Establishment and growth of the rhizome of Alstroemeria as affected by temperature and the root system

The effects of temperature and the root system on growth and establishment were assessed for rhizomes of three Alstroemeria cultivars.-The numbers of lateral rhizomes, aerial shoots and tubers produced by rhizome ‘splits’ were not significantly affected by increase in temperature in the range of 8 to 18°C. Significant increases in root system and rhizome dry weight were seen for cv. Butterfly and in aerial shoot dry weight for all three cultivars used. Increasing temperature significantly decreased the number of plants establishing from rhizome ‘splits’ obtained from plants grown in glasshouse soil. For maximum rhizome production a temperature of between 13 and 18°C was found to be required. A second experiment employing rhizome ‘splits’ from container-grown plants showed no effect of temperature on plant establishment. The presence or absence of the rootsystem on the ‘splits’ at planting was found to be important in plant establishment, with removal of the root system producing a significant decrease in the number of plants establishing. It is suggested that the rootsystem, and damage sustained by it prior to planting, are more important than temperature for the successful establishment of plants of Alstroemeria.     

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

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

Rapid plant regeneration protocol for cluster bean (Cyamopsis tetragonoloba L. Taub.)

Summary

An efficient in vitro regeneration procedure using thidiazuron (TDZ) has been developed to allow high frequency, multiple shoot induction from cotyledonary node explants of cluster bean (Cyamopsis tetragonoloba). Shoot bud induction occurred on Murashige and Skoog (MS) medium after 4 weeks in the presence of TDZ, followed by transfer onto shoot multiplication and elongation media containing MS salts, B5 vitamins, and different combinations of auxins and cytokinins. Multiple shoots were induced at all levels of TDZ in the medium, but the best proliferation capacity occurred at 5 µM TDZ. Combinations of auxins and cytokinins showed a stimulatory effect on shoot multiplication and also on the length of the newly formed shoots. Maximum shoot induction [i.e., the highest number of shoots (16.0 ± 0.94) per explant] was obtained on agar-solidified medium containing 5 µM benzyladenine (BA) with 0.5 µM indole-3-acetic acid (IAA). Rooting of in vitro-regenerated shoots was achieved in ex vitro conditions by a pulse treatment with 300 µM indole-3-butyric acid (IBA) for 15 min. Rooted plantlets were transferred to soil where 70 – 75% attained sexual maturity and produced viable seeds under greenhouse conditions. The present regeneration system is efficient and can be used in various in vitro manipulation studies.     

Comparison of shoot induction ability of different explants in herbaceous peony (Paeonia lactiflora Pall.)

Shoot induction ability of explants of herbaceous peony was investigated in semisolid MS medium containing BA, TDZ and GA₃. Callus was readily induced from stem without node and petiole explants within 2 days of culture but failed to generate shoots. Adventitious shoots were successfully produced from meristematic regions only: bud eyes on nodal stem sections, and junctions of petioles and petiolules. No shoots were induced from internode sections, petiole without junctions, or leaf sections. Nodal sections were the most efficient explants. There were up to 20 shoots in one explant generated within 20 days of culture. TDZ was more effective than BA to induce shoots. The 100% shoot induction rate was obtained in medium containing 0.1–3 mg L⁻¹ of TDZ. However, higher concentrations of TDZ inhibited shoot elongation and only large leaf clusters were produced. Combinations of BA and TDZ failed to increase shoot induction rates but caused shoots shorter. The 2–60-min pretreatment of explants with 20 mg L⁻¹ TDZ solution was very effective to induce adventitious shoots directly, but both shoot number and shoot length tended to decrease as treatment time increased. GA₃ was beneficial for shoot and stem elongation.     

Micropropagation of fastigiate bird cherry (Prunus padus L.) and adventitious shoot formation from leaves

A shoot tip of a mature clone of fastigiate bird cherry (Prunus padus L.) was successfully established in vitro. Culture of shoot tip explants on a Murashige and Skoog (MS) based medium with phloroglucinol (PG) resulted in micropropagation, but the clonal line gradually became hyperhydric on this medium. This problem was overcome using PG- free medium based on either MS or Driver and Kuniyuki Walnut medium (DKW). Heavier cultures with more shoots were obtained on DKW medium with fructose or glucose rather than sucrose or sorbitol. Leaf explants placed on DKW basal medium with benzyladenine (BA) produced adventitious shoots. Addition of 1-naphthaleneacetic acid to media with BA increased regeneration. More leaves produced shoots on medium with sucrose or sorbitol rather than glucose or fructose. Adventitious shoots were excised and micropropagated. Shoots were rooted by insertion into DKW medium with indol-3yl- butyric acid, followed by transfer to hormone-free DKW. PG increased the proportion of shoots that produced adventitious roots.     

