Seed germination and tissue culture of Cymbidium giganteum Wall. ex Lindl.

Efficient protocols were established for in vitro seed germination, neo-formation of secondary (2°) protocorms from primary (1°) protocorms and multiple shoot buds and protocorm-like body (PLB) induction from pseudo-stem segments of in vitro-raised seedlings of Cymbidium giganteum. Four nutrient media, namely Murashige and Skoog (MS), Phytamax (PM), Mitra et al. (M), and Knudson ‘C’ (KC) were evaluated for seed germination and early protocorm development. In addition, the effects of peptone, activated charcoal (AC) and two plant growth regulators [6-benzylaminopurine (BAP) and 2,4-dichlorophenoxyacetic acid (2,4-D)] were also studied. Both M and PM supplemented with 2.0 g l⁻¹ peptone or 1.0 mg l⁻¹ BAP resulted in ∼100% seed germination. Media supplemented with 2.0 g l⁻¹ AC could effectively induce large protocorms (1.6 ± 0.1 mm in diameter). Neo-formation of 2° protocorms from 1° protocorms was achieved in liquid and agar-solidified PM medium fortified with different concentrations and combinations of auxins (α-naphthalene acetic acid (NAA) and 2,4-D) and cytokinins [BAP and kinetin (KN)]. The highest number of 2° protocorms was obtained in liquid medium (10.7 ± 0.9/1° protocorm) supplemented with 2.0 mg l⁻¹ BAP + 1.0 mg l⁻¹ NAA. Although protocorms proliferated profusely in liquid medium, these did not develop further unless transferred to agar-solidified medium within 6–8 weeks. Multiple shoot buds and PLBs were induced from pseudo-stem segments on agar-solidified PM medium fortified with different concentrations and combinations of BAP and NAA and the maximum number of PLBs (6.00 ± 0.20) was recorded when BAP and NAA were applied at 2.0 mg l⁻¹ each. A solid root system was induced from PLBs and shoot buds when these were transferred to half-strength PM or M media fortified with 0.5 mg l⁻¹ indole-3-acetic acid. Well-rooted plants were transferred to the greenhouse with 95% survival.     

Plant regeneration of pansy (Viola wittrockiana) ‘Caidie’ via petiole-derived callus

An in vitro plant regeneration protocol for pansy (Viola wittrockiana) cultivar ‘Caidie’ from petioles was established as following: callus induction on a half-strength MS medium supplemented with 0.45 μmol l⁻¹ 2,4-d plus 8.9 μmol l⁻¹ BA, callus subculture on medium F (1/2MS with 4.5 μmol l⁻¹ 2,4-d, 2.7 μmol l⁻¹ NAA and 0.44 μmol l⁻¹ BA) and then on medium T (1/2MS with 4.5 μmol l⁻¹ 2,4-d, 2.7 μmol l⁻¹ NAA and 2.2 μmol l⁻¹ BA), shoot regeneration on medium D3 (MS media supplemented with 2.9 μmol l⁻¹GA₃, 23.6 μmol l⁻¹ AgNO₃, 0.02% active charcoal and 4.5 μmol l⁻¹ TDZ), shoot multiplication on medium M (half-strength MS medium containing NAA 1.1 μmol l⁻¹, TDZ 9.1 μmol l⁻¹ and GA₃ 8.7 μmol l⁻¹), and then shoot elongation and rooting on medium R (MS medium supplemented with 1.1 μmol l⁻¹ NAA and 1.1 μmol l⁻¹ BA). Subculture on appropriate medium was found to be important for successful shoot regeneration.     

