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.     

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.     

Regeneration of Plants from Alginate-encapsulated Shoot Tips of Myrtle (Myrtus communis L.)

Myrtle growing naturally in the Mediterranean Region in Turkey, is among the economically important plant species. The present study emphasized on in vitro conservation of M. communis by encapsulating regenerated shoot tips. In this research we reported synthetic seed production and subsequent conversion of encapsulated shoot tips into plantlets comparing with nonencapsulated shoot tips for myrtle. Two different myrtle genotypes were used for synthetic seed production. Sodium alginate solution at the rate of 3.0% and 100?mM calcium chloride solution were prepared for encapsulation. Encapsulation was accomplished by mixing shoot tips into sodium alginate solution and dropping these in calcium chloride solution for 25–30?min. Encapsulated and nonencapsulated shoot tips were cultured in MS (Murashige and Skoog) media supplemented with different BAP (6-Benzylaminopurine) concentrations (0, 0.5, 1, 2?mg L^?1). After six weeks, all shoots were transferred to MS media containing 1?mg L^?1 IBA for in vitro rooting. As a result, the highest germination rate was obtained on the BAP-free MS media. The best BAP concentrations were detected as 0.5 and 1?mg L^?1 for micropropagation. Genetic stability of plants coming from encapsulated and nonencapsulated shoot tips was tested by ISSR markers. Based on the results, there were no genetic differences among the samples.

     

Possible role of non-structural carbohydrates in flower induction in strawberry

Summary

Flower-induction is the event that initiates the transition of a vegetative apex into a floral apex in response to an environmental or developmental cue. Under flower-inducing conditions, biochemical or physiological changes can be recognised. One possible change that could occur is in sugar content. In this study, levels of non-structural carbohydrates (e.g., sucrose, glucose, fructose and starch) were measured in shoot tips, leaves and roots of strawberry (Fragaria ananassa cv. ‘Kordestan’) under flower bud-inducing conditions, and compared with non-induced plants. Runner plants were potted and grown for 4 weeks under non-inducing conditions (31º/25°C day/night; 16 h daylength). Half of the plants were then put under flower-inducing conditions (25°/15°C day/night; 8 h daylength) for 3 weeks. Samples for carbohydrate analysis were taken from induced and non-induced plants every 3 d over 3 weeks, and sucrose, glucose and fructose contents were determined by HPLC, and starch concentrations by the anthrone method. The most abundant soluble sugar, in all organs tested, was sucrose. Sucrose levels in shoot tips and leaves decreased at the beginning of the induction treatment, but soon increased to the levels recorded in non-induced plants. Fructose increased markedly in shoot tips of induced plants 3 d after the start of the short-day treatment, and declined thereafter. Starch contents in shoot tips, leaves and roots of non-induced strawberry plants were higher than those in induced plants on most sampling dates. From the results of this study, it appears that soluble carbohydrate contents in different organs of June-bearing strawberry may have a decisive role on flower-bud induction.     

In vitro corm production in Gloriosa superba L., an Ayurvedic medicinal plant

Summary

In vitro production of corms from Gloriosa superba L. using three kinds of explants: dormant, non-dormant corm buds and 30 d old in vitro derived multiple shoots is reported. Excellent responses was obtained in terms of corm production using MS medium supplemented with B₅ vitamins, 6% sucrose, 2iP, ADS and ANL for corm formation from dormant corm buds, using Kin, ADS and ANL for corm formation from non-dormant corm buds and using BAP, ADS and ANL for callus derived multiple shoot corm formation. Well developed corms were obtained from the tissue cultured plants with no dormancy breaking requirement.     

