An efficient protoplast-to-plant system for the hybrid ornamental shrub,Weigela ×florida cv. Bristol Ruby (Caprifoliaceae)

Strategies were developed for the successful isolation of large numbers of highly viable protoplasts from the leaves, stems and roots of axenic plants of the hybrid ornamental shrubWeigela ×florida cv Bristol Ruby. Protoplasts, of all sources, were cultured on different media, leading to the establishment of sustained divisions, and coupled with the production of multi-celled (>50 cells) colonies. However, those colonies derived from mesophyll protoplasts only were capable of a further proliferation to the callus stage. Upon transfer to a regeneration medium consisting of MS salts and organics plus a range of concentrations of NAA and BAP, such calli underwent caulogenesis, with optimum responses for a medium with 1.0 mg l⁻¹ NAA and 1.0 mg l⁻¹ BAP. The protoplast-derived shoots thus obtained were multiplied on MS medium with 0.1 mg l⁻¹ IBA, 0.5 mg l⁻¹ BAP and 0.1 mg l⁻¹ GA₃. Individual shoots were subsequently rooted on a half-strength MS medium plus 3.0 mg l⁻¹ IBA, and complete protoplast-derived plants were finally transferred to the glasshouse for acclimatization.     

A protoplast to plant system for the chrysanthemum Dendranthema zawadskii x D. grandiflora

Summary Plantlets were regenerated from protoplasts of in vitro shoot cultures and leaf-derived de novo shoots of the chrysanthemum Dendranthema zawadskii x D. grandiflora. Isolated protoplasts reformed cell walls and then began to divide within 24 hours of culture in streaky plate agarose lenses surrounded by liquid V-KM medium. Twenty one days after isolation, 1 mm diameter callus clumps were transferred to shoot regeneration medium. After a further 33 days leaves became visible. Elongated shoots were rooted on half strength hormone-free MS medium.Abbreviations BAP 6-benzylaminopurine - IAA 3-indoleacetic acid - MS Murashige and Skoog (1962) - NAA 1-naphthylacetic acid - Pfr Photon fluence rate - V-KM Binding and Nehls (1977)     

VARIATIONS OF MORPHOGENETIC BEHAVIOR IN PLANT TISSUE CULTURES I. CICHORIUM ENDIVIA

Several-years-old callus tissue derived from mature embryos of endive (Cichorium endivia Linn., Compositae) was grown on synthetic liquid and/or agar nutrient media. Incorporation of yeast extract or high concentrations of inositol, kinetin, casein hydrolysates (pancreatic and acid hydrolysates), etc., improved growth and organ formation. Rosettes of leaves, shoots and roots were differentiated on synthetic media. On agar media shoots arose first and were from marginal meristematic areas, while the roots arose later and were from pockets of meristematic tissue located in the deeper regions of the callus. In liquid media embryoids from single cells were formed which first developed roots and then shoots.     

Hybridisation experiments with protoplasts from chlorophyll-deficient mutants of some Solanaceous species

Following fusion between protoplasts from two different chlorophyll-deficient diploid mutants of Datura innoxia Mill. it was possible to select 33 green hybrid calli on agar culture medium. Half of the somatic hybrids gave rise to leaves and some to shoots. The chromosome number of 20 somatic hybrids was determined: five were tetraploid, eight hexaploid, three octoploid, and four showed an aneuploid chromosome number. After transfer of the shoots of the five tetraploid hybrids to soil they developed roots. In control experiments in which protoplasts of the two mutants were cultured either as a mixture without being treated with the fusion agent, or cultured separately, no green callus could be obtained. Similar experiments involving protoplasts from one chlorophyll-deficient mutant of Datura innoxia, on the one hand, and those from similar mutants of Nicotiana sylvestris Spegazz. et Comes and Petunia hybrida, on the other, yielded no green somatic hybrid although hybrid protoplasts could be detected.     