6-Benzylamino purine stimulates in vitro shoot organogenesis in Eucalyptus erythronema, E. stricklandii and their interspecific hybrids

In vitro bud and shoot organogenesis was investigated for the ornamental plants Eucalyptus erythronema var. erythronema, E. stricklandii and their interspecific hybrids cv. ‘Urrbrae Gem’ and ‘Hybrid 2.5’ by using 0.0, 0.1, 0.25, 0.5 or 1.0 μM BAP on apex and leaf explants. Callus developed on all explants and increased with all concentrations of BAP without significant differences between BAP concentrations. Buds formed on apex and leaf explants of E. erythronema and E. cv. ‘Urrbrae Gem’ especially with 1.0 μM BAP, but these buds rarely developed into shoots. Bud clusters formed on E. erythronema and E. cv. ‘Urrbrae Gem’ apex and leaf explants whereas E. stricklandii and ‘Hybrid 2.5’ produced fewer, individual buds on the explant. Shoots regenerated from apex explants of all genotypes with all levels of BAP, whereas few shoots of any genotype regenerated from leaf explants regardless of the number of buds formed. Shoots from apex explants could be multiplied successfully. Light microscopy showed meristems developed within the callus, and at the callus and bud surfaces. However, few shoots developed considering the level of bud and meristem formation. This report is the first for successful shoot organogenesis and multiplication in an ornamental eucalypt.     

Studies on the induction of flowering in juvenile Prunus avium L.

Juvenile Prunus avium shoot apices produced flowering shoots after grafting to mature trees irrespective of treatment of the apices in the season before grafting with G A or G A + cytokinin. Scions grown from mature shoot apices grafted to seedling stocks failed to flower, again irrespective of prior hormone treatment. Similar treatment of mature shoot apices with these hormones, or with zeatin, inhibited concurrent floral initiation, but G A or GA + zeatin treatments increased flowering of scions grown from the treated apices after grafting to untreated mature trees. Localized shoot tip or root drench treatment of one or two year old seedlings with (2RS, 3RS)-paclobutrazol failed to induce flowering, but treatment of three or four year old plants did stimulate flowering. Branch or stem girdling or root flooding applied alone or in combination to three year old plants did not affect flowering. Floral initiation by three or four year old plants was inhibited by treatment with GAs. The results were consistent with the presence in seedlings of a root- produced, xylem transported, graft-transmissible inhibitor(s), which control the initiation of otherwise competent meristems, and which could include factors other than GAs. Juvenile meristem competence to flower was not affected by prior GA treatment.     

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

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

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.     

An efficient adventitious shoot regeneration system on excised leaves of micropropagated lingonberry (Vaccinium vitis-idaea L.)

Summary

An efficient system to regenerate shoots in vitro on excised leaves of lingonberry (Vaccinium vitis-idaea L.) was developed. Leaf explants from shoot-proliferating cultures produced multiple shoots without an intermediary callus phase on zeatin (ZN)-containing shoot induction media within 3–4 weeks of culture initiation. Cultivars Regal and Splendor, and one clone from a natural stand in Estonia (ECL1), were used in the first experiment. Young expanding leaves with the adaxial side touching the culture medium, and maintained for 7 d in darkness, produced the best results. There were significant genotypic differences in adventitious shoot formation. A second experiment studied the effects of ten concentrations of three cytokinins: ZN at 5, 10, 20, 30 and 40 μM; 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea (thidiazuron, TDZ) at 0.1, 1, 5 and 10 μm; and 6-(γ-γ-dimethylallylamino) purine (2ip) at 25 μM were compared with leaf segments of different polarity in ‘ECL1’. Zeatin was found to be more effective than TDZ or 2iP as an inductive signal for regenerating many vigorous shoots. Zeatin induced multiple shoot formation at all concentrations tested, but maximum morphogenic response was observed at 20 to 30 μM. The media containing TDZ generally promoted more callus formation and suppresed shoot elongation. In a third experiment with the lingonberry cultivar Erntedank and the clone ‘ECL1’, a new medium developed for lingonberry shoot culture proved more effective than the modified Murashige and Skoog medium for regenerating shoots on leaf explants. Elongated shoots were excised and rooted directly on a 2 peat:1 perlite (v/v) medium after dipping in 0.8% indole-3-butyric acid. Rooted plantlets were acclimatized under greenhouse conditions to evaluate somaclonal variation.     

