GROWTH REGULATOR EFFECTS ON BUD INITIATION IN CALLUS CULTURES OF MEDICAGO SATIVA

Influence of growth regulators on bud initiation in callus of alfalfa (Medicago sativa L.) was studied by varying levels and combinations in the first medium of a two-medium sequence used to obtain whole plants. Callus of tetraploid clone S-4 (cv. ‘Saranac‘) was initiated from immature ovaries on a modified Blaydes' basal medium containing all combinations of six concentrations (0–36 μM) of kinetin (K), six concentrations (0–44 μM) of naphthaleneacetic acid (NAA), and seven concentrations (0–36 μM) of 2,4-dichlorophenoxyacetic acid (2,4-D). After 28 days the callus was challenged to form buds by transfer to the modified Blaydes' medium containing 2.0 g/liter yeast extract and 0.57 mM inositol. No buds were produced in the absence of 2,4-D in the first medium, and the frequency of bud formation on the second medium was directly proportional to the 2,4-D concentration in the range 2.3–54 μM in the preceding medium. Buds were produced in the absence of kinetin in the first medium, but its presence in the range 2.3–36 μM markedly increased bud formation. NAA was not required for bud formation, and the budding frequency increased only slightly with increasing NAA concentration in the first medium. Budding of callus of two other alfalfa clones was also influenced by the 2,4-D concentration in the initial medium. There were several indications that many of the buds were initiated on the first medium and completed development on the second medium. These included the differential effect on budding of combinations of 2,4-D, NAA and kinetin in the callus initiation medium, the specific media sequence required, and the presence of embryoids on the callus which after transfer to the yeast extract-inositol medium produced buds.     

Direct development of plantlets from immature panicles of rice in vitro

Cultured immature panicles of rice formed plantlets from spikelets without callus or embryoid formation on MS and HE media containing 2 mg/l each of NAA and kinetin. Developmental stage, ploidy of explant and plant growth regulators in the medium are the major factors affecting the frequency of spikelet budding in young panicle culture. It is suggested that spikelet budding occurs by the reversion of floral primordia to vegetative stage or by the formation of adventitious buds from epidermal cells.     

Node-derived cultures with high-morphogenic competence in barley and wheat

A successful regeneration system is presented for elite cultivars in barley and wheat based on nodes. Nodal explants were excised from in vitro and ex vitro grown plants. A combination of 8.28 μM 4-amino-3,5,6-trichloropicolinic acid and 4.54 μM to 22.71 μm thidiazuron (TDZ) used in MS-based medium containing 60 g l⁻¹ maltose favoured induction of clumps of multiple shoots/buds with or without callus formation in the primary cultures. Within 8–10 weeks upon further subcultures, the proliferation into callus with rapid and continuously forming adventitious buds containing clusters of meristemoids, termed meristematic bulk tissue (MBT) was obtained. Lowering the levels of growth regulators resulted in redifferentiation of shoots, which elongated, rooted, developed into morphologically normal plants and set seeds normally. With a frequency ranging between 37 and 82% the nodes raised from in vitro grown plants were proliferated into MBT independent of TDZ concentration, cultivar and species. The average number of shoots per responding node in different cultivars was 7–15 in barley and 1–6 in wheat after 12–14 weeks. Nodes from greenhouse grown plants mainly responded for callus formation with poor development of MBT.     

Effects of ionizing radiation (60Co gamma rays) on growth and morphogenesis ofHaworthia mirabilis,haw. Callus tissue

Summary The effects of γ-radiation on growth and morphogenesis ofHaworthia callus in vitro were determined. The doses ranged from 100 to 5000 rads. Survival, growth pattern, growth rate, and differentiation of vegetative buds and roots in both irradiated and nonirradiated callus were compared. Growth data up to 24 weeks for irradiated and control cultures were analyzed. The dose range between 800 to 2500 rads produced compact callus as compared to the controls which were friable. After 12 weeks all control cultures differentiated vegetative buds with roots, whereas callus exposed to 800 to 2500 rads continued to grow with little or no organogenesis. However, it was observed that the wet and dry weights of callus receiving 1000 to 1500 rads ultimately exceeded those of nonirradiated controls.     