The impact of within-row spacing on the productivity of glasshouse roses grown in two planting systems

Summary

Stem yield and quality of roses for cut flower production were evaluated. The plants were grown in two planting systems as an alternative to the traditional ``vase-shaped'' system. In the trellised system, two cultivars of Rosa hybrida (cvs Gabrielle and Kardinal) were planted in a commercial glasshouse in 3.m sections of bed. Within-row spacing was varied to give 6–16 plants m<sup>–2</sup>. After a three-month establishment phase the basal shoots were bent to an angle of 308 above horizontal and restrained with a trellis wire. Flowering shoots sprouting from axillary buds along a basal shoot were harvested at their lowest node, minimizing branching. Compared with ``vase-shaped'' rose plants at the same density, trellised roses produced 24% more basal shoots, 46% more flowering shoots (cv. Gabrielle) and approximately 46% less blind shoots per plant over five months. Phenotypic variation was greater in cv. Gabrielle than in cv. Kardinal in response to within-row spacing, as indicated by the number of basal shoots formed. Within-row spacing, over the range explored, did not affect the number of flowering shoots per basal shoot. Trellising rose plants increased stem yield and quality, but production over the long-term requires further investigation. The single shoot planting system contained a mixed population of single-stemmed rose plants of Rosa hybrida (cvs Gabrielle and Gerdo). It was grown in a field over a range of within-row spacings to give 20–105 plants m^–2. Over three harvests, increasing the number of plants by 10 plants m^–2 reduced the proportion of flowering shoots by 4.4%. Expressed on a unit area basis, a five-fold increase in plants m^–2 produced a three-fold increase in stem production.     

Biological control of the shoot disease of Amaranthus hybridus caused by Choanephora cucurbitarum with Bacillus subtilis

Summary

Isolates of Bacillus subtilis from soil and ogili (a local food condiment) controlled choanephora shoot disease of the vegetable crop plant, Amaranthus hybridus, in the greenhouse. Disease developed on plants inoculated simultaneously with the pathogen Choanephora cucurbitarum and the ‘ogili’ isolate, but not the soil isolate, of B. subtilis. Application of either bacterial strain to plants one day before a challenge with the pathogen prevented disease development. A single application of either strain prevented disease development from a subsequent challenge with the pathogen at any time over a thirty day period. Viable counts of the microflora on the shoot tips of the treated plants indicated strongly that the inoculated bacteria multiplied and colonized the extending shoot tip whether or not the shoots were covered with polythene bags. Both strains of Bacillus subtilis inhibited mycelial extension growth as well as conidial germination in vitro.     

In vitro regeneration and Agrobacterium mediated transformation in gladiolus

Summary

In vitro regeneration and transformation studies were conducted on two cultivars of gladiolus. Cormels of 1.0 to 1.5 cm diameter cut into 2–3 mm thick slices of top, middle and bottom, and in vitro derived bisected shoot tips were used as explants on MS medium supplemented with 18.6 μM kinetin for multiple shoot induction. Amongst the cormel slices, the top slice gave better shoot induction response of 89% with an average of 2.4 shoots per explant over both cultivars. In vitro derived bisected shoot tips were inoculated on the medium oriented cut-side up, cut-side down and vertically both with and without the cormel base attached. Bisected shoot tips without attached cormel base and inoculated in the cut-side down orientation showed an average of 90% shooting response. In vitro derived shoot tips were used as explants for transformation. Explants were wounded by scalpel and particle bombardment with 1.6 μm naked gold particles by the biolistic delivery system. The wounded explants, after 3 d of recovery period, were co-cultivated with Agrobacterium strain LBA4404 harbouring the binary vectors pBI141 and pTOK233 which contained gus reporter gene with rice actin and 35S promoters respectively. GUS expression frequencies of 5.3% and 23% was obtained from scalpel and particle bombardment wounded explants, respectively. Particle wounded explants showed an average of 63 and 103 GUS spots when co-cultivated with pBI141 and pTOK233 binary vectors respectively. Explants co-cultivated with pBI141, after three weeks of selection on antibiotic containing medium showed blue streaks of GUS expression. It was concluded that Agrobacterium could infect the monocot gladiolus and transform the tissue eficiently when tissues were prewounded with naked gold particles delivered by particle gun.     

In vitro propagation of Blandfordia grandiflora (Liliaceae)

Multiple shoots were obtained when single shoot tips of Blandfordia grandiflora were exposed to different concentrations of kinetin, N6~ benzylaminopurine (BAP) and N6- isopentenylaminopurine (2iP). The best multiplication results were obtained when explants were subjected to kinetin at 8 (iM BAP at 0.5,2 and 8 (iM and 2iP at 2,8,32 and 128 |iM. Preliminary rooting trials were performed with three different auxins: indo- lebutyric acid (IB A), naphthalene acetic acid (NAA) and indole acetic acid (I A A). IB A at a concentration of 8 |iM and IAA at a concentration of 32 |xM gave highest root number, but when transplanted to the glasshouse, the best surviving plantlets were those treated with 2 |xM of IBA or 0.5 μM of IAA.     