Direct regeneration of plants derived from in vitro cultured shoot tips and leaves of three Lysimachia species

The regenerability of three ornamental species—Lysimachia christinae, Lysimachia rubinervis and Lysimachia nummularia ‘Aurea’, were investigated using in vitro leaves and shoot tips. 6-Benzylaminopurine (BAP) and α-naphthalene acetic acid (NAA) added to Murashige and Skoog (MS) medium were tested for their effect on organogenesis. On the medium, shoot regeneration occurred directly without callus formation. In these species, L. christinae developed the highest regeneration rate and numbers of shoots/explant from shoot tips (100%, 12.25) and leaf bases (100%, 13.01) on the MS medium containing 3.0 mg l⁻¹ BAP and 0.1 mg l⁻¹ NAA. For L. rubinervis, the highest shoot induction rate and number of shoots/explant were obtained from shoot tip (100%, 16.87–17.20) on the MS medium with 0.1 mg l⁻¹ NAA and 3.0–5.0 mg l⁻¹ BAP. L. nummularia ‘Aurea’, however, showed the highest regeneration rate and number of shoots/explant (100%, 12.73) from leaf bases on MS medium supplemented with 1.0 mg l⁻¹ BAP and 0.1 mg l⁻¹ NAA. All in vitro shoots rooted well on half macronutrient MS medium containing 0.1 mg l⁻¹ NAA. After acclimatization, transplanted plantlets grew normally and flowered in the field.     

Factors affecting in vitro adventitious shoot formation on internode explants of Citrus aurantium L. cv. Brazilian

The in vitro formation of newly formed adventitious buds and shoots from internodal branch segments was studied on 12-month-old plants of Citrus aurantium L. cv. Brazilian. The effects of 6-Benzyladenine (BA) and α-Naphthalene acetic acid (NAA) treatments were evaluated on adventitious bud and shoot regeneration. High rates of bud initiation and shoot development were obtained both with BA supplemented medium, in the range from 1 mg L⁻¹ to 3 mg L⁻¹, and with 0.1 mg L⁻¹ NAA supplemented medium. NAA concentrations above 1 mg L⁻¹ significantly reduced bud initiation and shoot elongation. The results obtained using different in vitro culture vessels such as Petri dishes, tubes and glass culture jars were compared. The highest adventitious bud induction was observed in Petri dishes for internodes cultured in 2 mg L⁻¹ BA supplemented medium, with 95% responsive explants forming 9.0 ± 2.4 adventitious buds. The adventitious buds observed in Petri dishes reached a maximum height of 1 mm, with no further development, while some of the adventitious shoots cultured in tubes and glass culture jars grew over 1 cm in height. A shoot regeneration gradient of the internodes collected along the branch axis was noticed, with basal ones exhibiting higher regeneration frequency.     

Development of a plant regeneration system from seed-derived calluses of centipedegrass [Eremochloa ophiuroides (Munro.) Hack]

The objective of this study is to establish plant regeneration system with the seed of the new Chinese selection “E-126”of centipedegrass [Eremochloa ophiuroides (Munro.) Hack] as explant. In present study, the following results were obtained: (1) The medium formulation most suitable for calluses induction was identified to be MS with 1.0 mg l⁻¹ 2,4-D + 30 g l⁻¹ mannitol + 50 ml l⁻¹ coconut milk and the ratio of calluses induction was 96.0%, including 5.2% of yellow granule calluses induction. The above medium formulation was adopted for subculture. (2) The rate of shoot regeneration from yellow granular calluses was 98.0% by MS optimum medium formulation with 2.0 mg l⁻¹ KT + 50 ml l⁻¹ coconut milk. The differentiated rate retained as high as 88.0% even after 5 times of subculture and 18.6% after 15 times of subculture. The optimum medium formulation for shoot growth was identified to be MS medium plus 2.0 mg l⁻¹ BAP, 0.8 mg l⁻¹ NAA and 50 ml l⁻¹ coconut milk. (3) The optimum medium for shoot rooting was identified to be MS medium with 0.6 mg l⁻¹ NAA + 50 ml l⁻¹ coconut milk, and the rooting rate to be 98.0%. The survival rate of transplanted plantlets from tubes to basin with soil was 92.0%. In conclusion, the plant regeneration system was successfully developed in this study, which may provide basic reference for screening of somaclonal variants and genetic transformation of centipedegrass.     

