Micropropagation of Dendrobium draconis Rchb. f. from thin cross-section culture

An efficient procedure is outlined for rapid and mass in vitro propagation of an orchid, Dendrobium draconis Rchb. f. through in vitro culture of thin cross-sections (TCSs) derived from young stems. The TCS explants were excised along the stem from the base to shoot tip of 6-month-old plantlets and cultured on Murashige and Skoog (MS) medium supplemented with 20 g/l sucrose and different concentrations of N⁶-benzyladenine (BA), kinetin (Kn) and 1-naphthaleneacetic acid (NAA), either individually or in combination. Protocorm-like bodies (PLBs) were directly induced from the TCS explants and completely developed into shoots within 6–7 weeks. The optimal growth regulators combination for maximal PLB development was 2 mg/l BA and 1.0 mg/l NAA, giving rise to 68% of responding explants with an average 11 PLBs per explant. Shoot development was best achieved on MS medium containing sucrose and coconut water. Plantlets, 6–8 cm height were transplanted into coconut husk peat with 92% survival rate in a nursery.     

Effect of explants source on shoot proliferation and adventitious regeneration in 10 Buddleia cultivars

Viable shoot cultures and weaned plants were obtained from cultured apical meristems with 10 Buddleia cultivars giving viabilities of 32–72%. The number of shoots produced, the micropropagation rate and the root number produced in vitro was higher in meristem derived shoots compared to those derived from shoot-tips. The subsequent growth rate of meristem derived plants, in the greenhouse, was also greater. The number of roots produced by conventional cuttings collected from meristem derived plants was significantly higher than in cuttings which were collected from plants derived from shoot-tips or from the original stock plants.Endogenous bacteria were not detected in either shoot cultures derived from meristems or in 10-week-old weaned plants derived from meristems whereas those derived from shoot-tips showed the presence of endogenous bacteria when sterilized explants were cultured on nutrient agar or on tryptic soy broth.Factors affecting adventitious bud and shoot production in leaf and internode explants was determined for ‘Lochinch’, ‘Border Beauty’, ‘Ile de France’ and ‘Pink Delight’ using meristem derived shoot cultures. Adventitious shoots appeared after 4 weeks of culture, in both types of explant when cultured on MS supplemented with 0.5–5.0 μM TDZ. The highest percentage regeneration was achieved from bisected internode explants cultured on 0.5 μM TDZ, with 93–100% regeneration among the cultivars whereas BA was less effective. The best response was obtained using 5.0 μM TDZ which gave over 10–11 shoots per explant in all bisected explants for all cultivars.     

Direct somatic embryogenesis and plant regeneration from leaf cultures of ornamental species of Dianthus

Protocol for direct somatic embryogenesis from leaf explants of economically important species of Dianthus, viz. D. caryophyllus, D. barbatus and D. chinensis has been developed. Murashige and Skoog’s (MS) liquid medium supplemented with 2,4-D (1 mg/l) was used for direct induction of somatic embryogenesis without an intervening callus phase. Initially globular structures were observed after 21 days of culture of leaf explants in liquid medium. Development of embryos to heart and torpedo stages was achieved in the liquid medium incorporated with polyethylene glycol (PEG 6000) at a concentration of 2.5%. Embryo maturation was further promoted by addition of casein hydrolysate (CH) (200 mg/l) in MS liquid medium. Embryos germinated to form plantlets on solid MS medium supplemented with GA₃ (1 mg/l). Regenerated plants with well-developed root and shoot systems were successfully transferred to field conditions.     

