In vitro propagation of ‘Troyer’ citrange from epicotyl segments

Isolated epicotyl, root meristem and root segment tissues of ‘Troyer’ citrange [Poncirus trifoliata (L.) Rat. × Citrus sinensis (L.) Osbeck] were established in continuous culture to compare their regeneration potential. Callus was obtained from these explants on a Murashige—Skoog (MS) medium containing NAA (10 mg l^?1) and BAP (0.1–10 mg l^?1). Formation of shoots from root segments was direct without callus formation on MS medium containing BAP (10 mg l^?1) and NAA (1 mg l^?1). Shoot formation from epicotyl callus occurred on MS medium containing 0.25 mg l^?1 BAP and 0.1 mg l^?1 NAA. Formation of shoots from epicotyl segments occurred on MS medium containing BAP (0.5 mg l^?1) and NAA (0.1–1.0 mg l^?1), while rooting of regenerated shoots occurred in treatments containing 2.0 mg l^?1 NAA alone. This system provides a rapid method for propagation of ‘Troyer’ citrange.     

In vitro somatic embryogenesis from Mangifera indica L. callus

The nucellus and globular adventitious proembryos were removed from 2-month-old fruits of mango (Mangifera indica L.) cultivars ‘Ono’ and ‘Chino’, and were cultured on sterile, solid Murashige and Skoog (MS) medium that had been modified as follows: half-strength major salts and chelated iron; 20% (v/v) coconut water (CW); 6% sucrose; 100 mg l^?1 ascorbic acid and 400 mg l^?1 glutamine. Embryogenic explants were sub-cultured after 4–6 weeks in liquid modified MS medium containing 2 mg l^?1 2,4-dichlorophenoxyacetic acid (2,4-D) instead of CW. Rapidly growing cultures were established and were sub-cultured monthly. Somatic embryogenesis was induced following sub-culture from MS medium with 2,4-D to MS without growth regulators and with or without activated charcoal (0.5%). Germination of somatic embryos appeared to be enhanced by 1 mg l^?1 benzyladenine (BA); however, most of the germinating embryos became embryogenic.     

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.     

Rapid propagation of white cabbage by tissue culture

A tissue culture technique has been devised to produce plants of white cabbage (Brassica oleracea v. capitata L.f. alba) from heads stored at 0.5 ± 0.5°C for 8 months. Meristem-tips (0.5–2 mm diameter), excised from heads of 11 accessions, were grown initially on MS medium containing 2.56 mg l^?1 of kinetin and then induced to proliferate shoots on MS medium with 12.8 mg l^?1 kinetin. Subsequent transfer to a kinetin-free medium resulted in root development in 1–2 weeks. Rooted plantlets were readily established in soil. Plantlets obtained in this way from parent cabbages containing turnip mosaic virus remained infected.     

Meristem culture and micropropagation of a variety of ginger (Zingiber officinale Rosc.) with a high yield of oleoresin

Summary

Meristems of ginger with or without leaf primordia were induced to form shoots on three-quarter strength Murashige-Skoog’s (MS) medium containing sucrose 6%, coconut milk (CM) 20%, ascorbic acid (AA) 100 mg l^?1, glutamine (GL) 400 mg l^?1, activated charcoal (AC) 250 mg l^?1, 6-benzylaminopurine (BAP) 0.5 mg l^?1, indolebutyric acid (IBA) 0.4 mg l^?1 and agar 0.8%. Meristem-derived shoots exhibited consistent multiplication on three-quarter strength MS medium containing sucrose (3%), AA (100 mg l^?1), AC (100 mg l^?1), BAP (4–5 mg l^?1) and agar (0.8%). Liquid media (agitated or static) were less effective than a solid (agar-gelled) medium for micropropagation. Kinetin and naphthalene acetic acid (NAA) incorporated at various levels (0.01–0.8 mg l^?1) with or without added BAP and IBA neither improved plantlet formation nor enhanced shoot multiplication. The in vitro plants were successfully established in vivo and the rhizome yield was comparable with that of plants grown by conventional methods.     

In vitro vegetative multiplication of Fuchsia hybrida

Rapid development of axillary buds from shoot-tips and nodes of 18 cultivars of Fuchsia hybrida was obtained on solid Murashige and Skoog medium with BAP (1 mg l^?1 and an auxin (0.1 mg l^?1). NAA as the auxin appeared to be more active than IAA or IBA. Vegetative shoots were subsequently isolated and developed up to 15 supplementary axillary shoots on the same solid medium. Agitated and non-agitated liquid media of the same composition were less effective. One-cm long shoots could be rooted in 20 days in the presence of IBA before being transferred to soil.     

