Black Currant Leaf Spot: II. Laboratory Tests of Fungicides for the Prevention of Sporing of Pseudopeziza Ribis on Overwintered Leaves

Summary

We have developed a two-step procedure for rooting of tea microshoots in vitro. The effectiveness of different auxin treatments for root formation was found to differ. Among the auxins tested, 25 μM -naphthalene acetic acid (NAA) gave the best results, with 100% rooting, compared to 25 μM indole-3-butyric acid (IBA) or 25 μM indole-3-acetic acid (IAA), which induced 17% and 58% rooting, respectively. Incubation of tea microshoots on 0.33 Murashige and Skoog (MS) medium, supplemented with 25.0 μM NAA or 175.0 μM IBA for 10 d, followed by transfer to auxin-free 0.33 MS medium resulted in 100% rooting, whereas 50.0 μM IAA induced 91.7% rooting. Besides the different auxin treatments, the strength of the MS medium, the duration of incubation of microshoots in auxin-containing medium, the sucrose concentration, the gelling agent, the pH of the medium, the incubation temperature, the light intensity, and the quality of the shoots also played a significant role during in vitro rooting of micropropagated tea shoots. Among the combinations tested, the most effective results were obtained when green microshoots were incubated on 0.33 MS medium supplemented with 25.0 μM NAA, 50.0 mM sucrose, pH 5.5, gelled with 0.2% (w/v) Phytagel^TM for 10 d at 25° – 30°C at a light intensity of 40 μmol m^–2 s^–1, followed by transfer of shoots to auxin-free 0.3 MS medium. This resulted in 100% rooting and, on average, 11 long roots were formed per shoot. Anatomical changes during adventitious rooting of micropropagated tea shoots in vitro were also studied to understand the process of rooting.     

Effect of glucose on in vitro rooting of mature plants of Bambusa nutans

A high-frequency in vitro rooting method was developed for Bambusa nutans, one of the economically important bamboos in India. Two clones of approximately 10-year-old B. nutans were used for axillary bud proliferation. The effect of carbohydrate source (glucose and sucrose) and auxins (IAA, IBA and NAA) on the in vitro rooting response was evaluated. Rooting percent, mean number of roots and root length were recorded after 14 days of treatment. Successful treatment (49.0 μM IBA and 88 mM glucose in the induction phase for 3 days followed by MS salts with 88 mM sucrose) was repeated four times each with 100 shoots to ascertain the practical utility of the protocol. Addition of glucose along with 49.0 μM IBA during the root induction phase gave 85% rooting success. Anatomical studies in the nodal regions were conducted to determine the effect of glucose on root primordial development and elongation. It was observed that the presence of glucose in the root induction medium is required to recruit more number of root initials. A simple protocol was developed for the large-scale production of B. nutans, where rooting of microshoots developed from mature tissues is always difficult. This study showed that glucose as carbon source and auxin type IBA is essential for in vitro root formation in the cultures raised from mature tissues.     

Effects of auxins on soluble carbohydrates,starch and soluble protein content in Aechmea blanchetiana (Bromeliaceae) cultured in vitro

We investigated the content of soluble carbohydrates, starch and soluble proteins after treatment with auxins indole-3-butylic acid (IBA) and 1-naphthaleneacetic acid (NAA) during the development of Aechmea blanchetiana (Bromeliaceae) plantlets cultured in vitro, to determine the contribution of these compounds in plant propagation. Fresh and dry mass of roots increased more than fivefold with NAA (5.37 and 16.11 μM) compared to those with the auxin-free control. After 70 days of culture concentration of soluble protein concentration content was higher in the roots and leaves of plantlets treated with IBA and similar to auxin-free control, while a maximum accumulation of sugars was observed at 90 days in the roots of plantlets treated with NAA (0.54 μM) or IBA (0.49 and 4.9 μM). The major carbon reserves in the roots were soluble carbohydrates, suggesting that they play an important role in the growth of in vitro-cultured epiphytic bromeliads.     