Micropropagation of Prunus scoparia,a Suitable Rootstock for Almond under Drought Conditions

Prunus scoparia is a wild deciduous shrub, usually living on dry calcareous soils of the rocky mountains and has been used as a grafting rootstock for domesticated almonds to provide drought resistance. In the current study, micropropagation ability of P. scoparia was investigated using cytokinin and auxin. Uniform nodal shoot pieces (3–5 cm in length) of seedlings were used as explants. The explants were disinfected with 10% sodium hypochlorite solution. For adventitious shoot induction and proliferation, Murashige and Skoog (MS) media containing 7.00 g/l agar and 30.00 g/l sucrose containing five concentrations of benzyl adenine (BA) (0.00, 0.50, 1.00, 2.00, and 4.00 mg/1) and also containing six concentrations of Thidiazuron (TDZ) (0.00, 0.50, 1.00, 2.00, 5.00, and 7.00 mg/1) were compared. For rooting, in vitro shoots (2–3 cm) were transferred into ½ MS medium supplemented with 30 g/l sucrose, 7.50 g/l agar, and different concentrations of IBA (0.00, 0.25, 0.50, and 1.00 mg/l) and NAA (0.00, 0.25, 0.50, and 1.00 mg/l). Based on the results obtained for shoot proliferation, only 2.00 and 4.00 mg/l BA and 2.00 mg/l TDZ concentrations generated shoots, while other treatments did not show shoot proliferation. Among the three treatments that generated shoots, the best results for shoot number, leaf number, and leaf color quality were observed in media containing 2.00 mg/l TDZ. Based on the results obtained for rooting, the effect of IBA concentrations on the rooting percentage, root number, and root length was significant. Among IBA concentrations, only 0.50 mg/l IBA induced rooting, while there was no rooting in the media containing other IBA concentrations. None of the NAA concentrations showed rooting. In conclusion, MS culture medium supplemented with 2.00 mg/l TDZ and ½ MS culture medium supplemented with 0.50 mg/l IBA are suggested for in vitro shoot proliferation and rooting of P. scoparia, respectively. The results presented herein could be used for in vitro selection and micropropagation of P. scoparia.     

In vitro propagation of Lychnis senno Siebold et Zucc., a rare plant with potential ornamental value

Lychnis senno is a rare and valued ornamental plant. Seed propagation is not efficient because of the low germination rate. To grow commercially L. senno in China, a protocol for in vitro germination and propagation of this species was developed. Various germination rates were obtained by treating seeds with GA₃ during 1–6 months storage period. The highest germination rate reached 19.4% when seeds were treated with 250 mg/l GA₃ and stored for 5 months at 4 °C. Axillary shoot proliferation was induced in the nodal segments of the seedlings on medium containing specific concentrations of BA and NAA [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant 15, 473–497]. Maximum number of shoots was developed on a medium supplemented with 5 mg/l BA and 0.5 mg/l NAA, while the higher shoots were observed on a medium supplemented with 0.5 mg/l BA and 0.05 mg/l NAA. Rooting was induced in 91.7% of the regenerated explants on a half-strength MS medium supplemented with 0.5 mg/l NAA. The plantlets grew well and flowered after transfer to the greenhouse. The chromosome numbers of seedlings and propagated plants were also determined to be 2n = 2x = 24.     

In vitro micropropagation of the ornamental prickly pear cactus Opuntia lanigera Salm–Dyck and effects of sprayed GA3 after transplantation to ex vitro conditions