Influence of growth regulators on setting,retention and weight of fruits in two cultivars of litchi

Investigations were undertaken to explore the possibility of improving setting, retention and weight of fruits in ‘Early Seedless’ and ‘Calcuttia’ cultivars of lichi (Litchi chinensis) by means of growth regulators. Indole acetic acid (IAA) at 20, 40 and 80 mg l^?1, 2,4-dichlorophenoxy acetic acid (2,4-D) at 2,4 and 8 mg l^?1 and gibberellic acid (GA₃) at 50, 100 and 150 mg l^?1 were sprayed on panicles in the first fortnight of April, when 50–100% flowers had opened. All 3 growth regulators caused a favourable effect on fruit setting, fruit retention and weight of individual fruits, but IAA at 20 mg l^?1 proved the best for enhancing setting, GA₃ at 50 mg l^?1 for increasing retention and GA₃ at 100 mg l^?1 for improving fruit weight. IAA and GA₃ should, therefore, be used in combination. Between the 2 cultivars tested, ‘Calcuttia’ proved superior to ‘Early Seedless’ in fruit setting, fruit retention and weight of individual fruits.     

Adventitious shoot formation from sections of sweet potato grown in vitro

Sweet potato (Ipomoea batatas Lam.) root sections were obtained from the cultivars ‘Centennial’, ‘Redmar’ and ‘Jewel’ with a No. 6 cork borer. These sections were cut into 2–3-mm discs and explanted on to modified Murashige and Skoog (MS) medium consisting of MS high mineral salts, myo-inositol (100 mg l^?1), Staba vitamins, 6-benzyl-aminopurine (2.0 mg l^?1), naphthaleneacetic acid (0.1 mg l^?1), sucrose (30 g l^?1) and agar (10 g l^?1). Root discs from internal regions of the tuberous roots gave rise to calli and meristematic bud-like centers (MBLC's). A small percentage of ‘Centennial’ MBLC's burst open to reveal plantlets which grew and rooted well on the medium. Some of the ‘Jewel’ MBLC's contained only roots, while those of ‘Redmar’ did not differentiate. MBLC-formation occurred most often on discs taken from fresh (unstored) roots of ‘Centennial’. Petiole sections taken from in vitro-cultured plants of all 3 cultivars developed plants quite readily on the medium. Shoots of all 3 cultivars grew rapidly, to yield whole rooted plants which could easily be moved to soil and grown in the greenhouse and field.     

Production of genetically uniform plants from shoot tips of Aconitum heterophyllum Wall. – a critically endangered medicinal herb

We report the successful micropropagation of a critically endangered medicinal plant Aconitum heterophyllum Wall., using low concentrations of plant growth regulators (PGRs) and molecular validation of the clonal stocks. The maximum rate of in vitro shoot multiplication was obtained on 1.0 × Murashige and Skoog (MS) medium containing 0.25 mg L^?1 Kinetin (Kn) plus 0.25 mg L^?1 Indole acetic acid (IAA). Up to 100% rooting was obtained 15 for shoots cultured on 1.0 × MS medium supplemented with 1.0 mg L^?1 IAA. Adding 0.25 mg L^?1 2,4-dichlorophenoxyacetic acid (2, 4-D) to 1.0 × MS medium resulted in 100% callus formation, while adding 0.25 mg L^?1 IAA plus 0.25 mg L^?1 Kn to 1.0 × MS medium containing 0.25 mg L^?1 2,4-D resulted in 100% generation of embryogenic callus. Inter-simple sequence repeat (ISSR) marker analysis was carried out to check for possible somaclonal variation in the plantlets obtained after three consecutive sub-cultures. Of the 15 ISSR primers used, 10 were found to be monomorphic, with 95–98% similarity, and were used for cluster analysis by the unweighted pair group using arithmetic averages (UPGMA) method. The results revealed that in vitro-regenerated plantlets did not exhibit any genetic polymorphism.     

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.     

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.     