Micropropagation of Pinus heldreichii

Micropropagation by organogenesis from mature embryos of Pinus heldreichii Christ. was achieved. The frequency of adventitious bud induction was higher on embryos grown on Gresshoff and Doy medium than on Von Arnold and Eriksson, or Murashige and Skoog medium. The greatest number of buds and developed shoots was obtained after induction with benzyladenine at 2.22 or 4.40 μM for four weeks. Shorter induction time was less effective for bud induction, but subsequent shoot elongation was accelerated. Shoots elongated on half-strength, growth regulator-free medium supplemented with activated charcoal. After pulse treatment with 1 mM indole-3-butyric acid twenty shoots were rooted, while agar-solidified medium supplemented with α-naphthaleneacetic acid (0.27 or 1.08 μM), or indole-3-butyric acid (0.25 or 0.98 μM) induced callus formation only. This revised version was published online in June 2006 with corrections to the Cover Date.     

Polyamine metabolism and in vitro cell multiplication and differentiation in leaf explants of Chrysanthemum morifolium Ramat

Arginine decarboxylase (ADC), ornithine decarboxylase (ODC), diamine oxydase (DAO) free amine and conjugated amine titers were estimated in leaf explants of Chrysanthemum morifolium Ramat. var. Spinder cultivated in vitro in relation to hormone treatment. Addition of benzyladenine (BA) to a basal medium caused the formation of buds on the explants. BA plus 2,4 dichlorophenoxyacetic acid (2,4 D) caused callus formation and proliferation. Formation of roots was obtained by addition of indolylacetic acid (IAA). Arginine decarboxylase (ADC) ornithine decarboxylase (ODC) and diamine oxidase (DAO) activities increased during the first days of culture when cell multiplication was rapid, followed by a sharp decline as the rate of cell division decreased and differentiation took place. DAO activities increased rapidly in proliferating and growing organs and decreased during maturity. This increase was concomitant with ADC and ODC activities and polyamine content (free and conjugated polyamines). The biosynthesis and oxidation of polyamines which occurred simultaneously in physiological states of intense metabolism such as cell division or organ formation were directly correlated. In callus cultures DAO activity was blocked throughout development and regulated neither the cellular levels of polyamines nor polyamine conjugates. Levels of polyamine conjugates were high in callus cultures throughout development. In foliar explants cultivated on a medium promoting callus, inhibition of ODC activity by DFMO (-DL-difluoromethylornithine, a specific enzyme-activated ODC inhibitor) resulting in an amide deficiency facilated the expression of differentiated cell function; substantial activation of DAO was observed until the emergence of the buds. On a medium promoting bud formation, -OH ethylhydrazine (DAO inhibitor) promoted callus formation without differentiation. In this system DAO activity was blocked and there were high levels of polyamines, especially polyamine conjugates, throughout the culture period. The relationship among free and conjugated polyamines related biosynthetic enzyme activities, DAO activities, cell division and organ formation is discussed.Abbreviations ADC = arginine decarboxylase - ODC = ornithine decarboxylase - DOA = diamine oxidase - DFMA = -DL-difluoromethylarginine - DFMO = -DL-difluoromethylornithine - Put = putrescine     

Influence of putrescine on anthocyanin production in callus cultures of <Emphasis Type="Italic">Daucus carota</Emphasis> mediated through calcium ATPase