We established the conditions to micropropagate the ornamental prickly pear cactus Opuntia lanigera Salm–Dyck through axillary shoot development from isolated areoles. For the shoot proliferation stage different explant orientation (vertical and horizontal), type of cytokinin (BA, DAP and K), and concentrations (0, 1.25, 2,5, 5.0 and 7.5 mg/L) were evaluated. Media [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco cultures. Phys. Plant. 15, 473–497: 50 and 100%], and carbohydrate concentration (0.25, 0.5, 0.75 and 1.0%) were studied to optimize individual shoot growth and elongation. Following micropropagation and plantlet acclimatization, the effects of GA₃ on plant growth were determined by spraying a series of increasing concentrations (0, 150, 300 and 450 ppm). A reliable and efficient protocol of micropropagation was established for this particular plant species. The greatest propagation ratio (shoot proliferation) was obtained when explants were cultured in vertical orientation (4.975 shoots per explant) as compared to horizontal position (3.692 shoots per explant). The addition of BA to the media resulted in increased shoot number per explant (8) in comparison to K and DA, which produced only 2 shoots in average. However, after 42 days of culture, significantly higher shoot length was obtained with DAP (14 mm) compared to K and BA (4 mm). After the shoot proliferation stage, an elongation subculture was performed prior rooting in which shoot growth was enhanced when crowns of shoots were cultured in 50% of basal salt formulation of Murashige and Skoog (1962) and low sucrose concentration (2.5 and 5%). Exogenous application of GA₃ after plantlet acclimatization on glasshouse conditions increased spine-hair (developed from areoles in young plants) length as part of short-term effects. However, significantly higher values were obtained in plantlets treated with 300 ppm of GA₃ when compared with the rest of the treatments. At the end of the study, the most important long-term effect produced by GA₃ was the suppression of total shoot growth. The micropropagation protocol described here and the conditions to grow the plants through fertigation plus the application of GA₃ that induced changes in the phenotype may be used in commercial exploitations to regenerate 12,500 plantelts in average after 12 months of culture and produce healthy plants with better ornamental characteristics and higher commercial value.     

Optimizing shoot regeneration and transient expression factors for Agrobacterium tumefaciens transformation of sour cherry (Prunus cerasus L.) cultivar Montmorency

An efficient, adventitious shoot regeneration protocol was devised, and transient expression studies were carried out to enable Agrobacterium-mediated stable transformation of sour cherry (Prunus cerasus L.) cultivar Montmorency. Leaves, from in vitro stock cultures, with the petiole removed and four partial cuts made transversely and equidistant through the midrib area were found to be the optimum explant type. A 24 h liquid TDZ-pretreatment (0.05, 0.10 or 0.25 mg/l) in MS medium [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Plant Physiol. 15, 473–497.] of leaf explants stimulated shoot formation upon subsequent culture on QL medium [Quoirin, M., Lepoivre, P., 1977. Improved media for in vitro culture of Prunus sp. Acta Hort. 78, 437–442.] supplemented with 3.0 mg/l BAP and 0.5 mg/l NAA. A frequency of 38.9–54.4% of the explants produced at least one shoot with the maximum mean number of shoots, 4.5 per explant with the 0.10 mg/l TDZ pretreatment. The shoot regeneration scheme was subsequently linked with inoculation with Agrobacterium tumefaciens strains EHA105, GV3101 or LBA4404, each harboring the binary plasmid pBISN1. PBISN1 contains an intron interrupted ß-glucuronidase (GUS) gene (gusA) under control of the chimeric super promoter (Aocs)₃AmasPmas. Blue stained leaf cells were observed after co-cultivation with all three strains. Co-cultivation for 4 days with 19.6 mg/l acetosyringone (AS) and assay by GUS indicated over 90% of the leaf explants were infected with an average 7.5–8.8 blue foci per explant. No differences were observed in regard to A. tumefaciens strain used.     

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.     

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.     

De novo differentiation of shoot buds from leaf-callus of Dianthus caryophyllus L. and control of hyperhydricity

A method is described for producing de novo shoots from leaf derived callus of carnation (Dianthus caryophyllus L.). Plants were regenerated in four steps, viz. callus induction, shoot regeneration, removal of hyperhydricity from regenerated shoots and root development. Callus induction medium contained 2,4-D and BAP. Shoot buds were formed when the callus was further subcultured on 2,4-D- and BAP-containing medium, or MS medium without any growth regulators. The shoots so formed were hyperhydric, bushy in appearance with reduced stem length and watery leaves. The normal conformation of shoots was restored by culturing the hyperhydric shoots onto medium supplemented with GA₃ and bactopeptone. The recovered shoots were rooted on MS medium added with NAA (1 mg/l) or IBA (2 mg/l). Regenerated plants with well-developed root and shoot systems were successfully transferred to field conditions after initial acclimation.     

