Factors affecting the quality and in vitro germination capacity of strawberry pollen

Summary

Poor pollen quality and germination capacity curtails early yield in strawberry. The aim of this study was to establish a reliable method for in vitro assessment of strawberry pollen germination ability and to investigate further the effects of photoperiod and gibberellin on pollen germination and quality. In the first part of the study, pollen from seven strawberry cultivars (Chandler, Selva, Tudla, Camarosa, Eris, Pajaro and Irvine) was collected and its germination capacity and incidence of deformed pollen grains assessed in vitro using the hanging-drop technique. Highest germination rates, in ‘Selva’, were observed in a nutrient medium of 10% sucrose. Addition of calcium nitrate to the medium decreased the germination percentages of all cultivars. There was no significant difference, on average, between the germination rate at 20° and 25°C. Genetic factors affected the incidence of deformed pollen grains significantly, with ‘Pajaro’ showing the highest percentage (76%). In the second part, groups of young strawberry plants, cultivar Seascape, grown either under natural early spring conditions or under long-day or short-day conditions were sprayed once with GA₃ at 0, 50, or 200 mg l^–1. Pollen germination and deformation and stamen length were assessed three months later. In plants of the first group, GA₃ at 50 mg l^–1 increased pollen germination and decreased the incidence of deformed pollen grains, while GA₃ at 200 mg l^–1 decreased pollen germination without affecting the formation of deformed pollen grains. Plants of the second group showed a higher rate of pollen germination under long than under short days. GA₃ at 200 mg l^–1 decreased pollen germination under either short- or long-day conditions compared with the controls but doubled the percentage of deformed pollen only under short days. Stamens in control plants grew four times as long under long- than short-day conditions. GA₃ did not affect stamen length under long days but significantly enhanced their growth under short days.     

Antagonistic effect of indole-3-acetic acid on kinetin-stimulated amaranthin accumulation in the cotyledons of Amaranthus mangostanus seedlings

Light triggered the initiation of amaranthin biosynthesis in cotyledons of Amaranthus mangostanus L. seedlings. Cytokinin induced amaranthin synthesis in the dark and increased the accumulation of amaranthin under light irradiation. No studies have explored whether indole-3-acetic acid (IAA) can affect kinetin-induced amaranthin accumulation in seedlings of A. mangostanus L. In this study, we found that IAA inhibited both the kinetin- and light-induced synthesis of amaranthin. In the dark, 10.0 mg l^?1 IAA caused a 68% reduction in amaranthin production after induction by 5.0 mg l^?1 kinetin. In the presence of light, 10.0 mg l^?1 IAA resulted in a 50% decrease in amaranthin synthesis following induction by 5.0 mg l^?1 kinetin. In addition, IAA could reverse kinetin-induced amaranthin accumulation under red, blue, or far-red light conditions. Our results suggest that IAA had an antagonistic effect on the light-induced or cytokinin-stimulated accumulation of amaranthin in the cotyledons of A. mangostanus L. seedlings.     

In vitro micropropagation of Ephedra

The in vitro micropropagation of eleven species of Ephedra was investigated. Shoot nodal explants of E. fragilis were cultured on Murashige and Skoog medium supplemented with 3% sucrose, 0.05 jiM 3-indolebutryic acid and 0.0-0.5 |iM kinetin, zeatin or 6-ben- zylaminopurine. In general, the average number of shoots produced per explant increased and the average shoot length decreased with increasing cytokinin concentration. Substitution of 3-indolebutryic acid with 2,4-dichlorophenoxyacetic acid caused callusing and distorted shoot growth. Shoot cultures of ten other species were grown on 0.05 |iM 3-indolebutyric acid with 0.05 kinetin. Indole-3-acetic acid gave healthy rooting. E. equisitina, E. gerardiana, E. minima and E. saxatilis were successfully micropropagated using a single-stage protocol in which shoots were grown and rooted on Sorbarods using half strength Murashige and Skoog medium supplemented with 1% sucrose and 5.0 (iM indole-3-acetic acid. Healthy plantlets were weaned in John Innes No. 1: Perlite (1:1) following treatment with Captan (1.9 g I'1).     