The influence of Putrescine (Put) on the growth and elicitation of anthocyanin in callus cultures of Daucus carota var. Nantes scarlet was investigated through the use of α-DL-difluoromethylarginine (DFMA), the polyamine (PA) biosynthetic inhibitor. It was observed that the addition of Put (0.05 mM) resulted in enhancement of growth and anthocyanin content. The anthocyanin content was found to be enhanced by 1.68 fold on the 21^st day as compared to the untreated controls. The PA inhibitor was found to result in lowering of the growth and the anthocyanin accumulation, which could be partially restored by the addition of Put in combination with this inhibitor. The levels of Ca²⁺ ATPase were also found to be elevated in treatment with Put suggesting the involvement of calcium in the elicitation of anthocyanin. The endogenous titres of PAs and the ethylene production under these treatments were also studied. The treatment with DFMA resulted in lower levels of endogenous PAs and higher levels of ethylene. Lowering of ethylene by putrescine treatment shows that PA treatment also inhibited ethylene formation, which would also imply that endogenous ethylene does not influence anthocyanin production in carrot callus cultures.     

Direct shoot regeneration from leaf explants of Jatropha curcas in response to thidiazuron and high copper contents in the medium

Leaf explants of Jatropha curcas cultured on Murashige and Skoog’s (MS) medium supplemented with thidiazuron (TDZ; 0.90 μM) in combination with indole-3-butyric acid (IBA; 0.98μM) produced adventitious shoot buds directly on the surface of the explants without formation of intervening callus while shoot bud formation was accompanied with callus formation on medium supplemented with 6-benzylaminopurine (BAP; 13.3 μM) and IBA (2.46 μM). TDZ treatment resulted in more than twice higher rate of shoot bud induction than BAP. Shoot buds were multiplied and elongated following repeated transfers to medium containing BAP (2.22 μM) and gibberellic acid (GA₃; 1.44 μM). The effect of copper sulphate on differentiation of shoot buds from leaf segments was also investigated. Both shoot induction and multiplication media were supplemented with different levels of CuSO₄ (0–5 μM). Significant improvement in shoot bud induction was observed when the concentration of CuSO₄ was increased to 10 times the normal MS level. Healthy elongated shoots were rooted on half strength MS medium supplemented with IBA (2.46 μM). Rooted plantlets were transferred to field and survived. Histological analysis revealed direct formation of shoot buds from leaf explants.     

Organogenesis, shoot regeneration, and flowering response of Vernonia cinerea to different auxin/cytokinin combinations

Summary A viable protocol has been developed for direct and indirect shoot regeneration of Vernonia cinerea. To establish a stable and high-frequency plant regeneration system, leaf and stem explants were tested with different combinations of α-naphthalene acetic acid (NAA), indole-3-acetic acid (IAA), and benzylaminopurine (BA). Lateral buds on nodal explants grew into shoots within 2 wk of culture in Murashige and Skoog (MS) basal medium supplemented with 20.9 μM BA. Excision and culture of nodal segments from in vitro-raised shoots on fresh medium with the same concentration of BA facilitated development of more than 15 shoots per node. Similarly leaf, nodal, and internodal explants were cultured on MS basal medium supplemented with different concentrations of BA, NAA, and IAA either alone or in combinations for callus induction and organogenesis. Shoot buds and/or roots were regenerated on callus. Shoot buds formed multiple shoots within 4 wk after incubation in induction medium. Adventitious buds and shoots proliferated when callus was cut into pieces and subcultured on MS basal medium containing 20.9 μM BA and 5.3 μM NAA. This combination proved to be the best medium for enhanced adventitious shoot bud multiplication, generating a maximum of 50 shoots in 4 wk. This medium was also used successfully for shoot proliferation in liquid medium. Root formation was observed from callus induced in medium containing 8.05–13.4 μM NAA. Regenerated shoots exhibited flowering and root formation in MS basal medium without any growth regulators. Plantlets established in the field showed 85% survival and exhibited identical morphological characteristics as the donor plant.     