In vitro propagation using adventitious buds technique as a source of new variability in chrysanthemum

Eleven cultivars of Chrysanthemum × grandiflorum (Ramat.) Kitam.: ‘Richmond’ and its 10 radiomutants, representing the Lady group, were propagated in vitro with shoot tips and leaves as explants. The aim of this study was to investigate if the explant type used for micropropagation affects the genotype and phenotype of chrysanthemums. Plants grown from shoot tips and adventitious buds formed on leaves were rooted in vitro, acclimatized and cultivated in glasshouse up to full-flowering. The colour and shape of inflorescences of plants obtained from two different explant types were compared within the cultivars. All plants derived from shoot-tip explants showed the inflorescence colour and shape typical for the cultivars. Inflorescence colour of plants derived from adventitious buds were true-to-type in four cultivars: ‘Richmond’, ‘Lady Amber’, ‘Lady White’ and ‘Lady Yellow’. All plants of ‘Lady Apricot’ (originally: golden beet) and ‘Lady Salmon’ (salmon) propagated from adventitious buds technique showed altered inflorescence colour (respectively: purple gold; pink and white). ‘Lady Bronze’ (originally: reddish brown), ‘Lady Orange’ (orange brown) and ‘Lady Rosy’ (purple gold) propagated with adventitious buds had both typical and changed inflorescence colours (respectively: yellow; yellow and red; reddish pink). ‘Lady Vitroflora’ showed altered number of ligulate florets grown into tubes in inflorescence when propagated with shoot tips and leaves as explants. Those changes might be an effect of either chimeral structure or somaclonal variation of the plants investigated. The variation appears only if non-meristematical explants were used. The adventitious buds technique might be useful in chrysanthemum breeding as a source of a new variability.     

Changes in the competence of Quercus robur ‘Fastigiata’ to grow in vitro as affected by seedling rootstocks and differential pruning

Three physiologically distinct forms of the same genotype of Quercus robur ‘Fastigiata’, all derived from adult budwood, were identified following the growth of severely-pruned hedge plants grafted onto juvenile seedling rootstocks. Only explants from the form displaying the juvenile-like characteristic of over-winter leaf retention were capable of establishing sustainable cultures in vitro, although growth was not as rapid as those from a two year old seedling source. Pruning to release correlative inhibition of buds low on the scion framework produced vigorous shoot growth, but explants from these failed to show sustained growth in vitro. Withholding light from stockplants reduced excessive phenolic oxidation in explants taken from flushing unripe shoots, however, despite a beneficial effect on culture initiation, later growth was dependent upon the juvenile- or adult-like physiology of the original explant. A new medium was devised for this subject, which in addition to promoting growth generally, also stimulated the flushing of shoot apices. The relevance to in vitro culture of expression of juvenile habit in the stockplant is discussed.     

Vegetative and yield component characteristics of micropropagated muscadine grape (Vitis rotundifolia Michx.)

SUMMARY

Evaluations were made of the field performance of micropropagated (tissue cultured axillary buds) versus conventionally rooted softwood cuttings of muscadine grape, Vitis rotundifolia Michx. Trunk cross-sectional area did not differ between treatments during three years of field evaluation following planting. There were no differences in leaf area and dry weight or shoot number in the two propagation types; leaf morphology was normal with no apparent juvenile characteristics. Yield components including flower number per shoot and inflorescence, fruit number, total fruit weight and yield efficiency were greater in micropropagated plants during the second year, the first year of cropping; differences diminished and yield components were not different in year three. Performance of tissue cultured plants was therefore as good as, or surpassed, conventionally propagated plants during early vine establishment.     

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.     

