Effect of CPPU on in vitro axillary shoot proliferation and adventitious shoot regeneration in kiwifruit

Abstract

The effect of CPPU, N‐(2‐chloro‐4‐pyridyl)‐N‐phenylurea, on the development of axillary buds and on adventitious shoot regeneration was investigated in mature leaves of in vitro‐cultured shoots of Actinidia chinensis Planch (Sel. K190) and Actinidia deliciosa A. Chev. cultivars Hayward and Tomuri. In the multiplication phase, 2.2 mM CPPU induced proliferation rates comparable to 4.4 mM benzyadenine (BA) both in Hayward and in Tomuri, while a higher CPPU concentration reduced proliferation. In A. chinensis, significant differences in multiplication rates were not detected between BA and CPPU. However, shoots developed on CPPU appeared hyperhydric, and had very short internodes, reduced leaf laminas, higher water, carotenoid and phenol contents and considerably lower chlorophyll level in comparison with the BA‐grown shoots. On the other hand, in adventitious shoot regeneration CPPU was more effective than zeatin (ZEA) and BA in A. deliciousa cultivars and the best morphogenic response was obtained with the lowest concentration (10 mM) in cv Hayward, while 16 mM was the most efficient treatment in cv Tomuri. In A. chinensis, CPPU was as efficient as ZEA when the highest concentration was used.     

Micropropagation of three berry fruit species using nodal segments from field-grown plants

Plant micropropagation of blueberry (Vaccinium corymbosum L. cv. Berkeley), blackberry (Rubus sp. cv. Smoothstem) and raspberry (Rubus idaeus L. cv. Gradina) was carried out from nodal segments of adult field‐grown plants. Hardwood and softwood cuttings were studied as explant sources. The cultures successfully established were softwood from all three species, and hardwood only from blueberry. Shoot‐bud establishment from blueberry was achieved by culturing explants in WPM salts with MS vitamins for 15 days, and then 30 days in the same medium with 18 mM Zeatin. The best results of multiplication were obtained in the same medium with 25 mM 2iP. For blackberry, shoot‐bud establishment was achieved by culturing explants in MS medium for 15 days, and then in the same medium with 4 mM BA and 0.25 mM IBA. This medium was also the best for blackberry multiplication. Raspberry explants (cvs Gradina and Willamette) were cultured in MS medium for 15 days and then transferred to MS medium supplemented with 4 μm BA and 0.25 mM IBA. After 30 days of culture, only ‘Gradina’ explants survived, from which shoot‐bud establishment was obtained in a modified MS medium (Anderson's macronutrients except calcium, with Sequestrene as the iron source) with the same growth regulators. Multiplication was achieved by subculturing explants in the same medium either with 4 mM BA plus 0.25 mM IBA or with 8 mM BA plus 0.25 mM IBA. Shoots of at least 1 cm in length from all species were rooted ex vitro in a mixture of peat and Perlite (1:1, v/v) in a mist chamber, and 100% of rooting plants were acclimated. Bacterial, fungal and viral diseases were detected in stock plants, while tests carried out in both shoots and regenerated plants revealed the absence of any kind of disease.     

Endogenous levels of abscisic acid,indole-3-acetic acid and benzyladenine during in vitro bud growth induction of Wild cherry (Prunus avium L.)

Levels of endogenous growth substances (abscisic acid: ABA; indole-3-acetic acid: IAA) and applied benzyladenine (BA) were quantified during the eight first days of in vitro propagation of Wild Cherry (Prunus avium L.). Axillary buds from the middle part of the explants started to grow at day 2, thus were released from apical dominance. Hormone levels were measured in the apical, median and basal parts of the explants using an avidin-biotin based enzyme-linked immunosorbent assay (ELISA) after a purification of the extracts by high performance liquid chromatography (HPLC). All hormones showed rapid and considerable changes during the first eight days of growth. Exogenous IBA was probably transformed into IAA mainly in the basal part of the explant, and BA penetrated quickly. ABA levels were transiently enhanced in the apical part of the explants bearing young leaves. These phenomena are discussed in connection with the axillary bud reactivation.     