Embryogenic callus culture of Tribulus terrestris L. a potential source of harmaline, harmine and diosgenin

In the present study, a simple one medium formulation protocol for callus culture, somatic embryogenesis and in vitro production of β-carboline alkaloids and diosgenin in Tribulus terrestris L. was developed. Extensive callus induction and proliferation was obtained in leaf explant on Murashige and Skoog (MS) medium supplemented with 5.0 μM 6 benzyl adenine (BA) and 2.5 μM α-naphthaleneacetic acid (NAA). The embryogenic callus was maintained on subculture to fresh parental medium at 4-week intervals over a period of 28 months. The frequency of embryo formation was at a maximum (18.1 ± 0.9 per g of callus) on MS medium containing 5.0 μM BA and 2.5 μM NAA together with 75 mg l⁻¹ casein hydrolysate. Globular embryo developed into torpedo stage embryo under the influence of starvation. The accumulation of β-carboline alkaloids (harmaline and harmine) and steroidal saponin (diosgenin) in non-embryogenic and embryogenic callus culture derived from leaf explant was compared with root, leaf, stem, and fruit of the mother plant. The embryogenic callus accumulated equivalent amounts of harmaline (66.4 ± 0.5 μg/g dry weight), harmine (82.7 ± 0.6 μg/g dry weight), and diosgenin (170.7 ± 1.0 μg/g dry weight) to that of the fruit of T. terrestris. The embryogenic callus culture of this species might offer a potential source for production of important pharmaceuticals.     

烟草花柄愈伤组织花芽分化的能力(简报)

来源于开花植株的外植体(如花柄、花序轴等)具有在离体培养条件下直接分化花芽的能力,这一现象已在数十种植物的组织培养中得到证实。但是,这种成花能力能否保留在由这些外植体形成的愈伤组织之中?已有报道在风信子、布罗瓦利亚花、石龙芮、大蒜、矮通泉草等值物的愈伤组织中得到无     

In vitro plant regeneration from callus of shoot apices in apple shoot culture

A new reliable protocol for the induction of adventitious shoot formation and plant regeneration from apple callus has been developed. High regeneration frequency was obtained with this method in four different genotypes (Jork9, M26, Gala and McIntosh) and callus maintained regeneration ability for several months. The procedure consists of inducing vegetative shoot apices, excised from in vitro shoots, for 20 days in darkness on an MS medium without glycine, supplied with 17.8 μM BA, 2.7 μM NAA and 250 mg l⁻¹ cefotaxime. The explants are then transferred to a fresh auxin-free medium and given light. Histological studies revealed that all the regenerated shoots originated from callus. Regenerated shoots were multiplied, rooted and successfully established in soil. Received: 2 April 1999 / Revision received: 10 November 1999 / Accepted: 15 November 1999     

Organogenesis and somatic embryogenesis from callus cultures in Muscari armeniacum Leichtl. ex Bak.

Summary Establishment of fast-growing, highly regenerable callus cultures was examined in Muscari armeniacum Leichtl. ex Bak. in order to develop an efficient genetic transformation system. High-frequency callus formation was obtained from leaf explants of cv. Blue Pearl on media containing 2,4-dichlorophenoxyacetic acid (2,4-D), α-naphthaleneacetic acid (NAA) or 4-amino-3,5,6-trichloropicolinic acid (picloram, PIC). Fast-growing, yellowish nodular callus lines and white friable callus lines containing a few somatic embryos were established on initiation medium supplemented with 4.5 μM 2,4-D and with 54 μM NAA, respectively. The yellowish nodular calluses vigorously produced shoot buds after transfer to media containing 0.44–44 μM 6-benzyladenine (BA), whereas the white friable calluses produced numerous somatic embryos upon transfer to plant growth regulator-free (PGR-F) medium. Histological observation of shoot buds and somatic embryos indicated that the former consisted of an apparent shoot meristem and several leaf primordia, and the latter had two distinct meristematic regions, corresponding to shoot and root meristems. Both shoot buds and somatic embryos developed into complete plantlets on PGR-F medium. Regenerated plants showed no observable morphological alterations. High proliferation and regeneration ability of these calluses, were maintained for over 2 yr.     