Adventitious rooting of Jatropha curcas L. is stimulated by phloroglucinol and by red LED light

An efficient root induction system has been established for in vitro-regenerated Jatropha curcas L. shoots. Callus formation on shoots transferred to auxin containing medium was found to be a prominent and recurrent problem for rooting of in vitro-cultivated J. curcas. In particular, the type of auxins and cytokinins applied in the culture media were shown to strongly influence the severity of callus formation. Shoots cultivated on meta-methoxytopolin riboside (MemTR) were free of callus and produced elongated stems and well-developed leaves in comparison to the cytokinins benzyl adenine, zeatin, and thidiazuron. Subsequent root induction experiments were performed with shoots precultured on MemTR-containing medium. Shoots were excised and transferred to Murashige and Skoog (MS) medium supplemented with different concentrations of indole-3-butyric acid (IBA), indole-3-acetic acid (IAA), and α-naphtaleneacetic acid (NAA). The induction of excessive callus formation was avoided only on IBA-containing medium. The optimum rooting medium with good root induction (35%) and 1.2 roots per shoot contained half-strength MS salts supplemented with 2.5 μM IBA. The same medium supplemented with 0.25% (w/v) activated charcoal produced 46% rooted shoots. Further improvement of rooting was obtained by transferring in vitro grown shoots to woody plant medium containing phloroglucinol (PG). In the presence of 2.5 μM IBA and 238 μM PG, 83% of the shoots rooted with on average 3.1 roots per shoot. We also analyzed the impact of light quality on the rooting capacity of Jatropha in vitro grown shoots. In general, light-emitting diodes (LEDs) light sources were less efficient for root induction. Red LED light provided the most favorable growth conditions, inducing a rooting response in 65% of the shoots, which produced on average 5.5 roots per shoot. These results indicate that adventitious rooting in J. curcas is under control of photoreceptors and that optimal rooting requires fine-tuning of the salt concentration, auxin, and cytokinin balance and application of synergistic compounds.     

紫云英叶片及叶肉原生质体的培养

本工作研究了豆科植物紫云英的叶片及叶肉原生质体的培养。叶片培养实验表明,诱导愈伤组织的最适培养基为MS加1.0-2.0毫克/升2,4-D和0.25毫克/升KT;诱导根分化需加1.0—5.0毫克/升NAA和0.5毫克/升BA;而苗分化则以0—0.5毫克/升IAA和0.5毫克/升BA为好。高浓度的NAA有利于根分化而抑制茎芽形成;高浓度的IAA对根和芽分化都有抑制作用。叶肉原生质体分离和培养试验表明,紫云英叶肉原生质体的释放及其培养活力受叶龄、植株生理状态和酶浓度的影响。叶肉原生质体在改良的KM8P培养基中能分裂。用改良KM8细胞培养基定期稀释,可使分裂持续进行而得到细胞团。BA和2,4-D为诱导紫云英叶肉原生质体分裂所必需。其最佳组合激素为BA 0.21毫克/升和2,4-D 1.13毫克/升。葡萄糖作为渗透压稳定剂时,其浓度明显影响原生质体的存活率。弱光条件下培养比黑暗培养有利于叶肉原生质体分裂。由叶肉原生质体形成的愈伤组织能形成瘤状结构和根。     

Shoot multiplication in cultures of mature <Emphasis Type="Italic">Alnus glutinosa</Emphasis>

Shoot cultures were initiated from mature trees of Alnus glutinosa. On medium containing 1–5 μM 6-benzylamino purine (BAP), the shoots elongated without branching, formed heavy callus at the base of the stems and readily formed roots. The possibility that these characteristics could be attributed to the strong influence of endogenous auxin was tested on media that contained two auxin transport inhibitors, 1-N- naphthylphthalamic acid (NPA) and 2,3,5-triiodobenzoic acid (TIBA), at concentrations of 0.1–3 μM, in combination with 2 μM BAP. On these media, shoots produced numerous branches, less callus and no roots. After 30 weeks (five subcultures) on this medium, leaves were smaller and showed signs of vitrification. These problems were resolved without detriment to shoot proliferation, by reverting to medium without NPA or TIBA. Shoots rooted readily after transfer to medium without growth regulators and were successfully acclimatised after transfer to soil.     