Efficient in vitro multiplication in Ornithogalum ulophyllum Hand.-Mazz. from twin scale explants

Ornithogalum ulophyllum Hand.-Mazz. with beautiful white flowers is an important medicinal and ornamental plant of the Middle Eastern countries and need exploitation for commercial propagation. The study reports in vitro mass proliferation of bulblets achieved from twin scales and “in vitro regenerated bulblet” explants on MS medium supplemented with various concentrations of BAP–NAA. The best regeneration on twin scales and “in vitro regenerated bulblets” was obtained on MS medium containing 2 mg l⁻¹ BAP–0.5 mg l⁻¹ NAA and 2 mg l⁻¹BAP–1 mg l⁻¹ NAA, respectively. However, bulb scales seemed to be more potent for bulblet regeneration. A large number of the developing bulblets rooted on the regeneration medium. Remaining non-rooting bulblets were rooted on MS medium containing 1 mg l⁻¹ NAA. All plants were acclimatized in the environmental chamber for 4 weeks and were transferred to the greenhouse for flowering. Regenerated bulblets developed into morphologically normal plants.     

High-frequency embryogenesis,regeneration of broccoli (Brassica oleracea var. italica) and analysis of genetic stability by RAPD

High-frequency somatic embryogenesis and shoot regeneration of broccoli (Brassica oleracea var. italica) were achieved. Cotyledon and hypocotyl explants from four varieties of broccoli were cultured on MS and modified MS media (mMS, supplemented with PG-96 organic components) with different combinations of growth regulator. The effects of genotypes, different explants, growth regulator combinations, organic components and AgNO₃ on induction of calli and shoots were evaluated. The optimal media for inducting calli/shoots and roots were mMS medium containing 3% (w/v) sucrose and 0.8% (w/v) agar supplemented with NAA at 0.5 mg l⁻¹, 6-BA at 3.0 mg l⁻¹, AgNO₃ at 4.0 mg l⁻¹ and MS medium containing 3% sucrose and 0.8% (w/v) agar supplemented with NAA at 0.2 mg l⁻¹, respectively. The callus induction percentages were over 90% in all four varieties; shoot induction percentage was 92.5% and the average number of shoot per explant was 4.1 from cotyledon explant in variety Bishan. In this study, we established high-efficient embryogenesis and shoot regeneration system of broccoli and analyzed genetic stability of regenerants at DNA level using RAPD molecular marker. Out of 62 arbitrary primers screened using PCR amplification, 79 polymorphic bands were amplified from 20 primers. The results demonstrated the genetic stability of regenerants from the same variety.     

Regeneration and transformation system in Mirabilis jalapa

Protocols for in vitro regeneration and production of in vitro-propagated plants and a transformation system were developed for Mirabilis jalapa (Nyctaginaceae). Among the types of explants and the different media tested, consistent shoot regeneration was obtained only from nodal segments grown in a regeneration medium consisting of Murshashige and Skoog medium supplemented with 2 mg l⁻¹ 6-benzyladenine, 2 mg l⁻¹ zeatin and 1 mg l⁻¹ indole acetic acid. Regeneration efficiency was dependent on the type of plant – white or pink flowers – used as the source of explants. Stable transformation was obtained following inoculation of nodal segments with Agrobacterium tumefasciens strain EHA105, which harbours the binary plasmid pAD1339 containing both nptII and gus genes under the control of the 35S promoter. Transformation was confirmed by PCR and Southern blot analysis of genomic DNA from mature regenerated plants. β-Glucuronidase (GUS) activity was observed only in tissues regenerated from in vitro-grown plants and not in tissues originating from greenhouse-grown plants. GUS expression was not uniform in regenerated leaves and showed a chimera pattern.     