Comparative salt responses at cell and whole-plant levels of cultivated and wild tomato species and their hybrid

SUMMARY

Response to increased salinity was compared in whole plants and calli from leaf, stem and root of Lycopersicon esculentum Mill. cv. P-73, Lycopersicon pennellii (Correll) D'Arcy ac PE-47 and their interspecific hybrid. Three NaCl treatments were applied (0, 70 and 140 raM) for 28 d. In both calli and whole plants, L. pennellii was more salt-tolerant than the cultivated species and the hybrid according to the growth responses, although different degrees of salt tolerance were generally found between plants and calli. The Na⁺ and CI" accumulations with salinity were higher in L. pennellii than in L. esculentum at both levels oforganization. The interspecific hybrid showed an accumulation ability for Na⁺ and CI^" intermediate to its parents in the shoot of the whole plants and similar to L. pennellii in the callus tissues. Either no decrease or a small decrease of K⁺ concentrations with salinity were found in both whole plants and callus tissues of L. esculentum. However, K⁺ concentrations decreased in the organs and calli of L. pennellii and the hybrid with increasing salinity. Only at the whole plant level did L. pennellii have a Na7K⁺ ratio higher than L. esculentum, showing the hybrid to have values between those of its parents in the shoot.     

Auxin-dependent development and habituation of highbush blueberry (Vaccinium × covilleanum But. et Pl.) ‘Herbert’ in vitro shoot cultures

The influence of IAA (1.0 mg dm⁻³), and IBA (1.16 mg dm⁻³), on the development of highbush blueberry (Vaccinium × covilleanum But. et Pl.) ‘Herbert’ in vitro shoot cultures was examined. Depending on the kind of auxin and 2iP concentration in vitro cultures consisted of various number of axillary (AX) and adventitious (AD) shoots. Three different categories of AD shoots were found: leaf shoots (AD-L), node-adjoin shoots (AD-P), and base-adjoin shoots (AD-M, madshoots). The AX shoots were the least habituated (towards auxins, cytokinins and vitamins) whereas the AD-M shoots (madshoots) the most. In comparison to IAA, IBA caused dying or callusing higher number of initial explants. However, IBA generally suppressed development of AD shoots, especially madshoots whereas slightly weakened multiplication of AX shoots. IBA significantly enhanced elongation of AX shoots also. Axillary shoots obtained on IBA-media had relative long internodes and rigid, well-developed leaves. The adventitious shoots, especially base-adjoin (AD-M) ones, were easily distinguishable as were more thin and fragile, more or less vitrified, and had short internodes and smaller, sometimes unfolded leaves. Development of blueberry in vitro cultures on auxin-free and IAA-supplemented media was similar. AX shoots grown on such media resembled AD shoots. 2iP applied in higher doses along with IAA promoted much proliferation of AD than AX shoots. In contrast, 2iP applied in higher doses together with IBA stimulated significantly only growth of AX shoots whereas in general, development of adventitious shoots was not affected. Micropropagation carried out through routine method based on subculturing of shoot explants or shoot clumps on the medium supplemented with IAA (4 mg dm⁻³) and 2iP (10–15 mg dm⁻³) as well as stimulation of shoot elongation on the blank medium causes in fact the propagation of highbush blueberry through highly habituated adventitious madshoots. Replacement of IAA by IBA facilitates micropropagation of highbush blueberry cv. Herbert through axillary shoots.     

The response of chilli (Capsicum annuum L.) plants to early inoculation with mycorrhizal fungi at different levels of phosphorus

Summary

Chilli plants cv. Pusa Jwala were inoculated in the nursery beds with three mycorrhizal fungi, Gigaspora calospora, G. margarita and Glomus fasciculatum and transplanted to plots supplied with 0, 25 and 50% of a recommended P level (65 kg ha^?1). Uninoculated plants were also transplanted at 0 (Control) and 100% of.the recommended P level. Plant growth, height, shoot dry weight, uptake of P, root colonization and yield of green chillies were measured. Chilli responded well to inoculation and P. G. margarita best improved growth and yield. Inoculation plus 25 and 50% P were equally better than the control and equal to uninoculated plants at 100% P. As much as 50–75% P can be dispensed with by inoculation with mycorrhizal fungi and this represents a considerable economic saving in cost.     

