Micropropagation of red raspberry and the influence of phloroglucinol

Micropropagation of 12 raspberry seedling selections and the cultivar ‘Malling Jewel’ has been achieved. A basic culture medium (Linsmaier and Skoog, 1965) supplemented with benzylaminopurine (BAP), 1.0 mg l^?1, and indol-3-yl butyric acid (IBA), 0.1 mg l^?1, was optimal for shoot proliferation. The presence of phloroglucinol (PG) at a concentration of 162 mg l^?1 significantly increased shoot number at all auxin: cytokinin concentrations. Removal of the cytokinin and increasing the concentration of IBA to 1.0 mg l^?1 resulted in adventitious root formation. PG synergistically promoted the number of roots per rooted culture but did not significantly increase the percentage rooting. Viable plants were produced from all genotypes when transplanted to soil.     

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

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

Rapid propagation of Cucurbita pepo L. by culture of meristem tips

A tissue culture technique has been developed for the rapid multiplication of pumpkin (Cucurbita pepo L.) clones. Meristem-tips from seedlings of cultivar ‘Cinderella’ were grown initially on MS medium containing 2.56 mg l^?1 Kinetin and 8 mg l^?1 IAA, and then transferred to experimental media. Maximum shoot proliferation occurred on MS medium containing 1 mg l^?1 BA and no auxin. Cultures were rooted after 2–3 weeks on MS medium containing 8 mg l^?1 IAA and no cytokinin.     

Rejuvenation influences indirect organogenesis from leaf explants of Pomegranate (Punica granatum L.) ‘Kandhari Kabuli’

Sequential subculturing leads to a gradual physiological change in cells that may be termed ‘rejuvenation’. The effect of repetitive subculturing on callus induction and shoot regeneration from leaf explants of Punica granatum L. ‘Kandhari Kabuli’ were investigated. Surface-sterilised leaves were cultured on 1.0× Murashige and Skoog (MS) medium supplemented with 4.0 mg l^–1 α-naphthaleneacetic acid (NAA) and 2.0 mg l^–1 6-benzyladenine (BA) for callus induction. Shoots were regenerated from callus on 1.0× MS medium supplemented with 1.5 mg l^–1 BA, 0.5 mg l^–1 kinetin, and 0.25 mg l^–1 NAA. Subculturing of callus onto fresh medium maintained the rate of shoot formation and substantially increased the production of shoot buds up to the second subculture. Following further subculture passages, a lower shoot regeneration potential from callus was observed. A maximum shoot bud induction from callus of 63.9% was observed at the second subculture passage. The rate of multiplication of in vitro shoots increased until the fourth subculture, then became constant. Similarly, in vitro rooting of micro-shoots increased up to the third subculture, followed by a decline during further subculturing.     

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.     

Increased haploid production in Saintpaulia ionantha by anther culture

Anthers of Saintpaulia ionantha containing late-uninucleate stage pollen produced callus from the anther interior after 3–4 weeks culture on Murashige and Skoog medium supplemented with 1 mg l^?1 naphthylene acetic acid (NAA) and 0.5 mg l^?1 1,6-benzylaminopurine (BAP). Shoot regeneration occurred rapidly and up to 200 shoots could be recovered from callus derived from a single anther within 10 weeks. Examination of roottip mitoses from transplanted, established plants demonstrated that, with few exceptions, the plants were haploid, thus indicating that the callus was pollen derived. Exposure of buds to low temperature prior to anther excision was inhibitory to callus production. Shoot regeneration was studied by scanning-electron microscopy.     

Influence of BAP Concentrations and Nutrient Medium Composition on <Emphasis Type="Italic">In Vitro</Emphasis> Regeneration of ‘Öküzgözü’ and ‘Boğazkere’ (<Emphasis Type="Italic">Vitis vinifera</Emphasis> L.) Cultivars

This study has been conducted with the aim to determine the type of nutrient medium that can be used in micropropagation studies for ‘Öküzgözü’ and ‘Bo?azkere’ and to specify BAP concentrations. In the study where ejectors with a length of 0.7–0.8?cm that are obtained with single-node culture are used, it was focused on four different nutrient media such as MS, DKW, QL and WPM and on six different concentrations such as 0.2–0.4–0.6–0.8–1.0–1.5 mg l^?1 BAP. Single-node suspension explants which will be used in initiating the culture, are taken into culture in MS nutrient medium and the nutrient medium is supported with 30?g l^?1 sucrose, 6?g l^?1 agar and 1?mg l^?1 BAP. In the trial environment, parameters such as number of shoots, shoot length (cm), number of nodes and callus ratio have been investigated. For both grape varieties, the best outcome was obtained with MS nutrient medium with respect to number of shoots, shoot length, and number of nodes. These values were found as 4.66, 1.24 and 6.39 for ‘Öküzgözü’ variety respectively, whereas they are determined as 6.28, 1.15 and 6.81 for ‘Bo?azkere’ variety respectively. In both grape varieties in DKW nutrient medium, starting from the 2nd week of culture, obscuration began to appear on the shoots and after this stage no other development has taken place.     