Clonal propagation of Rosa clinophylla Thory. through axillary bud culture

A protocol for clonal propagation of Rosa clinophylla, a rare and endangered species but very important for breeding purposes had been standardized through in vitro axillary bud culture. Although cytokinins alone were able to induce shoot buds, but their proper growth and number could be increased only when they were used in combination with GA₃. However, there was shoot tip necrosis and leaf fall in the proliferated shoots. AgNO₃ at 58.85 μM proved effective to avoid shoot necrosis and yellowing of leaves. Activated charcoal (AC) at 250 mg l⁻¹ was found necessary at all the stages of shoot multiplication as well as rooting. Ninety percent rooting could be achieved in 1/2 MS medium supplemented with 4.92 μM IBA and 250 mg l⁻¹ AC. Rooted plantlets were hardened and transferred to the field successfully with 80% survival rate.     

In vitro high frequency direct plant regeneration from whole leaves of blackberry

High frequency and direct (without callus) plant regeneration was achieved from whole leaf explants of thornless blackberry (Rubus hybrid) cv. Black Satin (EC No. 381258; PI No. 553272) in vitro. Leaf blade explants from 1-, 3- and 5-month-old mother cultures were cultured on Murashige and Skoog (MS) medium with thidiazuron (TDZ), N⁶-benzylaminopurine (BAP), indol-3-butyric acid (IBA) and α-naphthalene acetic acid (NAA), alone or in combination. Three-month explants cultured on 0.02 mg l⁻¹ TDZ produced a high regeneration frequency (91.7%) and the most shoots/leaf explant (17.3). The shoot primordia developed within 3 weeks from the point of detachment of the petiole from the leaf blade. The age of the explant source significantly affected the shoot regeneration potential of the leaf explants. Leaves excised from 3-month-old in vitro-cultured shoots performed better than those from 1- and 5-month-old shoots. Shoots rooted best on half-strength MS basal medium with 0.5 mg l⁻¹ IBA and 90% of the plantlets survived acclimatization. The regenerated plantlets were morphologically similar to the mother plants.     

Medium-term conservation of redwood (Sequoia sempervirens (D. Don.) Endl.) in vitro shoot cultures and encapsulated buds

This study evaluated the survival and recovery of non-encapsulated and encapsulated shoots of Sequoia sempervirens after storage at 4 °C in the dark for up to 15 months on four different culture media. Survival and regrowth of encapsulated shoots declined within 3 months, regardless of the storage medium composition. By contrast, no significant decrease in survival and regrowth was noted with non-encapsulated shoots after 12 months of storage on Quoirin and Lepoivre medium supplemented, or not, with 1 mg l⁻¹ benzyladenine. Regrowth dropped to 60–61% after 15 months of storage on the same media. Medium-term conservation of S. sempervirens germplasm is therefore possible using in vitro storage of non-encapsulated shoot cultures.     

Direct plant regeneration from mature leaf explants of pistachio, Pistacia vera L.

A protocol was developed for direct shoot and plantlet regeneration from in vitro regenerated leaf explants of male Pistacia vera L. cv. ‘Atl?’. Leaves excised from axenic shoot cultures of pistachio were used to induce organogenesis on a Murashige and Skoog (MS) medium with Gamborg vitamins supplemented with combinations of different concentrations of BAP and IAA. The highest adventitious shoot regeneration in 35% of the explants, with the number of shoots ranging from 2 to 3 per explant, occurred in the explants cultured during the establishment phase in the medium with 1 mg l⁻¹ IAA and 2 mg l⁻¹ BAP. For shoot multiplication, the highest number of new microshoot/explants (5.76) was obtained in a culture medium supplemented with 1 mg l⁻¹ BAP, but it was not significantly different from the number obtained at 2 mg l⁻¹ BAP. A high rooting frequency (84%) for microshoots was recorded on a medium supplemented with 2 mg l⁻¹ IBA. In vitro rooted plantlets were transferred to pots filled with a mixture of soil, sand and peat (1:1:1). They were weaned in a growth room and finally moved to a greenhouse. This protocol could be utilized for in vitro clonal propagation of this economically important plant.     