High multiplication frequency and genetic stability for commercialization of the three varieties of micropropagated tea plants (Camellia spp.)

RAPD and microsatellite markers were used to determine the genetic fidelity of micropropagated plants from the three varieties of tea plant derived from explants of field grown mother bush as well as in vitro germinated seedlings. Rate of shoot multiplication was better from nodal explants than from shoot tips. A maximum of 32–33 shoots was observed in cotyledonary node in 1/2 MS medium with BAP (6 mg/l), GA₃ (1 mg/l) and IBA (0.5 mg/l). 90% of the in vitro derived microshoots were micrografted into rootstocks. The micropropagated plantlets showed both cytological and genetical stability. SSR primers showed complete stability among the regenerants. The results convinced that plants derived from axillary as well as adventitious mode of propagation can be genetically true to type. This cost effective technique would help in fast clonal propagation at a commercial scale.     

Cryopreservation of in vitro almond shoot tips by vitrification

Summary

Shoot tips of two almond scion cultivars, ‘Ne Plus Ultra’ and ‘Nonpareil 15-1’, and one almond/peach hybrid rootstock were successfully cryopreserved using a one-step vitrification technique. Three week old in vitro cultures were cold-hardened at 4°C on the multiplication medium (Murashige and Skoog for ‘Ne Plus Ultra’ and the hybrid rootstock; Almehdi and Parfitt for ‘Nonpareil 15-1’) for three weeks. Shoot tips, 2–2.5 mm long, were excised and precultured for 1 d at 4°C on the same basal medium, without plant growth regulators, supplemented with 0.7 M sucrose. After the preculture, the shoot tips were incubated in vitrification solution at 25°C for 45 min for the almond scion cultivars and 60 min for the hybrid rootstock, and then stored under liquid nitrogen (LN) for at least 3 d. After rapid thawing at 30°C, the shoot tips were washed with the appropriate liquid basal medium containing 1.0 M sucrose and then cultured on the same basal medium, solidified with agar, but excluding NH₄NO₃ or (NH₄)₂SO₄. Shoot regeneration was usually observed within 2–3 weeks. Survival after LN, recorded as the percentage of shoot tips that produced at least one new shoot four weeks after thawing, was 87.5, 60.0 and 72.5% for ‘Ne Plus Ultra’, ‘Nonpareil 15–1’ and the hybrid rootstock respectively. The one-step vitrification method is a promising simple technique for cryopreserving almond scion and rootstock shoot tips from in vitro cultures.     

Determination of essential oil contents and micropropagation of Gaultheria fragrantissima,an endangered woody aromatic plant of India

Summary

Leaves of mature of Gaultheria fragrantissima Wall. plants (Indian wintergreen) were collected from various locations in the Eastern Himalayan region on the Indo-China border and were analysed by steam distillation and gas chromatography to identify an elite line that contained 1.79% (v/v) essential oils, 98% of which was methyl salicylate. Subsequently, a highly reproducible micropropagation protocol using adult shoot tips from this elite genotype was developed in order to conserve this highly-valued, endangered, woody oil-bearing aromatic shrub in India. Among several plant growth regulator (PGR) combinations tested for in vitro multiplication, up to 35 shoots per explant could be induced within 14 weeks of culture on woody plant medium (WPM) fortified only with 0.22 mg l^–1 thidiazuron (TDZ). These shoots could elongate on WPM containing 1.0 mg l^–1 kinetin. Rooted plantlets were acclimatised ex vivo, with 70% success. Random amplified polymorphic DNA (RAPD) analysis indicated the genetic uniformity of both the micropropagated plantlets and the donor plants. This is the first report on in vitro micropropagation of G. fragrantissima.     

An efficient adventitious shoot regeneration system on excised leaves of micropropagated lingonberry (Vaccinium vitis-idaea L.)