High frequency plant regeneration through adventitious multiple shoot organogenesis in epicotyl explants of Indian gooseberry (Emblica officinalis Gaertn)

Shoots regenerated adventitiously on epicotyl segments from in vitro seedlings of Emblica officinalis var. ‘Kanchan’. Epicotyls derived from 2-week-old aseptic seedlings were most responsive and produced a maximum number of 303 shoots per explant in Murashige and Skoog (1962) medium (MS) augmented with 8.8 μM N⁶-benzyladenine (BA) + 1.425 μM indole-3-acetic acid (IAA). Shoots readily elongated in MS lacking growth regulators and rooted in half-salt-strength MS (1/2 MS) supplemented with indole-3-butyric acid (IBA) or α-naphthalene acetic acid (NAA). The highest rooting response was recorded in 1/2 MS containing 14.7 μM IBA. Plantlets were acclimatized inside the green house and 80% of the plantlets survived on transfer to garden soil.     

Restoration of rooting competence in a mature plant of Garcinia indica through serial shoot tip grafting in vitro

Apical and axillary buds from a high yielding, early fruiting elite tree (more than 20 years old) were cultured in woody plant medium (WPM) supplemented with 0.9 μM N⁶-benzyl adenine (BA). Multiple shoots were obtained on WPM basal medium containing 8.9 μM BA and 0.5 μM thidiazuron (TDZ). Elongation of axillary shoots was obtained in half-strength WPM medium supplemented with 0.4 μM BA. For root initiation, the elongated shoots were transferred to half strength WPM basal medium containing 2.5–245 μM indole-3-butyric acid (IBA) or 2.7–268.5 μM α-naphthaleneacetic acid (NAA) or the shoots were subjected to 2.5–53.9 mM IBA, 2.7–59.1 mM NAA dip for (30 s–30 min) and then transferred to half strength WPM basal medium. However, rooting was never achieved even after 2 months of culture.     

Direct shoot organogenesis on hypocotyl explants from in vitro germinated seedlings of Psidium guajava L. cv. Allahabad Safeda

Multiple shoots were generated via direct organogenesis on hypocotyl segments excised from in vitro germinated seedlings (45-day-old) of Psidium guajava L. cv. Allahabad Safeda. Modified basal Murashige and Skoog (MMS) medium supplemented with 6-benzylaminopurine (BAP), zeatin or thidiazuron with or without α-naphthalene acetic acid (NAA) were tried. Thidiazuron (0.1 μM) along with 0.54 μM NAA gave the highest response (44.6%) with the regeneration of 3.6 shoots per original explants. These shoots upon subculture gave rise to about 5.0 shootlets per explant in shoot proliferation medium, i.e. MMS supplemented with 2.22 μM BAP + 2.32 μM kinetin. The regenerated microshoots were elongated using a quick dip of gibberellic acid (GA₃; 1.44 mM) prior to culture on MMS medium supplemented with 0.88 μM BAP and adenine sulphate (54.29 μM) for 2 weeks. Rooting of microshoots was achieved best on half strength MMS medium supplemented with 4.90 μM indole-3-butyric acid along with 100 mg l⁻¹ activated charcoal.     

Micropropagation and accumulation of essential oils in wild sage (Salvia fruticosa Mill.)

A protocol for in vitro propagation of the wild three-lobed sage (Salvia fruticosa Mill.) (Synonym, Salvia triloba L.) was developed. Shoot tips were excised from in vitro seedlings and established on MS, Nitch and Nitch (NN), or B5 medium. For shoot proliferation, in vitro nodal and apical explants were cultured on MS medium containing 0.25–2 μM 6-benzylaminopurine (BA), 6-furfurylaminopurine (kinetin), or thidiazuron (TDZ). Proliferated microshoots were rooted on MS medium supplemented with 2.7–11.4 μM indole-3-butyric acid (IBA), indole-3-acetic acid (IAA), or α-naphthaleneacetic acid (NAA). Results indicated that shoots established at 100% regardless of media type, however, shoot height, nodes per shoot, and leaf number were highest for explants established on MS medium compared to NN or B5. Number and height of proliferated shoots, nodes per shoot, and leaf number were highest for nodal explants cultured on a medium containing 0.75 μM BA. Microshoots cultured on a medium supplemented with 2.7 μM IBA exhibited the highest rooting percentage compared to those cultured with IAA or NAA. Essential oil composition in microshoots and shoots of greenhouse-grown plants was determined by gas chromatography/mass spectrometry. The major essential oils detected in both plant materials were α-pinene, 1,8-cineole, camphor, and borneol. No α-thujone or β-thujone was detected. The content of essential oils, camphor, and borneol were higher in the microshoots than in shoots of greenhouse-grown plants.     