In vitro micropropagation of an endemic fruit tree Syzygium alternifolium (Wight) walp

Shoot proliferation was obtained from shoot tip and nodal bud explants ofSyzygium alternifolium (Wight) walp on modified Murashige and Skoogs medium (MS) supplemented with either BA, KN or AD alone or BA in combination with either IAA, NAA or IBA. A combination of BA and auxins produced more shoots from both types of explants than on the medium containing only cytokinins. The highest multiplication rate was achieved with nodal bud explants in presence of 17.7M BA and 2.6M NAA. Excised shoots were rooted on half-strength MS medium with either IAA or IBA. The regenerated plantlets have been successfully acclimatized and transferred to soil. About 70% of plantlets have survived underex vitro conditions.Abbreviations BA-N⁶ Benzyladenine - KN Kinetin - AD Adenine - IAA Indole-3-aceticacid - IBA Indole-3-butyricacid - NAA-1 Naphthalene aceticacid - MS Murashige and Skoogs medium (1962)     

Organogenesis and somatic embryogenesis in Codiaeum variegatum (L.) Blume cv. “Corazón de Oro”

Summary Adventive organogenesis and somatic embryogenesis were induced from leaf explants taken from in vitro or in vivo plants of Codiaeum variegatum cv. “Corazón de Oro.” Shoot multiplication occurred with N⁶-benzyladenine (BA) alone, where the simultaneous production of adventitious buds and somatic embryos occurred at the fourth subculture, and on leaves not in contact with the medium. A medium with BA and 2,4 dichlorophenoxy acetic acid (2,4-D) produced the largest organogenic response, for both in vivo- and in vitro-produced explants. Somatic embryogenesis was only induced when such explants were transferred to a medium lacking 2,4-D. Thus, a medium with BA only produced the largest percentage of explants with shoots and embryos. Replacing BA with thidiazuron induced up to 100% bud regeneration on in vitro-produced explants by 60 d, but was slower for in vitro-grown explants. Both types of embryos exhibited growth arrest that was partially overcome by transfer to hormone-free basal medium with activated charcoal. Rooted plants from all explants were successfully obtained on a medium with indole-butyric acid (IBA).     

Plantlets from somatic callus tissue of the East Indian Rosewood (Dalbergia latifolia Roxb.)

Callus-mediated shoot bud formation was demonstrated in Dalbergia latifolia Roxb. (East Indian Rosewood). Cultures were raised from shoot explants of six year-old plants on Murashige and Skoog (MS) medium supplemented with naphthaleneacetic acid (NAA) and benzyladenine (BA). A sequential treatment of callus with increasing BA levels and decreasing NAA ensured shoot bud induction. Rooting of shoots was achieved by a three-step culture procedure involving 1) White's(W) liquid medium containing indoleacetic acid (IAA), naphthaleneacetic acid and indolebutyric acid (IBA), 2) half-strength MS agar-solidified medium with charcoal (0.25%) and 3) half-strength MS liquid medium.Abbreviations BA Benzyladenine - IAA Indoleacetic acid - IBA Indolebutyric acid - MS Murashige and Skoog - NAA a-naphthaleneacetic acid - PVP Polyvinylpyrrolidone - W White's medium - 2,4-D 2,4-dichlorophenoxyacetic acid     

Multiple shoot induction by benzyladenine and complete plant regeneration from seed explants of chickpea (Cicer arietinum L.)

The efficacy of benzyladenine (BA) to induce multiple shoots from seed explants of chickpea (Cicer arietinum L.) was assessed. Shoot differentiation was influenced by the type of seed explant, genotype and concentration of BA. Orientation of the explant also strongly influenced the shoot regeneration response. The optimum BA concentration for shoot/shoot bud regeneration was genotype dependent. Two types of BA-induced response were observed: (1) at less than 7.5 gm BA, direct shoot differentiation (2 to 4-cm-long shoots) was observed within 30 days; (2) at higher BA concentrations (75–100 m), shoot/shoot bud differentiation was achieved in 45–90 days. A high BA concentration inhibited subsequent rooting of shoots. Roots, however, could be easily induced on shoots derived from <12.5 m BA. Following transfer to soil, 80% of the regenerants developed into morphologically normal and fertile plants.Abbreviations BA Benzyladenine     

Uptake, Distribution, and Metabolism of 1-Naphthaleneacetic Acid and Indole-3-Acetic Acid During Callus Initiation From Actinidia deliciosa Tissues