In vitro regeneration from mature leaf explants of Pelargonium rapaceum (L.) L’Hérit

Methods to regenerate whole plants from mature leaf explants of Pelargonium rapaceum (L.) L’Hérit were established. To optimize shoot induction, leaf explants were cultured on media containing different types and combinations of plant growth regulators. Growth was initiated within 17–24 days culture, and included callus formation, and root or shoot organogenesis ranging from 20 to 100% regeneration. Shoots were induced only when explants were cultivated on MS medium containing a combination of NAA and kinetin, NAA and BAP, IAA and Kinetin, or IAA and BAP. On media containing NAA and BAP, dark incubation was critical for efficient direct shoot regeneration from explants. Direct shoot formation and the highest number of shoots per explant (17.6) were obtained from leaf explants cultured in the dark for 30 days on MS medium containing 0.1 mg l⁻¹ NAA and 0.1 mg l⁻¹ BAP. Shoots cultured on MS medium containing 0.1 mg l⁻¹ NAA formed tuberous roots with microtubers within 42 days. Healthy regenerated plants were acclimated and transferred to a greenhouse.     

Production of protocorm-like bodies with bioreactor and regeneration in vitro of Oncidium ‘Sugar Sweet’

To produce mass propagules of Oncidium ‘Sugar Sweet’, we tested the feasibility of producing protocorm-like bodies (PLBs) using 5 l balloon type air-lift bioreactors, and selected an optimal culture medium for shooting and rooting in vitro. The results showed that liquid bioreactor cultures were more efficient for PLBs proliferation when compared to solid- and liquid-agitated flask cultures. The maximum PLBs biomass (326.3 g per bioreactor, FW) was obtained in the immersion bioreactor culture, with the growth ratio reaching 10.9 after 50 days of culture. This was obviously higher than the ebb and flood bioreactor culture. During bioreactor culture, sucrose and electric conductivity (EC) in the culture medium were negatively correlated with PLBs growth; the highest PLBs fresh and dry biomass was obtained 40 days after culture. An inoculation density of 20 g (FW) was optimal for PLBs growth in a 3 l working volume of 5 l bioreactor. Furthermore, MS medium supplemented with 2.0 mg l⁻¹ BA for shooting and 0.5 mg l⁻¹ IBA for rooting was optimal during in vitro culture, the plantlets were successfully established in the potting substrate.     

Effect of ABA and sucrose on germination of encapsulated somatic embryos of guava (Psidium guajava L.)

Present study demonstrates the effect of sucrose and ABA on germination of encapsulated somatic embryos of guava (Psidium guajava L.). Sucrose and ABA at different concentrations were also evaluated for their effects on maturation and germination of somatic embryos. Mature somatic embryos developed on MS medium containing high concentration of sucrose (10%) or ABA (1.0 mg l⁻¹) showed inhibition in germination if they continued to be in same medium for 4 weeks. With increasing concentrations of sucrose (3–9%) or ABA (0.01–1.0 mg l⁻¹) in medium, percent germination of encapsulated somatic embryos decreased significantly. Encapsulated somatic embryos after storage on MS medium supplemented with 9% sucrose or 1 mg l⁻¹ ABA for different duration (0–60 days) germinated when they were transferred to medium containing 3% sucrose. About 20.8% and 37.5% encapsulated somatic embryos germinated after storage on ABA (1 mg l⁻¹) or sucrose (9%) for 60 days, respectively. Temporarily suppression in germination of encapsulated somatic embryos by high concentration of sucrose or ABA may be important for short-term conservation of elite genotype of guava.     