Studies of cotyledon protoplast cultures from B napus,B. campestris and B. oleracea. II: Callus formation and plant regeneration

Cotyledons from twelve cultivars of Brassica; B. napus (Westar, Eureka, Global, Pivot and Narc 82); B. campestris: (Arlo, Sonja, Bunyip and Wonk Bok) and B. oleracea (Phenomenal Early, Sugar Loaf and Earliball) were used for protoplast isolation and culture in a comparative study of cell colony and callus formation, and plant regeneration. The formation of cell colonies and callus from protoplast cultures were significantly influenced by the light conditions of seed germination. All twelve cultivars showed callus formation from protoplast cultures derived from cotyledons of seedlings grown in dark for 3 days followed by 1 day dim light (dark/dim light-grown). Callus was obtained in all five liquid media used: modified K8P(1), modified K8P(2), modified MS, modified B and modified NN. In contrast, only six cultivars exhibited callus formation from the protoplasts isolated from cotyledons of seedlings germinated under light conditions for 7 days (light-grown) and in only three media: modified K8P(1), modified MS, modified B.Callus, derived from protoplast cultures isolated from dark/dim light-grown cotyledons and grown on K3 or MS series solid media for about 1 month, could develop shoots when further transferred onto MS series regeneration media. All five cultivars of B. napus, three of the four cultivars of B. campestris (Arlo, Sonja and Bunyip) and one of the three cultivars of B. oleracea (Sugar Loaf) exhibited shoot regeneration from protoplast cultures within 2–3 months after protoplast isolation. The frequency of shoot regeneration ranged among 1–22.5%. A high degree of reproducibility was observed in cultivars Westar, Eureka, Global, Arlo, Bunyip and Sugar Loaf. In contrast, among the six cultivars that formed callus in protoplast culture derived from light-grown cotyledons, only three cultivars from B. napus (Westar, Eureka, Global) exhibited shoot regeneration 5.5 months after protoplast isolation. Regenerated shoots from cultivars Westar, Eureka and Bunyip and Sugar Loaf, which derived from protoplasts of dark/dim light germinated seedling and were induced to root on rooting media, survived in soil and grew to produce silique and set seeds.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - BA benzylaminopurine - EDTA ethylenediaminetetraacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - KT kinetin - GA₃ gibberellic acid - MS Murashige and Skoog medium - NAA -naphthaleneacetic acid - PAR photosynthetically active radiation     

Protoplast isolation and culture for somatic hybridization of <Emphasis Type="Italic">Lupinus angustifolius</Emphasis> and <Emphasis Type="Italic">L</Emphasis>. <Emphasis Type="Italic">subcarnosus</Emphasis>

A method for isolation and shoot regeneration from electrofused protoplasts of L. angustifolius and L. subcarnosus was developed. Viable protoplasts were isolated from leaves of in-vitro grown seedlings at an average yield of 6 × 10⁵ protoplasts g⁻¹ fresh weight. Liquid and agarose solidified B5 media were used for protoplast culture. In the liquid-culture system, all tested media, VKM, P1 and KM8p, were applicable for inducing cell division (84% of all tested petri dishes at four weeks) and colony formation. Media containing additional carbohydrates were suitable to produce compact calli with green and brown pigmentations in different combinations. Analysis of callus with molecular markers allowed to identify six somatic hybrids. However, none of the parental-protoplast derived cell colonies could develop shoots. This is the first report on protoplast fusion of L. angustifolius and L. subcarnosus with subsequent shoot regeneration.     

SHOOT AND TREE PRODUCTION FROM ASPEN TISSUE CULTURES

Trees were produced from firm white callus tissue of triploid quaking aspen (Populus tremuloides Michx.), initiated on a modified Wolter and Skoog defined medium and subcultured monthly for two years. When subcultured to medium without auxin, kinetin or supplements, but containing 0.15 mg/liter BAP (6-benzylaminopurine), multiple stunted shoots appeared on most inocula. However, at 0.05 mg/liter BAP, only a few vigorous shoots per piece were initiated, but seven rooted on the callus: two in the dark with BAP and five in 200 ft-c of light with 0.04 mg/liter 2,4-D. After proliferation of the roots on the medium surface, four shoots elongated and were planted in semi-sterilized soil, then were given 3100 ft-c of light for rapid growth into trees. Both light sources were on for 16 hr/day. Two trees were also grown from stunted shoots excised from the callus and rooted in soil.     