Large-scale in vitro propagation of giant reed (Arundo donax L.), a promising biomass species

Summary

Giant reed (Arundo donax L.), a promising energy crop, is vegetatively-propagated from fragments of stems and rhizomes. This may limit large-scale cultivation, since it is time-consuming and involves considerable cost and effort. Tissue culture is an alternative to conventional methods of vegetative propagation and may represent a useful tool for large-scale propagation of plants for biomass production programmes. This report describes a protocol for the large-scale in vitro propagation of giant reed by adventitious bud formation. Stem nodes with dormant buds proved to be the most effective to initiate in vitro cultures giving the highest percentage of differentiated shoots (77%) compared to the other plant fractions tested. A sterilisation procedure using 5 g l^–1 HgCl₂ enabled the production of sterile explants. Moreover, early results indicated that late Autumn excision dates not only gave a higher percentage of well-developed shoots ( > 80%), but also a lower level of bacterial contamination (15 – 20%). 6-Benzylaminopurine (BAP) at 3.0 mg l^–1 was most effective in promoting shoot multiplication when added to a basal medium containing Murashige and Skoog (MS) macro- and micro-nutrients, Morel’s vitamins, 30 g l^–1 sucrose, and 7.0 g l^–1 bacteriological agar supplemented with 1.0 mg l^–1 indole-3-acetic acid (IAA) and 0.05 mg l^–1 gibberellic acid (GA₃). Rooting was successfully induced on the same basal medium used for proliferation, modified by halving the MS macro-nutrients and replacing BAP and the other growth regulators with 2.0 mg l^–1 1-naphthaleneacetic acid (NAA). Successful acclimatisation (> 95% survival) of plantlets was carried out, even in late Winter, in a cold greenhouse or under simpler facilities such as shade nets.     

Influence of endogenous IAA levels and exogenous IBA on rooting and quality of leafy cuttings of Prunus ‘GiSelA 5’

Summary

The influence of exogenously applied indole-3-butyric acid (IBA) on root and shoot development of leafy cuttings was analysed in Prunus cerasus P. canescens ‘GiSelA 5’, a dwarfing cherry rootstock, in two successive years. Compared to control cuttings, IBA application (4 g l^–1 in 2003; 2.5 g l^–1 in 2004) caused higher indole-3-acetic acid (IAA) accumulation in the cutting bases, but that did not influence the percentage of rooted cuttings, nor their survival in either year. However, IBA inhibited callus formation and, consequently, influenced the quality of the developed cuttings. Callus formation impeded root development, reducing the number of main roots, and inhibited the growth of the cuttings, reducing the average total length of shoots formed by individual cuttings. Callus formation was most reduced in the cuttings in the second experimental year, with high initial IAA concentrations.     

Rapid micropropagation of Psoralea corylifolia L. using nodal explants cultured in organic additive-supplemented medium

Summary

The influence of different growth regulators and additives on shoot multiplication from nodal explants of Psoralea corylifolia was investigated. Prolific shoot multiplication was achieved within 4 weeks of culture on Murashige and Skoog (MS) medium supplemented with 5 μM benzyladenine (BA), 5 μM ascorbic acid (AA), 100 mg l^–1 casein hydrolysate (CH) and 5% (v/v) coconut water (CW). Shoots elongated on half-strength MS basal medium devoid of inositol, but containing 5 μM 2-isopentenyladenine (2iP), 10 g l^–1 sucrose and 8 g l^–1 agar. Elongated shoots rooted on half-strength MS basal medium supplemented with 3 μM indole-3-butyric acid (IBA), 10 g l^–1 sucrose and 7 g l^–1 agar within 5 d of culture. The in vitro-raised plants were established successfully in 2:1:1 (v/v/v) garden soil:farmyard soil:sand, and maintained in a growth chamber with 100% survival. Acclimatised plants were transferred to a glasshouse and established successfully in the field. Flowers and fruits appeared after 4 months and resembled those on source plants. This system could be used for rapid commercial propagation of P. corylifolia for conservation strategies and to produce phytomedicines.     