In vitro morphogenesis and adventitious shoot mass regeneration of Vriesea reitzii from nodular cultures

Micropropagation systems based on nodular cultures (NCs), are considered as an intermediary in vitro morphogenetic route, diverging from regenerative systems based on organogenesis and somatic embryogenesis. The aim of this study was to establish a regenerative protocol based on the induction and development of NCs in Vriesea reitzii, an endangered bromeliad from the Atlantic forest which also has ornamental value. Additionally structural analyses were performed in order to better understand this in vitro morphogenetic route. NCs were regenerated in MSB culture medium free of PGR or supplemented with different levels of NAA alone or in combination with in combination with 2-iP. The subculture of these NCs on MSB medium supplemented with 10 μM of GA₃ promoted the synchronized shoot elongation. A regenerative efficiency of 12.4 g g⁻¹ of NCs was obtained, and this results in 5300 microshoots after 10 weeks in culture. The structural analyses of the NCs revealed that the regenerative process occurs from the proliferation of meristematic cell groups resulting in the development of multiple shoot meristems and buds. The development of NCs leads to the formation of monopolar structures called microshoots, which evolve to elongated shoots. Intermediary features shown in NCs are consistent with their classification as an intermediary system among organogenesis and somatic embryogenesis.     

Chromosome doubling of Lychnis spp. by in vitro spindle toxin treatment of nodal segments

Lychnis (Caryophyllaceae) consists of about 30 species distributed throughout the temperate regions of the Northern Hemisphere, from East Asia to Europe. Many Lychnis spp. have high ornamental value and cultivated as pot or garden plants. In the present study, in vitro chromosome doubling of several Lychnis spp. was examined in order to widen their variability in horticultural traits. Initially effect of various spindle toxin treatments [100, 500 or 1000 mg l⁻¹ colchicine (COL), 10, 20 or 50 mg l⁻¹ oryzalin (ORY), or 1, 5, 10 mg l⁻¹ amiprophos-methyl (APM)] of nodal segments of a triploid genotype of L. senno (3x) was investigated on survival of nodal segments and chromosome doubling in nodal segment-derived plantlets. Significantly higher percentage (75.0%) of surviving segments after spindle toxin treatment was obtained in 10 mg l⁻¹ ORY treatment. Flow cytometry (FCM) analysis of leaf tissues showed that 9.4–13.8% of plantlets, which were derived from 10 to 20 mg l⁻¹ ORY, or 5 mg l⁻¹ APM treatments, were hexaploid (6x) or ploidy chimera (3x + 6x, 4x + 6x, 5x + 6x, 3x + 4x + 6x). The results obtained by FCM analysis were confirmed by chromosome observation in root tip cells. Thus 10 mg l⁻¹ ORY treatment of nodal segments is suitable for in vitro chromosome doubling of triploid L. senno. Efficient chromosome doubling was also achieved in diploid L. fulgens (2x) and L. sieboldii (2x) by treating nodal segments with 10 mg l⁻¹ ORY: 68.9–88.7% of nodal segments survived after ORY treatment, and 24.7–26.5% of plantlets derived from ORY-treated nodal segments were tetraploid (4x) or ploidy chimera (2x + 4x) in both species. These results indicate that the in vitro chromosome doubling method established for triploid L. senno may be applicable to a wide range of Lychnis spp. Tetraploid L. fulgens and L. sieboldii showed a compact plant form, and had thick stems and deep green leaves compared with the diploid mother plants. On the other hand, hexaploid L. senno showed very poor growth and died before flowering.     

Plant regeneration from in vitro cultured leaves of Lanzhou lily (Lilium davidii var. unicolor)

Lanzhou lily (Lilium davidii var. unicolor) is one of the best lilies which are edible in China but the efficient shoot regeneration system has not been developed. The purpose of the present study is to establish an efficient and reproducible protocol for induction of shoots in vitro from L. davidii var. unicolor leaves. Shoot regeneration from in vitro cultured leaves of L. davidii var. unicolor was tested on the 26 media based on NN [Nitsch, J.P., Nitsch, C., 1969. Haploid plants from pollen grains. Science 163, 85–87] basal medium, containing different concentrations of thidiazuron (TDZ) in combination with different concentrations of α-naphthaleneacetic acid (NAA). Shoot organogenesis occurred directly from the leaves without forming callus. Shoot regeneration mainly occurred from the cuts across the midvein and the base of the leaf explants. The highest frequency of regeneration (93.3%) and the largest number of shoots per leaf (3.83) were obtained on NN basal medium supplemented with 0.5 mg l⁻¹ TDZ and 1.0 mg l⁻¹ NAA. All the regenerated shoots formed complete plantlets on half-strength MS [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 15, 473–497] basal medium containing 0.1–0.5 mg l⁻¹ indole-3-butyric acid (IBA) with in 30 days, and 92% of the regenerated plantlets survived in the soil. This study will be useful for Agrobacterium-mediated transformation and exploitation of somaclonal variation of Lanzhou lily.     