Comparative assessment of the efficacy of bacterial and cyanobacterial phytohormones in plant tissue culture

Efficient callus and explant regeneration medium, using microbial extract (SPE purified) or supernatant has been formulated for Brassica oleracea L. var. capitata. Two cyanobacterial strains (Anabaena sp. Ck1 and Chroococcidiopsis sp. Ck4) and two bacterial strains, (Pseudomonas spp. Am3 and Am4) known to produce a number of cytokinins, tZ, cZ, ZR, DHZR and IAA were selected for the media formulation. Supernatant from strains with high cytokinin to IAA ratio, including Pseudomonas aeruginosa Am3 (2.08) and Chroococcidiopsis sp. Ck4 (0.8) efficiently induced compact calli which were turned green upon exposure to light. The strains producing lower cytokinins to IAA ratio (0.11–0.13) on the other hand induced friable callus which were unable to regenerate on the selected media combinations. Leaf, stem and root explants of Brassica oleracea L. regenerated on MS medium supplemented with phytohormones from microbial origin with efficiency comparable to standard cytokinins and IAA. Supplements from cyanobacterial origin proved to be the best for induction of adventitious roots and shoots on internodal and petiolar segments. Hypocotyl explants however, responded well on MS supplemented with bacterial metabolites. Induction of adventitious shoots on root explants was supported by phytohormones from both origin equally well. Callus induction on the seeds and regeneration of shoots on calli was also observed. Cyanobacteria based media were more efficient to induce calli capable of regeneration upon exposure to light. Internodal explants were highly amenable to regenerate shoot and roots simultaneously. Root explants were the less successful to regenerate shoots.     

Winged-bean protoplasts: Isolation and culture to callus

A system was developed for protoplast isolation and culture from suspension cultured cells of winged bean,Psophocarpus tetragonolobus. Cells from a three-day-old suspension were incubated in an enzyme mixture containing 6% cullulysin, 1% Macerase, 1% desalted Rhozyme, 0.4M sorbitol, and 0.1M CaCl₂ at pH 5.5. Average yields of protoplasts were 6.5 × 10⁶ per gram fresh weight of cells. Protoplasts were cultured in modified B5 medium containing 68.4 g/l glucose, 250 mg/l xylose, 0.1 mg/l 2,4-D, 0.5 mg/l BAP, 250 mg/l N-Z amine type AS, and 20 ml/l coconut water. After 24 h of culture, the protoplasts had synthesized a new wall, and in three days had begun division. The optimum plating density was 1–2 × 10³ protoplasts/ml. The division frequency ranged between 40%–60% for most experiments with a high of 72% in one experiment. After three weeks, cell colonies could be transferred to solid MS medium containing N-Z amine and coconut water where callus developed. This protoplast system is technically comparable to soybean for experiments concerned with genetic manipulation involving legumes.     

Plant regeneration from mesophyll protoplasts of Centaurea cyanus,Senecio x hybridus and Callistephus chinensis

Protoplasts were isolated from leaves of glasshouse-grown plants of Centaurea cyanus and axenic shoot cultures of Senecio x hybridus. Upon culture, using modified MS-based media, protoplasts of both systems entered division to produce callus, followed by plant regeneration. Leaf protoplasts of Callistephus chinensis entered sustained division only following the preconditioning for 24h of peeled leaf tissues on agar-solidified MS-based medium. Protoplasts were also isolated from cell suspensions of C. chinensis and divided in MS-based or KM media. However, only leaf mesophyll protoplasts of Callistephus produced callus, which developed shoots.The establishment of protoplast-to-plant protocols for these ornamental species has provided a basis for broadening their gene pools through somatic hybridisation.Abbreviations BAP 6-benzylaminopurine - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) - KM Kao and Michayluk (1975) - g.f.wt. gram fresh weight     

IN VITRO CULTURE OF GRAIN AND VEGETABLE AMARANTHS (AMARANTHUS SPP.)