1-Naphthaleneacetic acid (NAA) and 6-benzyladenine (BA) were required for in vitro callus formation at the basal edge of kiwifruit (Actinidia deliciosa [A. Chev] Liang and Ferguson, cv. Hayward) petioles. The uptake, metabolism, and concentration of NAA and indole-3-acetic acid (IAA) content were examined in the explants during the callus initiation period. After 1, 6, 12, 24, 48, and 96 h of culture in the presence of [H³]NAA, petioles were divided into apical, middle, and basal portions and analyzed. Except for a high IAA level measured at 12 h, IAA content decreased in tissues during a culture period of 96 h. NAA uptake was higher in petiolar edges than in the middle portion, and NAA was rapidly conjugated with sugars and aspartic acid inside the tissues. The amide conjugation was triggered in apical and basal portions from 12 h and in the middle part from 48 h, with α-naphthylacetylaspartic acid being the major metabolite. Free-NAA concentration in cultured petioles achieved an equilibrium with the exogenously applied NAA (0.27 μm) from 12 h, and it remained constant thereafter. The relationships between the role attributed to NAA and BA in the initiation and the maintenance of disorganized growth of callus in kiwifruit cultures are discussed. Received December 21, 1998; accepted July 20, 1999     

Shoot Bud Proliferation from Axillary Nodes and Leaf Sections of Non-toxic <Emphasis Type="Italic">Jatropha curcas</Emphasis> L.

Protocols for in vitro propagation of non-toxic variety of J. curcas through axillary bud proliferation and direct adventitious shoot bud regeneration from leaf segments have been established. Shoot bud proliferation from axillaries was assessed on an initial basal Murashige and Skoog (MS) salt medium supplemented with different concentrations of benzyladenine (BA), kinetin and thidiazuron (TDZ) followed by subculture to medium with 4.4-8.9 μM BA. Regardless of the concentration of BA in the subculture medium, shoot multiplication rate was optimum (10–12.3) with primary culture on medium supplemented with 2.3–4.5 μM TDZ. Efficient adventitious shoot regeneration from leaf tissues was achieved with culture on medium with 8.9–44.4 μM BA + 4.9 μM indole-3-butyric acid (IBA) followed by transfer to medium supplemented with 8.9 μM BA + 2.5 μM IBA. Similarity index between toxic Indian variety and the non-toxic variety based on 435 RAPD markers was 96.3%. Crossing studies followed by phorbol ester quantitation revealed that outcrosses with toxic J. curcas do not affect the phorbol ester content of seeds borne on the non-toxic variety.     

High-frequency plant regeneration from coleoptile tissue of indica rice (Oryza sativa L.)

Summary An efficient method was established for high-frequency embryogenic callus induction and plant regeneration from 3-,4-, 5- and 7-d-old coleoptile segments of Indica rice (Oryza sativa L. cv. Kasturi), Compact and friable callus developed from the cut ends and also on the entire length of the coleoptile segments cultured on Murashige and Skoog (MS) basal medium (1962) supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D, 4.50–18.0 μM), kinetin (2.32 μM) and sucrose (3%, w/v). High frequency embryogenic callus induction and somatic embryo development was achieved when embryogenic calluses were transferred to MS medium supplemented with 2.25 μM 2,4-D, 2.32 μM kinetin, 490 μM L-tryptophan and 3% (w/v) sucrose. Plant regeneration was achieved by transferring clumps of embryogenic callus onto MS medium containing 2.85 μM indole-3-acetic acid (IAA), 17.77 μM 6-benzylaminopurine (BA) and 3% (w/v) sucrose. Histological observations of embryogenic calluses revealed the presence of somatic embryos and also plant regeneration via multiple shoot bud formation. Three, 4- and 5-d-old coleoptile segments showed a significantly (P<0.05) higher frequency of plant regeneration and mean number of plantlets per explant in comparison to 7-d-old coleoptile segments. The highest frequency (73.5%) of plant regeneration and mean number of plantlets (11.9±1.0) was obtained from 4-d-old coleoptile segments. Regenerated shoots were rooted on MS basal medium containing 4.92 μM indole-3-butyric acid (IBA) and plants were successfully transferred to soil and grown to maturity.     

Rapid clonal propagation of <Emphasis Type="Italic">Curculigo orchioides</Emphasis> Gaertn., an endangered medicinal plant

An efficient protocol was developed for in vitro clonal propagation of Curculigo orchioides Gaertn. through apical meristem culture. Multiple shoots were induced from apical meristems grown on Murashige and Skoog (MS) basal medium supplemented with 1.5 mg l⁻¹ 6-benzyladenine (BA), 100 mg l⁻¹ adenine sulfate (Ads) and 3% sucrose. Inclusion of indole-3-butyric acid (IBA) or indole-3-acetic acid (IAA) in the culture medium improved the formation of multiple shoots. The highest frequency of multiplication was obtained on MS medium supplemented with 1.5 mg l⁻¹ BA, 100 mg l⁻¹ Ads, 0.25 mg l⁻¹ IBA and 3% sucrose. Rooting was achieved upon transferring the micro-shoots to half-strength MS medium containing 0.25 mg l⁻¹ IBA and 2% sucrose. Micropropagtated plantlets were hardened in the greenhouse and successfully established in soil.     