Morphological and cytological diversity of regenerants derived from half-anther cultures of anthurium

Anthurium anther culture was successfully established using half-anthers as explants. Explants were cultured on Winarto–Teixeira basal medium (WT-1) containing 0.01 mg/l α-naphthalene acetic acid (NAA), 0.5 mg/l thidiazuron (TDZ), and 1.0 mg/l 6-benzylaminopurine (BAP), or on New Winarto–Teixeira basal medium (NWT-3) supplemented with 0.02 mg/l NAA, 1.5 mg/l TDZ, and 0.75 mg/l BAP for callus initiation. Regenerated calli produced multiple shoots on WT-1, which were then rooted in NWT-3 supplemented with 1% activated charcoal. Plantlets were acclimatized ex vitro using a mixture of burned rice husk, rice husk, and bamboo peat (1:1:1, v/v/v) as the potting medium. There was considerable morphological and cytological diversity of regenerants derived from anther culture, which are described in detail in this study. The callus cluster color ranged from green to light green and had a high regeneration capacity (7.3 and 4.8 shoots/callus cluster), light reddish-yellow callus showed moderate regeneration (2.6 shoots/callus cluster), while reddish-yellow callus had the lowest regeneration capacity (1.5 shoots/callus cluster). Morphological variations clearly observed in regenerants derived from this technique included alterations in plant size, peduncle length, spathe position compared to leaves, the type and number of buds, spathe and spadix color, and spadix length. There were also cytological variations in both in vitro and ex vitro regenerants of anther culture with 23–29% haploids, 5–10% aneuploids, 56–69% diploids, and 3–4% triploids. The results strengthen other studies in which the development of anther cultures, especially via callus formation, resulted in morphological and cytological alterations. These variations have been discussed to great length in this paper.     

Production of triploid plants of mulberry (Morus alba L) by endosperm culture

 A continuously growing callus was obtained from immature endosperm of Morus alba L Cv S-36 cultured on Murashige and Skoog medium containing 5 μm 2,4-dichlorophenoxyacetic acid. Shoot buds were produced when the callus was subcultured on a medium containing a cytokinin or a cytokinin and 1-naphthaleneacetic acid (NAA). The maximum number of shoots was formed on the medium containing thidiazuron (1 μM), or benzylaminopurine (5 μM) and NAA (1 μM). Shoots were multiplied by forced axillary branching and rooted in vitro. Endosperm-derived plants were established in soil. Each of the ten plants examined cytologically was triploid (3 n=42). Received:17 February 1999 / Revision received: 4 May 1999 / Accepted: 19 May 1999     

Callus Growth and Proline Accumulation in Response to Sorbitol and Sucrose-Induced Osmotic Stress in Rice

This study investigated the influence of osmotic stress, induced by sorbitol and sucrose combinations, on growth and proline accumulation in callus cultures of rice (Oryza sativa L.). Dehusked mature seeds, cv. Hassawi, were induced to callus on MS medium supplemented with 4.52 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.32 µM 6-furfurylaminopurine (kinetin). The medium also contained 29.2, 58.4, 87.6, and 116.8 mM sucrose combined with 0, 54.9, 109.8, and 164.7 mM sorbitol. Callus formation was observed in about 35 % of the cultured seeds irrespective of the sugar treatment. An increase in callus mass was observed as sucrose concentration increased reaching a maximum growth at 87.6 mM. Callus growth was enhanced in response to 54.9 mM sorbitol but at higher concentration it was inhibitory. Best callus growth was obtained on a medium containing 54.9 mM sorbitol combined with 87.6 mM sucrose. Increasing osmotic stress, as a consequence of increasing sucrose and sorbitol concentrations, induced proline accumulation and the highest concentration of proline, 5.8 µmol g^–1(f.m.), was obtained on 164.7 mM sorbitol combined with 116.8 mM sucrose.     