Regeneration of plants from Fraxinus pennsylvanica hypocotyls and cotyledons

An adventitious shoot regeneration and rooting protocol was developed for green ash (Fraxinus pennsylvanica) seedling explants. The best regeneration medium for freshly isolated hypocotyls and cotyledons was Murashige and Skoog (MS) supplemented with 13.3 μM 6-benzylaminopurine (BA) plus 4.5 μM thidiazuron (TDZ), and 22.2 μM BA plus 4.5 μM TDZ, respectively. Seventy-six percent of hypocotyl segments and 24% of cotyledon segments produced adventitious shoots, with a mean number of adventitious shoots per explant of 2.7 ± 0.5 and 2.3 ± 1.3, respectively. The effect of in vitro-germinated seedling age on adventitious shoot regeneration from hypocotyl and cotyledon explants was also studied. Results showed that hypocotyl and cotyledon explants from freshly isolated embryos exhibited a higher organogenesis potential than 4–15-day-old explants. Adventitious shoots from hypocotyls and cotyledons were established as proliferating shoot cultures following transfer to MS basal medium with Gamborg B5 vitamins supplemented with 10 μM BA plus 10 μM TDZ. A high rooting percentage (73–90%) was achieved when in vitro shoots were rooted on woody plant medium (WPM) containing 4.9 μM indole-3-butyric acid (IBA) and IAA (0, 2.9, 5.7, or 8.6 μM) with a combination of 10-day dark culture period followed by a 16-h photoperiod. The highest rooting (90%) of adventitious shoots or the number of roots per shoot (3.0 ± 1.0) was obtained on WPM with 4.9 μM IBA plus 5.7 μM IAA. Rooted plants were successfully acclimatized to the greenhouse and 100% survived after overwintering in cold storage. This regeneration system using hypocotyls and cotyledons provides a foundation for Agrobacterium-mediated genetic transformation of F. pennsylvanica for resistance to the emerald ash borer.     

A comparison of conventional cloning options for annatto (Bixa orellana L.)

Summary

Annatto (Bixa orellana L.) is a tree indigenous to tropical America that has been naturalised throughout the Indian sub-continent. The plant is chiefly valued for its carotenoid pigments, annatto. Methods for the clonal propagation of hardwood and softwood cuttings, air-layering, budding, and grafting of annatto were investigated. Hardwood cuttings collected during the wet Summer months (June-July) were treated with indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), or naphthaleneacetic acid (NAA) at varying concentrations. The highest percentage rooting (63.4%), along with highest number of roots (8.33 per cutting) were achieved by hardwood cuttings after 12 h of 2.5 mM IAA treatment. Among the various auxin treatments of softwood cuttings, 5.0 mM IBA in combination with 5.0 mM NAA for 5 min resulted in a significant (P < 0.05) rooting percentage (56.7%). The responses of softwood cuttings to an auxin analogue (boric acid) and/or to thiamine hydrochloride (vitamin B) were inferior to synthetic auxins. When subjected to air-layering, hardwood branches gave 100% rooting without any application of hormone. Among the different types of budding methods attempted (patch, T, or I), patch budding produced the highest efficiency (78%) of propagation. Splice-grafting could also be used for conventional propagation of annatto, with a 50% survival rate. Annatto can therefore be cloned by adopting these methods. Propagation based on softwood cuttings facilitated moderate-scale cloning of this valuable, elite germplasm.     