Effects of nitrogen and phosphorus nutrition on the growth of asparagus seedlings

N was applied at 50, 100 or 150 mg l^?1 in factorial combination with P at 7.5, 15 or 22.5 mg l^?1 to asparagus seedlings. There were 6 successional harvests. N and P increased shoot dry weight by increasing mean dry weight and number of shoots. Increasing P had no effect on shoot growth at 50 mg l^?1 N. N increased root dry weight (crown and roots) by increasing root number, whereas P decreased root dry weight due to a decrease in mean root dry weight. N increased total plant dry weight, but P had no effect. N and P increased the partitioning of dry weight to the shoots, while partitioning to the roots increased with time. Plant analysis revealed that 2.6–2.7% N and 0.29–0.36% P, on a dry-weight basis, were present in the shoots at the later harvests with the higher concentrations of N and P. 100–150 mg 1^?1 N in combination with 15 mg l^?1 P produced a seedling suitable for transplanting into commercial fields at 6 weeks from emergence.     

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.     

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

Summary

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

Nitrogen and calcium nutrition of Petunia × hybrida ‘Coral Sea’

The effects of N and Ca nutrition on plant growth and shoot elemental content of Petunia × hybrida Hort. Vilm. - Andr. ‘Coral Sea’ were evaluated. Nitrogen and Ca were applied separately or in combination in three experiments: (1) N at 0, 100, 200 or 400 mg l^?1; (2) Ca at 0, 75, 150 or 300 mg l^?1; (3) N at 0 or 100 mg l^?1 and Ca at 0 or 150 mg l^?1 combined factorially. Shoot and root dry weights, branch length and flower number were highest when plants received 100 mg l^?1 N. Plants treated with 150 mg l^?1 Ca had the highest shoot and root dry weights. Branch length was maximal at 300 mg l^?1 Ca.Nitrogen and Ca interacted to increase shoot dry weights, branch number and length, leaf area and flower number. Increasing N concentrations increased N and decreased P, Mn and Zn shoot contents. Calcium content of shoots increased while N, P and Mg decreased in response to increasing applications of Ca to petunia plants. Minimal N and Ca tissue concentrations for optimal P. × hybrida growth were 3.3 and 0.67%, respectively.     

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 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.     

Castanea sativa plantlets proliferated from axillary buds cultivated in vitro

Chestnut plants were proliferated in vitro from axillary buds of juvenile shoots. N6-Benzyl-aminopurine (BAP) at 0.1?0.5 mg l^?1 was optimal for shoot multiplication. The important role played by the macronutrient formula on shoot multiplication, and especially on the rooting-stage, is emphasized. The MS ($\text{1}\text{2}$ NO₃) macronutrients gave the best rooting percentage as well as the highest number of roots per rooted shoot. In these experiments, shoots remained in the 3 mg l^?1 indole-3-butyric acid (IBA) medium for 12 days, after which they were transferred to an auxin-free medium where roots developed fully. Optimum rooting was achieved by immersing the 1 cm basal end of shoots in concentrated IBA solutions (0.5?1 mg ml^?1) for periods ranging from 2 to 15 min.     

Effects of explant type, culture media and growth regulators on callus induction and plant regeneration of Chinese jiaotou (Allium chinense)

To establish an efficient protocol of shoot regeneration from callus, effects of explant type, culture media and plant growth regulators on callus induction and shoot regeneration of Chinese jiaotou (Allium chinense) were evaluated. The results showed that basal plate was the best explant for callus induction (47.5%) when cultured on B5 medium supplemented with 0.1 mg l⁻¹ 6-benzylaminopurine (BA) and 1.0 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D), and B5 was the best medium to induce callus formation with 49.3% of the explants forming callus. The highest callus induction (65.2%) was achieved culturing basal plate on B5 medium supplemented with 0.1 mg l⁻¹ BA and 1.0 mg l⁻¹ 2,4-D after 8 weeks of culture. The best callus proliferation was observed on B5 medium with 1.5 mg l⁻¹ 2,4-D. Shoots regenerated at the highest frequency of 58.8% with 4.5 shoots when calli were cultured on B5 medium with 0.1 mg l⁻¹ BA and 1.0 mg l⁻¹ a-naphthaleneacetic acid (NAA). This protocol provides a basis for future studies on genetic improvement and could be applied to large-scale multiplication systems for commercial nurseries of Allium chinense.     