Development of a medium for in vitro culture of Galanthus species based on the mineral composition of bulbs

Summary

To optimise conditions for micropropagating Galanthus species, a basal medium (G) was developed based on mineral analyses of G. nivalis, G. nivalis ‘Flore Pleno’ and G. elwesii bulbs. Compared with Murashige and Skoog (MS) medium, the main features of G medium were increased concentrations of Cu ( 30.4), P ( 3.6), Ca ( 1.9), Mg ( 1.3) and S ( 1.2) and reduced levels of Mn ( 0.07), Zn ( 0.59) and K ( 0.65). The efficacy of G medium in supporting bulblet initiation on bulb chip explants, bulblet multiplication (on media supplemented with 30 g l^–1 sucrose, 1.0 mg l^–1 6-benzylaminopurine and 0.1 mg l^–1 naphthalene acetic acid), and bulblet growth (on plant growth regulator-free media with 60 g l^–1 sucrose and 5 g l^–1 activated charcoal) was compared with MS medium over a range of dilutions (full-, ¹?2-, ¹?4-, and ¹?8-strength). Bulblet initiation was superior on G medium for G. nivalis and G. nivalis ‘Flore Pleno’, but inferior for G. elwesii. The choice of basal medium did not influence bulblet multiplication, although multiplication was reduced on both media diluted to ¹?8-strength. G medium supported bulblet growth and rooting better than MS medium, while dilution of either medium reduced bulblet growth and rooting. Using G medium in place of MS medium during bulblet multiplication greatly reduced hyperhydration with G. elwesii, as did dilution of either of the basal media.     

The effect of sucrose,silver thiosulphate and 8-hydroxyquinoline citrate on the quality of Lilium inflorescences cut at the bud stage and stored at low temperature

A continuous supply of sucrose together with 8-hydroxyquinoline citrate to cut Lilium Asiatic hybrid ‘Prima’ inflorescences resulted in buds opening satisfactorily and increased their longevity. The best results were obtained using 30 g l^?1 sucrose. Cut Lilium inflorescences could be stored at 1°C for 4 weeks without a great loss in potential vase-life and decorative value when the inflorescences were pre-treated with silver thiosulphate + 100 g l^?1 sucrose for 24 h before cold storage, kept in a cold room in a solution containing 50 mg l^?1 silver nitrate, and after cold storage kept in a solution containing 30 g l^?1 sucrose and 200 mg l^?1 8-hydroxyquinoline citrate. Such treatment greatly improved bud opening, increased the diameters of individual flowers and prolonged their life.     

In vitro propagation of garlic by shoot proliferation

Shoot buds (5–8 mm long), excised from dormant cloves of the New Zealand commercial garlic (Allium sativum L.) and a virus-free French cultivar ‘Rose-de-Kakylis’, proliferated both axillary and adventitious shoots on B-5 basal medium supplemented with 0.5 mg l^?1 isopentenyladenine (2-ip) and 0.1 mg l^?1 naphthaleneacetic acid (NAA). An 8-fold increase in shoot number occurred every 6 weeks. Shoots were readily rooted in B-5 + 0.01 mg l^?1 2-ip + 0.2 mg l^?1 NAA and transferred to pots, where about 70% of the shoots formed established plants. The plants raised by this shoot-proliferation method retained the diploid condition of the parents.     

Effects of growth regulators on growth and flowering in Hippeastrum hybridum hort

Soaking of bulbs in 3 concentrations of indoleacetic acid (IAA), gibberellic acid (GA₃), 2-chloroethyltrimethyl ammonium chloride (cycocel) or 2-chloroethylphosphonic acid (ethrel) showed various responses on growth and flowering. IAA increased the weight and number of bulblets, GA₃ increased bulb weight. Cycocel (1000 mg l^?1) increased the number of flowers, while GA₃ increased the diameter of the flowers.Application of IAA at 100 mg l^?1 and GA₃ at 10, 100 or 1000 mg l^?1 twice as foliar spray at an interval of 30 days promoted the number of bulblets on the treated plants, while high concentrations of cycocel and ethrel (1000 mg l^?1) increased the weight of bulblets. All concentrations of IAA, GA₃ and 1000 mg l^?1 cycocel increased the number and size of the flowers.     

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.     

Regulation of postharvest flower senescence in Zinnia elegans Jacq.