An improved micropropagation protocol for the recalcitrant plant Capsicum – a study with ten cultivars of Capsicum spp. (C. annuum,C. chinense,and C. frutescens) collected from diverse geographical regions of India and Mexico

Capsicum spp. is a commercially important crop of the Solanaceae family, well-known for its multipurpose use as a vegetable, spice, medicinal and ornamental plants. The genus Capsicum is a recalcitrant species in terms of in vitro morphogenesis and plant regeneration. An efficient method was developed for multiple shoot regeneration in 10 cultivars of Capsicum collected from diverse geographical regions of India and Mexico. Seeds germinated in vitro on a half-strength Murashige and Skoog (MS) medium supplemented with 3.0 % sucrose. Nodes of the in vitro germinated seedlings were used as explant for micropropagation. The combination of the 6-benzylaminopurine, indole-3-acetic acid, and spermidine was found to be the best for multiple shoot induction. However, the optimum responcse varied accompanied by different cultivers with maximum 8.9 ± 0.52 (Capsi-10) to 15.3 ± 0.69 (Capsi-5) multiple shoot per explant. Depending on the cultivar, multiplied shoots were successfully rooted with maximum 18.4 ± 0.20 (highest for Capsi-9) to 36.8 ± 0.29 (highest for Capsi-5) roots per shoot on half-strength MS medium supplemented with 2.0 mg l^?1 indole-3-butyric acid, 1.0 mg l^?1 α-naphthalene acetic acid, and 1.5 mM spermidine. Finally, the micropropagated plantlets were acclimatized with 40.0–86.7 % survival rate, depending on different cultivars.     

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.     

Cost-effective tissue culture media for large-scale propagation of three commercial banana (Musa spp.) varieties

Cost-effective tissue culture protocols have been established for the commercial multiplication of three banana varieties, ‘Rasthali’ (AAB – Silk), ‘Grand Naine’ (AAA – Cavendish), and ‘Udhayam’ (ABB – Pisang Awak). Reverse osmosis water and 3% (w/v) table sugar were used as the low-cost water and carbon source, respectively. Six different gelling agent treatments were tested: sago alone (T1), Isabgol alone (T2), sago + agar (T3), Isabgol + agar (T4), sago + Isabgol (T5), and agar alone as a control (T6). Full-strength Murashige and Skoog (MS) medium supplemented with 3 mg l^–1 6-benzylaminopurine (BAP) and 1 mg l^–1 indole-3-acetic acid (IAA) were used for culture initiation and subculturing. Rooting was accomplished on low-cost MS medium containing 1.0 mg l^–1 α-napthaleneacetic acid (NAA), 1.0 mg l^–1 indole-3-butyric acid (IBA), and 250 mg l^–1 activated charcoal. Statistical analysis indicated that sago + Isabgol (T5) produced the maximum number of shoots (10 per explant) in ‘Udhayam’ and ‘Rasthali’, while sago alone (T1) produced the maximum number of shoots (6 per explant) in ‘Grand Naine’. The genetic stability of tissue-cultured banana plantlets produced using these low-cost substitutes was assessed using inter-simple sequence repeat (ISSR) markers. The results indicated that the ISSR profiles of the five treatments and the control (T6) were similar, indicating genetic stability using these cost-effective tissue culture protocols. Reductions in cost over the control (l^–1 of MS medium) ranged from 65% to 86%, while the per plant production cost was reduced by 12.5%–20.0%. Adoption of these treatments (T1–T5) as low-cost tissue culture protocols for in vitro propagation would reduce production costs significantly, leading to an expansion of the area planted with tissue-cultured banana, thereby increasing productivity.     

Factors affecting tissue culture of Damask rose (Rosa damascena Mill.)