Effect of genotype,explant size,position, and culture medium on shoot generation of Gerbera jamesonii by receptacle transverse thin cell layer culture

An unique procedure for the mass shoot propagation of Gerbera using receptacle transverse thin cell layer (tTCL) culture procedure was developed. Genotype, flower bud age, explant size, position of receptacle tTCLs and culture media were found to affect the success of culture. Ten interspecific crosses of Gerbera showed different shoot regeneration rates and callus induction via receptacle tTCL culture, all of which had shoot regeneration rates higher than 57%. Flower buds collected on the 10th day resulted in 91% shoot regeneration after 6 weeks of culture on basal MS medium [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol. Plant. 15, 475–497] supplemented with 0.02 mg l⁻¹ thidiazuron (TDZ), 0.8 mg l⁻¹ adenine and 10% (v/v) coconut water (CW). This was significantly higher than those from flower buds on the 7th and 14th days (22% and 54%), respectively. Shoot regeneration rate was the highest (94–100%) in the middle layers of the receptacle. For mass shoot propagation, shoot clusters were subcultured on half-strength MS medium supplemented with 0.5 mg l⁻¹ indole-3-butyric acid (IBA), 0.5 mg l⁻¹ 6-benzyladenine (BA) and 2.0 mg l⁻¹ kinetin after every 4 weeks. Plantlets formed when single shoots were cultured on half-strength MS medium containing 1 mg l⁻¹ IBA. All plantlets acclimatized well in the greenhouse.     

Ethylene influences development and flowering of Ptilotus spp. in vitro and ex vitro

The genus Ptilotus has immense potential for ornamental horticulture but its commercial development has been hindered by propagation limitations. Poor seed quality and germination are reported. Cutting propagation is limited by cutting supply as the juvenile phase of Ptilotus is short. Micropropagation has been used in an attempt to overcome these difficulties but explants become floral in vitro and this causes plantlets to elongate. Ethephon has been used to control flowering of stock plants of many ornamental species. This study investigated the effect of ethephon applied to young (3-week-old, deflasked from tissue culture) and mature (1-year-old) Ptilotus plants in a greenhouse. A system of applying gaseous ethylene at 0, 100, 200 and 300 mg l⁻¹ to the headspace of in vitro plantlets in glass jars was developed and the response of in vitro plantlets to ethylene studied. One-year-old Ptilotus plants were treated with 500 mg l⁻¹ ethephon 2 days before pruning or 1 or 2 weeks after pruning. Ethephon application 2 days before pruning decreased the number of inflorescences and increased the number of shoots (compared to the control) but was phytotoxic. Ethephon applications of 150 or 300 mg l⁻¹ applied weekly or fortnightly to 3-week-old plants deflasked from tissue culture reduced plant height and number of inflorescences and at low concentrations increased the number of new shoots. A fortnightly application at 150 mg l⁻¹ is recommended. Previous reports on the effects of ethylene on inflorescence production on plantlets in vitro are limited. Our study showed that exposure of in vitro plantlets of P. nobilis to ethylene gas at 100 mg l⁻¹ for 1 h significantly increased the number of shoots and plant height but this did not occur for plantlets of P. spicatus. Plantlets of P. spicatus exposed to transient ethylene at 200 and 300 mg l⁻¹ showed significantly greater rooting (52.4%) than the control (13.6%).