Summary

An efficient system to regenerate shoots in vitro on excised leaves of lingonberry (Vaccinium vitis-idaea L.) was developed. Leaf explants from shoot-proliferating cultures produced multiple shoots without an intermediary callus phase on zeatin (ZN)-containing shoot induction media within 3–4 weeks of culture initiation. Cultivars Regal and Splendor, and one clone from a natural stand in Estonia (ECL1), were used in the first experiment. Young expanding leaves with the adaxial side touching the culture medium, and maintained for 7 d in darkness, produced the best results. There were significant genotypic differences in adventitious shoot formation. A second experiment studied the effects of ten concentrations of three cytokinins: ZN at 5, 10, 20, 30 and 40 μM; 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea (thidiazuron, TDZ) at 0.1, 1, 5 and 10 μm; and 6-(γ-γ-dimethylallylamino) purine (2ip) at 25 μM were compared with leaf segments of different polarity in ‘ECL1’. Zeatin was found to be more effective than TDZ or 2iP as an inductive signal for regenerating many vigorous shoots. Zeatin induced multiple shoot formation at all concentrations tested, but maximum morphogenic response was observed at 20 to 30 μM. The media containing TDZ generally promoted more callus formation and suppresed shoot elongation. In a third experiment with the lingonberry cultivar Erntedank and the clone ‘ECL1’, a new medium developed for lingonberry shoot culture proved more effective than the modified Murashige and Skoog medium for regenerating shoots on leaf explants. Elongated shoots were excised and rooted directly on a 2 peat:1 perlite (v/v) medium after dipping in 0.8% indole-3-butyric acid. Rooted plantlets were acclimatized under greenhouse conditions to evaluate somaclonal variation.     

An assessment of the in-vitro multiplication rates of fourteen black currant cultivars

Summary

The in-vitro multiplication rates of fourteen black currant (Ribes nigrum) cultivars from a range of geographical origins were compared. Shoot tips and axillary buds were cultured for 21 days in a medium supplemented with 2.2 mg l^?1 6-benzylaminopurine. The highest shoot multiplication rate achieved was by cv. Ben Lomond, where the mean rate was 3.53 shoots from a single initial expiant. Mean rates between 1.50 and 3.33 were obtained from other cultivars. The results are discussed in relation to the parentage of each cultivar.     

The culture of shoot tips of hop (Humulus lupulus L.) to eliminate viruses

The culture of hop shoot tips has proved highly effective in eliminating Prunus necrotic ringspot and hop mosaic virus from eleven varieties of hop. The culture technique was successful even when plants were raised from tips as long as 5 mm.     

Nitrogen-enriched ground kenaf (Hibiscus cannabinus L.) stem core as a component of soilless growth media

Summary

Four experiments showed that stem core (xylem of kenaf (Hibiscus cannabinus L.) in combination with sphagnum peat moss and fertilizer nutrients was a satisfactory growth medium for plants of ‘Toy Boy’ tomato (Lycopersicon esculentum Mill.). Greater shoot growth was achieved in media containing 20 to 35% kenaf by volume than 50%, and with fine kenaf (2–4 mm diameter) than coarser grades. In the absence of weekly solution fertilization, N-enrichment of the kenaf was necessary to support greater shoot growth than occurred in commercial growth media. Soaking the kenaf in solutions of increasing nitrogen (N) concentration (0 to 15,000 mg N l^?1) increased shoot growth, but urea ammonium nitrate (UAN, 30N–0P–OK) generally resulted in greater shoot growth than 20N–4.4P–16.6K at the same N concentration. The soaking time for kenaf in UAN was considerably less than in the complete fertilizer to produce similar shoot dry weights. Only a small portion of the kenaf in the growth media required pre-plant N enrichment provided the N concentration of the soak solution was increased in ‘ proportion to the volume reduction. Increasing the N concentration of weekly solution fertilization (20N–4.4P–16.6K) from 0 to 500 mg N l^?1 increased shoot growth irrespective of the N concentration of the kenaf soak solution. In media receiving 0 or 100 mg N l^?1 weekly solution fertilization, shoot growth increased with increasing N concentration of the kenaf soak solution.