The present study was conducted to evaluate the regeneration ability of Damask rose. Single-node explants were surface sterilised with 10% chlorox for 15 min and cultured on Murashige and Skoog (MS) medium supplemented with various concentrations of N⁶-benzyladenine (BA) or kinetin (Kin) separately or in combination with different concentrations of indole-3-butyric acid (IBA). Combination of 2.5–3 mg/l BA and 0.1 mg/l IBA was the most suitable treatment for proliferation. In vitro derived shoots were subcultured four times on the fresh medium within a 4-week period. Other treatments such as explant orientation (horizontal, vertical and oblique) and explant wounding were also examined but did not affect shoot multiplication rate significantly. Several experiments were carried out to stimulate in vitro rooting of Damask rose. Application of different media such as MS, 1/2 MS, 1/3 MS and 1/4 MS with different concentrations of indole-3-acetic acid (IAA), IBA and naphthaleneacetic acid (NAA) did not produce satisfactory results. Quick-dip method using sterilised 0–2000 mg/l IAA, IBA and NAA solutions was also studied. Other treatments such as using various concentrations of abscisic acid (ABA) in combination with various concentrations of IAA, IBA and NAA, and using various concentrations of sucrose and agar did not produce any roots. The best treatment for rooting of shoots was 2.5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) for 2 weeks in MS medium and then transferring the explants to MS medium without any growth regulator. Plantlets were acclimatised in a soil mixture consisting of peat moss and sand 1:1 (v/v) and successfully transferred to the greenhouse after 3 weeks.     

An improved protocol for adventitious shoot regeneration and plant formation in Psoralea corylifolia L.

An effective protocol was developed for in vitro regeneration of Psoralea corylifolia through enriched cotton moistened-liquid (CML) and solid culture systems. Prolific adventitious shoot buds were achieved from hypocotyl explants of 2-week-old cultures on enriched CML Phillips and Collins (L2) medium supplemented with different concentrations and combinations of thidiazuron (TDZ), benzyladenine (BA), kinetin (KIN), naphthalene acetic acid (NAA), indole-3-acetic acid (IAA) and bavistin (BVN). Combination of 2 μM TDZ, 0.5 μM BA, 100 mg l⁻¹ BVN and 2 μM NAA produced a greater number of adventitious shoots per explant (93.5) when transferred to half-strength enriched solid L2 medium. Regenerated shoots (40–50 mm in length) were exposed simultaneously for rooting as well as hardening in moistened (1/8-L2 basal salt solution with 5 μM IBA and 100 mg l⁻¹ BVN) soil mixture and vermiculite (3:1, v/v). The plants were subsequently established in the field. The survival percentage differed with seasonal variations.     

Effects of chlorocholine chloride and prohexadione-Ca on rhizome growth and lateral bud production in Iris germanica L.

Iris germanica L. is a popular perennial flower worldwide, but its use is limited in China due to an inadequate availability of propagules. To accelerate rhizome growth and lateral bud production using plant growth retardants (PGRs), chlorocholine chloride (CCC; at 1,500 or 3,000 mg l^?1) or prohexadione-Ca (at 700 or 1,500 mg l^?1) were applied to uniform plants of I. germanica. Time-course measurements of changes in morphogenesis, carbohydrate metabolism, total soluble protein (TSP) concentrations, and endogenous phytohormone concentrations in rhizomes were conducted to test the efficacy of CCC or prohexadione-Ca for increasing rhizome growth and lateral bud production. The results showed that both PGRs increased the fresh weights of rhizomes at 2, 4, and 6 weeks after treatment (WAT). Overall, 700 mg l^?1 prohexadione-Ca was most effective at promoting the formation of lateral buds which increased by 183.5% at 12 WAT relative to untreated control plants. Concentrations of sucrose and starch in PGR-treated rhizomes increased at 2, 4, and 6 WAT, while a decline was observed by 12 WAT. TSP concentrations increased during rhizome enlargement, then decreased during lateral bud germination after prohexadione-Ca treatment. In general, concentrations of endogenous phytohormones, such as gibberellins, indole-3-acetic acid, jasmonic acid, and zeatin riboside, decreased significantly in rhizomes at 4 WAT, then increased at 12 WAT. Our study indicated that prohexadione-Ca promoted rhizome growth and the accumulation of sucrose and starch before summer dormancy, then significantly accelerated the production of lateral buds.     