Direct regeneration of plants derived from in vitro cultured shoot tips and leaves of three Lysimachia species

The regenerability of three ornamental species—Lysimachia christinae, Lysimachia rubinervis and Lysimachia nummularia ‘Aurea’, were investigated using in vitro leaves and shoot tips. 6-Benzylaminopurine (BAP) and α-naphthalene acetic acid (NAA) added to Murashige and Skoog (MS) medium were tested for their effect on organogenesis. On the medium, shoot regeneration occurred directly without callus formation. In these species, L. christinae developed the highest regeneration rate and numbers of shoots/explant from shoot tips (100%, 12.25) and leaf bases (100%, 13.01) on the MS medium containing 3.0 mg l⁻¹ BAP and 0.1 mg l⁻¹ NAA. For L. rubinervis, the highest shoot induction rate and number of shoots/explant were obtained from shoot tip (100%, 16.87–17.20) on the MS medium with 0.1 mg l⁻¹ NAA and 3.0–5.0 mg l⁻¹ BAP. L. nummularia ‘Aurea’, however, showed the highest regeneration rate and number of shoots/explant (100%, 12.73) from leaf bases on MS medium supplemented with 1.0 mg l⁻¹ BAP and 0.1 mg l⁻¹ NAA. All in vitro shoots rooted well on half macronutrient MS medium containing 0.1 mg l⁻¹ NAA. After acclimatization, transplanted plantlets grew normally and flowered in the field.     

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.     

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

Summary

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

Influence of yeast extract and casein hydrolysate on callus multiplication and somatic embryogenesis of date palm (Phoenix dactylifera L.)

Complex organic additives are known to improve growth and differentiation of in vitro plant cultures. The present investigation was conducted to determine the effect of various concentrations of yeast extract (YE) and casein hydrolysate (CH) on callus growth and somatic embryogenesis in date palm cultivar Nabout Saif. Callus induced from shoot tip explants was grown on callus multiplication medium supplemented with either YE or CH at 0.0, 0.1, 0.25, 0.5, and 1 g l⁻¹. To induce somatic embryogenesis, callus was transferred to a hormone-free medium containing the corresponding concentration of additives. The results have shown that callus weight and the number of somatic embryos were directly proportional to increases in the concentration of organic additives tested. Callus growth was best achieved when 1 g l⁻¹ of either YE or CH was added to the culture medium. At this concentration of YE, callus growth was double that of the control medium. On CH-containing media growth was 2.3 times that of the control. This indicates that CH is more effective in enhancing callus growth. However, YE was more effective in enhancing somatic embryogenesis. The data show that the best somatic embryo formation was obtained on either 1 g l⁻¹ YE or 0.5 g l⁻¹ CH which produced 45 and 30 embryos per culture, respectively, as compared to 20 embryos produced in the control treatment. Resultant somatic embryos successfully rooted and regenerated plantlets which exhibited normal growth in the greenhouse. Enhanced plant regeneration, an essential criterion for commercial micropropagation, was achieved.     

皇帝蕉薄片外植体愈伤组织的诱导及植株再生

将皇帝蕉试管苗茎段徒手切成厚约1mm的薄片,经无菌的0.5%柠檬酸溶液处理片刻后,接入各种培养基中。结果表明:(1)适度的暗培养预处理有利于愈伤组织诱导,合适暗培养天数为4d;(2)诱导愈伤组织最佳培养基为:B5+2,4-D13.572μmol/L+IBA4.921μmol/L+NAA5.371μmol/L+KT13.94μmol/L+椰乳5%+PP3330.0001mg/L,愈伤组织诱导率为86.0%;(3)最佳愈伤组织分化芽培养基为:改良MS培养基+BA13.6831μmol/L+NAA0.537μmol/L,芽分化率达到87.0%;(4)诱导芽生根的最佳培养基是:1/2MS+IBA0.492μmol/L(或+NAA0.537μmol/L),生根率达到100%。