Shoot tip culture of Ribes nigrum in vitro

A procedure of shoot tip culture for commercial production of plantlets of Ribes nigrum is described. An average multiplication rate of 4.7 proliferated shoots was achieved within 21 days of shoot tip (1-2 mm) cultures on a Murashige and Skoog (MS) medium supplemented with 1-2 mg 1“1 6-benzylaminopurine and 0.1 mg T1 indole-3-acetic acid. Following 1-3 d of dark treatment with three proliferated shoots per culture tube on half-strength MS medium, 83-96% of these shoots rooted. When these rooted shoots were transferred to wooden boxes with vermiculite as supporting medium for hardening, 97% survived. Plantlets grew well after transplanting to the nursery field. It is concluded that the use of (i) smaller shoot tip explants during shoot profileration stage, (ii) initial three days of dark treatment during the root initiation stage, and (iii) vermiculite as a supporting medium for plantlets during the hardening stage, are economic, efficient and practical procedures for commercial production of plantlets of R. nigrum by shoot tip cultures.     

The effect of light,darkness and temperature on micropropagation of the pear rootstock BP10030

There was no effect of irradiance level on surviving percentages of shoot tip explants of the pear rootstock BP10030, but low irradiance stimulated the initial growth of the explant. Irradiance had a strong effect on shoot multiplication. With an increase in photosynthetic photon flux (PPF) from 10 to 80 μmol m^?2s^?1, shoot number and length and shoot fresh and dry weights increased. The greatest number of shoots and the longest ones were obtained with a 16 h photoperiod, while the highest fresh and dry weight of shoots were produced with a 24 h photoperiod. Rooting percentage and the number of roots were markedly promoted under 80 μmolm^?2s^?1 PPF. Photoperiods of 8, 16 and 24 h produced similar effects on rooting percentages and the numbers of roots. Four to seven days of darkness were the optimum for rooting. Rooting percentage and the number of roots increased with increased temperature during darkness between 5 and 25°C. A further increase in dark temperature up to 30°C reduced rooting percentage and root number.     

Adventitious shoot regeneration from in vitro leaves of adult Camellia reticulata

Experiments were conducted with Camellia reticulata cv. Captain Rawes to develop an adventitious regeneration system. Leaves were taken from axillary shoot proliferation cultures in WPM medium that had been established from mature trees. They were sectioned, and then plated in Petri dishes on media containing various combinations of cyto- kinins and auxins; the best response was induced by 2 mg I“1 BA + 1 mg I-1 IBA. The shoots obtained were multiplied by axillary branching on WPM + 2 mg I-1 BA + 2 mg I-1 Z + 2 mg I-12iP + 0.01 mg I-1 IBA. There was no significant difference in multiplication coefficient (the product of the proportion of explants forming axillary shoots and the mean number of new segments per explant) between shoots of adventitious and axillary origin, but there was between the various types of explant used in the multiplication stage: shoot tips (ST1) and nodal segments (ns) of harvested shoots longer than 14- 15 mm, and whole harvested shoots 7-10 mm long (ST2). The best results were achieved with ns and ST2 explants. Shoots of adventitious origin rooted very poorly in comparison with those of axillary origin under the same culture conditions.     

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.     