Gibberellins and <Emphasis Type="Italic">N</Emphasis>-(2-chloro-4-pyridyl)-<Emphasis Type="Italic">N′</Emphasis>-phenylurea improve retention force and reduce water core in pre-mature fruit of Japanese pear cv. Housui

A study of the effect of a gibberellin A₃ + A₄ mixture (GAs) on pre-harvest fruit drop of Japanese pear cv. Housui is reported. The GAs was applied alone or in combination with N-(2-Chloro-4-pyridyl)-N′-phenylurea (CPPU) in a lanolin paste to the abscission zone at the spur-end of the pedicel. The results showed that the GAs and CPPU combination treatment increased the pedicel–spur retention force relative to that of the untreated controls. Histological studies showed an accelerated rate of cambium division and the development of secondary xylem in the abscission zone near the spur-end of pedicels treated with the GAs plus CPPU paste, which delayed the formation of the abscission zone. Fruit quality (sugar, acid, firmness, color) was not adversely affected by the application of GAs plus CPPU, although the application of GAs alone promoted ripening. In contrast, the addition of CPPU to the two GAs delayed fruit ripening, which was measured as ethylene efflux. All treatments were without adverse effects on return bloom, measured as bud size. The CPPU plus GAs treatment also suppressed the incidence of water core, whereas the application of the GAs alone accelerated water core in this water core susceptible pear variety.     

Micropropagation and encapsulation of medicinally important Chonemorpha grandiflora

Summary This study describes a protocol for in vitro propagation of Chonemorpha grandiflora (Roth) S.M. & M.R. Almeida (Apocynaceae) through axillary bud proliferation and rooting. For shoot induction, the combination of 13.3 &#x03BC;M N ⁶-benzyladenine (BA) and 2.46 &#x03BC;M indole-3-butyric acid (IBA) in Murashige and Skoog (MS) medium was better than other growth regulators. Liquid medium was superior over solid medium for root formation and growth. IBA was more effective than other auxins for root induction. Addition of silver nitrate in the presence of IBA significantly increased the number, length, and branching of roots. Shoot tips encapsulated in medium containing 0.49 &#x03BC;M IBA and 11.7 &#x03BC;M silver nitrate exhibited 95% conversion to plantlets. This protocol enables the production of 250 plantlets from a single nodal explant within 7 mo. Plantlets showed 90% survival after acclimatization in soil and were morphologically similar to the source plant under field conditions.     

adventitious shoot regeneration from petiole explants of Heracleum candicans wall

Summary A method for adventitious shoot induction from petiole explants of Heracleum candicans is reported. Shoot buds were induced on Murashige and Skoog (MS) medium with 4.4μM 6-benzylaminopurine (BA) and 1.1 μM 2,4-dichlorophen-oxyacetic acid (2,4-D). A wound response in the presence of BA and 2,4-D at the time of culture was necessary for inducing shoot buds. The shoot bud regeneration was significantly influenced by size, type and orientation of explants on the culture medium. These shoot buds developed into 4–5 cm shoots upon transfer to a medium containing 1.1μM BA and 0.5 μM α-naphthaleneacetic acid (NAA). The regenerated shoots formed rooted plantlets on MS medium supplemented with 4.9 μM indole-3-butyric acid (IBA). About 15 plants were established in the field for further evaluation.     

Production of solamargine by in vitro cultures of Solanum paludosum

Callus cultures of Solanum paludosum were established from roots, hypocotyles, cotyledons and leaf limbs of plantlets cultivated in sterile conditions on a Murashige and Skoog's modified medium. Non organogenous calluses were obtained with addition of BA or kinetin (10^-5M to 10^-6M) as the cytokinin and 2,4-d or NAA (10^-5M to 10^-6M) as the auxin. These calluses permitted the establishment of a cell suspension culture with BA (10-6M) and 2,4-d (10^-6M). Zeatin (10^-6M) with IAA (10^-6M) gave rise to organogenous calluses. These organogenous callus cultures developed multiple shoots which either proliferated if they were cultivated on a medium containing zeatin with IAA or IBA or were able to regenerate into whole plants when zeatin was used as the only hormone. The different plant material produced solamargine, the main steroidal glycoalkaloid present in the unripe fruits. The best production was obtained with the fruits of regenerated plants from organogenous callus cultures after reintroduction of these plants in their brasilian biotope. The solamargine content of the two types of plant materials was about 0.06% and 2.5% (dry weight) respectively for the callus cultures and the fruits from in vitro plants. The fruits were harvested a year after the beginning of the plantlet regeneration step.Abbreviations HPTLC high performance thin layer chromatography - HPLC high performance liquid chromatography - 2,4-d 2,4-dichlorophenoxyacetic acid - BA benzylaminopurine - IAA 3-indolebutyric acid - NAA -naphthaleneacetic acid - IBA 3-indolebutyric acid - IPA isopentenyladenine     

