Adventitious shoot regeneration from leaf segments of in vitro cultured shoots of the apple rootstock Jork 9

SUMMARY

Adventitious shoot formation was investigated using leaf segments of in vitro cultured shoots of the apple rootstock Jork 9. Regeneration capacity was influenced by the pretreatment of the mother shoots, macroelements, hormone concentrations, the gelling agent and the carbohydrate source. The highest regeneration rate and most shoots per leaf explant resulted from young leaves on a medium based on MS macroelements supplemented with 22 µM BAP and 0.1 µM_NAA together with sorbitol, at concentrations of 165 mM or 220 mM. Sorbitol was more effective than sucrose, glucose, fructose or a combination of these sugars. A cold and dark pretreatment of the shoots enhanced the formation of adventitious shoots.     

Propagation of black currants by single-noded cuttings

Single-noded cuttings were taken from four regions of one-year-old black currant shoots in September 1966, before winter chilling had begun, and stored at 1 °C for 0, 4, 8 or 12 weeks before being inserted in seed trays and placed in a growth room at 20 °C, with continuous illumination. Cuttings from the lower half of shoots rooted and grew well with or without chilling, but cuttings from the upper middle quarter of shoots rooted better after 8 or more weeks in cold storage. Few cuttings from the top region rooted, even after 12 weeks of cold. Cold storage accelerated subsequent bud burst, as did 2 minutes’ treatment with GA₃ at 100 ppm.

In the field, dormancy of buds on intact plants increased after September and was pronounced in December. However, cuttings from all regions of shoots taken from plants in the field in January and immediately placed in the growth room rooted and grew well     

Plum Rootstock Trials at East Malling

Summary

Dormant axillary buds excised from crowns of pineapple (Ananas comosus L., Merr.) cultured on growth regulator free Nitsch medium sprouted after 8–10 d. Sprouted buds produced multiple shoots (7–10 shoots per bud) upon transfer to solidified Murashige and Skoog medium supplemented with 9.67 μM NAA, 9.84 μM IBA and 9.29 μM KIN. Each isolated shoot upon subculture to liquid medium of the same composition further proliferated to form more multiple shoots (60–65 shoots) and were maintained on a gyratory shaker (90–100 rpm). In vitro grown shoots were rooted on White medium supplemented with 0.54 μM NAA and 1.97 μM IBA. In vitro plantlets were established in cups with soilrite and hardened for four weeks. Phenotypic variants such as albinos, white streaked shoots and shoots with elongated internodes were observed in in vitro cultures. Approximately 520 in vitro produced plantlets were established in the field and these plants exhibit somaclonal variation. Thirty-eight plants were found to be yellowish, spineless with anthocyanin streaks and three were anthocyanin rich, spined plants.     

Anthocyanins from tubers and shoots of the purple potato,Solanum tuberosum

Summary

The anthocyanin content of tubers and shoots of the purple potato, Solanum tuberosum cv. Congo has been determined using various chromatographic and spectroscopic techniques to be petanin and the novel 3-O-[6-(4-ferulyl-O-α-rhamnopyranosyl)-β-glucopyranoside]-5-O-β-glucopyranosides of petunidin and malvidin. Acylated pigments constitute more than 98% of the total anthocyanin content both in tubers and shoots.     

The Influence of Some Genetic and Environmental Factors on the Concentration of L-Ascorbic Acid in the Tomato Fruit

Summary

An efficient, reproducible protocol has been developed for in vitro multiplication of Sida cordifolia L. High-frequency, multiple shoots (90%) were obtained indirectly from nodal explants. Callus was induced when nodal explants were cultured on Murashige and Skoog (MS) medium supplemented with 0.5 mg l^–1 Kinetin (Kn). These nodal calli were then cultured in order to differentiate multiple shoots on MS medium supplemented with 0.5 mg l^–1 Kn plus 0.5 mg l^–1 naphthaleneacetic acid (NAA). Roots were induced from these multiple shoots following culture on MS medium supplemented with 0.8 mg l^–1 NAA for 4 weeks. Finally, these in vitro plantlets were hardened, acclimatised, and successfully transferred to the field.     