Regeneration and characterization of plants derived from leaf in vitro culture of two sweet orange (Citrus sinensis (L.) Osbeck) cultivars

In the current work attempts were made to investigate culture of leaf explants derived from in vitro seedlings of two sweet orange (Citrus sinensis (L.) Osbeck) cultivars, Bingtangcheng and Valencia. Effects of several factors, including culture medium, lighting condition, explant age and genotype on regeneration response were examined based on three parameters, percentage of explants producing shoots, mean number of shoots per explant and shoot forming capacity. Culture of the explants on shoot-inducing media (SIM) composed of MT salts supplemented with different growth regulators gave rise to disparate shoot regeneration, in which SIM₁ (MT + 0.5 mg L⁻¹ BA + 0.5 mg L⁻¹ Kinetin + 0.1 mg L⁻¹ NAA + 3% sucrose + 0.8% agar, pH 5.8) was shown to be the most effective medium for direct induction of shoots from leaf explants. Highly significant difference in the response of shoot bud regeneration was noted between the two cultivars, with Bingtangcheng being more responsive than Valencia. Culture of explants from fully developed leaves led to better shoot regeneration capacity in comparison to undeveloped ones. However, the two lighting conditions used herein did not cause significant difference in shoot regeneration. Phenotypic observation and randomly amplified polymorphic DNA (RAPD) analysis confirmed that all the regenerated plants from both genotypes were genetically identical to their donor plants, suggesting absence of detectable genetic variation in the regenerated plants. The data presented here demonstrated that direct initiation of plants from leaf explants has been successfully accomplished. To our knowledge, this is the first report on direct regeneration of shoots from leaf explants in Citrus, which will provide an alternative source for citrus genetic manipulation in the future.     

The Incorporation of Growth Hormones in Seed Dressings

Summary

The consequences of using ex vitro, single-node explants from different topophysical positions in chrysanthemum (Chrysanthemum grandiflorum /Ramat./ Kitam) were determined. In particular, how explant topophysis affected the rate of propagation, which is important for the successful micropropagation of chrysanthemum. Uniform shoots of five cultivars of chrysanthemum, cultured in vitro, were each divided into three equal zones: distal, central, and proximal. Two single-node explants were isolated from each zone and cultured on MS medium without any added growth regulators. After 10 weeks of culture, 50% of the shoots that had developed from axillary buds on each single-node explant were excised and measurements were taken in order to compare those shoots that had developed from explants from the different topophysical zones. The remaining shoots were sub-cultured on rooting medium. After 4 weeks, the numbers of roots per plantlet, and the total fresh weight (FW) of roots were recorded. The cultivars fell into two groups. ‘Lady Amber’, ‘Lady Orange’, and ‘Lady Vitroflora’ explants were topophysis-dependent, while ‘Lady Bronze’ and ‘Lady Rosy’ explants were topophysis-independent. For the three topophysis-dependent cultivars, the propagation rate, growth rate, shoot length, internode length, single leaf weight, and total plantlet FW values were highest for those shoots derived from the central and proximal zones. Topophysis failed to affect the number of leaves per shoot or the number of days between the appearance of two successive leaves. The effects of topophysis on the number of roots per plantlet and on root FW were inconsistent. The unequal growth of chrysanthemum plantlets during in vitro micropropagation can be an effect of topophysis, and this phenomenon is cultivar-specific in chrysanthemum.     

Assessment of clonal fidelity of micropropagated gerbera plants by ISSR markers

True-to-type clonal fidelity is one of the most important pre-requisites in micropropagation of crop species. Genetic fidelity of in vitro raised 45 plants of gerbera (Gerbera jamesonii Bolus) derived from three different explants, viz., capitulum, leaf and shoot tips, was assessed by 32 ISSR markers, for their genetic stability. Out of 32 ISSR markers, 15 markers produced clear, distinct and scorable bands with an average of 5.47 bands per marker. The markers designed from AG motif amplified more number of bands. The markers anchored at 3′ ends produced high number of consistent bands than unanchored markers. Fifteen ISSR markers generated a total of 3773 bands, out of which 3770 were monomorphic among all the clones. The Jaccard's similarity coefficient revealed that out of 45 clones derived from different explants, 44 were grouped into a single large cluster alongwith the mother plant with a similarity coefficient value of 1.00, whereas one clone (C38) remained ungrouped. The clones derived from capitulum and shoot tip explants did not show any genetic variation, whereas, one of the leaf-derived clones exhibited some degree of variation.     