Adventitious root formation in Grevillea (Proteaceae), an Australian native species

Grevillea (Proteaceae) is a native Australian plant genus with high commercial value as landscape ornamentals. There has been limited research on the culture and propagation of Australian native species. The effect of indole-3-butyric acid (IBA) on the rooting of G.‘Royal Mantle’ and G.‘Coastal Dawn’ in winter, spring and summer was evaluated at University of Queensland Gatton, Southern Queensland in order to determine the rooting ability of this species in different seasons. Both Grevillea cultivars showed seasonal rooting. The more difficult-to-root G.‘Coastal Dawn’ had a reduced response to IBA application than G. ‘Royal Mantle’. Stem and leaf indole-3-acetic acid (IAA) levels were not different between cultivars, therefore rooting ability between the two cultivars does not appear to be due to the differences in endogenous IAA levels.     

Micropropagation of the strawberry tree,Arbutus unedo L.

Actively growing shoots of potted greenhouse-grown strawberry tree (Arbutus unedo L.) were initially sterilised and established in basal woody plant medium containing 11.1 μM BA. Optimum shoot proliferation was achieved on a basal WPM containing MS vitamins, sucrose, agar and 22.2 μM BA. Microshoots rooted successfully in basal in vitro medium containing 10 μM IBA or IAA, but their survival rate during acclimatisation was low. Addition of a mixture 1 part peat:4 parts perlite in the basal in vitro rooting medium (1:1 v/v) containing 10 μM IAA resulted in high rooting percentage and plantlets with branched roots. These plantlets were successfully acclimatised. This novel rooting medium can be exploited further due to its potential in commercial applications.     

Factors affecting tissue culture of Damask rose (Rosa damascena Mill.)

The present study was conducted to evaluate the regeneration ability of Damask rose. Single-node explants were surface sterilised with 10% chlorox for 15 min and cultured on Murashige and Skoog (MS) medium supplemented with various concentrations of N⁶-benzyladenine (BA) or kinetin (Kin) separately or in combination with different concentrations of indole-3-butyric acid (IBA). Combination of 2.5–3 mg/l BA and 0.1 mg/l IBA was the most suitable treatment for proliferation. In vitro derived shoots were subcultured four times on the fresh medium within a 4-week period. Other treatments such as explant orientation (horizontal, vertical and oblique) and explant wounding were also examined but did not affect shoot multiplication rate significantly. Several experiments were carried out to stimulate in vitro rooting of Damask rose. Application of different media such as MS, 1/2 MS, 1/3 MS and 1/4 MS with different concentrations of indole-3-acetic acid (IAA), IBA and naphthaleneacetic acid (NAA) did not produce satisfactory results. Quick-dip method using sterilised 0–2000 mg/l IAA, IBA and NAA solutions was also studied. Other treatments such as using various concentrations of abscisic acid (ABA) in combination with various concentrations of IAA, IBA and NAA, and using various concentrations of sucrose and agar did not produce any roots. The best treatment for rooting of shoots was 2.5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) for 2 weeks in MS medium and then transferring the explants to MS medium without any growth regulator. Plantlets were acclimatised in a soil mixture consisting of peat moss and sand 1:1 (v/v) and successfully transferred to the greenhouse after 3 weeks.     

Significance of explant preparation and sizing in Aloe vera L.—A highly efficient method for in vitro multiple shoot induction

The present study was carried out to assess the effect of explant preparation and sizing for in vitro micropropagation of Aloe vera L. The stem nodal explants and shoot tips were cultured on modified Murashige and Skoog's medium (1962) supplemented with different concentrations of 6-benzylaminopurine (BA), kinetin (KIN), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and α-naphthaleneacetic acid (NAA) either singly or in combination. The best media composition was found to be MS medium supplemented with IAA (11.42 μM), IBA (9.8 μM) and BA (8.88 μM). The explants were divided into 2 sets, with and without ensheathing leaf base. Explant sizing, pruning and retention of mother tissue was highly significant in induction of multiple shoots and roots. The stem nodal explants with leaf base performed much better than those without such covering. A very high number of shoots and roots grew from these explants. The rooted plantlets were successfully acclimatized and transferred to the green house conditions and finally to field conditions.     

Protocol for rapid propagation,and to overcome delayed rhizome formation in field established in vitro derived plantlets of Kaempferia galanga L.