Regeneration and transformation of the premier UK apple (Malus × pumila Mill.) cultivar Queen Cox

Summary

A number of factors were assessed for their effects on in vitro shoot proliferation and adventitious shoot regeneration. More in vitro leaves of a quality suitable for use in regeneration and transformation experiments were obtained from shoots on DKW proliferation medium compared with MS medium, and also on MS and DKW media containing phloroglucinol. Compared with MS medium, shoot proliferation was greater on MS with halved levels of NH₄NO₃ and KNO₃. Adventitious shoots were hyperhydric on MS-based but not on DKW-based regeneration medium. More adventitious shoots regenerated on media solidified with Sigma Agargel than on media with Sigma Phytagel or Gelcarin (FMC). Viable transformed shoots were recovered on Sigma Agar or Agargel but not Phytagel. Wounding of leaf explants by stabbing with needles, and stabbing combined with scoring with a scalpel, increased the number of calli regenerating, and these methods, as well as solely scoring with a scalpel, increased the number of calli regenerating shoots compared with the control. Combined stabbing and scoring resulted in more calli producing shoots than solely scoring or stabbing. Vortexing leaf explants with silicon carbide whiskers increased the percentage of subsequently formed calli that regenerated shoots compared with the control. Transformed shoots were regenerated following co-cultivation with Agrobacterium tumefaciens EHA101 harbouring the binary vector pSCV1.6 (with selectable marker gene npt II and GUS reporter gene uid A). The number of transformed shoots as a percentage of explants varied from 0.5% to 2.2%. Molecular analysis of the four extant transformed lines confirmed integration of the transgene and indicated that in three lines there was one integration site, and in one line there were four sites of integration.     

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.     

Micropropagation of guava (Psidium guajava L.)

Summary

Guava (Psidium guajava L.) is difficult to propagate using conventional asexual techniques, with most growers using seedling planting stock. However, these seedlings are highly variable. We therefore developed an in vitro technique to clonally propagate guava. Various concentrations of BAP (6-benzylaminopurine) and TDZ (thidiazuron) were used to regenerate and micropropagate plants. Two explant sources were compared: greenhouse grown plants (GHRP) and in vitro-harvested axillary buds (IVDS). GHRP with BAP at 2 mg l^–1 gave 3.7 shoots per single node cutting with an average length of 0.7 cm. Shoots 3.0 cm long were obtained with 0.5 mg l^–1 BAP, however only 2.1 shoots per explant were produced. For IVDS, the largest number of shoots (3.9 per explant) was obtained with BAP at 0.25 mg l–1 , with an average shoot length of 1.6 cm. Generally, lower concentrations of BAP gave fewer but longer shoots. The highest number of roots and longest roots per shoot (5.4 and 2.0 cm, respectively) were obtained with 1 mg l^–1 indole-3-butyric acid (IBA). A protocol for producing clonal plants over eight weeks is described.     

Micropropagation of bay laurel (Daphne gnidium L.)

Summary

Procedures have been developed for the micropropagation of Daphne gnidium, a shrub species of ecological interest, using explants of juvenile and adult origin. Shoot proliferation rates were significantly affected by both salt formulation and benzylade- nine concentration. Best results were obtained on WP medium with 5 µM BA. The presence of indoleacetic acid in the induction medium improved BA-induced axillary bud proliferation from juvenile explants. Rooting of shoots produced in culture was difficult, especially those of adult origin. Besides an absolute requirement for auxin, calcium concentration and the pH of the medium affected the formation of adventitious roots from regenerated shoots of D. gnidium.     

In vitro colchicine-induced polyploid plantlet production and regeneration from leaf explants of the diploid pear (Pyrus communis L.) cultivar, ‘Fertility’

Summary

Polyploid plantlets, including triploid, tetraploid, and mixoploid, were induced from the European pear (Pyrus communis L.) cultivar ‘Fertility’ by in vitro colchicine treatment of leaf explants. The leaf explants were incubated in 0.4% (w/v) colchicine for 24, 48, or 72 h, then transferred to adventitious shoot-induction medium. Regenerated shoots were pre-selected according to their morphological characteristics when compared to control shoots from untreated shoot proliferation cultures. Shoots with putative polyploid morphological characteristics were maintained and proliferated. The ploidy levels of all putative polyploid individuals were analysed by flow cytometry and identified by chromosome counts of shoot tip tissue squashes. Polyploid shoots were rooted, and the resulting plantlets were transferred to the field. Polyploid plantlets had a higher specific leaf mass and larger stomata than those of diploid plantlets.     