High-efficiency colony formation and whole plant regeneration from mesophyll protoplasts of Pelargonium × hortorum ‘Panaché Sud’

Summary

This study aimed to establish a plant regeneration system from protoplasts of Pelargonium hortorum, efficient enough to be used for further direct gene transfer experiments. A rapid and efficient system that allowed high efficiency colony formation (40%) and whole plant regeneration (83%), as well as rooting within 4 months, was established using mesophyll protoplasts of the cultivar ‘Panaché Sud’. Protoplast culture in liquid medium was found to be better than culture on solid medium both for cell division and colony formation. The optimum density for high colony formation (31-40%) from viable cultivated protoplasts was 3 – 5 10⁴ protoplasts ml^–1. Reducing the osmotic pressure and increasing the macronutrient and sucrose contents of the culture medium after the first week of culture facilitated the rapid development of colonies. The transfer of microcalli to mannitol-free callus-induction medium produced green calli in all cases. The highest frequency of bud and shoot regeneration from protoplast-derived calli (83%; 6.6 per callus) was obtained at a density of 3 10⁴, on medium containing 0.2 mg l^–1 indole-3-acetic acid (IAA), 1.0 mg l^–1 zeatin and 0.1 mg l^–1 thidiazuron (TDZ). The best results were obtained when the medium was gelled with Gelrite^® and cultures were maintained under low light (12 µmol s^–1 m^–2). Sixty-five percent of protoplast-derived calli underwent bud and shoot regeneration and 2.6 rootable plantlets were obtained per callus after 3 weeks on elongation medium. All acclimatised plants grew normally and gave fertile flowers. However, flow cytometry on 42 plants showed that 40 of these were tetraploids, and only two were diploids, like the mother plant. This protocol can now be used in transformation experiments and applied to other genotypes to improve regeneration.     

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.     

Microsporogenesis and anther culture in carob tree (Ceratonia siliqua L.)

An in vivo study was made on male flowers of carob tree (Ceratonia siliqua L.), in order to establish a correlation between the flower and anther development, and microsporogenesis. In addition studies were conducted to find which phase is more appropriate for anther culture and haploid production. During the development of male flowers, six stages were identified. The male gametophytic cycle begins when flowers are in developmental phase 0, with the formation of the epidermis, endothecium, primary sporogeneous tissue, primary parietal cells and pollen mother cells. During developmental phase I we observed the formation of pollen mother cells, the microspore tetrads, and uni- and binucleate pollen grains. At developmental phase II, uni- and binucleate microspores, and completely formed pollen grains were observed. In developmental phase III we could observe mature pollen grains ready to be released from the anthers as single binucleate pollen grains. Anthers from flowers at developmental phases I and II, with microspores at late uninucleate to early binucleate stage, were cultured in semi-solid Murashige and Skoog's (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) combined with one of the citokinins: N⁶-benzyladenine (BA), kinetin (Kin), zeatin (Zea) and thidiazuron (TDZ). To obtain embryogenic calli anthers should be collected from flowers in developmental phase I. High frequencies of callogenesis were obtained, and the best medium for calli induction was MS supplemented with 0.5 mg l⁻¹ 2,4-D + 4 mg l⁻¹ TDZ. The frequency of haploid cells was found to be 17.2%.     

Effect of gibberellic acid (GA3) on elongation and rooting of ‘St. Julien A’ rootstock in vitro

‘St. Julien A’ (Prunus instititia L.) rootstock was induced to proliferate shoots on a modified half-strength Murashige and Skoog (MS) medium. Cultures treated with 12.5 mg l^?1 gibberellic acid (GA₃) produced elongated shoots suitable for rooting. Elongated shoots were placed in media with indolebutyric acid (IBA) or indole-3-acetic acid (IAA) with or without a 16-day dark incubation. Light (16-h photoperiod) inhibited rooting. IAA (4 mg l^?1) was ineffective in promoting rooting. Rooting was best when shoots were incubated in the dark with IBA (4 mg l^?1). GA₃ was deleterious to shoots, causing chlorosis and apical die-back. Light regime interacted with auxin treatments in affecting shoot condition. Shoot condition was better on shoots treated with IBA and dark-incubated; while those treated with IAA were better when light-incubated.     

Development of an in-vitro rooting bioassay using juvenile-phase stem cuttings of Persea americana Mill.

Summary

A rooting test was developed with shoot cuttings taken from aseptically germinated avocado seed. The rooting required treatments in two steps: (1) three days in a medium containing indole-3-butyric acid (IBA) (25 mg l^?1) and (2) four to eight weeks in an auxin-free medium. Rooting was accomplished with both media containing 0.3× strength Murashige and Skoog salts, 3% sucrose, 0.4 mg l^?1 thiamine hydrochloride, 100 mg l^?1 i-inositol, and 0.8% ‘TC’ agar. The auxin-free medium also contained 1 gl^?1 activated charcoal.     

