experiments) survived this transfer. It was necessary to acclimatise the regenerated plants first using cutting mix in a mist chamber before transferring them in potting mix (as detailed in Materials and Methods) under glasshouse conditions. Regenerated plants were uniform with normal leaf, flower, shape and colour. No morphological variation was observed. In conclusion, we have developed an efficient method for shoot regeneration from callus derived from leaf and root explants of Scaevola phlebopetala F. Muell (Table 3). Development of regeneration protocols for horticulture important species will facilitate access to natural and induced variations. Variability is desirable in ornamental horticulture plants as the market is always looking for novel plants with different flower colours and shapes. Acknowledgements .

Our sincere thanks to Ms. Nicole Smith for statistical analysis of the data, Pro£ Scott Russell, University of Oklahoma, USA for critical reading of the manuscript and Dr. lnes Swoboda for her invaluable help during preparation of the manuscript. Financial assistance from the Australian Research Council (ARC) to PLB is also gratefully acknowledged. References

BHOJWANI, S. S. and M. K. RAzoAN: Plant tissue culture: theory and practice. Elsevier Science Publishers, Amsterdam, The Netherlands (1983).

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SALM, T. P.M., C. J. G. TooRN, C. H. HANISCH TEN CATE, and J. DoNES: Somatic embryogenesis and shoot regeneration from excised adventitious roots of the rootstock Rosa hybrida L. 'Moneyway'. Plant Cell Reports 15, 522-527 (1996). STEFFEN, K.: A hanging basket plant with future- Scaevola aemula. Gaertnermeister 92, 944-945 (1989). VoN HENTIG, W. U. and D. EHLERS: Mutterpflanzenhaltung und Stecklingsvermehrung bei Scaevola aemula. Deutscher Gartenbau 6, 359-361 (1991). VoN HENTIG, W. U.: Effect of light and temperature on flower development. Gartenbau Magazin 2, 56-57 (1993). WRIGLEY, R. J. and M. FAGG: Australian native plants, 3rd edition. W. Collins Pty Ltd., Sydney, Australia, 1988.