Partitioning sources of rooting potential in plum hardwood cuttings

SUMMARY

Rooting of leafless winter hardwood cuttings of the plum rootstock Prunus insititia ‘Pixy’ increased as the location from which the shoots were taken from within specially grown stockplants decreased in height above ground, associated with a parallel reduction in shoot thickness. However, the actual height of the least-ready-rooting crown cuttings had no effect on rooting, suggesting that relative rather than absolute position is important. Rooting was unaffected by bark-ringing and trunk incision distal to the shoot position, suggesting that such treatments did not interfere with a basipetally translocated root promotor which might have accounted for improved rooting of cuttings in the lower parts of the hedge. The rooting of crown cuttings above a bark ring was reduced considerably compared with that of cuttings from normal bushes, and this was associated with increased thickness of shoots distal to the ring. Delaying pruning in spring until after growth had started resulted in thinner crown shoots compared with those from plants pruned normally while dormant, and the rooting of these thinner crown shoots was much higher than that of the normal crown cuttings. It was shown by covariance analysis that shoot thickness accounted for part of the rooting response but could not account for the total effect due to shoot position within the bush, ringing, or time of pruning. Competence to root appears to develop independently in individual shoots, modified by a shoot thickness factor which favours the subordinate shoots induced in the shoot hierarchy of severely pruned hedges.     

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.     

Water Salinity and Initial Development of Pitaya (Hylocereus undatus)

ABSTRACT

Salinity is an important environmental problem, especially in arid and semiarid regions of the world. An experiment was conducted to evaluate the effects of water salinity on the initial development of pitaya (Hylocereus undatus)using five levels of irrigation water with electrical conductivity (EC_w) levels: 0; 1.0; 2.0; 3.0 and 4.0 dS m^_1. Salinity inhibited plant height, stem diameter, root length, number of additional stems and the dry weight of roots and shoots. Tissue collapse on the stems was also noted. At the end of the experiment it was observed that 50% of plants irrigated with EC_w 4.0 dS mr ¹ died. Roots of pitaya were as sensitive as shoots to saline effects.     

Regeneration from in-vitro leaves of ‘Conference’ and other pear cultivars (Pyrus communis L.)

Summary

Regeneration of shoots from in-vitro grown leaf tissues of the pear cv. Conference was achieved at rates of up to 40%. The highest regeneration percentage was achieved using a phytohormone combination of benzylamino purine (5 mg 1^?1) and 1-naphthalene acetic acid (1.0 mg 1^?1) on a basal medium of Murashige and Skoog, to which had been added 200 mg I^?1 of the antibiotic cefotaxime. The percentage regeneration was reduced when a higher concentration of cefotaxime (400 mg 1^?1) was used. Cefotaxime had little beneficial effect on regeneration at a lower concentration of 1-naphthalene acetic acid (0.5 mg l^?1). Regenerated shoots were easily micropropagated, rooted and transplanted to soil. To date, the plants have a true-to-type phenotypic appearance. Regeneration was also achieved with other cultivars—‘Abbé Fetel’, ‘Doyenne d’Hiver’, ‘Doyenne du Comice’ and ‘Passé Crassane’—but percentage regeneration was lower and, apart from ‘Passé Crassane’, the regenerated shoots were weak and could not be subcultured.     

Production of four commercially cultivated Echinacea species by different methods of in vitro regeneration

Summary

Efficient in vitro procedures for mass propagation of four commercially important Echinacea species have been deveoped. Plants of E. angustifolia, E. pallida, E. paradoxa and E. purpurea were regenerated by three methods, namely axillary bud proliferation, adventitious shoot formation and somatic embyrogenesis. Shoot tips obtained from in vitro germinated seedlings, adventitious shoots or somatic embryo-derived plantlets, when cultured on Murashige and Skoog medium enriched with 1 μM 6-benzylaminopurine, 2 μM kinetin, 0.5 μM indole-3-butyric acid and 4 mg^–1 paclobutrazol multiplied three-fold within 3–4 weeks in culture. Incorporation of paclobutrazol in the shoot multiplication medium was necessary to recover healthy and robust shoots suitable for rooting. Direct, high-frequency shoot formation on intact leaves of shoots grown on 6-benzylaminopurine and kinetin-supplemented media, an unusual and novel observation made in this study, occurred in all the species studied. Rooting of in vitro developed shoots was achieved relatively easily with Murashige and Skoog basal medium rather than with auxin-enriched media. Culturing of hypocotyl explants on medium containing 3,6-dichloro-o-anisic acid (commonly known as dicamba), or 2,4-dichlorophenoxyacetic acid, resulted in direct somatic embryogenesis in all the species examined. The presence of cytokinin was required for somatic embryo germination, but further development of germinated somatic embryos into normal plantlets occurred in Murashige and Skoog medium. We conclude that the procedures described here could be used for rapid propagation as well as genetic transformation of commerically cultivated Echinacea species.     