Adventitious bud formation in Alhagi graecorum

Various parts of seedlings and in vitro propagated shoots of Alhagi graecorum Boiss were cultured on different media with different 6-benzyladenine (BA) and kinetin (KIN) concentrations to compare their potential to regenerate shoots. Murashige and Skoog (MS) medium with 2.5 μM BA and hypocotyl gave the best results. Callus was obtained from stem segments on MS medium supplemented with 2.5 μM BA, 5 μM 1-naphthaleneacetic acid (NAA) and 0.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Shoot formation from callus occurred upon its transfer to MS medium supplemented with 2.5 μM BA. Mature explants which showed a relatively low potential for adventitious buds or callus formation, regenerated shoots abundantly using the tiny-mature-explant method. The regenerated shoots were rooted on half strength MS medium supplemented with 5 μM 3-indolebutyric acid (IBA). This revised version was published online in June 2006 with corrections to the Cover Date.     

Micropropagation ofLarix decidua Mill. andpinus sylvestris L.

Shoot multiplication of Larixdecidua was achieved using axillary and adventitious buds. The formation of axillary buds was stimulated on shoot tips soaked in a cytokinin solution (BAP 10-50 mg 1⁻¹ for 2–4 h. Adventitious buds were induced on cotyledons, needles and vegetative buds cultured on WPM or QL medium supplemented with cytokinin (BAP 1–3 mg 1⁻¹). The shoot formation from induced axillary and adventitious buds was promoted on WPM or QL medium containing a low concentration of auxin (IBA 0.1 mg 1⁻¹). Shoot multiplication of Pinussylvestris was stimulated on WPM, MS, and QL media supplemented with a low concentration of cytokinin (BAP 0.2 mg 1⁻¹) and auxin (IBA 0.1 mg 1⁻¹). Shoot segments produced 2–5 new axillary shoots within 4–5 weeks. Root initiation was stimulated on larch and pine shoots cultured first on WPM supplemented with auxins (NAA and IBA) and later transferred to auxin-free medium.     

In vitro plant regeneration from callus of niger (Guizotia abyssinica Cass.) cv. Sahyadri

Callus formation was achieved with root, hypocotyl, and cotyledon explants of niger (Guizotia abyssinica Cass.) cultivar Sahyadri on Murashige and Skoog medium containing 0.5 mg l^–1 β-indoleacetic acid + 1.5 mg l^–1 6-benzylaminopurine (BAP). Hypocotyl and cotyledon-derived calli when transferred onto a medium with 0.5 mg l^–1 BAP produced an average of 12–32 shoots/ callus culture. The callus retained its potential for shoot regeneration for more than 19 months. The shoots formed an extensive root system and were transferred to pots kept in a greenhouse, where the survival rate was 98%. The plantlets flowered in vitro if transfer to fresh medium or to soil was delayed by 40–50 days. All regenerants were diploid with 2n=30. Received: 13 March 1997 / Revision received: 17 May 1997 / Accepted: 5 July 1997     

Levels of endogenous abscisic acid and indole-3-acetic acid influence shoot organogenesis in callus cultures of rice subjected to osmotic stress

Osmotic stress and endogenous hormone levels may have a role in shoot organogenesis, but a systematic study has not yet to investigate the links. We evaluated the changes of the endogenous indole-3-acetic acid (IAA) and abscisic acid (ABA) levels in rice (Oryza sativa L. cv. Tainan 5) callus during shoot organogenesis induced by exogenous plant growth regulator treatments or under osmotic stress. Non-regenerable callus showed low levels of endogenous ABA and IAA, with no fluctuation in level during the period evaluated. The addition of 100 μM ABA or 2 mM anthranilic acid (IAA precursor) into Murashige and Skoog basal induction medium containing 10 μM 2,4-D enhanced the regeneration frequency slightly, to 5 and 35%, respectively, and their total cellular ABA or IAA levels were increased significantly, correspondingly to the treatments. However, the regeneration frequency was greatly increased to 80% after treatment with 0.6 M sorbitol or 100 μM ABA and 2 mM anthranilic acid combined. Both treatments produced high levels of total cellular ABA and IAA at the callus stage, which was quickly decreased on the first day after transfer to regeneration medium. Thus, osmotic stress-induced simultaneous accumulation of endogenous ABA and IAA is involved in shoot regeneration in rice callus.