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.     

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.     

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.     

Double-phase in vitro culture using sorbitol increases shoot proliferation and reduces hyperhydricity in Japanese pear

This study aimed to improve in vitro shoot proliferation efficiency without inducing hyperhydricity in Japanese pear. The shoot number increased at 2.5–10.0 mg l⁻¹ benzyladenine (BA), while shoot fresh mass increased at 1.0 and 2.5 mg l⁻¹ BA. Different macroelement formulation did not affect shoot proliferation, but adding activated charcoal (AC) to the medium inhibited markedly the production of axillary shoots and biomass and many shoots were hyperhydric. Different carbon sources (CS) significantly increased the shoot number and fresh mass, with the best results for shoot proliferation at 20–30 g l⁻¹ sorbitol. With gelling agents, the shoot number increased at 0.4 and 0.6% agar and 0.3% gellan gum, while fresh mass increased at 0.4% agar. The hyperhydric explants were more than 30% at 0.4–0.6% agar and at any concentration of gellan gum. The improved culture (woody plant (WP) supplemented with 20 g l⁻¹ sorbitol, 0.1 mg l⁻¹ 3-indolyl-butyric acid (IBA), 2.5 mg l⁻¹ BA and 0.8% agar) and double-phase culture (the same medium using a double-phase liquid-gelling agent solidified culture system) produced a higher number of axillary shoots than the conventional culture (1/2MS supplemented with 0.1 mg l⁻¹ IBA, 1.0 mg l⁻¹ BA, 30 g l⁻¹ sucrose and 0.8% agar), moreover, double-phase culture had a higher fresh mass than the other cultures.     

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.     

Effect of agar,galactomannan and indole-butyric acid on in vitro rooting of the pear cultivar ‘Durondeau’ and apple rootstock cultivar ‘Marubakaido’

Summary

The influence of partial substitution of agar by galactomannans in culture media supplemented with different concentrations of indole-3-butyric acid (IBA) was studied on in vitro rooting of pear (Pyrus communis L.) cultivar ‘Durondeau’ and apple rootstock (Malus prunifolia Borkh.) cultivar ‘Marubakaido’. The galactomannans applied were obtained from Cassia fastuosa (cassia) and Cyamopsis tetragonolobus (guar gum) seeds. The results obtained with mixtures of agar and galactomannan (3 g l^–1 each) were compared with those from media solidified with a standard concentration of agar (6 g l^–1). The rooting of pear shoots was enhanced significantly in the presence of a mixture of agar plus cassia galactomannan compared to medium solidified with agar only. The modified media promoted a higher number of roots than the control, and increased the percentage of rooted shoots. A maximum of 84.8% rooting was obtained on half-strength MS medium (¹?2MS) supplemented with 0.49 µM IBA and solidified with a blend of agar plus cassia galactomannan. For the apple rootstock, only the number of roots per shoot was influenced significantly by the addition of galactomannan to the rooting medium. The highest number of roots per shoot was 16.67 on ¹?2MS medium gelled with a mixture of agar plus guar galactomannan supplemented with 4.90 µM IBA. The behaviour of the agar-galactomannan gel and the possibility of reduced costs when compared with systems containing only agar, suggest new biological and commercial applications for galactomannans.     

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.     

Effect of gibberellic acid (GA3) on elongation and rooting of ‘St. Julien A’ rootstock in vitro

‘St. Julien A’ (Prunus instititia L.) rootstock was induced to proliferate shoots on a modified half-strength Murashige and Skoog (MS) medium. Cultures treated with 12.5 mg l^?1 gibberellic acid (GA₃) produced elongated shoots suitable for rooting. Elongated shoots were placed in media with indolebutyric acid (IBA) or indole-3-acetic acid (IAA) with or without a 16-day dark incubation. Light (16-h photoperiod) inhibited rooting. IAA (4 mg l^?1) was ineffective in promoting rooting. Rooting was best when shoots were incubated in the dark with IBA (4 mg l^?1). GA₃ was deleterious to shoots, causing chlorosis and apical die-back. Light regime interacted with auxin treatments in affecting shoot condition. Shoot condition was better on shoots treated with IBA and dark-incubated; while those treated with IAA were better when light-incubated.     

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.     

Shoot production from onion callus tissue cultures

Shoots have been produced on callus derived from onion set and from seedling radicle tissue. Whilst callus of set origin responded optimally to medium containing the cytokinin 6-(3-methyl-2-buten-1-ylamino)-purine (2iP) at 2.00 mg l^?1 and naphthaleneacetic acid (NAA) at 0.06 mg l^?1, seedling radicle callus showed a range of response. The medium used for callus initiation, the age of callus, and the provision of a dark period following inoculation on to the organogenesis medium have been shown to be critical for shoot formation. A limited number of embryoids have been produced from cultures; their occurrence was usually associated with an increase in shoot numbers for the generative callus tissue. Meristemoid areas have been observed in light micrographs of callus cultured upon organogenesis media.     

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.     