Callus and axillary-bud culture of Vitis vinifera ‘Sylvaner Riesling’

Healthy growth of serially subcultured callus of the grape Vitis vinifera cultivar ‘Sylvaner’ was obtained by incubation at 30° C in continuous light in a defined culture medium containing 2% w/v sucrose, 1.0 mg l^?1 1-naphthaleneacetic acid (NAA) and 0.2 mg l^?1 kinetin (K). Organogenesis was not induced in this callus by alteration in the absolute or relative levels of NAA and K.Continued shoot initiation was obtained by culture of axillary buds in a medium containing 10^?5 M Benzyladenine (BA). Plantlets could be generated from these shoot buds by transfer to media containing 10^?7 M BA or lacking a cytokinin.     

Rapid in vitro germination of zygotic embryos of fluted pumpkin (Telfairia occidentalis Hook. f.)

Summary

Fluted pumpkin, Telfairia occidentalis, is becoming an important regional vegetable for its food and medicinal uses. The recalcitrant nature of its seed makes conservation difficult and in vitro techniques may be a viable option for conservation. A pilot study was conducted on the effects of different concentrations of a commercial bleach [3.85% (w/v) sodium hypochlorite] for surface sterilisation of T. occidentalis seed. The optimum concentration [25% (v/v)] was then used as a basis to investigate the responses of mature embryonic axes of T. occidentalis to different concentrations of kinetin (Kin; 0, 1.0, or 2.0 mg l^–1) and 1-naphthaleneacetic acid (NAA; 0, 0.5, or 1.0 mg l^–1) combined in a factorial design. The results of the first experiments indicated that commercial bleach at 25% (v/v) resulted in the lowest contamination of explants (10%), with no evident injury to the embryonic axes. The results revealed that root emergence started 3 d after initiation (DAI) only on Murashige and Skoog medium (MS) with no added plant growth regulator (PGR), and that, by 12 DAI, all media supported the rooting of explants. The highest rooting percentage (69%) was observed at 15 DAI on MS medium with 0.5 mg l^–1 NAA, without Kin. However, shoot emergence started at 9 DAI on PGR-free MS medium, on MS with 0.5 mg l^–1 NAA, or on MS plus 1.0 mg l^–1 Kin. The highest shooting percentage (91%) of explants was observed with 0.5 mg l^–1 NAA at 21 DAI. Considering all other growth parameters, MS medium supplemented with 0.5 mg l^–1 NAA was found to be best for the germination of embryonic axes of T. occidentalis.     

Influence of high concentrations of cytokinins on the production of somatic embryos by germinating seeds of tomato,aubergine and pepper

Summary

Somatic embryos of tomato, aubergine and pepper were initiated from intact seedlings when seeds were cultured on medium containing 6-benzylaminopurine (BAP) or thidiazuron (TDZ). The percentage of explants producing somatic embryos was highest for aubergine on media containing low concentrations of BAP, i.e. 0–10 mg l^–1, for tomato at 15–20 mg l^–1 and for pepper at 40–80 mg l^–1. Aubergine and tomato produced fewer somatic embryos per responsive seedling when cultured on medium containing TDZ, and pepper did not produce any somatic embryos on media containing 0–20 mg l^–1 1 TDZ. Morphogenesis of the seedlings producing somatic embryos was similar for all the genotypes, i.e. the seedlings were dwarf, only the cotyledons expanded, development of the apical meristem and the root were suppressed and a ring-like crown of nodular tissue developed at the base of the hypocotyl from which somatic embryos were initiated. Co-cultivation of tomato and aubergine seeds with seeds of pepper in media containing 0, 5, 10, 15 and 20 mg l^–1 BAP inhibited somatic embryogenesis in tomato and aubergine instead of assisting somatic embryogenesis in pepper. This is discussed in relation to the recent findings for the induction of somatic embyrogenesis in peanut (Arachis hypogea L.) and the role of BAP and TDZ.