We have developed in vitro culture systems for both “grain” and “vegetable” species of the genus Amaranthus. Leaf discs and hypocotyl segments from 2- to 3-wk-old seedlings of A. hypochondriacus, A. cruentus, and A. tricolor were cultured in B5 and MS media supplemented with 2,4-dichlorophenoxyacetic acid, α-naphthaleneacetic acid, benzyladenine and zeatin in various combinations. Rapidly growing callus and abnormal roots formed on leaf discs of A. hypochondriacus and A. cruentus in the presence of 0.1-1.0 mg/l 2,4-D. At higher levels (1.0-10.0 mg/l) of 2,4-D, embryo-like structures arose from the surface and veins of the leaf discs. Shoots formed on hypocotyl-derived callus of both grain species in B5 medium + 0.1 mg/l NAA and 0.1-1.0 mg/l zeatin. Lower ratios of zeatin/NAA stimulated root formation from hypocotyl segments. Viable mesophyll protoplasts were isolated from primary leaves of all three species, with an enzyme solution of 1% Cellulysin and 0.05% Pectolyase Y-23, producing a yield of more than 10⁶ protoplasts/g fresh weight.     

Protoplast Culture in Agarose Media with Particular Emphasis to Streaky Culture Lenses*

Isolated protoplasts of 60 species of the Magnoliatae and 19 species of the Liliatae were embedded in droplets of agarose-containing media in such a way that streaks of locally high cell densities were obtained. For comparison, agarose cultures with dispersed protoplasts and liquid cultures were prepared. Fast and reliable regeneration was obtained in several taxa. After transfer to low osmolar media, shoots were formed in 18 species belonging to the genera Arabidopsis, Asparagus, Biscutella, Brassica, Cichorium, Clianthus, Hypericum, Nicotiana, Petunia, Reseda, Senecio, and Solanum. Only protoplast-derived callus which did not organize shoots was grown in species of Agrimonia, Daucus, Cucumis, Duchesnea, Helianthus, Potentilla, Prunus, Reseda, Solanum, Sorbus, Spinacia, and Vicia.     

Isolation and culture of protoplasts from mesophyll tissue of the legume Cyamopsis tetragonoloba L.

Protoplasts of Cyamopsis tetragonoloba were isolated from leaves of in vitro grown plants. The yield of the protoplasts, their viability and subsequent divisions were greatly influenced by the pH of the media used for isolation and culture of protoplasts. Sustained divisions of the cultured protoplasts were best supported by a modified Kao and Michayluk (1975) nutrient medium containing glucose (0.4 M), NAA (4 mgl^–1), 2,4-D (1 mgl^–1) and KIN (2 mgl^–1 ). The protoplast derived cells developed calli on transfer to Murashige and Skoog (1962) medium supplemented with 1 mgl^–1 each of 2,4-D, NAA and KIN.     

An Evaluation of Some Parameters Required for the Enzymatic Isolation of Cells and Protoplasts with CO2 Fixation Capacity from C3 and C4 Grasses

Variable factors affecting the enzymatic isolation of mesophyll protoplasts from Triticum aestivum (wheat), a C₃ gras, and mesophyll protoplasts and bundle sheath strands from Digitaria sanguinalis (crabgrass), a C₄ grass, have been examined with respect to yields and also photosynthetic capacity after isolation. Preparations with high yields and high photosynthetic capacity were obtained when small transverse leaf segments were incubated in enzyme medium in the light at 30°C, without mechanical shaking and without prior vacuum infiltration. Best results were obtained with an enzyme medium that included 0.5 M sorbitol, 1 mM MgCl₂, 1 mM KH₂PO₄, 2% cellulase and 0.1% pectinase at pH 5.5. In gerneral, leaf age and leaf segment size were important factors, with highest yields and photosynthetic capacities obtained from young leaves cut into segments less than 0.8 mm. To facilitate the cutting of such small segments, a mechanical leaf cutter is described that uniformly (± 0.05 mm) cuts leaf tissue into transverse segments of variable size (0.4–2 mm). Isolations that required more than roughly 4 h gave poor yields with reduced photosynthetic capacity; however, using the optimum conditions described, functional preparations could be roughly 2 h. High rates of light dependent CO₂ fixation by the C₄ mesophyll protoplasts required the addition of pyruvate and low levels of oxalacetate, while isolated bundle sheath strands and C₃ mesophyll protoplasts supported CO₂ fixation without added substrates. Rates of CO₂ fixation by isolated wheat protoplasts generally exceeded the reported rates of whole leaf photosynthesis. Wheat mesophyll protoplasts and crabgrass bundle sheath strands were stable when stored at 4°C while C₄ mesophyll protoplasts were stable when stored at 25°C.     