生长素与乙烯在沙田柚上胚轴不定芽再生中的作用

结果表明,6 BA只能诱导较低频率的不定芽再生,IBA的加入可以促进不定芽的再生。从再生率与单位外植体再生芽数综合考虑,以高浓度IBA(1.5mg·L 1)与低浓度6 BA(0.5mg·L 1)配合的效果最佳,加入生长素极性运输的调节剂TIBA与Flavone可进一步提高不定芽的再生频率。乙烯抑制上胚轴不定芽的再生,而乙烯生理作用的拮抗剂Ag+与生物合成抑制剂AOA、Co+可以显著提高不定芽再生频率。水杨酸(SA)也抑制不定芽的再生。     

Interfamilial cell fusion among leaf protoplasts of Populus alba, Betula platyphylla and Alnus firma: assessment of electric treatment and invitro culture conditions

The optimal conditions for fusion of leaf protoplasts of Populus alba, Betula platyphylla, and Alnus firma by electric treatment were alternate current (AC) 200 V cm⁻¹ in 2.5 mM CaCl₂ for a pearl chain formation and direct current (DC) pulse of 100 μs at 2 kV cm⁻¹ After interfamilial cell fusion treatment, colonies were obtained using liquid media containing 2,4-D or NAA as an auxin and BA or CPPU as a cytokinin at 0.1, 1, or 10 µM in MS (Murashige and Skoog (1962) Physiol. Plant. 15: 473--497), 1/2salt MS, or NH₄NO₃-free MS containing 0.6 M mannitol and 3% sucrose (totaling 147 combinations). Two shoots after electric cell fusion treatment between P. alba and B. platyphylla, and 12 regenerated plants after electric cell fusion between P. alba and A. firma, were obtained from colonies induced on agar medium containing NAA, IBA, CPPU, and BA. Seven lines of the latter 12 plants which were regenerated later cultured in vitro had serrated leaves different from those of P. alba.     

In vitro clonal propagation of an aromatic medicinal herb Ocimum basilicum L. (sweet basil) by axillary shoot proliferation

Summary An efficient protocol for in vitro propagation of an aromatic and medicinal herb Ocimum basilicum L. (sweet basil) through axillary shoot proliferation from nodal explants, collected from field-grown plants, is described. High frequency bud break and maximum number of axillary shoot formation was induced in the nodal explants on Murashige and Skoog (1962) medium (MS) containing N⁶-benzyladenine (BA). The nodal explants required the presence of BA at a higher concentration (1.0 mg·l⁻¹, 4.4 μM) at the initial stage of bud break; however, further growth and proliferation required transfer to a medium containing BA at a relatively low concentration (0.25 mg·gl⁻¹, 1.1 μM). Gibberellic (GA₃) at 0.4 mg·l⁻¹ (1.2 μM) added to the medium along with BA (1.0 mg·l⁻¹, 4.4 μM) markedly enhanced the frequency of bud break. The shoot clumps that were maintained on the proliferating medium for longer durations, developed inflorescences and flowered in vitro. The shoots formed in vitro were rooted on half-strength MS supplemented with 1.0 mg·l⁻¹ (5.0 μM) indole-3-butyric acid (IBA). Rooted plantlets were successfully acclimated in vermi-compost inside a growth chamber and eventually established in soil. All regenerated plants were identical to the donor plants with respect to vegetative and floral morphology.     

In vitro propagation of banana (Musa spp.): initiation,proliferation and development of shoot-tip cultures on defined media

In vitro multiplication of banana (Musa spp.) from shoot-tip explants isolated from lateral suckers is described. Using explants with apical domes, a total of 22 banana cultivars were successfully cultured on a modified Murashige and Skoog's medium containing 6-benzylaminopurine (BA) and indolebutyric acid (IBA). Shoot-tip explants could be induced to produce multiple shoot initials in the presense or absence of apical domes, but the survival rates were higher if apical domes were retained. Cultivars varied widely in their multiplication rates in response to cytokinins, BA being consistently more effective than kinetin (Kn). Although Kn was less effective in this regard, it stimulated vigorous root growth. Rooted plantlets were successfully established in soil.