Conventional and in Vitro Propagation of Lechenaultia Macrantha

HORTSCIENCE 48(1):108–114. 2013. the cuttings to enhance survival and en- courage growth. Additionally, many woody Australian species do not form adventitious Conventional and In Vitro Propagation roots on stem cuttings readily, which hinders propagation (Dawson and King, 1994). Auxins of Lechenaultia macrantha are used in vegetative propagation to stimu- late adventitious root growth. Because the (Goodeniaceae) ratios of endogenous plant growth regulators vary between different stem tissues, applica- Guochen K. Png1, Katherine S. Downes, and Beng H. Tan tion of excessive levels of exogenous auxin Department of Environment and Agriculture, Curtin University, Building to the cuttings may suppress the growth of 311, Kent Street, GPO Box U1987, Perth, WA 6845, Australia shoots and/or roots (Hartmann et al., 2002). Therefore, it is necessary to consider the con- Additional index words. dormancy, micropropagation, ornamental Australian endemic, seed centration of exogenous auxin applied to stem germination, stem cuttings, tissue culture cuttings to optimize propagation success. Shoot micropropagation, the most com- Abstract. Lechenaultia macrantha K. Krause (Goodeniaceae) is a species with great mon in vitro propagation system, provides an horticultural potential that is endemic to the sandy and gravelly soils of central, alternative method of propagating plants that southwestern Australia. The effectiveness of several conventional and in vitro propaga- is time-consuming or difficult to propagate tion techniques were assessed. Seeds possessed non-deep physiological dormancy, and otherwise (Offord et al., 2009). The feasibility a combination of seed nicking and imbibition in 10% (v/v) smoke water for 24 hours of shoot micropropagation of L. macrantha resulted in 81% germination after 33 days of incubation. Softwood stem cuttings has yet to be investigated. The provision of produced adventitious roots readily without the need for exogenous auxin application. exogenous cytokinins to the explant through In vitro microcuttings produced shoots in response to cytokinins. However, optimal or the culture medium at the shoot multiplica- suboptimal cytokinin concentrations resulted in hyperhydric shoots, which poses an tion stage stimulates the formation of micro- obstacle to successful micropropagation. The use of auxin was necessary for the initiation shoots (Hartmann et al., 2002). Thidiazuron of adventitious roots in vitro. This information will assist in the horticultural devel- (TDZ; N-phenyl-N-1, 2, 3-thiadiazol-5-ylurea), opment of this species. a potent synthetic diphenylurea cytokinin, exhibits a higher cytokinin activity and persistency than synthetic aminopurine cyto- Lechenaultia macrantha K. Krause and those seeds can be germinated. The kinins at equimolar concentrations and initi- (Goodeniaceae), commonly known as the germination of many taxa within the Good- ates microshoots that are unresponsive to Wreath Lechenaultia, is endemic to central, eniaceae is constrained by poorly understood aminopurine cytokinins such as kinetin [N- southwestern Australia (Morrison, 1992). dormancy mechanisms and/or germination (s-furnylmethyl)-1H-purin-6-amine] and 6- Owing to its intriguing ‘‘wreath-like’’ form, stimulant requirements (Bell et al., 1993; Merritt benzyladenine (BA) in other species (Hutteman L. macrantha is one of the main wildflower et al., 2007). To better understand how to and Preece, 1993; Lu, 1993). Although in- attractions of the Shires of Morawa and overcome dormancy, it is instructive to de- creasing cytokinin concentration is generally Mullewa in Western Australia (WA) and is termine the type of seed dormancy (Baker associated with increasing shoot initiation, it of interest horticulturally (Wrigley and Fagg, et al., 2005a) using a widely accepted clas- also increases callus growth, which promotes 2003). Although the species is not currently sificationsystemproposedbyBaskinand the indirect development of adventitious shoots classified as threatened (Western Australian Baskin (2004). and reduces the direct development of axil- Herbarium, 1998), increasing pressures from The seeds of L. macrantha are enclosed in lary microshoots (Hutteman and Preece, illegal collection (D.G. Williams, personal a hard woody endocarp and are often referred 1993). The high rate of adventitious shoot communication; Kelly et al., 2003), agricul- to as ‘‘woody articles’’ (Jabaily et al., 2012). production is undesirable where clonal fidel- ture (Hobbs, 1993), and associated land frag- If these seeds are unable to imbibe water, they ity is required, because adventitiously formed mentation (Wilcox and Murphy, 1985) may have physical dormancy. If they can imbibe shoots may be somaclonal variants (Hutteman pose threats to the species’ long-term survival. water, they may possess a physiological block and Preece, 1993). Supraoptimal levels of Despite its outstanding ornamental horticul- that reduces the capacity of the embryo to cytokinin activity also adversely affect the tural potential, there is a dearth of knowledge push through the surrounding hard woody multiplication process through the inhibition regarding the propagation of this species. Any endocarp (Baskin and Baskin, 2004). Some of shoot elongation (Hutteman and Preece, research leading to its successful propagation methods used to overcome physical dormancy 1993; Lu, 1993); the intensification of callus- will contribute to its horticultural development include removal or scarification of the outer ing (Hutteman and Preece, 1993); the inhibi- as well as its conservation. layers of the seed and acid scarification (Baskin tion of root development in the root formation Seed propagation is one of the most cost- and Baskin, 2004). Seeds with non-deep phys- stage (Hutteman and Preece, 1993; Lu, 1993); efficient means of multiplying plants if there iological dormancy can also be artificially and the elevation of hyperhydricity in a is a sufficient supply of good quality seeds induced to germinate by seedcoat nicking concentration-dependent manner (Debergh et al., or more naturally through dry afterripening, 1992; Ivanova et al., 2006; Kevers et al., 2004). cold/warm stratification (Baskin and Baskin, Hyperhydricity (previously known as vitrifi- Received for publication 5 Sept. 2012. Accepted 2004), and/or wet/dry alternations (Baker et al., cation) refers to the malformed and watery for publication 8 Nov. 2012. 2005b). Smoke application is another method appearance of plantlets resulting from ab- We thank Lydia Kupsky, Peter Mioduszewski, that has enhanced the germination of many normal physiological development, which William Parkinson, Kevin Seaton (DAFWA), WA species, including several closely related impedes the multiplication, growth, and ac- Chris McMullan (DAFWA), Joyce Ho, and Minh Goodeniaceae species (Dixon et al., 1995). climatization of plantlets, resulting in signif- Hien To for logistic/technical assistance, the Shire Vegetative propagation through stem cut- icant losses in micropropagation (Debergh of Morawa WA for access to plants, and Byron tings is a commercially important technique et al., 1992). Because shoot formation response Lamont, Christine Cooper, and Kelly Shepherd for used to produce large numbers of clones of to the different cytokinins is species-specific, it comments on the manuscript. Stem cuttings were collected with permission from the Western Australia plants in horticultural systems (Dawson and is necessary to assess the efficacy and quality of Department of Environment and Conservation King, 1994). However, the use of stem cuttings L. macrantha microshoots produced by the (license no. SW014016). is generally more costly than seed propagation different cytokinin types and concentrations. 1To whom reprint requests should be addressed; because additional infrastructure (e.g., fog or In micropropagation, a high auxin-to- e-mail [email protected]. mist systems) is required to protect and nurture cytokinin concentration ratio is typically used 108 HORTSCIENCE VOL. 48(1) JANUARY 2013 in the culture medium to induce root formation water, filter paper, and vermiculite were auto- placed on a propagation bench (bottom heat (Hartmann et al., 2002). However, supraop- claved before use. After treatment, seeds of each maintained at 27 °C) in a glasshouse main- timal concentrations of auxin can suppress replicate were placed in a sterile petri dish on top tained at 99% humidity. Overhead fine- root growth (de Klerk, 2000; Riov and Yang, of two layers of moistened filter paper on misters were programed to spray for 1 min 1989). Hence, investigating the optimal auxin a vermiculite substratum. Petri dishes were at 5-min intervals. After 6 weeks, the rooting concentration required for L. macrantha root wrapped in foil to impose darkness as per response, number of roots produced per stem, induction would assist in the micropropaga- Dixon et al. (1995) and placed in a room with and the root length per root (measured to the tion of this species. an ambient temperature maintained at 24 °C. nearest 0.02 mm using a pair of electronic The aims of this study were to enhance the This temperature was used for incubation as it calipers) of stems in each of the three IBA ability to propagate L. macrantha for horti- is between the temperature ranges recommen- concentrations were assessed. cultural and conservation purposes by com- ded by Bell et al. (1995; 15 to 23 °C) and Stem anatomy. Transverse slices from the paring the relative effectiveness of some Merritt (2006; 25 to 35 °C) for the germination

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