Establishment of in vitro tissue cultures from Echinacea angustifolia D.C. adult plants for the production of phytochemical compounds

The establishment of in vitro cultures of Echinacea angustifolia D.C. was obtained directly from sections of flower stalks of adult plants. The shoot formation was obtained from this plant material placed on a modified MS basal medium named CH supplemented with 0.5 mg L⁻¹ 6-benzylaminopurine (BA). The in vitro propagation procedure of E. angustifolia consisted of three distinct phases: an initial regeneration phase from stalk sections (IP shoots on basal medium with 0.25 mg L⁻¹ BA), an elongation phase on active charcoal and an axillary proliferation of the shoots (AP shoots on basal medium with 0.5 mg L⁻¹ BA).Regenerating calli were established from leaves of in vitro shoots cultured on CH medium supplemented with 3 mg L⁻¹ BA and 0.5 mg L⁻¹ indole-3-butyric acid (IBA). Developed shoots from the callus cultures were subcultured on the CH medium with 0.5 mg L⁻¹ BA (leaf regenerated shoots: LR shoots). The secondary metabolite content of the in vitro plant material was compared with that of the greenhouse growing plants. The quali-quantitative LC-DAD-ESI-MS analysis on the extracts from axillary proliferation shoots (AP shoots) showed significant production of caffeic acid derivatives while leaf callus and LR shoots, accumulated mainly alkamides. These results showed that the proper choice of the procedures for in vitro multiplication allowed us to obtain plant biomass able to produce the active compounds typical of E. angustifolia plants.     

Development of micropropagated plants from different clones of Senecio x hybridus in relation to BAP concentration and temperature in vitro

Summary

Selected mature seedling plants were used as stock plants and about half of them were successfully micropropagated. There was no correlation between the ability of petiole expiants to form adventitious shoots and the capacity of these shoots to multiply. Growth and development varied between the micropropagated clones, originating from one seedling each. The colour of the flowers was always constant within a clone. Developmental time, foliage height and the numbers of shoots, leaves and flowers were influenced by the BAP concentration used in vitro. The temperature during the in vitro phase affected the development time and the height of foliage and inflorescences. Increased BAP concentration (from 0 or 0.1 mg l^?1 to 1 mg H) resulted in plants with a longer development time to anthesis and more shoots, leaves and flowers. Plants raised on 5 mg l^?1 BAP developed slowly, resulting in stunted growth, low foliage height, few leaves and flowers. Consequently, it is possible to use the BAP concentration in vitro to regulate the growth of the progeny crop. After transfer to soil in a growth chamber, plants grown at 10°C in vitro flowered earlier, and had lower foliage and higher inflorescences than plants grown at 21°C. This observation indicates that flowering may be promoted by low temperatures in vitro.     

Efficient plant regeneration via organogenesis in “Egusi” melon (Colocynthis citrullus L.)

Plant regeneration protocol of “Egusi” melon (Colocynthis citrullus L.) was established using three local (“Ejagham”, “Sewere” and “Barablackedge”) and one improved (NHC1-130) cultivars. Cotyledonary explants of different lengths (1/2, 1/4 and 1/6) excised from 4- or 8-day-old seedlings germinated in vitro were cultured on MS medium supplemented with different concentrations of 6-benzylaminopurine (BA). The best results were obtained when cotyledons from 4-day-old seedlings were cut into 2 (1/2) halves. Plant regeneration was optimal on medium containing 5 mg/l BA, yielding 86.3%, 77.0% and 76.3% shoot induction frequencies amongst the three local cultivars of “Ejagham”, “Sewere” and “Barablackedge”, respectively. In NHC1-130, the highest shoot induction frequency (85%) was obtained on medium containing 2 mg/l BA. Adventitious shoots were elongated on medium containing 0.1 mg/l BA and successfully rooted on hormone-free MS medium. Flow cytometric analysis revealed 70% of the plants to be diploid.