Effect of sequential subcultures on in vitro proliferation capacity and shoot formations pattern of pineapple (Ananas comosus L. Merr.) over different incubation periods

In vitro obtained shoots of pineapple (Ananas comosus L. Merr.) cv Smooth cayenne was cultured on MS medium enriched with 6-benzylaminopurine (BAP) at 3.25 mg l⁻¹ (14.43 μM) and indole acetic acid (IAA) at 1.75 mg 1⁻¹ (9.40 μM) and subcultured for four times at four different incubation periods (30, 45, 60 and 75 days). By the fourth subculture, irrespective of incubations periods, the explants lost 50% of its shoot formation capacity. Longer incubation resulted on higher rate and total shoots than a shorter incubation, however, the magnitude of that different varied over subcultures. The difference in shoots formation between the explants incubated for 30 and 75 days was 6 shoots at first, increased to 21 at the third and declined to 11 shoots at the fourth subculture. Over a period of 75 days, the majority of shoots formation occurred during the first 30 days (35%) at a rate of 1.5 shoot per week and the last 15 days (40%) at a rate of 3.8 shoot per week. In the period between 30 and 60 days, 15% of shoots at rate of 1.8 and 10% at rate of 1.0 shoot per week formed during the first and the second 15 days interval, respectively. Over four consecutive subcultures, explants incubated for 30 days produced the lowest total (1028 shoots) and that incubated for 75 days produced the highest total (120,917 shoots) from a single explant. No significant differences in total were observed between explants incubated for 45 and 60 days over the first three subcultures. However, by the fourth subculture the total of 60 days (14,288 shoots) was two times higher (7163 shoots) than that of 45 days long incubation.     

In vitro regeneration and conservation of wild species of Arachis

Summary

Wild species of Arachis are restricted to South America and generally occur in regions under intensive environmental disturbance. Both in situ and ex situ conservation strategies are required in order to maintain the availability of these genotypes. This work developed in vitro regeneration systems from seed explants of 17 wild species of Arachis from six Sections (Heteranthae, Caulorrhizae, Triseminatae, Erectoides, Procumbentes and Arachis). After seed disinfection, embryonic axes, leaflets and cotyledons were excised aseptically and cultured on Murashige and Skoog (MS) medium supplemented with 8.8 µM, 22 µM or 110 µM 6-benzylaminopurine (BAP). Cultures were maintained in a growth chamber at 28° ± 2°C with a 16 h photoperiod. Regeneration patterns from seed explants were similar among species from all Sections. Embryonic axes produced plants through meristematic amplification or multiple shoot formation, while cotyledons and embryonic leaflets produced shoots at significantly lower frequencies through direct and indirect organogenesis, respectively. Shoots obtained from all explants were transferred to MS medium without growth regulators to induce root formation.     

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.     

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 plant density and macronutrient nutrition on Delphinium shoot cultures

The effect of shoot density on the uptake of macronutrients from MS medium and growth rates of Delphinium shoot tissue cultures was determined. Multiplication rates and uptake of phosphate, nitrate and sugar per shoot increased with decreasing shoot density. Increasing the concentration of total nutrients significantly increased both fresh weight gain and multiplication rate at the high plant density (15 shoots 50 ml'1 medium) usually used for micropropagation. However, increasing the phosphate concentration (phosphate being the nutrient most rapidly depleted in the medium) resulted in higher fresh weight only, while the multiplication rates of shoots remained similar.     

In vitro high frequency direct plant regeneration from whole leaves of blackberry

High frequency and direct (without callus) plant regeneration was achieved from whole leaf explants of thornless blackberry (Rubus hybrid) cv. Black Satin (EC No. 381258; PI No. 553272) in vitro. Leaf blade explants from 1-, 3- and 5-month-old mother cultures were cultured on Murashige and Skoog (MS) medium with thidiazuron (TDZ), N⁶-benzylaminopurine (BAP), indol-3-butyric acid (IBA) and α-naphthalene acetic acid (NAA), alone or in combination. Three-month explants cultured on 0.02 mg l⁻¹ TDZ produced a high regeneration frequency (91.7%) and the most shoots/leaf explant (17.3). The shoot primordia developed within 3 weeks from the point of detachment of the petiole from the leaf blade. The age of the explant source significantly affected the shoot regeneration potential of the leaf explants. Leaves excised from 3-month-old in vitro-cultured shoots performed better than those from 1- and 5-month-old shoots. Shoots rooted best on half-strength MS basal medium with 0.5 mg l⁻¹ IBA and 90% of the plantlets survived acclimatization. The regenerated plantlets were morphologically similar to the mother plants.