Isolation,callus formation and plantlet regeneration from mesophyll protoplasts of <Emphasis Type="Italic">Tylophora indica</Emphasis> (Burm. f.) Merrill: an important medicinal plant

Protoplast culture and plant regeneration of an important medicinal plant Tylophora indica were achieved through callus regeneration. Protoplasts were isolated from leaf mesophyll cells and cultured at a density of 5 × 10⁵ protoplasts per gram fresh weight, which is required for the highest frequency of protoplast division (33.7%) and plating efficiency (9.3%). The first division was observed 2 d after plating and the second division after 4 d. Culture medium consists of Murashige and Skoog (MS) liquid medium with 4 μM 2,4-D, 0.4 M mannitol and 3% (w/v) sucrose with pH adjusted to 5.8. After 45 d of culture at 25°C in the dark, protoplasts formed colonies consisting of about 100 cells. The protoplast-derived microcalli were visible to the naked eye within 60 d of culture and reached a size of 0.2–0.4 mm in diameter after 90 d. Calli of 0.2–0.4-mm size were transferred to MS medium supplemented with 2,4-D (4 μM), 3% (w/v) sucrose and 0.8% (w/v) agar, formed friable organogenic calli (7-8 mm size) after 8 wk under incubation in normal light period supplemented with 200 μmol m⁻² S⁻¹ of day light fluorescent illumination. The calli were transferred to MS medium supplemented with thidiazuron (TDZ) (1–7 μM) and naphthalene acetic acid (NAA) (0.2–0.4 μM) for regeneration. The calli developed shoot buds after 3–4 wk, and the frequencies of calli-forming shoots varied from 5% to 44%. Optimum shoot regeneration occurred on MS medium supplemented with 5 μM TDZ and 0.4 μM NAA. On this medium, 44% cultures responded with an average number of 12 shoots per callus. Whole plants were recovered following rooting of shoots in 1/2 MS medium supplemented with 3 μM indole 3-butyric acid.     

Establishment of Camptotheca acuminata regeneration from leaf explants

Plantlet regeneration through shoot formation from young leaf explant-derived callus of Camptotheca acuminata is described. Calli were obtained by placing leaf explants on Woody plant medium (WPM) supplemented with various concentrations of 6-benzyladenine (BA) and naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D). Callus induction was observed in all media evaluated. On the shoot induction medium, the callus induced on the WPM medium containing 19.8 μM BA and 5.8 μM NAA was the most effective, providing high shoot regeneration frequency (70.3 %) as well as the highest number of shoots (11.2 shoots explant⁻¹). The good rooting percentage and root quality (98 %, 5.9 roots shoot⁻¹) were achieved on WPM medium supplemented with 9.6 μM indole-3-butyric acid (IBA). 96 % of the in vitro rooted plantlets with well developed shoots and roots survived transfer to soil.     

Clonal propagation byin vitro culture of Corchorus (jute)

Callus was produced on cotyledon, shoot tip, hypocotyl and root explants of twoCorchorus species on several media. Cytokinin was necessary for callus production on cotyledon explants. BothC.olitorius genotypes produced most callus on media with zeatin and either NAA or IAA, and theC.capsularis genotype produced most callus on media with IAA and either zeatin or BA. High frequencies of regenerated shoots were obtained from shoot tip explants of both species, from the apical meristem and from callus. Media with 2.0 mg 1⁻¹ BA were superior for both species, and media with zeatin were equally good forC.capsularis only. More regeneration was obtained for all genotypes after subculture of callus on media with 2.0 mg 1⁻¹ zeatin. Cotyledon callus produced less regeneration, also with differences between genotypes; explants of both genotypes ofC.olitorius produced regeneration on a medium with NAA and zeatin, and theC.capsularis genotype produced regeneration on a medium with IAA and BA. Limited regeneration from root explant callus was obtained forC.capsularis only on medium with BA and IAA. Regeneration was not obtained from hypocotyl callus. Further regeneration of shoots of both species was obtained from secondary callus after subculture, and from nodal segments of regenerated shoots and of seedling shoots cultured on basic MS medium without growth hormones. Roots were produced on about 80% of all shoots after transference to medium with 0.2 mg 1⁻¹ IBA, and rooted plantlets survived and flowered normally after transference to compost.