Single medium based efficient protocol for rapid propagation, and to overcome the delayed rhizome formation in field established in vitro derived plantlets of Kaempferia galanga L. through in vitro rhizome induction was achieved. MS medium with combination of 8.87 μM N⁶-benzyladenine (BA), and 2.46 μM indole-3-butyric acid (IBA) induced a mean of 6.2 shoots per explant. Addition of 11.7 μM silver nitrate to 8.87 μM BA and 2.46 μM IBA supplemented medium facilitated the highest number of shoots (mean of 8.3 shoots) as well as roots within 60 days. Subculture of isolated shoots on medium with the same concentration of BA, IBA and silver nitrate increased the number to a mean of 12.1 shoots. Silver nitrate enriched medium developed rhizome at the base of shoots. Increase of sucrose concentration (6–8%) in medium with BA, IBA and silver nitrate favoured the best rhizome development. Ninety five per cent of the plantlets survived in field conditions. The plantlets established in field without in vitro developed rhizome (from medium with BA and IBA) did not form rhizome even at 7 months after transplantation. Instead, they developed tuberous roots only. The plantlets with in vitro developed rhizome (on medium having BA, IBA, silver nitrate, and 6–8% sucrose), and that established from conventional way (through splitting of old rhizome) showed no difference in growth of the rhizome. The present study emphasizes the efficacy of silver nitrate and sucrose to develop rhizome in vitro, which enabled to overcome the delayed development of rhizome, and reduced yield of plantlets established in field without in vitro developed rhizome.     

Effect of phytohormones on in vitro shoot multiplication and rooting of lime Citrus aurantifolia (Christm.) Swing

Multiple shoots were produced from node explants of lime (Citrus aurantifolia (Christm.) Swing) on MS medium supplemented with 6-benzylaminopurine (BAP), 6-furfurylaminopurine (kinetin) and α-naphthaleneacetic acid (NAA). The highest number of shoots, nine shoots per node, were produced on a medium containing 2 mg l⁻¹ BAP (8.8 μM), 1 mg l⁻¹ kinetin (4.6 μM) and 1 mg l⁻¹ NAA (5.4 μM). Depending on the concentration of BAP and kinetin, NAA either inhibited, stimulated or did not affect shoot multiplication, which also depended on the cytokinin level. Maximum shoot length was obtained from treatments containing 0.5 mg l⁻¹ BAP (2.2 μM) combined with 1 mg l⁻¹ kinetin (4.6 μM) and 0.5 mg l⁻¹ NAA (2.7 μM). The largest leaves of resultant shoots were produced on a medium containing 0.5 mg l⁻¹ each of kinetin (2.3 μM) and NAA (2.7 μM). Transferring in vitro shoots to rooting media containing indole-3-butyric acid (IBA) and NAA produced complete plantlets. The highest rooting percentage was obtained on a medium containing either 1 mg l⁻¹ NAA (5.4 μM) alone or 0.5 mg l⁻¹ NAA (2.7 μM) combined with 2 mg l⁻¹ IBA (9.6 μM), whereas the highest number of roots were produced on a treatment containing both 2 mg l⁻¹ NAA (10.8 μM) and 2 mg l⁻¹ IBA (9.6 μM). Roots elongated most on treatments containing 0.5 mg l⁻¹ of either NAA (2.7 μM) or IBA (2.4 μM). Shoot growth associated with the rooting phase was the highest in response to 2 mg l⁻¹ IBA (9.6 μM) or 0.5 mg l⁻¹ NAA (2.7 μM). Plantlets that survived acclimatization, 82%, exhibited normal growth in soil under greenhouse conditions.     

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.     