In vitro induction of chromosome-doubling in cultured shoots of three cultivars of mint (Mentha canadensis L.) treated with colchicine

Summary

To improve the yield and quality of essential oils, chromosome-doubling in mint cultivars was induced by treating shoots with colchicine in vitro. Shoot tips of three mint cultivars (‘68-7’, ‘73-8’, and ‘HU 39’) were cultured in vitro and treated at 2 months using either of two methods to induce chromosome-doubling. Explants were immersed separately in each of three concentrations of colchicine [0.1, 0.2, or 0.3% (w/v)] for 24 h or 48 h. Alternatively, shoots were cultured on solid 1.0× MS (Murashige and Skoog) medium supplemented with one of five concentrations of colchicine (10, 20, 30, 40,, or 50 mg l^–1) for 30 d. After each treatment, ploidy levels were measured by flow cytometry. The results showed that high yields of 4n plants were induced by the immersion of shoots in 0.2% (w/v) colchicine for 24 h (13.3%), or by culturing shoots on MS medium containing 20 mg l^–1 colchicine for 30 d (17.3%). The immersion method, which gave a survival rate of 93.3%, was more convenient and less phytotoxic to produce 4n mint plants. Compared with untreated plants, we observed fewer but larger stomata in chromosome-doubled plants. We also observed significant differences in the size, internode length, leaf length, leaf width, and stem width in chromosome-doubled mint plants.     

An Experiment on the Winter-Killing of Vegetable Crops in Market Gardens

Summary

The development of an efficient methodology for the genetic transformation of orchids is needed in order to support thegenetic engineering of orchids. It is therefore important to identify those factors affecting the transformation process.Previously, we reported a convenient method for the transformation of Phalaenopsis amabilis using Agrobacterium tumefaciens, in which intact protocorms were used. We also found that embryos cultured on a medium containing tomato extract grew more rapidly than those cultured on a medium with coconut water. When we used protocorms grown on a medium containing tomato extract, we obtained regenerated shoots that had been transformed with a kanamycin resistance gene at relatively high frequencies (7 – 17%). These results suggest that the rate of growth of pre-cultured protocorms may be important for the successful regeneration of transformed shoots. We also obtained regenerated shoots that had been transformed with the green fluorescent protein (GFP) gene at a high frequency (10 – 14%). Both the presence and expression of these transgenes were confirmed in transformed plants by molecular analyses and by the detection of green fluorescence following excitation with blue light.     

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.     

The Internal Blackening of Potatoes Caused By Bruising

Summary

Pandorea jasminoides and P. pandorana are successful Australian endemic ornamental species of the family Bignoniaceae. Vigorous growth and spectacular floral display make them popular landscape plants. Application of tissueculture could facilitate Pandorea breeding and improvement programmes, however, there is no report on in vitro regeneration of the genus Pandorea. A protocol for de novo shoot production is described for commercial cultivars of P. pandorana and P. jasminoides. Amenability of explants under in vitro conditions was found to differ between the two species. Direct shoot organogenesis of P. pandorana was obtained using node explants while seedling (epicotyl) explants of P. jasminoides were found to be responsive for shoot regeneration. A maximum of 13 and 15 shoots per explant of P. pandorana and P. jasminoides, respectively, was obtained. Benzyladenine, BA, (8.8.mM) proved to be more effective for shoot regeneration than thidiazuron (TDZ) for P. pandorana while a combination of cytokinin (BA and N-(choloro-4-pyridyl)-N-phynyl urea (CCPU) and auxin is required for P. jasminoides regeneration. However, no significant difference in rooting of in vitro regenerated P. pandorana and P. jasminoides shoots was observed. Regenerated shoots were rooted on a medium containing indolebutyric acid (IBA) and plantlets were successfully established under glasshouse conditions.     

The effects of mechanically-induced stress and water stress on freezing resistance in lettuce and cauliflower seedlings

Summary

Mechanically-induced stress, applied by brushing young lettuce and cauliflower plants for 90 s each day, reduced the freezing resistance of cauliflower but had no effect on that of lettuce. Brushing reduced fresh weight in both species and smaller plants were less freezing-resistant than larger ones. The levels of abscisic acid (ABA) in the shoots of brushed cauliflowers were slightly less than in unbrushed plants, whereas in lettuce the levels were similar in the two treatments. ABA sprayed onto lettuce plants had no effect on freezing resistance. With both lettuce and cauliflower, freezing resistance, and the osmolarity of sap extracted from the shoots, increased following water-stress and declined progressively following rehydration.