Postharvest decorative life of Zinnia elegans flowers was prolonged by holding-solutions containing 8-hydroxyquinoline citrate (8-HQC) and sucrose. Flowers lasted longest in a solution of 200 mg l^?1 8-HQC and 1% sucrose. Flowers held in 2 or 3% sucrose and 200 mg l^?1 8-HQC developed necrotic lesions on ray florets and foliage. The decorative life of flowers held in 0.25 or 0.5% sucrose and 200 mg l^?1 8-HQC was extended beyond those in de-ionized water, but this extension was less than, or equal to, the postharvest life of those in 1% sucrose and 8-HQC (200 mg l^?1), depending on the cultivar. Postharvest life of flowers produced in May — June under natural photoperiod was significantly longer than that of flowers produced during February to April under a 14-h day provided by incandescent light.     

Propagating palms in vitro with special emphasis on the date palm (Phoenix dactylifera L.)

Tissue-culture methods are described for the vegetative propagation of several palm species either through shoot tip culture or plantlet differentiation via embryogenic callus. The influence of explant size, medium composition and physical environment required for the establishment of palm shoot tips in vitro was determined. Date palm (Phoenix dactylifera L.) seedling shoot tips of various sizes were cultured in either liquid or agar modified Murashige and Skoog (MS) medium containing 0.0–1.0 mg 1^?1 α-naphthaleneacetic acid (NAA) and 0.0–15.0 mg 1^?1 benzyladenine or N⁶-(Δ²-isopentenyl) adenine (2iP) in order to enhance shoot growth and induce axillary budding. Satisfactory date palm shoot tip growth and proliferation was obtained from explants that were 3 mm in length, consisting of the apical meristem region and 2–5 adjacent leaf primordia. Optimum shoot tip development and axillary budding was obtained by initially establishing explants on an agar medium for 2 weeks, then transferring to a liquid medium. Shoot tips from several palm species were cultured on MS media containing 100 mg 1^?1 2,4-dichlorophenoxyacetic acid (2,4-D), 3 mg 1^?1 2iP and 3 g 1^?1 activated charcoal, or on MS medium containing 1 mg 1^?1 NAA and charcoal, to determine their morphogenetic responses in vitro. Shoot tips of Metroxylon sp., Phoenix canariensis Hort. ex. Chabaud., P. dactylifera ‘Khalasa’, ‘Thoory’ and ‘Zahidi’, and P. roebelenii O'Brien planted on medium with 2,4-D and 2iP initiated callus, asexual embryos and free-living plantlets after 4–8 months in culture. Shoot tips from Erythea edulis S. Wats., P. canariensis, P. dactylifera ‘Khalasa’, Thoory' and ‘Zahidi’, Washingtonia filifera Wendl. and W. robusta Wendl. cultured on medium containing NAA developed into plantlets with well-developed leaves and adventitious roots within 2–6 months from the time of planting. In some cases, cultured date palm shoot tips gave rise to axillary buds.     

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.     

Factors affecting the shoot multiplication of Persian walnut (Juglans regia L.)

A number of experiments were conducted to identify suitable procedure for in vitro shoot multiplication of Persian walnut (Juglans regia L.). Three different nutrient media (DKW, MS and WPM) and three different gelling agents (Phytagel, Difco Bacto agar and a mixture of Phytagel and Difco Bacto agar) were studied in the first experiment. Driver and Kuniyuki walnut (DKW) medium solidified with 2.2 g l⁻¹ Phytagel was found optimum. Performance of explants was better on DKW medium than on MS and WPM. The DKW and MS media were not significantly different from each other, but both of them were significantly better than WPM, which was a very poor medium for this species. Phytagel alone was significantly better than Difco Bacto agar or Phytagel combined with Difco Bacto agar. In another experiment different concentrations of BA were studied. Medium containing 1.0 mg l⁻¹ BA and 0.01 mg l⁻¹ IBA was the best, although medium containing 0.6 and 0.8 mg l⁻¹ BA were also successful, and utilisation of 0.4 mg l⁻¹ BA and 0.01 mg l⁻¹ IBA was optimum for shoot elongation. Application of different kinds of auxins (IAA, IBA and NAA at 0.01 or 0.1 mg l⁻¹) with 1.0 mg l⁻¹ BA were also studied. Media containing IBA were significantly better than media containing IAA for shoot fresh weight, but neither of them was significantly different from media containing NAA. Application of 0.01 mg l⁻¹ or 0.1 mg l⁻¹ auxin, with 1.0 mg l⁻¹ BA, was not significantly different for shoot multiplication of Persian walnut. The morphology of shoots on media containing 0.01 mg l⁻¹ IBA was the best.