Collection time,cutting age,IBA and putrescine effects on root formation in Corylus avellana L. cuttings

The effects of collection time, cutting age, indole-3-butyric acid (IBA) and putrescine application on the rooting of cuttings of Italian hazelnut cultivars ‘Tonda Gentile Romana’, ‘Tonda di Giffoni’ and ‘Nocchione’ were investigated. Samples collected during late June, late July and early September from newly formed and 1-year old part of twigs to be utilized to produce leafy cuttings, after being treated with 1000 and 2000 ppm IBA, respectively. In addition, the September cuttings were also treated, respectively, with 1000 and 2000 ppm IBA and 1600 ppm putrescine. Rooting ability was evaluated 2 months after planting for each treatment and collection time. Satisfactory rooting of hazelnut leafy cuttings was observed when collection time occurred in June and September, whereas leafy cuttings collected in July showed a limited capacity of rooting in all cultivars tested. On average, the rooting of the newly formed leafy cuttings was more than the 1-year old cuttings. Rooting was also promoted by IBA treatments, mainly in ‘Nocchione’ and ‘Tonda di Giffoni’. In contrast, young cuttings collected from ‘Tonda Gentile Romana’ in early September rooted poorly when treated with IBA alone, but showed the best rooting (∼80%) after the application of a combination of 1000 ppm IBA and 1600 ppm putrescine. The current findings confirm that putrescine can be a useful substance for increasing rooting percentage and root quality in cuttings of some hazelnut cultivars as obtained from ‘Tonda Gentile Romana’ cultivar.     

An improved protocol for adventitious shoot regeneration and plant formation in Psoralea corylifolia L.

An effective protocol was developed for in vitro regeneration of Psoralea corylifolia through enriched cotton moistened-liquid (CML) and solid culture systems. Prolific adventitious shoot buds were achieved from hypocotyl explants of 2-week-old cultures on enriched CML Phillips and Collins (L2) medium supplemented with different concentrations and combinations of thidiazuron (TDZ), benzyladenine (BA), kinetin (KIN), naphthalene acetic acid (NAA), indole-3-acetic acid (IAA) and bavistin (BVN). Combination of 2 μM TDZ, 0.5 μM BA, 100 mg l⁻¹ BVN and 2 μM NAA produced a greater number of adventitious shoots per explant (93.5) when transferred to half-strength enriched solid L2 medium. Regenerated shoots (40–50 mm in length) were exposed simultaneously for rooting as well as hardening in moistened (1/8-L2 basal salt solution with 5 μM IBA and 100 mg l⁻¹ BVN) soil mixture and vermiculite (3:1, v/v). The plants were subsequently established in the field. The survival percentage differed with seasonal variations.     

Micropropagation of Capsicum frutescens L. using axillary shoot explants

A novel micropropagation protocol was established for Capsicum frutescens L. cv. ‘Uchithi’, a pungent chilli cultivar, through induction of axillary shoot proliferation of in vitro raised plantlets by decapitation and using the axillary shoots as explants for multiple shoot bud induction. About 2–6 axillary shoots were induced within 2 weeks when 4-week-old in vitro raised plantlets were decapitated. The axillary shoot-tip explants produced multiple shoot buds when cultured on Murashige and Skoog's (MS) medium containing 8.8–44.4 μM 6-benzylaminopurine (BAP) or 9.3–46.7 μM kinetin alone or 8.8–44.4 μM BAP with 4.6 μM kinetin or 5.7 and 28.5 μM indole-3-acetic acid (IAA). Maximum number of shoots (5.6) were induced on medium containing 22.2 μM BAP in combination with 4.65 μM kinetin. The separated shoots rooted and elongated on medium containing 2.8 μM IAA or 2.4–4.9 μM indole-3-butyric acid (IBA). Rooted plantlets were successfully established in the soil. Efficient mass multiplication of this important food crop was achieved.     

Auxin synergists in the rooting of mango cuttings

Preplanting application of phenolic compounds, p-hydroxybenzoic acid p-coumaric acid and ferulic acid, in most cases promoted auxin-induced rooting on cuttings of mango, a difficult-to-root fruit plant. The ethylene-releasing chemical CEPA (ethrel, ethephon) also stimulated rooting in the presence of auxins. Synergism was more pronounced in the presence of IBA than with IAA. To a certain extent, root-promoting effects of auxins and non-auxin chemicals were related to the utilization of carbohydrate fractions from the region of root formation. Utilization of reserve polysaccharides assumed greater importance only during the root-development phase.