Effect of genotype,explant size,position, and culture medium on shoot generation of Gerbera jamesonii by receptacle transverse thin cell layer culture

An unique procedure for the mass shoot propagation of Gerbera using receptacle transverse thin cell layer (tTCL) culture procedure was developed. Genotype, flower bud age, explant size, position of receptacle tTCLs and culture media were found to affect the success of culture. Ten interspecific crosses of Gerbera showed different shoot regeneration rates and callus induction via receptacle tTCL culture, all of which had shoot regeneration rates higher than 57%. Flower buds collected on the 10th day resulted in 91% shoot regeneration after 6 weeks of culture on basal MS medium [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol. Plant. 15, 475–497] supplemented with 0.02 mg l⁻¹ thidiazuron (TDZ), 0.8 mg l⁻¹ adenine and 10% (v/v) coconut water (CW). This was significantly higher than those from flower buds on the 7th and 14th days (22% and 54%), respectively. Shoot regeneration rate was the highest (94–100%) in the middle layers of the receptacle. For mass shoot propagation, shoot clusters were subcultured on half-strength MS medium supplemented with 0.5 mg l⁻¹ indole-3-butyric acid (IBA), 0.5 mg l⁻¹ 6-benzyladenine (BA) and 2.0 mg l⁻¹ kinetin after every 4 weeks. Plantlets formed when single shoots were cultured on half-strength MS medium containing 1 mg l⁻¹ IBA. All plantlets acclimatized well in the greenhouse.     

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

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

Cost-effective tissue culture media for large-scale propagation of three commercial banana (Musa spp.) varieties

Cost-effective tissue culture protocols have been established for the commercial multiplication of three banana varieties, ‘Rasthali’ (AAB – Silk), ‘Grand Naine’ (AAA – Cavendish), and ‘Udhayam’ (ABB – Pisang Awak). Reverse osmosis water and 3% (w/v) table sugar were used as the low-cost water and carbon source, respectively. Six different gelling agent treatments were tested: sago alone (T1), Isabgol alone (T2), sago + agar (T3), Isabgol + agar (T4), sago + Isabgol (T5), and agar alone as a control (T6). Full-strength Murashige and Skoog (MS) medium supplemented with 3 mg l^–1 6-benzylaminopurine (BAP) and 1 mg l^–1 indole-3-acetic acid (IAA) were used for culture initiation and subculturing. Rooting was accomplished on low-cost MS medium containing 1.0 mg l^–1 α-napthaleneacetic acid (NAA), 1.0 mg l^–1 indole-3-butyric acid (IBA), and 250 mg l^–1 activated charcoal. Statistical analysis indicated that sago + Isabgol (T5) produced the maximum number of shoots (10 per explant) in ‘Udhayam’ and ‘Rasthali’, while sago alone (T1) produced the maximum number of shoots (6 per explant) in ‘Grand Naine’. The genetic stability of tissue-cultured banana plantlets produced using these low-cost substitutes was assessed using inter-simple sequence repeat (ISSR) markers. The results indicated that the ISSR profiles of the five treatments and the control (T6) were similar, indicating genetic stability using these cost-effective tissue culture protocols. Reductions in cost over the control (l^–1 of MS medium) ranged from 65% to 86%, while the per plant production cost was reduced by 12.5%–20.0%. Adoption of these treatments (T1–T5) as low-cost tissue culture protocols for in vitro propagation would reduce production costs significantly, leading to an expansion of the area planted with tissue-cultured banana, thereby increasing productivity.     

In vitro propagation of ‘Troyer’ citrange from epicotyl segments

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

In vitro 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.     

Vegetative growth and nutritional status as influenced by auxins and gibberellic acid,and their effect on fruit yield in lemon

Application of 2,4-D and 2,4,5-T at concentrations ranging from 5 to 20 mg l^?1 to 5-year-old ‘Pant Lemon-1’ (Citrus limon Burm) trees reduced the vegetative growth in terms of height, spread, shoot length, number and size of the leaves in the autumn flush. Various NAA treatments (5–20 mg l^?1), however, enhanced growth, but not to the extent that was observed after GA₃ treatments. Application of GA₃ at 10–40 mg l^?1 significantly enhanced all aspects of growth, and the effects were most pronounced at 20 and 40 mg l^?1. Nutritional status of the leaves showed a slight variation in relation to vegetative growth under various treatments.Some 2,4-D- and 2,4,5-T-treatments increased the fruit yield over the control, which could suggest mobilization of foods even at the expense of reduced vegetative growth. On the other hand, NAA, particularly at 10 mg l^?1, increased both vegetative growth and yield, suggesting that the transport of the photosynthates from the leaves to the fruits was not at the expense of new growth extension. Due to excessive growth enhancement under higher concentrations of GA₃ (20 and 40 mg l^?1), comparatively fewer nutrients were translocated to the fruit “sinks”, thereby resulting in a non-significant decrease in yield.     

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.     

Clonal propagation of Rosa clinophylla Thory. through axillary bud culture

A protocol for clonal propagation of Rosa clinophylla, a rare and endangered species but very important for breeding purposes had been standardized through in vitro axillary bud culture. Although cytokinins alone were able to induce shoot buds, but their proper growth and number could be increased only when they were used in combination with GA₃. However, there was shoot tip necrosis and leaf fall in the proliferated shoots. AgNO₃ at 58.85 μM proved effective to avoid shoot necrosis and yellowing of leaves. Activated charcoal (AC) at 250 mg l⁻¹ was found necessary at all the stages of shoot multiplication as well as rooting. Ninety percent rooting could be achieved in 1/2 MS medium supplemented with 4.92 μM IBA and 250 mg l⁻¹ AC. Rooted plantlets were hardened and transferred to the field successfully with 80% survival rate.     

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.